Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/415—1,2-Diazoles
  • A61K31/416—1,2-Diazoles condensed with carbocyclic ring systems, e.g. indazole
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/21—Interferons [IFN]
  • A61K38/212—IFN-alpha
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • 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
• • 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
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• HCV Hepatitis C Virus
• the 9.6-kb positive single-stranded RNA HCV genome encodes a 3,000-amino-acid polyprotein that is proteolytically processed into structural proteins, which are components of the mature virus, and nonstructural proteins (NS), which are involved in replicating the viral genome (Curr Top Microbiol Immunol 242, 55-84 (2000)).
• NS nonstructural proteins
• HCV appears to replicate in association with intracellular membrane structures.
• the structures are referred to as the membranous web (J Virol 76, 5974-5984 (2002)), the formation of which is believed to be induced by the NS4B protein.
• NS4B is also used to assemble the other viral NS proteins within the apparent sites of RNA replication (J Virol 78, 11393-11400 (2004)). It is not known how viral RNA, especially the negative strand template used for production of progeny genomes, might be incorporated or maintained at these replication sites.
• embodiments of this disclosure include compounds, compositions, pharmaceutical compositions, methods of treating a host infected with a virus from the Flaviviridae family of viruses, methods of treating HCV replication in a host, methods of inhibiting the binding of NS4B polypeptide to the 3'UTR of HCV negative strand RNA in a host, methods of treating liver fibrosis in a host, and the like.
• the present invention provides a method of treating a subject infected with a virus from the Flaviviridae family.
• the method typically comprises administering to the subject a compound of the present invention, or a pharmaceutically acceptable salt, an isomer, a tautomer or a prodrug thereof, in an amount that is effective in reducing viral load of said virus in said subject.
• the virus of the Flaviviridae family can include a flavivirus; a pestivirus; a Hepatitis C virus; a ⁇ yellow fever virus; a Dengue virus; a Japanese Encephalitis virus; a Murray Valley Encephalitis virus; a St.
• Louis Encephalitis virus a West Nile virus; a tick-borne encephalitis virus; a Kunjin virus; a Central European encephalitis virus; a Russian spring-summer encephalitis virus; a Powassan virus; a Kyasanur Forest disease virus; a Omsk hemorrhagic fever virus; and their respective genotypes and subgenotypes.
• the present invention provides a method of inhibiting formation of a complex between NS4B polypeptide and hepatitis C viral (HCV) RNA in a cell.
• the method comprises administering to the cell a compound of the present invention, or a pharmaceutically acceptable salt, an isomer, a tautomer or a prodrug thereof, in an amount that is effective in reducing binding of NS4B polypeptide to HCV RNA.
• the present invention provides a method of treating liver fibrosis in a subject.
• the method comprises administering to the subject a therapeutically effective amount of a compound of the present invention, or a pharmaceutically acceptable salt, an isomer, a tautomer or a prodrug thereof.
• any of the methods of the present invention involves administration of a compound of the present invention (or a composition (e.g., pharmaceutical) including the compound or consisting essentially of the compound) having a structure of Formula II-a, or a pharmaceutically acceptable salt or an isomer thereof:
• Ri is selected from the group consisting of: -H and wherein V is selected from alkyl, cycloalkyl, heterocyclo, aryl, and heteroaryl;
• R 3 is -H, -OH, -0(CH 2 ) n X,or -(CH 2 ) n X, wherein n is 1, 2, 3, or 4, and X is -NH 2 , - NH(alkyl), -N(alkyl) 2 , -OH, -O-alkyl, -O-aryl, -SO 2 (alkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), NHSO 2 (alkyl), heteroaryl, N-attached heterocyclo, or C-attached heterocyclo; each Of R 4 -R 7 is independently selected from the group consisting of: -H, -Br, -Cl, -
• R 4 and R 5 , R 5 and R ⁇ , or R 6 and R 7 are joined together with a bond to form a 5, 6, or 7-membered ring; or, optionally, R 4 and R 5 , R 5 and Rg, or R 6 and R 7 are joined together to form a 1,2-(methylenedioxy)benzene ring system.
• X is selected from the group consisting of: -
• R 12 is hydrogen, hydroxy, alkoxy, alkyl, ( ) , ( ) y ( ) y or -SO 2 alkyl
• R 11 is hydrogen, ( ) y ( ) ( y ) y y y or - SO 2 N(alkyl) 2
• ring A is a 5-, 6-, or 7- membered ring
• j is O, 1, or 2
• h is 1, 2, or 3.
• R 3 is selected from the group consisting of:
• Ri 2 is hydrogen, hydroxy, alkoxy, alkyl, oxo, -(CH 2 ) n -OH, - C(O)alkyl, -C(O)aryl, or -SO 2 alkyl.
• the indazole compound employed in the subject method can have any of the structures disclosed herein, Ri is -CH 2 V, wherein V is selected from cycloalkyl, heterocyclo, aryl or heteroaryl.
• the treatment methods provided herein may further comprise administering one or more additional therapeutic agents (or a composition (e.g., pharmaceutical) including one or more of these or consisting essentially of the compound and one or more of these) selected from the group consisting of: an HCV NS3 protease inhibitor, an HCV NS5B RNA- dependent RNA polymerase inhibitor, a thiazolide, a sustained release thiazolide, a nucleoside analog, an interferon-alpha, a pegylated interferon, ribavirin, levovirin, viramidine, a TLR7 agonist, a TLR9 agonist, a cyclophilin inhibitor, an alpha-glucosidase inhibitor, an NS5A inhibitor, and an NS3 helicase inhibitor.
• additional therapeutic agents or a composition (e.g., pharmaceutical) including one or more of these or consisting essentially of the compound and one or more of these) selected from the group consisting of: an HCV NS
• the present invention also provides a compound (or a composition (e.g., pharmaceutical) including the compound or consisting essentially of the compound) having a structure of Formula II-a, or a pharmaceutically acceptable salt or an isomer thereof:
• Ri is selected from the group consisting of: -H and wherein V is selected from alkyl, cycloalkyl, heterocyclo, aryl or heteroaryl;
• R 3 is -H, -OH, -O(CH 2 ) n X,or -(CH 2 ) n X, wherein n is 1, 2, 3, or 4, and X is -NH 2 , - NH(alkyl), -N(alkyl) 2 , -OH, -O-alkyl, -O-aryl, -SO 2 (alkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), NHSO 2 (alkyl), heteroaryl, N-attached heterocyclo, or C-attached heterocyclo; each OfR 4 -R 7 is independently selected from the group consisting of: -H, -Br, -Cl, -
• R 4 and R5, R5 and R 6 , or R 6 and R 7 are joined together with a bond to form a 5, 6, or 7-membered ring; or, optionally, R 4 and R 5 , R 5 and R 6 , or R 6 and R 7 are joined together to form a 1,2-(methylenedioxy)benzene ring system; provided that the
• Ri is and V is aryl.
• R 3 is -H, -OH, - O(CH 2 ) n X,or -(CH 2 ) n X, wherein n is 1, 2, 3, or 4, and X is -NH 2 , -NH(alkyl), -N(alkyl) 2 , - OH, or N-attached heterocyclo.
• X of Formula II-a is selected from the group consisting of: -OCH 3 , -OCH 2 CH 3 , -OCH 2 CH 2 OH, -OCH 2 CH 2 OCH 3 ,
• R 3 of a compound of Formula II-a is selected from the group consisting of:
• Ri is -CH 2 V, wherein V is selected from cycloalkyl, heterocyclo, or heteroaryl; and/or R 4 and R 7 are both hydrogen, and R 5 and R 6 are both a substituent other than hydrogen.
• the present invention provides a compound (or a composition (e.g., pharmaceutical) including the compound or consisting essentially of the compound) of Formula III
• V is an unsubstituted or a monosubstituted phenyl, cyclohexyl, or a 6-membered heterocyclo group where the heterocyclo group contains 1 nitrogen atom;
• R 3 is -O-L-X
• L is an unsubstituted or a monosubstituted C 1 -C5 alkylene
• X is an unsubstituted or is a substituted 5, 6, or 7 membered non aromatic heterocyclo containing at least 1 nitrogen atom, -N(R 2 o) 2 , or 4-substituted phenyl;
• R5 is hydrogen, alkyl, halo, a substituted or an unsubstituted 5, 6, 7 membered heterocyclo, or -NR 21 R 22 ; each R 2 O is independently a substituted or an unsubstituted C 1 -C 3 alkyl.
• R 21 and R 22 are each independently selected from hydrogen, substituted or unsubstituted Ci-C 3 alkyl, C 3 -C 8 cycloalkyl, aryl, or heteroaryl group, -CORi 6 , or -SO 2 R 16 , or R 21 and R 22 together with the nitrogen atom to which they are attached form a 5-7 membered substituted or unsubstituted heterocyclo group;
• Ri6 is a substituted or an unsubstituted C 1 -C 3 alkyl.
• n is 1. In another embodiment, m is 2.
• the present invention provides a compound (or a composition (e.g., pharmaceutical) including the compound or consisting essentially of the compound) of Formula III wherein X is a 5, 6, or 7 membered non aromatic heterocyclo that is an unsubstituted or is a substituted with 1-2 -OH, Ci-C 3 alkoxy, -CO 2 Ri 7 , - CON(R 18 ) 2 , a substituted or an unsubstituted 5 or 6 membered aryl or heteroaryl group, Q- C 3 alkyl, or C 1 -C 3 alkyl substituted with -OH, Ci-C 3 alkoxy, -CO 2 R] 7 , -NR 23 R 245 -CO 2 H;
• Rn is a substituted or an unsubstituted Q-C ⁇ alkyl; each Ri 8 is independently selected from hydrogen or substituted or unsubstituted Q- C 3 alkyl;
• R 23 and R 24 are each independently selected from hydrogen, a substituted or an unsubstituted aryl, heteroaryl, C 1 -C 3 alkyl, or R 23 and R 24 together with the nitrogen atom they are attached form a substituted or an unsubstituted 5-7 membered non aromatic heterocycle.
• X is 1 -pyrrolidinyl that is an unsubstituted or a substituted with 1-2 -OH, Ci-C 3 alkoxy, a substituted or an unsubstituted 6 membered aryl, Ci -C 3 alkyl, or Cj-C 3 alkyl substituted with -OH, Ci-C 3 alkoxy, or -NR 23 R 24
• X is a substituted or an unsubstituted piperidinyl.
• X is a 7-membered non aromatic heterocyclo group where the 7-membered non aromatic heterocyclo group contains 1 nitrogen atom.
• the present invention provides a compound of Formula III wherein L is -(CH 2 ) n - and n is 1, 2, 3, or 4. In another embodiment, L is 3. In another embodiment, L is 2, in another embodiment, L is 1. In another embodiment, L is 4.
• the present invention provides a compound of Formula III wherein V is 4-chlorophenyl or 4-isopropylphenyl. In another embodiment, the present invention provides a compound of Formula III wherein V is 4-chlorophenyl.
• the present invention provides a compound of Formula III wherein R 5 is hydrogen, halo, substituted or unsubstituted 5, 6, 7 membered heterocyclo, or -NR 21 R 22 .
• R 5 is hydrogen.
• R 5 is halo.
• R5 is a substituted or an unsubstituted 5 membered heterocyclo.
• R 5 is a substituted or an unsubstituted 6 membered heterocyclo.
• R 5 is a substituted or an unsubstituted 7 membered non aromatic heterocyclo.
• R 5 is NR 21 R 22 .
• R 21 and R 22 are both a substituted or an unsubstituted Q-C 3 alkyl.
• R 2 ] is a substituted or an unsubstituted Ci-C 3 alkyl.
• R 2 i is hydrogen.
• R 22 is -COR16, or -SO 2 Ri6-
• R 22 is C 3 -C 8 cycloalkyl.
• R 22 is aryl or heteroaryl.
• the present invention provides a pharmaceutical composition comprising, or consisting essentially of, the compound of Formula III and a pharmaceutically accceptable carrier, excipient, or diluent.
• the present invention provides a method of treating a subject infected with a virus from the Flaviviridae family comprising administering to the subject the compound of Formula III or a pharmaceutical composition comprising, or consisting essentially of the compound of Formula III, in an amount that is effective in reducing viral load of said virus in said subject.
• a pharmaceutical composition comprising, or consisting essentially of, a compound of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer or prodrug thereof.
• a pharmaceutical composition comprising, or consisting essentially of, a compound of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer or prodrug thereof, and further comprising one or more additional anti-HCV therapeutic agents selected from the group consisting of: an HCV NS3 protease inhibitor, an HCV NS5B RNA-dependent RNA polymerase inhibitor, a thiazolide, a sustained release thiazolide, a nucleoside analog, an interferon-alpha, a pegylated interferon, ribavirin, levovirin, viramidine, a TLR7 agonist, a TLR9 agonist, a cyclophilin inhibitor, an alpha-glucosidase inhibitor, an NS5A inhibitor, and an NS3 helicase inhibitor.
• additional anti-HCV therapeutic agents selected from the group consisting of: an HCV NS3 protease inhibitor, an HCV NS5B RNA-dependent RNA polyme
• the present invention provides a method of treating a subject infected with a virus from the Flaviviridae family.
• the method comprises administering to the subject a compound (or a composition including the compound or consisting essentially of the compound) of the present invention, or a pharmaceutically acceptable salt, isomer, tautomer or prodrug thereof, in an amount that is effective in reducing viral load of said virus in said subject.
• a compound of the present invention that has similar activity to clemizole is administered to an HCV patient in a daily dose of at least about 200 mg, i.e., about 100 mg BID.
• this about 100 mg BID administration schedule when used with these agents, will be used in treatment regimens in which at least one additional drug is also administered to the patient, i.e., treatment regimens in which a compound of the present invention is co-administered with (i) ribavirin; (ii) interferon; or (iii) ribavirin (e.g., using weight-based dosing or dosing at 15 mg/kg/day) and interferon (e.g., alpha 2a or alpha 2b, and pegylated versions of the same).
• ribavirin e.g., using weight-based dosing or dosing at 15 mg/kg/day
• interferon e.g., alpha 2a or alpha 2b, and pegylated versions of the same.
• the patient can be a previously untreated ("na ⁇ ve") patient, a patient that has not responded to a prior treatment, such as standard of care (“SOC”) therapy, a post-transplant patient, or a patient co-infected with another virus.
• SOC standard of care
• Combination therapy e.g., and without limitation, administration of a compound of the present invention in combination with ribavirin and interferon alpha
• exemplary administration schedules include: 100 mg TID; 200 mg BID; 200 mg TID; 300 mg BID; 300 mg TID; 400 mg BID; 400 mg TID; 500 mg BID; and 500 mg TID.
• exemplary administration schedules include: about 100 mg TID; about 200 mg BID; about 200 mg TID; about 300 mg BID; about 300 mg TID; about 400 mg BID; about 400 mg TID; about 500 mg BID; and about 500 mg TID.
• genotype 1 For the more difficult to treat genotype, i.e., genotype 1, more frequent dosing or higher daily doses than that provided by about 100 mg po BID or about 200 mg po BID are preferred if a Clemizole Like Analog is administered as single agent therapy.
• a compound of the present invention can be administered in combination with another drug, including but not limited to (i) ribavirin (e.g., using fixed or weight-based dosing or dosing at 15 mg/kg/day); (ii) interferon; (iii) ribavirin (e.g., using fixed or weight- based dosing or dosing at 15 mg/kg/day) and interferon (e.g., alpha 2a or alpha 2b, and pegylated versions of the same, and for other interferons as described herein and such as, but not limited to, albuferon).
• ribavirin e.g., using fixed or weight-based dosing or dosing at 15 mg/kg/day
• interferon e.g., alpha 2a or alpha 2b, and pegylated versions of the same, and for other interferons as described herein and such as, but not limited to, albuferon.
• the patient can be a previously untreated (“naive") patient, a patient that has not responded to a prior treatment, such as standard of care (“SOC”) therapy, a post-transplant patient, or a patient co-infected with another virus.
• SOC standard of care
• Combination therapy e.g., administration of a compound of the present invention in combination with ribavirin and interferon alpha, or with other direct-acting specific antivirals
• additional therapeutic agent(s) including, without limitation, an HCV
• Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of synthetic organic chemistry, biochemistry, biology, molecular biology, recombinant DNA techniques, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
• Flaviviridae virus any virus of the Flaviviridae family, including those viruses that infect humans and non-human animals.
• the polynucleotide and polypeptides sequences encoding these viruses are well known in the art, and may be found at NCBI's GenBank database, e.g., as Genbank Accession numbers NC 004102, AB031663, Dl 1355, Dl 1168, AJ238800, NC OO 1809, NC_001437, NC_004355 NC_004119, NC_003996, NC_003690, NC_003687, NC_003675, NC_003676, NC_003218, NC_001563, NC_000943, NC_003679, NC_003678, NC_003677, NC_002657, NC 002032, and NCJ)01461, the contents of which database entries are incorporated by references herein in their entirety.
• treatment As used herein, the terms “treatment”, “treating”, and “treat” are defined as acting upon a disease, disorder, or condition with an agent to reduce or ameliorate the pharmacologic and/or physiologic effects of the disease, disorder, or condition and/or its symptoms.
• Treatment covers any treatment of a disease in a host (e.g., a mammal, typically a human or non-human animal of veterinary interest), and includes: (a) reducing the risk of occurrence of the disease in a subject determined to be predisposed to the disease but not yet diagnosed as infected with the disease (b) impeding the development of the disease, and (c) relieving the disease, i.e., causing regression of the disease and/or relieving one or more disease symptoms.
• Treatment encompasses delivery of a disease or pathogen inhibiting agent that provides for enhanced or desirable effects in the subject (e.g., reduction of pathogen load, reduction of disease symptoms, and the like).
• prophylactically treat and “prophylactically treating” refer completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
• the term "host,” “subject,” “patient,” or “organism” includes humans and mammals (e.g., mice, rats, pigs, cats, dogs, and horses). Typical hosts to which compounds of the present disclosure may be administered will be mammals, particularly primates, especially humans. For veterinary applications, a wide variety of subjects will be suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals particularly pets such as dogs and cats.
• livestock such as cattle, sheep, goats, cows, swine, and the like
• poultry such as chickens, ducks, geese, turkeys, and the like
• domesticated animals particularly pets such as dogs and cats.
• living host refers to a host noted above or another organism that is alive.
• living host refers to the entire host or organism and not just a part excised (e.g., a liver or other organ) from the living host.
• isolated compound and “purified compound” mean a compound which has been substantially separated from, or enriched relative to, other compounds with which it occurs in nature. Isolated compounds are usually at least about 80%, at least 90% pure, at least 98% pure, or at least about 99% pure, by weight. The present disclosure is meant to include diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
• unit dosage form refers to physically discrete units suitable as unitary dosages for human and/or animal subjects, each unit containing a predetermined quantity of a compound (e.g., an anti-viral compound, as described herein) calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
• a compound e.g., an anti-viral compound, as described herein
• the specifications for unit dosage forms depend on the particular compound employed, the route and frequency of administration, and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.
• a “pharmaceutically acceptable excipient”, “pharmaceutically acceptable diluent”, “pharmaceutically acceptable carrier”, and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and/or adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use and/or human pharmaceutical use.
• “A pharmaceutically acceptable excipient, diluent, carrier and/or adjuvant” as used in the specification and claims includes one or more such excipients, diluents, carriers, and adjuvants.
• a "pharmaceutical composition” and a “pharmaceutical formulation” are meant to encompass a composition suitable for administration to a subject, such as a mammal, especially a human.
• a “pharmaceutical composition” or “pharmaceutical formulation” is sterile, and preferably free of contaminants that are capable of eliciting an undesirable response within the subject (e.g., the compound(s) in the pharmaceutical composition is pharmaceutical grade).
• Pharmaceutical compositions can be designed for administration to subjects or patients in need thereof via a number of different routes of administration including oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intracheal, intramuscular, subcutaneous, inhalational and the like.
• terapéuticaally effective amount and "an effective amount” are used interchangeably herein and refer to that amount of an agent (which may be referred to as a compound, an inhibitory agent, and/or a drug) being administered that is sufficient to effect the intended application including but not limited to disease treatment.
• an effective amount of an inhibiting agent will relieve to some extent one or more of the symptoms of the disease, i.e., infection, being treated, and/or that amount that will prevent, to some extent, one or more of the symptoms of the disease, i.e., infection, that the host being treated has or is at risk of developing.
• the therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
• the term also applies to a dose that will induce a particular response in target cells, e.g. inhibiting viral replication in a target cell, and inhibiting NS4B binding to viral RNA.
• the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
• “Pharmaceutically acceptable salt” refers to those salts (organic or inorganic) that retain the biological effectiveness and optionally other properties of the free bases.
• Pharmaceutically acceptable salts can be obtained by reaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid, and the like.
• salts are within the scope of the present disclosure.
• Reference to an agent of any of the formulas herein is understood to include reference to salts thereof, unless otherwise indicated.
• the term "salt(s)", as employed herein, denotes acidic and/or basic salts formed with inorganic and/or organic acids and bases.
• zwitterions inner salts
• Pharmaceutically acceptable salts are preferred, although other salts are also useful, e.g., in isolation or purification steps which may be employed during preparation. Salts of the compounds of an agent may be formed, for example, by reacting the agent with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization.
• Embodiments of the agents that contain a basic moiety may form salts with a variety of organic and inorganic acids.
• Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides (formed with hydrochloric acid), hydrobromides (formed with hydrogen bromide), hydroiodides, 2-hydroxyethanesulfon
• Embodiments of the agents that contain an acidic moiety may form salts with a variety of organic and inorganic bases.
• Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines (formed with N 5 N- bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamines, N-methyl-D-glucamides, t- butyl amines, and salts with amino acids such as arginine, lysine, and the like.
• organic bases for example, organic amines
• organic bases for example, organic amines
• benzathines such as benzathines, dicyclohexylamines, hydrabamines (formed with N 5 N- bis(dehydroa
• Basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g., benzyl and phenethyl bromides), and others. Solvates of the agents of the disclosure are also contemplated herein.
• lower alkyl halides e.g., methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
• dialkyl sulfates e.g., dimethyl, diethy
• stereoisomers of the agents such as those that may exist due to asymmetric carbons on the various substituents, including enantiomeric forms (which may exist even in the absence of asymmetric carbons) and diastereomeric forms, are contemplated within the scope of this disclosure.
• Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers, or may be admixed, for example, as racemates or with all other, or other selected, stereoisomers.
• the stereogenic centers of the compounds of the present disclosure can have the S or R configuration as defined by the IUPAC 1974 Recommendations.
• prodrug refers to an inactive precursor of an agent that is converted into a biologically active form in vivo.
• Prodrugs are often useful because, in some situations, they may be easier to administer than the parent compound. They may, for instance, be bioavailable by oral administration whereas the parent compound is not.
• the prodrug may also have improved solubility in pharmaceutical compositions over the parent drug.
• a prodrug may be converted into the parent drug by various mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, NJ. (1962). Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977). Application of Physical Organic Principles to Prodrug Design in E. B. Roche ed.
• administration refers to introducing an agent of the present disclosure into a host.
• One preferred route of administration of the agents is oral administration.
• Another preferred route is intravenous administration.
• any route of administration such as topical, subcutaneous, peritoneal, intraarterial, inhalation, vaginal, rectal, nasal, introduction into the cerebrospinal fluid, or instillation into body compartments can be used.
• aliphatic group refers to a saturated or unsaturated linear or branched hydrocarbon group and encompasses alkyl, alkenyl, and alkynyl groups, for example.
• alk refers to straight or branched chain hydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms, such as methyl, ethyl, n- propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, hexyl, heptyl, n-octyl, dodecyl, octadecyl, amyl, 2-ethylhexyl, and the like.
• An alkyl group is optionally substituted, unless stated otherwise, with one or more groups, selected from aryl (optionally substituted), heterocyclo (optionally substituted), carbocyclo (optionally substituted), halo, hydroxy, protected hydroxy, alkoxy (e.g., Ci to C 7 ) (optionally substituted), acyl (e.g., C ⁇ to C 7 ), aryloxy (e.g., Ci to C 7 ) (optionally subsituted), alkylester (optionally substituted), arylester (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, urethane, sulfonyl, and the like.
• alkenyl refers to straight or branched chain hydrocarbon groups having 2 to 12 carbon atoms, preferably 2 to 4 carbon atoms, and at least one double carbon to carbon bond (either cis or trans), such as ethenyl.
• alkenyl group is optionally substituted, unless stated otherwise,with one or more groups, selected from aryl (including substituted aryl), heterocyclo (including substituted heterocyclo), carbocyclo (including substituted carbocyclo), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkylester (optionally substituted), arylester (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), cyano, nitro, amino, substituted amino, amido, lactam, urea, urethane, sulfonyl, and the like.
• alkylene refers to divalent saturated aliphatic hydrocarbon groups having from 1 to 12 carbon atoms and, in some embodiments, from 1 to 6 carbon atoms.
• the alkylene groups include branched and straight chain hydrocarbon groups.
• “Ci- C 6 alkylene” is meant to include methylene, ethylene, propylene, butylene, 2- methypropylene, pentylene, hexylene, and the like.
• Substituted alkylene refers to an alkylene group having from 1 to 5 and, in some embodiments, 1 to 3 or 1 to 2 substituents selected from aryl (optionally substituted), heteroaryl, heterocyclo (optionally substituted), carbocyclo (optionally substituted), halo, hydroxy, protected hydroxy, alkoxy (e.g., C ⁇ to C 7 ) (optionally substituted), acyl (e.g., Cj to C 7 ), aryloxy (e.g., C ⁇ to C 7 ) (optionally subsituted), alkylester (optionally substituted), arylester (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, urethan
• alkynyl refers to straight or branched chain hydrocarbon groups having 2 to 12 carbon atoms, preferably 2 to 4 carbon atoms, and at least one triple carbon to carbon bond, such as ethynyl.
• An alkynyl group is optionally substituted, unless stated otherwise, with one or more groups, selected from aryl (including substituted aryl), heterocyclo (including substituted heterocyclo), carbocyclo (including substituted carbocyclo), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkylester (optionally substituted), arylester (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), cyano, nitro, amino, substituted amino, amido, lactam, urea, urethane, sulfonyl, and the like.
• alkoxy refers to an alkyl group linked to oxygen thus: R-O-.
• R represents the alkyl group.
• An example is the methoxy group CH 3 O-.
• Organic groups may be functionalized or otherwise comprise additional functionalities associated with the organic group, such as carboxyl, amino, hydroxyl, and the like, which may be protected or unprotected.
• alkyl group is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, t-butyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, and the like.
• alkyl group includes ethers, esters, haloalkyls, nitroalkyls, carboxyalkyls, hydroxyalkyls, sulfoalkyls, and the like.
• Cyano refers to a -CN substituent.
• halo and halogen refer to the fluoro, chloro, bromo or iodo groups. There can be one or more halogen groups, which can be the same or different. In an embodiment, each halogen can be substituted by one of the other halogens.
• haloalkyl refers to an alkyl group as defined above that is substituted by one or more halogen atoms. The halogen atoms may be the same or different.
• dihaloalkyl refers to an alkyl group as described above that is substituted by two halo groups, which may be the same or different.
• trihaloalkyl refers to an alkyl group as describe above that is substituted by three halo groups, which may be the same or different.
• perhaloalkyl refers to a haloalkyl group as defined above wherein each hydrogen atom in the alkyl group has been replaced by a halogen atom.
• perfluoroalkyl refers to a haloalkyl group as defined above wherein each hydrogen atom in the alkyl group has been replaced by a fluoro group.
• cycloalkyl refers to a mono-, bi-, or tricyclic saturated ring that is fully saturated or partially unsaturated. Examples of such a group includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, adamantyl, cyclooctyl, cis- or trans decalin, bicyclo[2.2.1]hept-2-ene, cyclohex-1-enyl, cyclopent-1-enyl, 1,4-cyclooctadienyl, and the like.
• a cycloalkyl group is optionally substituted, unless stated otherwise, with one or more groups, selected from aryl (including substituted aryl), heterocyclo (including substituted heterocyclo), carbocyclo (including substituted carbocyclo), halo, hydroxy, protected hydroxy, alkoxy (e.g., C ⁇ to C 7 ) (optionally substituted), acyl (e.g., C ⁇ to C 7 ), aryloxy (e.g., Ci to C 7 ) (optionally subsituted), alkylester (optionally substituted), arylester (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, urethane, sulfonyl, and the like
• (cycloalkyl)alkyl refers to the above-defined cycloalkyl group substituted by an above defined alkyl group. Examples of such a group include (cyclohexyl)methyl, 3-(cyclopropyl)-n-propyl, 5-(cyclopentyl)hexyl, 6- (adamantyl)hexyl, and the like.
• a (cycloalkyl)alkyl group is optionally substituted, unless stated otherwise, with one or more groups, selected from alkyl (including substituted alkyl), aryl (including substituted aryl), heterocyclo (including substituted heterocyclo), carbocyclo (including substituted carbocyclo), halo, hydroxy, protected hydroxy, alkoxy (e.g., C 1 to C 7 ) (optionally substituted), acyl (e.g., Ci to C 7 ), aryloxy (e.g., C 1 to C 7 ) (optionally subsituted), alkylester (optionally substituted), arylester (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, ure
• substituted phenyl examples include a mono- or di(halo)phenyl group such as 2, 3 or 4-chlorophenyl, 2,6-dichlorophenyl, 2,5-dichlorophenyl, 3,4-dichlorophenyl, 2, 3 or 4-bromophenyl, 3,4-dibromophenyl, 3-chloro-4-fluorophenyl, 2, 3 or 4-fluorophenyl and the like; a mono or di(hydroxy)phenyl group such as 2, 3, or 4-hydroxyphenyl, 2,4- dihydroxyphenyl, the protected-hydroxy derivatives thereof and the like; a nitrophenyl group such as 2, 3, or 4-nitrophenyl; a cyanophenyl group, for example, 2, 3 or 4- cyanophenyl; a mono- or di(alkyl)phenyl group such as 2, 3, or 4-methylphenyl, 2,4- dimethylphenyl, 2, 3 or 4-(iso-
• substituted phenyl represents disubstituted phenyl groups wherein the substituents are different, for example, 3-methyl-4-hydroxyphenyl, 3-chloro-4- hydroxyphenyl, 2-methoxy-4-bromophenyl, 4-ethyl-2-hydroxyphenyl, 3-hydroxy-4- nitrophenyl, 2-hydroxy-4-chlorophenyl and the like.
• (substituted phenyl)alkyl refers to one of the above substituted phenyl groups attached to one of the above-described alkyl groups.
• Examples of (substituted phenyl)alkyl include such groups as 2-phenyl-l -chloroethyl, 2-(4'-methoxyphenyl)ethyl, 4- (2',6'-dihydroxy phenyl)n-hexyl, 2-(5'-cyano-3'-methoxyphenyl)n-pentyl, 3-(2',6'- dimethylphenyl)n-propyl, 4-chloro-3-aminobenzyl, 6-(4'-methoxyphenyl)-3-carboxy(n- hexyl), 5-(4'-aminomethylphenyl)-3-(aminomethyl)n-pentyl, 5-phenyl-3-oxo-n-pent-1-yl, (4-hydroxyn
• aromatic refers to aromatic homocyclic (i.e., hydrocarbon) mono-, bi- or tricyclic ring-containing groups preferably having 6 to 12 members such as phenyl, naphthyl and biphenyl.
• An aryl group is optionally substituted, unless stated otherwise, with one or more groups, selected from alkyl (optionally substituted alkyl), alkenyl (optionally substituted), aryl (optionally substituted), heterocyclo (optionally substituted), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkanoyl (optionally substituted), aroyl, (optionally substituted), alkylester (optionally substituted), arylester (optionally substituted), cyano, nitro, amino, substituted amino, amido, lactam, urea, urethane, sulfonyl, and the like.
• adjacent substituents, together with the atoms to which they are bonded form a 3- to 7-member ring.
• heteroaryl refers to optionally substituted five-membered or six- membered rings that have 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen atoms, either alone or in conjunction with, additional nitrogen, sulfur or oxygen ring atoms.
• the above optionally substituted five-membered or six-membered rings can optionally be fused to an aromatic 5-membered or 6-membered ring system.
• the rings can be optionally fused to an aromatic 5-membered or 6-membered ring system such as a benzene, pyridine or a triazole system.
• a heteroaryl group is optionally substituted, unless stated otherwise, with one or more groups, selected from one to three halo, trihalomethyl, amino, protected amino, amino salts, mono-substituted amino, di-substituted amino, carboxy, protected carboxy, carboxylate salts, hydroxy, protected hydroxy, salts of a hydroxy group, lower alkoxy, lower alkylthio, alkyl (optionally, substituted), cycloalkyl (optionally substituted), (cycloalkyl)alkyl (optionally substituted), phenyl (optionally substituted), phenylalkyl (optionally substituted).
• Substituents for the heteroaryl group are as heretofore defined, or in the case of trihalomethyl, can be trifluoromethyl, trichloromethyl, tribromomethyl, or triiodomethyl.
• lower alkoxy means a C 1 to C 4 alkoxy group
• lower alkylthio means a C 1 to C 4 alkylthio group.
• heterocycle refers to fully saturated or partially unsaturated or completely unsaturated, including aromatic (“heteroaryl”) or nonaromatic cyclic groups (for example, 3- to 13-member monocyclic, 7- to 17-member bicyclic, or 10- to 20-member tricyclic ring systems, preferably containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom- containing ring.
• aromatic heteroaryl
• nonaromatic cyclic groups for example, 3- to 13-member monocyclic, 7- to 17-member bicyclic, or 10- to 20-member tricyclic ring systems, preferably containing a total of 3 to 10 ring atoms
• Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
• the heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system.
• An N-attached heterocyclo is a heterocyclo moiety wherein the heterocyclo moiety is attached to a compound, e.g. a compound of Formula I through a nitrogen that forms part of the heterocyclo ring.
• N- attached heterocyclo examples include but are not limited to
• a C-attached heterocyclo is a heterocyclo moiety wherein the heterocyclo moiety is attached to a compound, e.g., a compound of formula II-a, b, or c through a carbon that
• Non-limiting examples include
• the rings of multi-ring heterocycles may be fused, bridged and/or joined through one or more spiro unions.
• Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2- oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, pyridyl, pyrazinyl, pyrimidinyl, pyri
• bicyclic heterocyclic groups include indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinuclidinyl, quinolinyl, tetra-hydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, benzofuranly, dihydrobenzofuranyl, chromonyl, coumarinyl, benzodioxolyl, dihydrobenzodioxolyl, benzodioxinyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl, or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydro
• Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like.
• a heterocyclo group is optionally substituted, unless stated otherwise, with one or more groups, selected from alkyl (including substituted alkyl), alkenyl, oxo, aryl (including substituted aryl), heterocyclo (including substituted heterocyclo), carbocyclo (optionally substituted), halo, hydroxy, alkoxy (optionally substituted), aryloxy (optionally substituted), alkanoyl (optionally substituted), aroyl (optionally substituted), alkylester (optionally substituted), arylester (optionally substituted), cyano, nitro, amido, amino, substituted amino, lactam, urea, urethane, sulfonyl, and the like where optionally one or more pair of substituents together with the atoms to which they are bonded form a 3- to 7-member ring.
• alkanoyl refers to an alkyl group (which may be optionally substituted as described above) linked to a carbonyl group (i.e. --C(O)-alkyl).
• aroyl refers to an aryl group (which may be optionally substituted as described above) linked to a carbonyl group (i.e., --C(O)-aryl).
• (monosubstituted)amino refers to an amino group with one substituent chosen from the group consisting of phenyl, substituted phenyl, alkyl (including substituted alkyl), C 1 to C 4 acyl, C 2 to C 7 alkenyl (including C 2 to C 7 substituted alkenyl), C 2 to C 7 alkynyl, C 7 to alkylaryl (including C 7 to Cj 6 substituted alkylaryl), and heteroaryl group.
• the (monosubstituted) amino can additionally have an amino-protecting group as encompassed by the term "protected (monosubstituted)amino."
• the term "(disubstituted)amino” refers to amino groups with two substituents chosen from the group consisting of phenyl, substituted phenyl, alkyl, substituted alkyl, Ci to C 7 acyl, C 2 to C 7 alkenyl, C 2 to C 7 alkynyl, C 7 to Ci 6 alkylaryl, C 7 to Ci 6 substituted alkylaryl and heteroaryl. The two substituents can be the same or different.
• heteroaryl(alkyl) refers to an alkyl group as defined above, substituted at any position by a heteroaryl group, as above defined.
• “Isosteres” are different atoms, molecules, or ions that have different molecular formulae but have similar or identical outer shell electron arrangements and also have similar properties (e.g., pharmacological properties (e.g., pharmacokinetic and pharmacodynamic)).
• “Moiety” refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
• Niro refers to the -NO 2 radical.
• Oxa refers to the -O- radical.
• “Sulfonyl” refers to the groups: -S(O 2 )-H, -S(O 2 )-(alkyl), -S(O 2 )-(cycloalkyl), -S(O 2 )-(amino), -S(O 2 )-(aryl), -S(0 2 )-(heteroaryl), and -S(0 2 )-(heterocycloalkyl).
• “Sulfonamidyl” or “sulfonamido” refers to a -S(O) 2 -NRR radical, where each R is selected independently from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon).
• the R groups in -NRR of the -S(O) 2 -NRR radical may be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6-, or 7-membered ring (-S(O 2 )-(heterocycloalkyl).
• each R in sulfonamido contains 1 carbon, 2 carbons, 3 carbons, or 4 carbons total.
• a sulfonamido group is optionally substituted by one or more of the substituents described herein for alkyl, cycloalkyl, aryl, heteroaryl respectively.
• a “sulfone” refers to a -S(O 2 )-(alkyl), -S(O 2 )-(aryl), -S(O 2 )-(heteroaryl), or -S(O 2 )-(heterocycloalkyl) (when the sulfone group is attached to a carbon atom in the heterocycloalkyl).
• a sulfonamido group is optionally substituted by one or more of the substituents described herein for alkyl, cycloalkyl, aryl, and heteroaryl.
• solvent each mean a solvent inert under the conditions of the reaction being described.
• Nonlimiting examples include benzene, toluene, acetonitrile, tetrahydrofuran (“THF”), dimethylformamide (“DMF”), chloroform, methylene chloride (or dichloromethane), diethyl ether, methanol, N-methylpyrrolidone (“NMP”), pyridine and the like.
• protecting group refers to chemical moieties that block some or all reactive moieties of a compound and prevent such moieties from participating in certain chemical reactions until the protective group is removed, for example, those moieties listed and described in T.W. Greene, P.G.M. Wuts, Protective Groups in Organic Synthesis, 3rd ed. John Wiley & Sons (1999). It may be advantageous, where different protecting groups are employed, that each (different) protective group be removable by a different means. Protective groups that are cleaved under totally disparate reaction conditions allow differential removal of such protecting groups. For example, protective groups can be removed by acid, base, and hydrogenolysis.
• Groups such as trityl, dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile and may be used to protect carboxy and hydroxy reactive moieties in the presence of amino groups protected with Cbz groups, which are removable by hydrogenolysis, and Fmoc groups, which are base labile.
• Carboxylic acid moieties may be blocked with base labile groups such as, without limitation, methyl, or ethyl, and hydroxy reactive moieties may be blocked with base labile groups such as acetyl in the presence of amines blocked with acid labile groups such as tert-butyl carbamate or with carbamates that are both acid and base stable but hydrolytically removable.
• ring can refer to a chemical moiety having a ring structure comprising 3 to 10 carbon atoms in which one or more carbon atoms may be optionally substituted with a heteroatom, such as N, O, or S.
• a ring may or may not be aromatic and thus may be completely unsaturated, completely saturated, or partially unsaturated; and a ring may refer to a ring within a fused system or an unfused ring. Unless otherwise stated, this definition of "ring” does not modify other definitions of rings provided herein.
• compositions comprising, “comprising,” “containing” and “having” and the like can have the meaning ascribed to them in U.S. Patent law and can mean “includes,” “including,” and the like; “consisting essentially of or “consists essentially” or the like, when applied to methods and compositions encompassed by the present disclosure refers to compositions like those disclosed herein, but which may contain additional composition components or method steps. Such additional composition components or method steps, etc., however, do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.
• NS4B a virus that encodes NS4B.
• virus includes any virus of the Flaviviridae family encompassing e.g., flaviviruses, pestiviruses and hepatitis C viruses.
• Other NS4B encoding viruses include yellow fever virus (YFV); Dengue virus, including Dengue types 1-4; Japanese Encephalitis virus; Murray Valley Encephalitis virus; St.
• the subject methods and compositions are particularly useful for treating or prophylactically treating HCV, including one or more genotypes 1, 2, 3, 4, 5, 6, and the like, as well as subtypes of an HCV genotype (e.g., Ia, Ib, 2a, 2b, 3a, and the like).
• the method of treating such viral infection comprises administering to a subject infected with a virus from the Flaviviridae family, an effective amount of an isostere of a benzoimidazole core structure, or a pharmaceutically acceptable salt, an isomer, a tautomer or a prodrug thereof.
• the isostere is an indazole.
• the subject method is effective in reducing viral load in the infected subject by e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95% or even higher as compared to the level of viral load present in the subject prior to such treatment.
• the reduction in viral load can be effected, in whole or in part, by reducing binding of NS4B polypeptide to the viral genome.
• a decrease in viral load upon administering a compound of the present invention can be attributable to, in part, a decrease in binding of NS4B polypeptide to HCV negative strand RNA, e.g., at a site on the 3'UTR.
• the subject methods can also utilize one or more other isosteres, including, but not limited to, Hl receptor antagonists that share structural similarity with clemizole and exhibit anti-viral activity.
• Hl receptor antagonists that share structural similarity with clemizole include, but are not limited to, the compounds in the classes known as alcoholamines ⁇ e.g., diphenhydramine, carbinoxamine, and clemastine), ethylenediamines (e.g., mepyramine and tripelennamine (clemizole is in this class)), alkylamines ⁇ e.g., triprolidine and chlorpheniramine), piperazines (e.g., meclizine and homchlorcyclizine), and phenothiazines (e.g., promethazine).
• alcoholamines ⁇ e.g., diphenhydramine, carbinoxamine, and clemastine
• ethylenediamines e.g., mepyramine and tripelennamine (cle
• the subject treatment methods can also employ prodrugs of the compounds provided herein.
• exemplary prodrugs can be activated by liver enzymes (e.g., cyclic-1,3- propanyl esters substituted with groups that promote an oxidative cleavage reaction by CYP3A, and the like.). These modifications can render the compounds of the present invention inactive or less active until it reaches the liver (see, Current Opinion in Investigational Drugs 2006 VoI 7 No 2, 109-117; J. Med. Chem. 2008, 51, 2328-2345; and Nucleosides, Nucleotides, and Nucleic Acids, 24 (5 - 7):375— 381, (2005) each of which is incorporated herein by reference for the corresponding discussion).
• an exemplary indazole compound has the structure of Formula II-a or Formular II-b:
• Ri is -H or
• V is selected from alkyl, cycloalkyl, heterocyclo, aryl or heteroaryl.
• alkyl moiety is an unsubstituted or a substituted.
• the alkyl moiety of Ri includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso- butyl, tert-butyl, n- pentyl, iso-pentyl, n-hexyl, septyl, heptyl, nonyl, and decyl.
• R 1 is , wherein the cycloalkyl moiety is unsubstituted or substituted.
• Nonlimiting exemplary Ri include the following formulae:
• the invention further provides compound of Formula II-a or II-b, wherein Ri is of
• An N- attached heterocyclo is a heterocyclo moiety wherein the heterocyclo moiety is attached to the compound of formula II-a or b through a nitrogen that forms part of the heterocyclo
• Non-limiting N- attached heterocyclo include, but are not limited to,
• an N- attached heterocyclo is a moiety having the following formula:
• h is 1, 2, or 3
• R 12 is hydrogen, hydroxy, alkoxy, alkyl, oxo, -(CH 2 ) n -0H, - C(O)alkyl, -C(O) 2 alkyl -C(O)aryl, or -S0 2 alkyl.
• Examples include, but are not limited to,
• a C- attached heterocyclo is a heterocyclo moiety wherein the heterocyclo moiety is attached to the compound of formula II-a or b through a carbon that forms part of the heterocyclo ring.
• a C- attached heterocyclo is a moiety of the following formula:
• R 11 is hydrogen, -C(O)alkyl, -C(O)aryl, -C(O)NH 2 , -C(O)NHalkyl, -C(O)N(alkyl) 2 , -SO 2 alkyl, -S0 2 aryl, -SO 2 NH 2 , -SO 2 NHalkyl, or -SO 2 N(alkyl) 2 ;
• ring A is a 5-, 6-, or 7- membered ring, and j is 0, 1, or 2. Examples include, but are not limited to,
• Non-limiting exemplary Ri include the following formulae:
• alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• Ri of Formula II-a or II-b is , wherein the aryl moiety is unsubstituted or substituted.
• the aryl is one of the following moieties:
• X 1 -X5 are each independently selected from the group consisting of: l
• Non-limiting exemplary Ri include the following formulae:
• R, of Formula II-a or II-b can be , wherein the heteroaryl moiety is unsubstituted or substituted.
• the heteroaryl moiety is a monocylic 5 membered heteroaryl.
• Monocyclic heteroaryl includes, but is not limited to, pyrrolyl, imidazolyl, thiazolyl, and pyrazolyl.
• Ri when Ri is heteroaryl may be a six membered hetereoaryl moiety.
• the six membered heteroaryl moiety includes, but is not limited to, 2- pyridyl, 3-pyridyl, 4-pyridyl, pyrimidinyl, pyrazinyl, or trianzinyl.
• the R1 heteroaryl is N-(2-aminoethyl)-2-aminoethyl
• X 6 is selected from the group consisting of: -H and X 7 and X 8 are independently selected from the group consisting of: H and CH 3 .
• R 1 inlcude the following formulae:
• the alkyl, cycloalkyl, heterocyclo, aryl or heteroaryl moiety of Rj may be substituted by one or more substituents selected from the group consisting of alkyl, aryl, heterocyclo, carbocyclo, halo, hydroxy, protected hydroxy, alkoxy, acyl, aryloxy, alkylester, arylester , alkanoyl , aroyl, carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, urethane, and sulfonyl.
• R 3 is -H, -OH, - 0(CH 2 ) n X,or -(CH 2 ) n X, wherein n is 1, 2, 3, or 4, and X is -NH 2 , -NH(alkyl), -N(alkyl) 2 , - OH, -O-alkyl, -O-aryl, -SO 2 (alkyl), -SO 2 NH 2 , -SO 2 NH(alkyl), NHSO 2 (alkyl), heteroaryl, N-attached heterocyclo, or C-attached heterocyclo.
• R 3 is -H or -OH.
• R3 is -O(CH 2 ) n X,or -(CH2) n X, wherein X is -NH 2 .
• Non limiting examples include: -OCH 2 CH 2 NH 2 and -CH 2 CH 2 NH 2 .
• R 3 is - 0(CH 2 ) n X, or -(CH 2 ) n X, wherein X is -NH(alkyl), wherein the alkyl is substituted or unsubstituted.
• Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• R 3 -0(CH 2 ) n NH(alkyl),or -(CH 2n NH(alkyl), include, but are not limited to, - OCH 2 CH 2 NHMe, -OCH 2 CH 2 CH 2 NHEt, -CH 2 CH 2 NH(iso-propyl), and -CH 2 CH 2 CH 2 NHMe.
• the invention also provides compounds of Formula II, II-a, and II-b, wherein R 3 is - 0(CH 2 n X,or -(CH 2 ) n X, wherein X is -N(alkyl) 2 ), wherein the alkyl is substituted or unsubstituted.
• Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• R 3 -O(CH 2 ) n N(alkyl) 2 ,or -(CH 2 ) n N(alkyl) 2 include, but are not limited to, - OCH 2 CH 2 (Me) 2 , -OCH 2 CH 2 CH 2 N(Et) 2 , -CH 2 CH 2 N(iso-propyl) 2 , and -CH 2 CH 2 CH 2 N(Me) 2 -
• R 3 is -O(CH 2 ) n X,or -(CH 2 )Nx, wherein X is -OH. Examples include, but are not limited to, -OCH 2 CH 2 OH, -OCH 2 CH 2 CH 2 OH, -CH 2 CH 2 OH, and - CH 2 CH 2 CH 2 OH.
• R3 is -O(CH 2 ) n X,or -(CH 2 ) n X, wherein X is O-alkyl, wherein the alkyl is unsubstituted or substituted.
• Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• R 3 is -O(CH 2 ) n O-alkyl, or -(CH 2 ) n 0- alkyl, include, but are not limited to, -OCH 2 CH 2 OMe, -OCH 2 CH 2 CH 2 OEt, -CH 2 CH 2 O(iso- propyl), and -CH 2 CH 2 CH 2 OEt.
• R 3 is -O(CH 2 ) n X,or -(CH 2 ) n X, wherein X is -O-aryl, wherein the aryl is substituted or unsubstituted.
• Aryl includes, but is not limited to, phenyl, naphthyl and fluorenyl.
• R 3 examples include, but are not limited to, -O(CH 2 ) n O-aryl,or - (CH 2 ) n O-aryl include, but are not limited to, -OCH 2 CH 2 O-phenyl, -OCH 2 CH 2 CH 2 O-(3- methoxy-phenyl), -CH 2 CH 2 O-(4-methyl phenyl), and -CH 2 CH 2 CH 2 O-phenyl.
• R 3 is -O(CH 2 ) n X,or -(CH 2 ) n X, wherein X is -SO 2 (alkyl) and alkyl is unsubstituted or substituted.
• Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• Examples include, but are not limited to, -OCH 2 CH 2 SO 2 Me, -OCH 2 CH 2 CH 2 SO 2 Et, -CH 2 CH 2 SO 2 (butyl) and -CH 2 CH 2 CH 2 SO 2 Me.
• R 3 is - O(CH 2 ) n X,or -(CH 2 ) n X, wherein X is -SO 2 NH 2 or-SO 2 NH(alkyl), and alkyl is unsubstituted or substituted.
• Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• Examples include, but are not limited to, -OCH 2 CH 2 SO 2 NH 2 , -OCH 2 CH 2 CH 2 SO 2 NHMe, - CH 2 CH 2 SO 2 NH 2 and -CH 2 CH 2 CH 2 SO 2 NHMe.
• R 3 is -O(CH 2 ) n X,or -(CH 2 ) n X, wherein X is NHSO 2 (alkyl), and alkyl is unsubstituted or substituted.
• Alkyl includes, but is not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n- pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl. Examples include, but are not limited to, -OCH 2 CH 2 NHSO 2 Me, - OCH 2 CH 2 CH 2 NHSO 2 Et, -CH 2 CH 2 NHSO 2 Me and -CH 2 CH 2 CH 2 SO 2 NHSO 2 Et.
• the invention also provides compounds of Formula II-a and II-b, wherein R 3 is - O(CH 2 ) n X,or -(CH 2 ) n X, wherein X is heteroaryl, and the heteroaryl is unsubstituted or substituted.
• the heteroaryl moiety is a monocylic 5 membered heteroaryl.
• Monocyclic heteroaryl includes, but is not limited to, pyrrolyl, imidazolyl, thiazolyl, and pyrazolyl. Additional nonlimiting monocyclic 5 membered heteroaryl moieties include the following formulae:
• Y is selected from the group consisting of: - O, -S, -NH, -N-alkyl, and -N-acyl
• X 3 is selected from the group consisting of: -H, -CH 3 , - Cl, -I, -F, CF 3 and -OCH 3
• X 4 and X 5 are, when present, independently selected from the group consisting of: H and CH 3 .
• heteroaryl may be a six membered hetereoaryl moiety.
• the six membered heteroaryl moiety includes, but is not limited to, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidinyl, pyrazinyl, or trianzinyl.
• Non limiting R 3 -O(CH 2 ) n -heteroaryl or - (CH 2 ) n heteroaryl include -OCH 2 CH 2 -pyridinyl, -OCH 2 CH 2 CH 2 -(4-methyl-pyridin-2-yI), - CH 2 CH 2 -(thiazolyl), and -CH 2 CH 2 CH 2 -pyrazinyl.
• R 3 is -0(CH 2 ) n X,or -(CH 2 ) n X, wherein X is N-attached heterocyclo, or C-attached heterocyclo, and the heterocyclo is unsubstituted or substituted.
• the heterocyclo includes, but is not limited to, azetidinyl, pyrrolidinyl, morpholinyl, piperidinyl, or piperazinyl.
• R 3 -O(CH 2 ) n -heterocyclo which includes both N- attached or C-attached heterocyclo
• -(CH 2 ) n heterocyclo which includes both ⁇ - attached or C-attached heterocyclo
• -OCH 2 CH 2 -morpholinyl include -OCH 2 CH 2 CH 2 -(4 ⁇ -methyl- piperazinyl), -CH 2 CH 2 -(pyrrolidin-2-yl), and -CH 2 CH 2 CH 2 -(4N-methyl-piperazinyl).
• the alkyl, aryl, heteroaryl and heterocyclo moiety forming all or part of X may be substituted by one or more substituents, which is selected from the group consisting of alkyl, aryl, heterocyclo, carbocyclo, halo, hydroxy, protected hydroxy, alkoxy, acyl, aryloxy, alkylester, arylester, alkanoyl, aroyl, carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, urethane, and sulfonyl. Additionally, the alkyl and heterocyclo moiety forming all or part of X may be substituted by oxo.
• each OfR 4 -R 7 is independently selected from the group
• the alkyl and aryl moieties are unsubstituted or substituted.
• the alkyl moieties that form part OfR 3 -R 7 include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-pentyl, iso-pentyl, n- hexyl, septyl, heptyl, nonyl, and decyl.
• the aryl moieties that form part OfR 3 -R 7 include, but are not limited to, phenyl, naphthyl and fluorenyl.
• the alkyl and aryl moieties that form part OfR 3 -R 7 may be substituted by one or more substituents, which is selected from the group consisting of alkyl, aryl, heterocyclo, carbocyclo, halo, hydroxy, protected hydroxy, alkoxy, acyl, aryloxy, alkylester, arylester, alkanoyl, aroyl, carboxy, protected carboxy, cyano, nitro, amino, substituted amino, (monosubstituted)amino, (disubstituted)amino, protected amino, amido, lactam, urea, urethane, and sulfonyl.
• At least one OfR 4 -R 7 is a hydrogen. In other embodiments, where R 3 is present, R 3 is hydrogen. In further embodiments, at least two OfR 4 -R 7 is a hydrogen. Alternatively, at least two OfR 4 -R 7 are hydrogen, and the remaining R 4 -R 7 groups (and R 3 , if present) are independently selected from the group consisting of: -Cl, -F, -CH 3 , and -OCH 3 .
• R 5 and Re are substituted, and the substituted moiety is, for each substituted position, independently selected from the group consisting of: -Cl, -F, -CH 3 , and -OCH 3 , while R 4 and R 7 (and R 3 if present) are hydrogen.
• R 4 and R 5 , R 5 and Re, or R 6 and R 7 are joined together with a bond to form a ring; or, optionally, R 4 and R 5 , R 5 and R 6 , or R 6 and R 7 are joined
• the ring is composed of a structure selected from the group consisting of:
• R 5 and R 6 are connected by one of the rings
• the compound of Formula II-a is not
• the present invention provides a compound of Formula II-a shown below.
• the compound of Formula II-a is the compound wherein R] is
• alkyl, aryl are each independently selected from the group consisting of: - is selected from the group consisting of: -O, -S, -NH, -N-alkyl, and -N-acyl;
• Xe is selected from the group consisting of: -H, -CH3, -I, -Cl, -F, CF 3 and -OCH3;
• X 7 and Xs are independently selected from the group consisting of: H and CH 3 ; each OfR 4 -R 7 is independently selected from the group consisting of: -H, -I, -Br, -Cl, -F, -CH 3 , -CN, -OH, -
• R 4 and R 5 , R 5 and R 6 , or R 6 and R 7 are joined together with a bond to form a 5, 6, or 7-membered ring; or, optionally, R 4 and R5, R5 and R 6 , or R 6 and R 7 are joined together to form a 1 ,2-(methylenedioxy)benzene ring system.
• the compound of Formula II-a is the compound wherein R 3 is wherein X is selected from the group consisting of:
• the compound of Formula II-a is the compound wherein Ri is selected from the group consisting of:
• the compound of Formula II-a is the compound, wherein Ri is wherein V is selected from the group consisting of: cycloalkyl, heterocyclo, aryl and heteroaryl; wherein X is selected from the group
• R 7 are both hydrogen, and R 5 and R 6 are both a substituent other than hydrogen.
• the compound of Formula II-a is the compound wherein
• Ri is selected from the group consisting of X ,
• the invention also provides compounds of Formula II-a wherein Ri wherein V is selected from cycloalkyl, heterocyclo, aryl or heteroaryl; (CH 2 ) n X, wherein X is selected from the group consisting of:
• R 4 is - NHSO 2 alkyl
• R 7 is hydrogen and R5 and R ⁇ are both a substituent other than hydrogen.
• the compounds of Formula II-a are also a compound wherein Ri is selected from
• R 3 is selected from: -
• R 7 is hydrogen and R 5 and R 6 are both not hydrogen.
• Table 1 shows structures of additional compounds (also referred to as “inhibiting agents”, (compounds and inhibiting agents are interchangeable as is appropriate for the particular usage herein)) of the invention and illustrative starting materials to prepare them
• Table 2a shows structures of additional inhibiting agents of the invention based on the structure below.
• Some nonlimiting illustrative compounds of the present invention having a structure of Formula II-a include those in which Ri is any Ri moiety described in Table 2a, in combination with any R 3 moiety described in Table 2b, and any R 4 R 5 , R 6 , and R 7 as described in Table 2c.
• a compound of Formula II-a includes any combination of Ri, R 3 , R 4 R 5 , Re, and R 7 . Additional exemplary compounds of Formula II-a are illustrated in Tables 3, 4 and 5.
• R 4 , R 5 , R 6 , and R 7 moieties of the compounds of Formula II-a include, but are not limited to, the following:
• Some non-limiting illustrative compounds of the present invention having a structure of Formula II-b include those in which Ri is any Ri moiety described in Table 2d, in combination with any R3 moiety described in Table 2e, and any R 4 R 5 , R 6 , and R 7 as described in Table 2f.
• a compound of Formula II-b includes any combination OfR 1 , R 3 , R 4 , Rs, R O , and R7. Additional exemplary compounds of Formula II-b are illustrated in Tables 3, 4, and 5.
• Ri moieties of Formula II-b include, but are not limited to, the following:
• R3 moieties of the compounds of Formula II-b include, but are not limited to, the following:
• Embodiments of the present invention include prodrugs of the compounds of Formula II-a or II-b.
• the present invention provides a compound of Formula III
• m is 1 or 2;
• V is an unsubstituted or a monosubstituted phenyl, cyclohexyl, or a 6-membered heterocyclo group where the heterocyclo group contains 1 nitrogen atom;
• R 3 is -O-L-X
• L is an unsubstituted or a monosubstituted Ci-C 5 alkylene
• X is an unsubstituted or is a substituted 5, 6, or 7 membered non aromatic heterocyclo containing at least 1 nitrogen atom, -N(R 20 ) 2 , or 4-substituted phenyl;
• R. 5 is hydrogen, alkyl, halo, substituted or unsubstituted 5, 6, 7 membered heterocyclo, or -NR21R22; each R 20 is independently substituted or unsubstituted C 1 -C3 alkyl.
• R 21 and R 22 are each independently selected from hydrogen, a substituted or an unsubstituted C 1 -C 3 alkyl, C 3 -C 8 cycloalkyl, aryl, or heteroaryl group, -COR) 6 , or -SO 2 Ri 6 , or R 21 and R 22 together with the nitrogen atom to which they are attached form a 5-7 membered a substituted or an unsubstituted heterocyclo group;
• Ri 6 is a substituted or an unsubstituted C 1 -C3 alkyl.
• n is 1. In another embodiment, m is 2.
• the present invention provides a compound of Formula III wherein X is a 5, 6, or 7 membered non aromatic heterocyclo that is an unsubstituted or is sa ubstituted with 1-2 -OH, C r C 3 alkoxy, -CO 2 Rn, -CON(R 1 g ) 2 , a substituted or an unsubstituted 5 or 6 membered aryl or heteroaryl group, C 1 -C 3 alkyl, or Ci-C 3 alkyl substituted with -OH, C 1 -C 3 alkoxy, -CO 2 R 17, -NR 23 R 24, -CO 2 H;
• R 17 is a substituted or an unsubstituted Ci-C 6 alkyl; each R 18 is independently selected from hydrogen or a substituted or an unsubstituted Ci-C 3 alkyl;
• R 23 and R 24 independently are hydrogen, a substituted or an unsubstituted aryl, heteroaryl, C 1 -C 3 alkyl, or R 23 and R 24 together with the nitrogen atom they are attached form a substituted or an unsubstituted 5-7 membered non aromatic heterocycle.
• X is 1-pyrrolidinyl that is an unsubstituted or a substituted with 1-2 -OH, Ci-C 3 alkoxy, a substituted or an unsubstituted 6 membered aryl, C 1 C 3 alkyl, or C 1 -C 3 alkyl substituted with -OH, C 1 -C 3 alkoxy, or -NR 23 R 24
• X is a substituted or an unsubstituted piperidinyl.
• X is a 7-membered non aromatic heterocyclo group where the 7-membered non aromatic heterocyclo group contains 1 nitrogen atom.
• the present invention provides a compound of Formula III wherein L is -(CH 2 ) n - and n is 1, 2, 3, or 4. In another embodiment, L is 3. In another embodiment, L is 2, in another embodiment, L is 1. In another embodiment, L is 4.
• the present invention provides a compound of Formula III wherein V is 4-chlorophenyl or 4-isopropylphenyl. In another embodiment, the present invention provides a compound of Formula III wherein V is 4-chlorophenyl. In another embodiment, the present invention provides a compound of Formula III wherein R 5 is hydrogen, halo, a substituted or an unsubstituted 5, 6, 7 membered heterocyclo, or -NR 21 R 22. In another embodiment, R 5 is hydrogen. In another embodiment, R 5 is halo. In another embodiment, R5 is a substituted or an unsubstituted 5 membered heterocyclo. In another embodiment, R 5 is a substituted or an unsubstituted 6 membered heterocyclo.
• R5 is a substituted or an unsubstituted 7 membered non aromatic heterocyclo.
• R5 is NR 21 R 22 -
• R 21 and R 22 are both a substituted or an unsubstituted C 1 -C 3 alkyl.
• R 21 is a substituted or an unsubstituted C 1 -C3 alkyl.
• R 21 is hydrogen.
• R 22 is -COR16, or -SO 2 Ri6- In another embodiment, R 22 is C 3 -C 8 cycloalkyl.
• R 22 is ary! or heteroaryl.
• the present invention provides the compunds disclosed in Table 6 below. In another embodiment, the present invention provides the compunds disclosed in Table 7 below.
• the compounds provided herein as viral inhibiting agents are generally capable of inhibiting viral replication in vitro and/or in vivo.
• a compoud of the present invention when contacted with an HCV-infected cell (e.g., an HCV-infected liver cell), reduces the amount of infectious HCV viral particles produced by the HCV-infected cell by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% or even higher, compared to the number of infectious HCV viral particles produced by the cell not contacted with the inhibiting agent.
• In vitro assay typically determines the number of viral particles present in the culture medium, wherein an in vivo assay typically measures the viral titer present in a bodily fluid of an infected subject.
• Bodily fluids suitable for viral titer measurement include, but are not limited to, blood, serum, plasma, saliva, semen, spinal fluid, urine, sweat, and cerebral spinal fluid.
• Commonly employed methods for detecting viral load in vitro or in vivo include quantitative polymerase chain reaction (PCR) and branched DNA (bDNA) test. Numerous quantitative assays for measuring the viral load (titer) of HCV RNA have been developed.
• RT-PCR quantitative reverse transcription PCR
• Amplicor HCV MonitorTM Roche Molecular Systems, New Jersey
• bDNA branched DNA (deoxyribonucleic acid) signal amplification assay
• bDNA branched DNA (deoxyribonucleic acid) signal amplification assay
• NAT nucleic acid test
• the compounds provided herein can also be characterized by their ability to inhibit binding of NS4B polypeptide to the 3'UTR of HCV negative strand RNA by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% or higher, compared to the binding of NS4B polypeptide to the 3'UTR of HCV negative strand RNA in the absence of the compound.
• the inhibiting agents of the present invention inhibit binding of NS4B polypeptide to the 3'UTR of HCV negative strand RNA with a 50% inhibitory concentration (IC 50 ) of about 100 ⁇ M to 50 ⁇ M, about 50 ⁇ M to 25 ⁇ M, about 25 ⁇ M to 10 ⁇ M, about 10 ⁇ M to 5 ⁇ M, about 5 ⁇ M to 1 ⁇ M, about 1 ⁇ M to 500 nM, about 500 nM to 400 nM, about 400 nM to 300 nM, about 300 nM to 250 nM, about 250 nM to 200 nM, about 200 nM to 150 nM, about 150 nM to 100 nM, about 100 nM to 50 nM, about 50 nM to 30 nM, about 30 nM to 25 nM, about 25 nM to 20 nM, about 20 nM to 15 nM, about 15 nM to 10 nM, about 10 nM to 5
• the inhibiting agents of the present invention lack substantial cross-reactivity with HERG K + channel.
• Drug-induced cardiac arrhythmia such as QT prolongation
• QT prolongation is a serious safety concern in the discovery, development and use of new medications.
• Drug-induced QT interval prolongation is an active field of research and has been reviewed (Pearlstein et al. J. Med. Chem. (2003), 46(11):2017-2022; Fermini et al., Annual Reports in Medicinal Chemistry (2004), 39:323; http://www.qtdrugs.org).
• a common cause of QT prolongation is the inhibition of the cardiac HERG K + channel by a drug. Drugs from widely different chemical classes and therapeutic utility have been shown to block HERG activity.
• an exemplary inhibiting agent of the present invention has a HERG IC50 of greater than about 100 nM.
• the inhibiting agent described herein has a HERG IC50 of greater than about 500 nM, 1,000 nM, 5,000 nM, 1 ⁇ M, 5 ⁇ M, 10 ⁇ M, 50 ⁇ M, 100 ⁇ M or even higher.
• the inhibiting agents provided can be made according to organic synthesis techniques known to those skilled in this art and/or according to the synthesis schemes provided herein.
• synthesis of the subject compound begins with commercially available chemicals and/or from compounds described in the chemical literature. "Commercially available chemicals” may be obtained from standard commercial sources including Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee WI, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co.
• reaction times and conditions are intended to be approximate, e.g., taking place at about atmospheric pressure within a temperature range of about -10 °C to about 110 °C over a period of about 1 to about 24 hours; reactions left to run overnight average a period of about 16 hours.
• the solvents used in the reactions described herein are inert organic solvents. Unless specified to the contrary, for each gram of the limiting reagent, one cc (or mL) of solvent constitutes a volume equivalent.
• Isolation and purification of the chemical entities and intermediates described herein caOn be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography or thick-layer chromatography, or a combination of these procedures.
• suitable separation and isolation procedures can be had by reference to the examples hereinbelow. However, other equivalent separation or isolation procedures can also be used.
• the (R)- and (S)-isomers of the compounds of the present invention may be resolved by methods known to those skilled in the art, for example by formation of diastereoisomeric salts or complexes which may be separated, for example, by crystallization; via formation of diastereoisomeric derivatives which may be separated, for example, by crystallization, gas-liquid or liquid chromatography; selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic oxidation or reduction, followed by separation of the modified and unmodified enantiomers; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support, such as silica with a bound chiral ligand or in the presence of a chiral solvent.
• a specific enantiomer may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one
• the compounds described herein can be optionally contacted with a pharmaceutically acceptable acid to form the corresponding acid addition salts.
• the compounds of the invention can be synthesized by an appropriate combination of known synthetic methods in the art and the instant disclosure.
• the discussion below is offered to illustrate certain of the diverse methods available for use in making the compounds of the invention and is not intended to limit the scope of reactions or reaction sequences that can be used in preparing the compounds of the present invention.
• Scheme 3 illustrates the synthesis of compounds of the invention.
• 3-indazolinone 3.1 can be alkylated in high yields by treatment with NaOH in EtOH, followed by treatment with a substituted benzyl halide to provide 3.2 (Japanese Patent JP49007278).
• Deprotonation followed by alkylation with a protected bromo-alcohol provides 3.3.
• Deprotection of the alcohol followed by activation and displacement with primary and secondary amines and/or alkoxides provides 3.4.
• Scheme 4 illustrates a method for the synthesis of compounds of the invention.
• Treatment of 3-indazilinone 4.1 with PBu 3 and diazodipiperidinylamide provides ether 4.2.
• Deprotonation with NaH followed by alkylation provides compounds of general structure 4.3.
• the synthesis of one or more of the inhibiting agents of the present invention may employ protecting groups and blocking groups. Allyl blocking groups are useful in the presence of acid- and base- protecting groups since the former are stable and can be subsequently removed by metal or pi-acid catalysts.
• an allyl-blocked carboxylic acid can be deprotected with a palladium(O)-catalyzed reaction in the presence of acid labile t-butyl carbamate or base-labile acetate amine protecting groups.
• Yet another form of protecting group is a resin to which a compound or intermediate may be attached. As long as the residue is attached to the resin, that functional group is blocked and cannot react. Once released from the resin, the functional group is available to react.
• Typical blocking/protecting groups are known in the art and include, but are not limited to, the following moieties.
• the present invention provides pharmaceutical compositions comprising one or more inhibiting agents disclosed herein with or without pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants.
• the pharmaceutical compositions are formulated to be substantially free of excipients.
• the present invention provides a pharmaceutical composition comprising or consisting essentially of the compound of Formula III and a pharmaceutically accceptable carrier, excipient, or diluent.
• inhibiting agents can be formulated with one or more pharmaceutically acceptable auxiliary substances.
• the inhibiting agent can be combined with another anti-viral agent to prepare a composition of the invention, and the composition can include one or more pharmaceutically acceptable excipients, diluents, carriers and/or adjuvants.
• the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
• pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
• the inhibiting agent is administered to the host using any means capable of resulting in the desired effect (e.g., reduction in viral load, reduction in liver fibrosis, increase in liver function, and the like).
• the inhibiting agent can be incorporated into a variety of formulations for therapeutic administration.
• the inhibiting agent can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.
• the inhibiting agent may be administered in the form of its pharmaceutically acceptable salts, or a subject active agent may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
• a subject active agent may be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds.
• the following methods and excipients are merely exemplary and are in no way limiting.
• the inhibiting agent can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
• conventional additives such as lactose, mannitol, corn starch or potato starch
• binders such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins
• disintegrators such as corn starch, potato starch or sodium carboxymethylcellulose
• lubricants such as talc or magnesium stearate
• Embodiments of the inhibiting agent can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
• an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
• solubilizers isotonic agents
• suspending agents emulsifying agents, stabilizers and preservatives.
• Embodiments of the inhibiting agent can be utilized in aerosol formulation to be administered via inhalation.
• Embodiments of the inhibiting agent agent can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen, and the like.
• embodiments of the inhibiting agent can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
• Embodiments of the inhibiting agent can be administered rectally via a suppository.
• the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
• Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions, may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibiting agents.
• unit dosage forms for injection or intravenous administration may comprise the inhibiting agent in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
• Embodiments of the inhibiting agent can be formulated in an injectable composition in accordance with the invention.
• injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
• the preparation may also be emulsified or the active ingredient (inhibiting agent) encapsulated in liposome vehicles in accordance with the invention.
• the inhibiting agent is formulated for delivery by a continuous delivery system.
• continuous delivery system is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.
• Mechanical or electromechanical infusion pumps can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852; 5,820,589; 5,643,207; 6,198,966; and the like.
• delivery of the inhibiting agent can be accomplished using any of a variety of refillable, pump systems. Pumps provide consistent, controlled release over time.
• the inhibiting agent is in a liquid formulation in a drug-impermeable reservoir, and is delivered in a continuous fashion to the individual.
• the drug delivery system is an at least partially implantable device.
• the implantable device can be implanted at any suitable implantation site using methods and devices well known in the art.
• An implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned.
• Implantation sites include, but are not necessarily limited to, a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body. Subcutaneous implantation sites are used in some embodiments because of convenience in implantation and removal of the drug delivery device.
• Drug release devices suitable for use in the disclosure may be based on any of a variety of modes of operation.
• the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system).
• the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug-impregnated polymeric material).
• the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, and the like.
• Drug release devices based upon a mechanical or electromechanical infusion pump can also be suitable for use with the present disclosure. Examples of such devices include those described in, for example, U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like.
• a subject treatment method can be accomplished using any of a variety of refillable, non-exchangeable pump systems. Pumps and other convective systems are generally preferred due to their generally more consistent, controlled release over time. Osmotic pumps are used in some embodiments due to their combined advantages of more consistent controlled release and relatively small size (see, e.g., PCT published application no. WO 97/27840 and U.S. Pat. Nos.
• Exemplary osmotically-driven devices suitable for use in the disclosure include, but are not necessarily limited to, those described in U.S. Pat. Nos. 3,760,984; 3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899; 4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442; 4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423; 5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like.
• the drug delivery device is an implantable device.
• the drug delivery device can be implanted at any suitable implantation site using methods and devices well known in the art.
• an implantation site is a site within the body of a subject at which a drug delivery device is introduced and positioned. Implantation sites include, but are not necessarily limited to a subdermal, subcutaneous, intramuscular, or other suitable site within a subject's body.
• an active agent is delivered using an implantable drug delivery system, e.g., a system that is programmable to provide for administration of the agent.
• implantable drug delivery system e.g., a system that is programmable to provide for administration of the agent.
• exemplary programmable, implantable systems include implantable infusion pumps.
• Exemplary implantable infusion pumps, or devices useful in connection with such pumps, are described in, for example, U.S. Pat. Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276; 6,241,704; 6,464,687; 6,475,180; and 6,512,954.
• a further exemplary device that can be adapted for the present disclosure is the Synchromed infusion pump (Medtronic).
• Suitable excipient vehicles for the inhibiting agent are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
• the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
• auxiliary substances such as wetting or emulsifying agents or pH buffering agents.
• compositions of the present invention include those that comprise a sustained- release or controlled release matrix.
• embodiments of the present invention can be used in conjunction with other treatments that use sustained-release formulations.
• a sustained-release matrix is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid-based hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids.
• a sustained- release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co- glycolide (copolymers of lactic acid and glycolic acid), polyanhydrides, poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxcylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
• biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co- glycolide (copolymers of lactic acid and glycolic acid), polyanhydr
• Illustrative biodegradable matrices include a polylactide matrix, a polyglycolide matrix, and a polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) matrix.
• the sustained release formulations of embodiments of the invention can help maintain viral-inhibiting concentrations over a longer time interval.
• the pharmaceutical composition of the present disclosure (as well as combination compositions) are delivered in a controlled release system.
• the inhibiting agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
• a pump may be used (Sefton (1987). CRC Crit. Ref.
• a controlled release system is placed in proximity of the therapeutic target, i.e., the liver, thus requiring only a fraction of the systemic dose.
• a controlled release system is placed in proximity of the therapeutic target, thus requiring only a fraction of the systemic. Other controlled release systems are discussed in the review by Langer (1990). Science 249:1527-1533.
• compositions of the present invention include those formed by impregnation of an inhbiting agent described herein into absorptive materials, such as sutures, bandages, and gauze, or coated onto the surface of solid phase materials, such as surgical staples, zippers and catheters to deliver the compositions.
• absorptive materials such as sutures, bandages, and gauze
• solid phase materials such as surgical staples, zippers and catheters.
• a compound of the present invention can be formulated in a unit dose form between about 10 mg to about 500 mg for treating viral infections, especially infections by a virus of the Flaviviridae family.
• a compound of the present invention is formulated in a unit dose form between about 25 mg to about 250 mg, between about 25 mg to about 100 mg, or between about 50 mg to about 100 mg.
• a compound of the present invention can be formulated in 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg or 200 mg unit dose form.
• the unit dose form is a tablet; in another, the unit dose form is a capsule.
• the tablet can be formulated as immediate release dose form or as sustained release form.
• the unit dose form is a liquid.
• the present invention provides a method of treating a subject infected with a virus from the Flaviviridae family comprising administering to the subject the compound of Formula III or a pharmaceutical composition comprising, or consisting essentially of the compound of Formula III, in an amount that is effective in reducing viral load of said virus in said subject.
• the treatment methods typically comprise administering to a subject infected with such virus a therapeutically effective amount of an inhibiting agent in one or more doses, alone or in combination with other agents.
• a virus of the Flaviviridae family such as Hepatitis Virus C
• the method of the present invention is generally effective in reducing the viral load over a period of a few days, a few weeks or a few months.
• the present invention also provides methods of prophylactically treating an infection by a virus of the Flaviviridae family of viruses comprising administering an effective amount of an inhibiting agent described herein to a subject in need thereof.
• Prophylactic treatment of infection by a virus of the Flaviviridae family is particularly important for patients who will be undergoing liver transplantation for HCV-associated end stage liver disease (ESLD). It has been reported that the new graft is nearly certain to be infected with HCV if viremia is present at the time of transplantation.
• Prophylactic treatment with the compounds of the present invention can be performed to reduce or eliminate HCV viral load prior to liver transplantation, and can help prevent the recurrence of HCV after transplantation.
• the administeration of the compounds of the present invention may also be advantageous for patients who cannot tolerate full doses of standard of care therapy (pegylated interferon and ribavirin).
• an isostere monotherapy or an isostere of the present invention in combination with regular or reduced doses of pegylated interferon and ribavirin
• a compound of the present invention in combination with nitazoxanide can be used to treat these patients, as can indazole plus nitazoxanide (or another thiazolide, or sustained formulations of either of these) plus standard of care medications, at reduced or regular doses, as tolerated.
• Clemizole administered via any of the above embodiments, can also be administered as suppressive (e.g., maintenance) or consolidation therapy, such after effective suppression of HCV by regimens containing clemizole or other agents.
• the inhibiting agent of the present invention and pharmaceutical composition comprising the same can be administered to a subject in one or more doses.
• the inhibiting agent can be administered in an amount of about 10 mg to 1000 mg per dose, e.g., about 10 mg to 20 mg, about 20 mg to 25 mg, about 25 mg to 50 mg, about 50 mg to 75 mg, about 75 mg to 100 mg, about 100 mg to 125 mg, about 125 mg to 150 mg, about 150 mg to 175 mg, about 175 mg to 200 mg, about 200 mg to 225 mg, about 225 mg to 250 mg, about 250 mg to 300 mg, about 300 mg to 350 mg, about 350 mg to 400 mg, about 400 mg to 450 mg, about 450 mg to 500 mg, about 500 mg to 750 mg, or about 750 mg to 1000 mg per dose.
• the amount of the inhibiting agent per dose is determined on a per body weight basis.
• the inhibiting agent can be administered in an amount of about 0.5 mg/kg to 100 mg/kg, e.g., about 0.5 mg/kg to 1 mg/kg, about 1 mg/kg to 2 mg/kg, about 2 mg/kg to 3 mg/kg, about 3 mg/kg to 5 mg/kg, about 5 mg/kg to 7 mg/kg, about 7 mg/kg to about 10 mg/kg, about 10 mg/kg to 15 mg/kg, about 15 mg/kg to 20 mg/kg, about 20 mg/kg to 25 mg/kg, about 25 mg/kg to 30 mg/kg, about 30 mg/kg to 40 mg/kg, about 40 mg/kg to 50 mg/kg per dose, about 50 mg/kg to 60 mg/kg, about 60 mg/kg to 70 mg/kg, about 70 mg/kg to 80 mg/kg, about 80 mg/kg to 90 mg/kg, or about 90 mg/kg to 100 mg/kg, or more than about
• dose levels can vary as a function of the specific inhibiting agent administered, the severity of the symptoms and the susceptibility of the subject to side effects. Preferred dosages for a given compound are readily determinable by those of skill in the art by a variety of means.
• multiple doses of the inhibiting agent are administered.
• the frequency of administration of the inhibiting agent can vary depending on any of a variety of factors, e.g., severity of the symptoms, and the like.
• the inhibiting agent is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid).
• the inhibiting agent is administered continuously.
• efficacious dosing of a compound of the invention can include dosing at about 200 mg po BID, 150 mg po BID, 75mg po BID, or 50 mg po BID.
• the total daily dose can also be split among multiple doses, which allows for a lower dose at each administration with less potential for sedation while maintaining sufficient efficacy.
• a more frequent dosing schedule can be applied for sever cases, for example, TID administration or administration every 4, 6, 8, or 12 hours of a 25 mg, 50 mg, 75 mg, 150 mg or higher dose.
• a sustained release formulation may be used.
• the duration of administration of the inhibiting agent can vary, depending on any of a variety of factors, e.g., patient response, and the like.
• the inhibiting agent can be administered over a period of time of about one day to one week, about two weeks to four weeks, about one month to two months, about two months to four months, about four months to six months, about six months to eight months, about eight months to 1 year, about 1 year to 2 years, or about 2 years to 4 years, or more.
• the practice of a method of the present invention typically involves administering an effective amount of an inhibiting agent or a pharmaceutical composition comprising such inhibiting agent.
• an effective amount of the inhibiting agent is an amount that, when administered in one or more doses to a host (e.g., human) in need thereof, reduces HCV viral load in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% or more, compared to the viral load in the individual not treated with the inhibiting agent.
• Viral load can be measured by measuring the titer or level of virus in serum. These methods include, but are not limited to, a quantitative polymerase chain reaction (PCR) and a branched DNA (bDNA) test. Quantitative assays for measuring the viral load (titer) of HCV RNA have been developed. Many such assays are available commercially, including a quantitative reverse transcription PCR (RT-PCR) (Amplicor HCV MonitorTM, Roche Molecular Systems, New Jersey); and a branched DNA (deoxyribonucleic acid) signal amplification assay (QuantiplexTM HCV RNA Assay (bDNA), Chiron Corp., Emeryville, California). See, e.g., Gretch et al. (1995) Ann. Intern.
• RT-PCR quantitative reverse transcription PCR
• bDNA branched DNA signal amplification assay
• NAT nucleic acid test
• an effective amount of the inhibiting agent is an amount that, when administered in one or more doses to a host (e.g., human) in need thereof, increases liver function in the individual by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, or more, compared to the liver function in the individual not treated with the inhibiting agent.
• a host e.g., human
• an effective amount of the inhibiting agent is an amount that, when administered in one or more doses to a host (e.g., a human) in need thereof, reduces liver fibrosis in the host by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%, or more, compared to the degree of liver fibrosis in the individual not treated with the inhibiting agent.
• Liver fibrosis reduction can be determined by analyzing a liver biopsy sample.
• An analysis of a liver biopsy comprises assessments of two major components: necroinflammation assessed by "grade” as a measure of the severity and ongoing disease activity, and the lesions of fibrosis and parenchymal or vascular remodeling as assessed by "stage” as being reflective of long-term disease progression. See, e.g., Brunt (2000) Hepatol. 31:241-246; and METAVIR (1994) Hepatology 20:15-20. Based on analysis of the liver biopsy, a score is assigned. A number of standardized scoring systems exist which provide a quantitative assessment of the degree and severity of fibrosis. These include the METAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.
• the METAVIR scoring system is based on an analysis of various features of a liver biopsy, including fibrosis (portal fibrosis, centrilobular fibrosis, and cirrhosis); necrosis (piecemeal and lobular necrosis, acidophilic retraction, and ballooning degeneration); inflammation (portal tract inflammation, portal lymphoid aggregates, and distribution of portal inflammation); bile duct changes; and the Knodell index (scores of periportal necrosis, lobular necrosis, portal inflammation, fibrosis, and overall disease activity).
• each stage in the METAVIR system is as follows: score: 0, no fibrosis; score: 1, stellate enlargement of portal tract but without septa formation; score: 2, enlargement of portal tract with rare septa formation; score: 3, numerous septa without cirrhosis; and score: 4, cirrhosis.
• Knodell's scoring system also called the Hepatitis Activity Index, classifies specimens based on scores in four categories of histologic features: I. Periportal and/or bridging necrosis; II. Intralobular degeneration and focal necrosis; III. Portal inflammation; and IV. Fibrosis.
• scores are as follows: score: 0, no fibrosis; score: 1, mild fibrosis (fibrous portal expansion); score: 2, moderate fibrosis; score: 3, severe fibrosis (bridging fibrosis); and score: 4, cirrhosis. The higher the score, the more severe the liver tissue damage. Knodell (1981) Hepatol. 1:431.
• the Ishak scoring system is described in Ishak (1995) J. Hepatol. 22:696-699. Stage 0, No fibrosis; Stage 1, Fibrous expansion of some portal areas, with or without short fibrous septa; stage 2, Fibrous expansion of most portal areas, with or without short fibrous septa; stage 3, Fibrous expansion of most portal areas with occasional portal to portal (P-P) bridging; stage 4, Fibrous expansion of portal areas with marked bridging (P-P) as well as portal-central (P-C); stage 5, Marked bridging (P-P and/or P-C) with occasional nodules (incomplete cirrhosis); stage 6, Cirrhosis, probable or definite.
• the benefit of a therapy provided by the invention can also be measured and assessed by using the Child-Pugh scoring system which comprises a multicomponent point system based upon abnormalities in serum bilirubin level, serum albumin level, prothrombin time, the presence and severity of ascites, and the presence and severity of encephalopathy. Based upon the presence and severity of abnormality of these parameters, patients may be placed in one of three categories of increasing severity of clinical disease: A, B, or C.
• the subject inhibiting agents and pharmaceutical compositions containing the agents can be used in combination of one or more other therapeutic agents for treating viral infection and other diseases.
• the inhibiting agents and pharmaceutical formulations provided herein can be employed in combination with other anti-viral agents to treat viral infection.
• an inhibiting agent that is used to treat a host infected by a Flaviviridae family viral infection is used in combination with one or more other anti-HCV agents to treat HCV infection.
• an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA also referred to herein as an "HCV NS4B antagonist" can be used in combination with one or more other anti-HCV agents to treat HCV infection.
• the inhibiting agents and pharmaceutical compositions containing the agents can be used in combination with another agent ⁇ e.g. an anti-viral agent) to prophylactically treat an infection with a virus from the Flaviviridae family of viruses including but not limited to HCV.
• an anti-viral agent e.g. an anti-viral agent
• Embodiments of the method involve administering to an individual in need thereof one or more inhibiting agents that inhibit binding of an NS4B polypeptide to the 3'UTR of HCV negative strand RNA.
• an inhibiting compound can be used in combination with these standard therapies to treat HCV infection.
• HCV protease inhibitors are in development for the treatment of HCV infection, and in accordance with the methods of the present disclosure, co-administration of an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA and an HCV protease inhibitor can be efficacious in the treatment of HCV.
• an interferon alpha and/or a nucleoside analog such as ribavirin is/are also employed in this combination therapy.
• Suitable HCV protease inhibitors include, but are not limited to, telaprevir (VX-950, Vertex), BILN 2061 and BI 12202 (Boehringer Ingelheim), boceprevir (SCH 503034, Schering Plough), ITMNl 91 (Roche/InterMune/Array BioPharma), MK- 7009 (Merck), TMC435350 (Tibotec/Medivir), ACH-1095 and ACH-806 (Achillion/Gilead), and other inhibitors of NS3/NS4A protease, including, but not limited to, compounds in development by Presidio.
• HCV RNA polymerase (NS5B) inhibitors are in development for the treatment of HCV infection, and in accordance with the methods of the present disclosure, co-administration of an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA and an HCV RNA polymerase inhibitor can be efficacious in the treatment of HCV.
• an interferon alpha and/or a nucleoside analog such as ribavirin and/or an HCV protease inhibitor is/are also employed in this combination therapy.
• Suitable HCV RNA polymerase inhibitors include, but are not limited to, valopicitabine (NM283, Idenix/Novartis), HCV-796 (WyethNiroPharma), Rl 626 (Roche), R7128 (Roche/Pharmasset), GS-9190 (Gilead), MK-0608 (Merck), PSI-6130 (Pharmasset), and PFE-868,554 (PFE).
• TLR toll-like receptor
• an NS4B antagonist that prevents the binding of NS4B to the 3'-UTR of HCV RNA and a TLR agonist can be efficacious in the treatment of HCV.
• an interferon alpha and/or a nucleoside analog such as ribavirin and/or an HCV protease inhibitor and/or an HCV RNA polymerase inhibitor is/are also employed in this combination therapy.
• Suitable TLR agonists include, but are not limited to, TLR7 agonists (i.e., ANA245 and ANA975 (Anadys/Novartis)) and TLR9 agonists (i.e., Actilon (Coley) and IMO-2125 (Idera)).
• TLR7 agonists i.e., ANA245 and ANA975 (Anadys/Novartis)
• TLR9 agonists i.e., Actilon (Coley) and IMO-2125 (Idera)
• a number of thiazolide derivatives are in development for the treatment of HCV infection, and in accordance with the methods of the present disclsoure, co-administration of an NS4B antagonist that prevents the binding of NS4B to the 3'-UTR of HCV RNA and a thiazolide, including, but not limited to, Nitazoxanide (Alinia, or other sustained release formulations of nitazoxanide or other thiazolides, Romark Laboratories) can be efficacious in the treatment of HCV.
• an NS4B antagonist that prevents the binding of NS4B to the 3'-UTR of HCV RNA
• a thiazolide including, but not limited to, Nitazoxanide (Alinia, or other sustained release formulations of nitazoxanide or other thiazolides, Romark Laboratories) can be efficacious in the treatment of HCV.
• an interferon alpha and/or a nucleoside analog such as ribavirin and/or an HCV protease inhibitor and/or an HCV RNA polymerase inhibitor and/or a TLR agonist is/are also employed in this combination therapy.
• co-administration of an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA and a cyclophilin inhibitor i.e., NIM-81 1 (Novartis) and DEBIO-025 (Debiopharm)
• a cyclophilin inhibitor i.e., NIM-81 1 (Novartis) and DEBIO-025 (Debiopharm)
• an alpha-glucosidase inhibitor i.e., Celgosivir (Migenix)
• one or more agents from one or more of the other classes of HCV therapeutic agents discussed herein is used to treat HCV infection.
• NS4B there are several targets within NS4B, and compounds that interact with these other targets can, in accordance with the methods of the present disclosure, be used in combination with an NS4B antagonist that prevents the binding of NS4B to the 3'-UTR of HCV RNA and, optionally, one or more of the other classes of inhibiting agents mentioned herein, to treat HCV infection.
• Such additional NS4B targets include: the N-terminal amphipathic helix (see PCT publication WO 2002/089731, incorporated herein by reference), the NS4B GTPase (see PCT publication WO 2005/032329, incorporated herein by reference), the second amphipathic helix, the PIP2 binding activity of the first amphipathic helix in NS4B (see US provisional patent application serial no. 60/057,188, incorporated herein by reference).
• agents targeting NS5A including, but not limited to, A-831 (Arrow Therapeutics), AZD2836 (Astra Zeneca), and agents in development by XTL/Presidio or BMS (see PCT publications WO 2006/133326 and WO 2008/021928, incorporated herein by reference); (ii) agents targeting TBC1D20 and/or NS5A's interaction with TBC1D20 (see PCT publication WO 2007/018692 and U.S. patent application serial no.
• agents targeting NS4B's GTPase activity see PCT publication WO 2005/032329 and US patent application publication 2006/0199174, incorporated herein by reference
• agents inhibiting membrane association mediated by the HCV amphipathic helices such as those found in NS5A, NS4B, and NS5B (see PCT publication WO 2002/089731, supra)
• agents targeting P1P2 or BAAPP domains in HCV proteins such as those found in NS4B and NS5A (see US provisional patent application 60/057,188, supra)
• agents targeting HCV entry, assembly, or release including antibodies to co- receptors
• agents targeting HCV NS3 helicase agents targeting HCV NS3 helicase
• an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA is used in combination with one or more drugs capable of treating an HIV infection to treat a patient that is co-infected with HIV and HCV.
• an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA is used in combination with one or more drugs capable of treating an HBV infection to treat a patient that is co-infected with HBV and HCV.
• an inhibiting agent that prevents the binding of NS4B to the 3'-UTR of HCV RNA is used in combination with a PD-Ll inhibitor to treat a viral infection.
• embodiments of the present include the administration of an inhibiting agent identified herein (or by using an embodiment of the screen of the invention) in conjunction with at least one additional therapeutic agent to treat a viral infection.
• additional therapeutic agents include, but are not limited to, ribavirin; a nucleoside analog (e.g., levovirin, viramidine, and the like.); an NS3 inhibitor; an NS5 inhibitor; an interferon; and a side effect management agent.
• the at least one additional suitable therapeutic agent includes ribavirin.
• Ribavirin l- ⁇ -D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide, available from ICN Pharmaceuticals, Inc., Costa Mesa, Calif., is described in the Merck Index, compound No. 8199, Eleventh Edition. Its manufacture and formulation is described in U.S. Pat. No. 4,211,771. The disclosure also contemplates use of derivatives of ribavirin (see, e.g., U.S. Pat. No. 6,277,830).
• the at least one additional suitable therapeutic agent includes levovirin.
• Levovirin is the L-enantiomer of ribavirin, and exhibits the property of enhancing a ThI immune response over a Th2 immune response. Levovirin is manufactured by ICN Pharmaceuticals.
• the at least one additional suitable therapeutic agent includes viramidine.
• Viramidine is a 3-carboxamidine derivative of ribavirin, and acts as a prodrug of ribavirin. It is efficiently converted to ribavirin by adenosine deaminases.
• Nucleoside analogs that are suitable for use in a combination therapy include, but are not limited to, ribavirin, levovirin, viramidine, isatoribine, an L-ribofuranosyl nucleoside as disclosed in U.S. Patent No. 5,559,101 and encompassed by Formula I of U.S. Patent No.
• the at least one additional suitable therapeutic agent can include HCV NS3 inhibitors.
• HCV non-structural protein-3 (NS3) inhibitors include, but are not limited to, a tri-peptide as disclosed in U.S. Patent Nos. 6,642,204, 6,534,523, 6,420,380, 6,410,531, 6,329,417, 6,329,379, and 6,323,180 (Boehringer-Ingelheim); a compound as disclosed in U.S. Patent No. 6,143,715 (Boehringer-Ingelheim); a macrocyclic compound as disclosed in U.S. Patent no. 6,608,027 (Boehringer-Ingelheim); an NS3 inhibitor as disclosed in U.S. Patent Nos.
• any of the NS3 protease inhibitors disclosed in WO 99/07733, WO 99/07734, WO 00/09558, WO 00/09543, WO 00/59929 or WO 02/060926 e.g., compounds 2, 3, 5, 6, 8, 10, 11, 18, 19, 29, 30, 31, 32, 33, 37, 38, 55, 59, 71, 91, 103, 104, 105, 112, 113, 114, 1 15, 116, 120, 122, 123, 124, 125, 126 and 127 disclosed in the table of pages 224-226 in WO 02/060926
• an NS3 protease inhibitor as disclosed in any one of U.S. Patent Publication Nos. 2003019067, 20030187018, and 20030186895; and the like.
• the NS3 inhibitor used in a combination therapy of the invention is a member of the class of specific NS3 inhibitors, e.g., NS3 inhibitors that inhibit NS3 serine protease activity and that do not show significant inhibitory activity against other serine proteases such as human leukocyte elastase, porcine pancreatic elastase, or bovine pancreatic chymotrypsin, or cysteine proteases such as human liver cathepsin B.
• the at least one additional suitable therapeutic agent includes NS5B inhibitors.
• Suitable HCV non-structural protein-5 (NS5; RNA-dependent RNA polymerase) inhibitors include, but are not limited to, a compound as disclosed in U.S. Patent No. 6,479,508 (Boehringer-Ingelheim); a compound as disclosed in any of International Patent Application Nos. PCT/CA02/01 127, PCT/CA02/01128, and PCT/CA02/01129, all filed on July 18, 2002 by Boehringer Ingelheim; a compound as disclosed in U.S. Patent No.
• an NS5B inhibitor as disclosed in WO 02/100846 Al or WO 02/100851 A2 both Shire
• an NS5B inhibitor as disclosed in WO 01/85172 Al or WO 02/098424 Al both Glaxo SmithKline
• an NS5B inhibitor as disclosed in WO 00/06529 or WO 02/06246 Al both Merck
• an NS5B inhibitor as disclosed in WO 03/000254 Japan Tobacco
• an NS5B inhibitor as disclosed in EP 1 256,628 A2 (Agouron); JTK-002 (Japan Tobacco); JTK- 109 (Japan Tobacco); and the like.
• the NS5 inhibitor used in the combination therapies of the invention is a member of the class of specific NS5 inhibitors, e.g., NS5 inhibitors that inhibit NS5 RNA-dependent RNA polymerase and that lack significant inhibitory effects toward other RNA dependent RNA polymerases and toward DNA dependent RNA polymerases.
• the at least one additional therapeutic agent is an interferon, e.g., interferon-alpha (IFN- ⁇ ).
• IFN- ⁇ interferon-alpha
• Any known IFN- ⁇ can be used in the treatment methods of the invention.
• the term "interferon-alpha" as used herein refers to a family of related polypeptides that inhibit viral replication and cellular proliferation and modulate immune response.
• the term "IFN- ⁇ " includes naturally occurring IFN- ⁇ ; synthetic IFN- ⁇ ; derivatized IFN- ⁇ (e.g., PEGylated IFN- ⁇ , glycosylated IFN- ⁇ , and the like); and analogs of naturally occurring or synthetic IFN- ⁇ ; essentially any IFN- ⁇ that has antiviral properties, as described for naturally occurring IFN- ⁇ .
• Suitable alpha interferons include, but are not limited to, naturally-occurring IFN- ⁇ (including, but not limited to, naturally occurring IFN- ⁇ 2a, IFN- ⁇ 2b); recombinant interferon alpha-2b such as Intron-A interferon available from Schering Corporation, Kenilworth, NJ. ; recombinant interferon alpha-2a such as Roferon interferon available from Hoffmann-La Roche, Nutley, N.
• interferon alpha-2C such as Berofor alpha 2 interferon available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, Conn.
• interferon alpha-nl a purified blend of natural alpha interferons such as Sumiferon available from Sumitomo, Japan or as Wellferon interferon alpha-nl (INS) available from the Glaxo-Wellcome Ltd., London, Great Britain
• interferon alpha-n3 a mixture of natural alpha interferons made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, Conn., under the Alferon tradename.
• IFN- ⁇ also encompasses consensus IFN- ⁇ .
• Consensus IFN- ⁇ (also referred to as “CIFN” and “IFN-con” and “consensus interferon”) encompasses, but is not limited to, the amino acid sequences designated IFN-coni, IFN-con 2 and IFN-COn 3 which are disclosed in U.S. Pat. Nos. 4,695,623 and 4,897,471 ; and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (e.g., Infergen®, InterMune, Inc., Brisbane, Calif.).
• IFN-coni is the consensus interferon agent in the Infergen® alfacon-1 product.
• the Infergen® consensus interferon product is referred to herein by its brand name (Infergen®) or by its generic name (interferon alfacon- 1). DNA sequences encoding IFN-con may be synthesized as described in the aforementioned patents or other standard methods.
• the at least one additional therapeutic agent is CIFN.
• fusion polypeptides comprising an IFN- ⁇ and a heterologous polypeptide can also be used in the combination therapies of the invention.
• IFN- ⁇ fusion polypeptides include, but are not limited to, Albuferon-alphaTM (a fusion product of human albumin and IFN- ⁇ ; Human Genome Sciences; see, e.g., Osborn et al. (2002) J. Pharmacol. Exp. Therap. 303:540-548).
• gene-shuffled forms of IFN- ⁇ See, e.g., Masci et al. (2003) Curr. Oncol. Rep. 5:108-113.
• Other suitable interferons include, Multiferon (Viragen), Medusa Interferon (Flamel Technology), Locteron (Octopus), and Omega Interferon (Intarcia/Boehringer Ingelheim).
• IFN- ⁇ also encompasses derivatives of IFN- ⁇ that are derivatized (e.g., are chemically modified relative to the naturally occurring peptide) to alter certain properties such as serum half-life.
• IFN- ⁇ includes glycosylated IFN- ⁇ ; IFN- ⁇ derivatized with polyethylene glycol ("PEGylated IFN- ⁇ "); and the like. PEGylated IFN- ⁇ , and methods for making same, is discussed in, e.g., U.S. Patent Nos. 5,382,657; 5,981,709; and 5,951,974.
• PEGylated IFN- ⁇ encompasses conjugates of PEG and any of the above-described IFN- ⁇ molecules, including, but not limited to, PEG conjugated to interferon alpha-2a (Roferon, Hoffman La-Roche, Nutley, N.J.), interferon alpha 2b (Intron, Schering-Plough, Madison, NJ. ), interferon alpha-2c (Berofor Alpha, Boehringer Ingelheim, Ingelheim, Germany); and consensus interferon as defined by determination of a consensus sequence of naturally occurring interferon alphas (Infergen®, InterMune, Inc., Brisbane, Calif.).
• the IFN- ⁇ polypeptides can be modified with one or more polyethylene glycol moieties, i.e., PEGylated.
• the PEG molecule of a PEGylated IFN- ⁇ polypeptide is conjugated to one or more amino acid side chains of the IFN- ⁇ polypeptide.
• the PEGylated IFN- ⁇ contains a PEG moiety on only one amino acid.
• the PEGylated IFN- ⁇ contains a PEG moiety on two or more amino acids, e.g., the IFN- ⁇ contains a PEG moiety attached to two, three, four, five, six, seven, eight, nine, or ten different amino acid residues.
• IFN- ⁇ may be coupled directly to PEG (i.e., without a linking group) through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group.
• HCV replication assays and/or animal studies can be performed in the presence of various combinations of the various anti-HCV agents. Increased inhibition of replication in the presence of an additional agent (above that observed with monotherapy) is evidence for the potential benefit of the combination therapy.
• side effect management agents can be used in the treatment methods of the invention, and these include agents that are effective in pain management; agents that ameliorate gastrointestinal discomfort; analgesics, antiinflammatories, antipsychotics, antineurotics, anxiolytics, and hematopoietic agents.
• embodiments of the invention contemplate the use of any compound for palliative care of patients suffering from pain or any other side effect in the course of treatment with a subject therapy.
• Exemplary palliative agents include acetaminophen, ibuprofen, other NSAIDs, H2 blockers, and antacids.
• the inhibiting agents and pharmaceutical compositions provided herein can be used to treat a variety of patients or hosts infected with a virus of the Flavirivirus family.
• the subject treatment methods may particularly benefit "treatment failure patients".
• Such patients include, but are not limited to, those who have failed to respond to previous therapy for HCV (referred to as “non-responders") or who initially responded to previous therapy, but in whom the therapeutic response was not maintained (referred to as "relapsers").
• the previous therapy generally can include treatment with any anti-viral agent other than an inhibiting agent of the present disclosure.
• Sucn individuals include na ⁇ ve individuals (e.g., individuals not previously treated for HCV). Individuals who are infected with HCV can be identified by detecting HCV RNA in their blood, and/or having an anti-HCV antibody in their serum.
• hosts suitable for treatments of the present invention have an HCV titer of at least about 10 5 , at least about 5 x 10 5 , or at least about 10 6 , genome copies of HCV per milliliter of serum.
• the patient may be infected with any HCV genotype (genotype 1, including Ia and Ib, 2, 3, 4, 6, and the like, and subtypes (e.g., 2a, 2b, 3a, and the like.)), particularly a difficult to treat genotype such as HCV genotype 1 and particular HCV subtypes and quasispecies.
• HCV-positive hosts as described above
• who exhibit severe fibrosis or early cirrhosis non-decompensated, Child 's-Pugh class A or less
• more advanced cirrhosis decompensated, Child 's-Pugh class B or C
• chronic HCV infection and who are viremic despite prior anti-viral treatment, or who have a contraindication to therapy with a known anti-viral agent.
• HCV-positive hosts with stage 3 or 4 liver fibrosis according to the METAVIR scoring system are suitable for treatment with the methods of the present disclosure.
• hosts suitable for treatment with embodiments of the present disclosure are patients with decompensated cirrhosis with clinical manifestations, including patients with far-advanced liver cirrhosis, including those awaiting liver transplantation.
• hosts suitable for treatment with embodiments of the present disclosure include patients with milder degrees of fibrosis including those with early fibrosis (stages 1 and 2 in the METAVIR, Ludwig, and Scheuer scoring systems; or stages 1, 2, or 3 in the Ishak scoring system).
• the use of appropriate diagnostic tests provided by the present invention can be of great benefit. For example, assessing the sensitivity of the specific virus found in a given patient to the contemplated therapy can help identify the best match between candidate patient and the corresponding appropriate therapy. In the case of compounds of the present invention identified herein, this can be done by isolating the NS4B sequence from a given patient's HCV isolate and determining the efficacy of the drug's inhibition of RNA binding by the patient's NS4B isoform.
• Combination therapy with a compound of the present invention in accordance with embodiments of the present invention includes, for example and without limitation, (1) treatment with indazole plus nitazoxanide, (2) treatment with indazole followed by nitazoxanide, (3) treatment with indazole plus nitazoxanide and a NS3 protease inhibitor, (4) treatment with indazole plus nitazoxanide plus a NS3 protease inhibitor plus a NS5B polymerase inhibitor, (5) treatment with indazole plus a N S3 protease inhibitor plus a NS5B polymerase inhibitor, (6) treatment with indazole plus nitazoxanide plus a NS3 protease inhibitor plus a NS4B second amphipathic helix inhibitor, (7) treatment with indazole plus nitazoxanide plus a NS4B second amphipathic helix inhibitor, (8) treatment with indazole plus a NS3 protease
• Nitazoxanide administration in accordance with the combination therapies of the invention can be, for illustration and without limitation, 500 mg po BID.
• Other doses, other thiazolides, or other formulations of nitazoxanide or another thiazolide, such as sustained release formulations, can also be used in the combination therapies of the invention.
• the inhibiting agents and pharmaceutical compositions thereof can be administered to a subject using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect.
• An active agent can be administered in a single dose or in multiple doses.
• Embodiments of the inhibiting agent can be administered to a host using available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes.
• routes of administration contemplated by the disclosure include, but are not limited to, enteral, parenteral, or inhalational routes.
• Parenteral routes of administration other than inhalation administration include, but are not limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, and intravenous routes, i.e., any route of administration other than through the alimentary canal.
• Parenteral administration can be conducted to effect systemic or local delivery of the inhibiting agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
• the inhibiting agent can also be delivered to the subject by enteral administration.
• Enteral routes of administration include, but are not limited to, oral and rectal ⁇ e.g., using a suppository) delivery.
• Methods of administration of the inhibiting agent through the skin or mucosa include, but are not limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
• a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
• Iontophoretic transmission may be accomplished using commercially available "patches" that deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
• indazole is administered by oral, intravenous, transdermal, sublingual, intramuscular, or rectal route.
• the present invention further provides an in vitro cell-free method of identifying agents (inhibiting agents) that modulate RNA binding to an RNA-binding protein.
• a test agent that inhibits binding of NS4B polypeptide to the 3'UTR of HCV negative strand RNA can be further tested for its ability to inhibit HCV replication in a cell-based assay.
• a test agent of interest can be contacted with a mammalian cell that harbors all or part of an HCV genome; and the effect of the test agent on HCV replication is determined.
• Suitable cells include mammalian liver cells that are permissive for HCV replication, e.g., an immortalized human hepatocyte cell line that is permissive for HCV.
• a suitable mammalian cell is Huh7 hepatocyte or a subclone of Huh 7 hepatocyte, e.g., Huh-7.5.
• Suitable cell lines are described in, e.g., Blight et al. (2002) J. Virol. 76:13001 ; and Zhang et al. (2004) J. Virol. 78:1448.
• the HCV genome in the cell comprises a reporter, e.g., a nucleotide sequence encoding luciferase, a fluorescent protein, or other protein that provides a detectable signal; and determining the effect, if any, of the test agent on HCV replication is achieved by detection of a signal from the reporter.
• test agents are organic moieties.
• test agents are synthesized from a series of substrates that can be chemically modified. "Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions.
• These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepines, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, and the like), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present methods.
• alkyl groups including alkanes
• Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c, subcutaneous(ly); and the like.
• Example 1 Chemical Synthesis Synthetic Method 1
• Indazole compounds can be synthesized according to methods disclosed in the literature, e.g., Lukin et al. J. Org. Chem. 2006, 71, 8166-8172 and Souers et al. J. Med. Chem. 2005, 48, 1318-1321.
• a mixture of 5-(trifluoromethyl)-1H-indazole 3a (0.46 g, 2.5 mmol), K 2 CO3 (1.04 g, 7.5 mmol) in DMF (8 rnL) was stirred at room temperature for 30 min.
• p-chlorobenzyl bromide (0.77 g, 3.75 mmol). The resulting mixture was heated at 60 °C for 6 h.
• Table 3 shown below illustrates the effects of certain indazole isosteres on HCV RNA replication (AV) and cell viability (Viab) using the Luciferase and Alamar Blue assays described herein. Compound activities were measured at two concentrations to determine whether the effects were dose-dependent. Numerical values represent the percent of normal activity (either viral replication or cell viability) remaining after compound treatment; these values have also been binned to provide a rough measure of relative activity.
• Cell viability is stated as a percentage of viable cells in a treated population of cells in comparison to an untreated population of same cell type.
• a suitable Ib HCV RNA replicon assay uses the Huh7 cell line, which contains an HCV Ib RNA replicon with a stable luciferase (LUC) reporter.
• Huh7 cell line which contains an HCV Ib RNA replicon with a stable luciferase (LUC) reporter.
• LUC reporter is used as an indirect measure of HCV replication.
• the activity of the LUC reporter is directly proportional to HCV RNA levels and positive control anti-viral compounds behave comparably using LUC endpoints.
• HCV assays suitable for use in demonstrating the anti-viral activity of the compounds useful in the methods of the invention include the Luciferase Assay for HCV Replicon Reporter Cell Lines and the MTT Assay for HCV Replicon Reporter Cell Lines described in this example.
• the embodiments of these assays described in this example were developed by Shanghai ChemPartner Co., Ltd., a corporation of China with its principal office located at 720 Cailun Road, Building No. 3, Shanghai 201203, China.
• Fresh growth medium is prepared just before use.
• the container used in the procedure is a 10 cm diameter culture dish.
• HCV replicon reporter cell lines are used.
• Prepare complete medium add FBS and appropriate additives as described in "Culture Media", below. Pre-warm the medium in a 37 °C thermostat water bath. Remove the dish from a 37 °C/C ⁇ 2 incubator. Check the cell name and complete medium and passage number marked on the dish. Aspirate the medium carefully and add 1 ml PBS to rinse the cells. Remove and discard the solution and add 1 ml of 0.25% Trypsin/0.02% EDTA. Rinse the cells with the added Trypsin/EDTA to ensure all the cells have been rinsed.
• Compounds are prepared or provided at 25 mM in 100% DMSO. This is the compound stock solution.
• the dilution procedure should be performed in a cell culture hood. Dispense the stock solution into the second column of a 96-well plate. Prepare 9-step (10 concentrations total), 5-fold serial dilutions by transferring 10 ⁇ l of the compound into the next well containing 40 ⁇ l of DMSO. Repeat for all compounds. Aspirate 2 ⁇ l of the above compound solution from each well and add into 198 ⁇ l complete media using a 12- channel pipetter to obtain the 10-fold concentration compound solution with 1% DMSO, mix well.
• the cell culture media is DMEM complete: DMEM (Life Technologies #41965- 039) supplemented with 10% FCS, 2 mM Glutamin (Life Technologies #25030-024), Penicillin (100 IU/ml) / Streptomycin (100 ⁇ g/ml) (Life Technologies #15140-114) and Ix nonessential amino acids (Life Technologies #11140-035).
• G418 ("Geneticin", Life Technologies): concentrations are given as weight per volume of the original substance. Specific activity of a typical batch is ca. 700 ⁇ g/mg as stated by the manufacturer. This value does not necessarily reflect the biological activity in a user's system. Therefore each new batch of G418 should be tested individually e.g., in an electroporation experiment using different selection conditions (0.2-1 mg/ml).
• the MTT assay (and the MTS assay) is a laboratory test and standard colorimetric assay (an assay which measures changes in color) for measuring the activity of enzymes that reduce MTT or MTS + PMS to formazan, giving a purple color. It can also be used to determine cytotoxicity of potential medicinal agents and other toxic materials, since those agents would result in cell toxicity and therefore metabolic dysfunction and therefore decreased performance in the assay. Yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in living cells.
• a solubilization solution (usually either dimethyl sulfoxide, an acidified ethanol solution, or a solution of the detergent sodium dodecyl sulfate in diluted hydrochloric acid) is added to dissolve the insoluble purple formazan product into a colored solution.
• the absorbance of this colored solution can be quantified by measuring at a certain wavelength (usually from 500 and 600 nm) by a spectrophotometer. The absorption maximum is dependent on the solvent employed.
• Culture medium, culture plates, and additives are prepared as described in part A of this example. Pre-warm the medium in a 37 °C thermostat water bath. Remove the dish from a 37 °C/C ⁇ 2 incubator. Check the cell name and complete medium and passage number marked on the dish. Aspirate the medium carefully and add 1 ml PBS to rinse the cells. Remove and discard the solution and add 1 ml of 0.25% Trypsin/0.02% EDTA. Rinse the cells with the added Trypsin/EDTA to ensure all the cells have been rinsed. Remove the Trypsin/EDTA with a vacuum pump and incubate at 37 °C for 3-5 minutes. Examine the cell morphology under an inverted microscope until single cell suspension is clearly visible.
• Compounds are prepared or provided at 25 mM in 100% DMSO. This is the compound stock solution.
• the dilution procedure should be performed in a cell culture hood. Dispense the stock solution into the second column of a 96-well plate. Prepare 9-step (10 concentrations total), 5-fold serial dilutions by transferring 10 ⁇ l of the compound into the next well containing 90 ⁇ l of DMSO. Repeat for all compounds. Aspirate 2 ⁇ l of the above compound solution from each well and add into 198 ⁇ l complete media using a 12- channel pipetter to obtain the 10-fold concentration compound solution with 1% DMSO, mix well. Remove the 96-well assay plate from 37 °C /5% CO 2 incubator, examine the cell morphology under an inverted microscope.
• the assays in parts A and B were used to generate the genotype 1 b inhibitory activity and related cell toxicity (viability) data supported herein. This assay has been used to generate the genotype 2a inhibitory activity data supported herein.
• Pulse the cells 82Ov, 5 pulses, 99 ⁇ sec, 220 ms interval, unipolar. 11) Allow cells to rest for 15 min. 12) Transfer cells using the Pasteur pipette in the cuvette package to medium. Make a common stock from all tubes. 13) Plate 10,000 cells/well in 96 well plates. 14) Rotate plate a little for even cell plating. 15) Incubate for 24 hr before treatment.
• Alamar blue assay - a) Include medium for background subtraction (and also for seeing change in color easily), b) Aspirate medium, c) Make a stock of medium plus 10% Alamar blue. Total volume per well is 100 ⁇ l. d) Incubate for 2-2.5 hrs at 37 °C (or until there is a color change), c) Read plates at flex station.
• Drugs belonging to different classes have been shown to be associated with QT prolongation and in some cases serious ventricular arrhythmias.
• the most common mechanism for these adverse events is the inhibition of one or more cardiac potassium channels, in particular hERG.
• This current is important for cardiac myocyte repolarization and is a common target for drugs that prolong the QT interval.
• Test articles in this study were therefore characterized to determine their ability to inhibit the hERG channel.
• Ion channel activity was measured using a stably transfected Chinese Hamster Ovary (CHO) cell line expressing the hERG mRNA. The pharmacology of this cloned channel expressed in the CHO cell line is very similar to that observed in native tissue.
• Cells AVIVA's CHO cell line, which stably expresses hERG channels, was used for the study. Cells were cultured in DMEM/F12 containing 10% FBS, 1% penicillin/streptomycin and 500 ⁇ g/ml G418. Before testing, cells were harvested using Accumax (Innovative Cell Technologies).
• External Solution 2 mM CaCI 2 ; 2 mM MgCl 2 ; 4 mM KCl; 150 mM NaCl; 10 mM Glucose; 10 mM HEPES; 310-320 mOsm; pH 7.4 (adjusted with IM NaOH).
• Internal Solution 140 mM KCl; 10 mM MgCl 2 ; 6 mM EGTA; 5 mM HEPESNa; mM ATP-Mg; 300-320 mOsm; pH 7.25 (adjusted with IM KOH).
• Electrophysiology Whole cell recordings were performed using PX 7000A (Axon Instruments) with VIVA's SealChipTM technology. Cells were voltage clamped at a holding potential of -80 mV. The hERG current was then activated by a depolarizing step to -50 mV for 300 ms. This first step at -50 m V was used as a baseline for measuring peak amplitude of the tail current. Next, a voltage step to +20 mV was applied for 5 s to activate the channels. Finally a step back to -50 mV for 5 seconds removed activation and the deactivating tail current was recorded.
• Compound Handling and Dilutions All compounds were prepared from either 10 or 30 mM DMSO stock solutions.
• Electrophysiology Procedures After achieving whole cell configuration, cells were monitored for 90 s to assess stability and then washed with External Solution for 66 s. The voltage protocol described above was then applied to the cells every 12 s throughout the procedure. Only stable cells with recording parameters above threshold (see Quality Control section) were allowed to enter the drug addition procedure. External solution containing 0.1% DMSO (vehicle) was applied to the cells to establish a baseline. After allowing the current to stabilize for 3 to 5 min, test articles were applied. Test article solutions were added to cells in 4 separate additions. Cells were kept in test solution until effect of the test article reached steady state, to a maximum of 12 min. Next, 1 ⁇ M cisapride (positive control) was added. Finally, washout with External Solution was performed until the recovery current reached a steady state.
• DMSO vehicle
• Data Analysis Data analysis was performed using DataXpress (Axon Instruments), Clampfit (Axon Instruments) and Origin (Originlab Corporation) software.
• Example presents the results of testing illustrative compounds of the invention in the assays described in Example 1.
• the compounds are divided into two tables: Table 6 presents "Ib Active Analogs"; and Table 7 presents "Clemizole Like Analogs". Each of these categories is discussed below.
• Table 6 presents results for compounds that demonstrate significant activity against HCV in the Ib replicon assay. Therefore, Table 6 provides certain non-limiting examples of Ib Active Analogs provided by the present invention. Many of these compounds also show activity in the 2a infectious clone assay; compounds that are active in the Ib replicon assay are typically active in the 2a infectious clone assay as well. In the Ib replicon assay, test compounds were tested at 3-fold serial dilutions from concentrations of 25 ⁇ M to 0.001 ⁇ M. The results are reported as micromolar activity against inhibition of 50% of replication of the Ib replicon (EC50).
• a high value of >25 ⁇ M means that >25 ⁇ M of test compound is required to inhibit 50% of replication of the Ib replicon.
• a low value of 1 ⁇ M means that 1 ⁇ M of test compound is required to inhibit 50% of replication of the Ib replicon.
• lower EC 50 values correspond to higher potency.
• compound EBP899 from Table 6 which shows for this assay an EC 50 value of 2.2 ⁇ M, indicating that this compound is a relatively potent inhibitor against Ib replicon replication.
• Table 6 lists only compounds with test results demonstrating cell viability > 10 ⁇ M, and activity in Ib replicon assay of ⁇ 10 ⁇ M.
• Table 7 presents results for compounds that demonstrate significant activity against HCV in the 2a infectious clone assay at 5 ⁇ M.
• Table 7 provides certain examples of Clemizole Like Analogs provided by the present invention.
• test compounds are tested at two concentrations of 5 ⁇ M and 10 ⁇ M. Only the results from testing at 5 ⁇ M are shown in Table 7. The results are reported as % replication activity of a control sample with no test compound present. A low value of 40% indicates that, at 5 ⁇ M of test compound, 40% of replication activity remains, indicative of a compound of high potency. A high value of 90% indicates that, at 5 ⁇ M of test compound, 90% of replication activity remains, indicative of a compound of low potency.
• Table 7 lists only compounds with test results demonstrating cell viability levels of >85% at 5 ⁇ M and >80% at 10 ⁇ M, and activity in 2a infectious clone assay of ⁇ 90%. Compounds were tested for cell toxicity at concentrations of 5 ⁇ M and 10 ⁇ M. The results are reported as % cell survival. High values of >85% at 5 ⁇ M and >80% at 10 ⁇ M indicate that at 5 ⁇ M, greater than 85% of cells are viable and at 10 ⁇ M, greater than 80% of cells are viable.
• a compound other than one of those described above can be used to treat HCV.
• one or more of the compounds above can be used in other embodiments of the present disclosure to treat HCV.
• ratios, concentrations, amounts, and other numerical data may be expressed herein in a range format. It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
• a concentration range of "about 0.1% to about 5%” should be interpreted to include not only the explicitly recited concentration of about 0.1 wt% to about 5 wt%, but also include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range.
• the term "about” can include ⁇ 1%, ⁇ 2%, ⁇ 3%, ⁇ 4%, ⁇ 5%, ⁇ 6%, ⁇ 7%, ⁇ 8%, ⁇ 9%, or ⁇ 10%, or more of the numerical value(s) being modified.

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