WO1999051619A1 - Arabinofuranosyl benzimidazoles comme agents antiviraux - Google Patents

Arabinofuranosyl benzimidazoles comme agents antiviraux Download PDF

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Publication number
WO1999051619A1
WO1999051619A1 PCT/US1999/007308 US9907308W WO9951619A1 WO 1999051619 A1 WO1999051619 A1 WO 1999051619A1 US 9907308 W US9907308 W US 9907308W WO 9951619 A1 WO9951619 A1 WO 9951619A1
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compound
group
benzimidazole
dichloro
compounds
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PCT/US1999/007308
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English (en)
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Leroy B. Townsend
John C. Drach
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The Regents Of The University Of Michigan
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Priority to AU33791/99A priority Critical patent/AU3379199A/en
Publication of WO1999051619A1 publication Critical patent/WO1999051619A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/052—Imidazole radicals
    • 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
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention pertains to the general field of benzimidazole nucleoside analogs and their use as antiviral agents. More particularly, this invention pertains to benzimidazole nucleoside analogs wherein the sugar group is an arabinofuranosyl group and derivatives thereof. The present invention also pertains to methods of making such benzimidazole nucleoside analogs and derivatives, compositions comprising such compounds, the use of such compounds in antiviral treatment.
  • viruses infect humans and animals include those belonging to herpes family, such as herpes simplex viruses (HSV) 1 and 2, varicella- zoster (which causes chickenpox and shingles); Epstein-Barr virus (EBV, which causes mononucleosis), and human cytomegalovirus (HCMV), as well as non-herpes viruses such as hepatitis B and C (HBV and HCV).
  • Animal viruses include bovine rhinotracheitis virus, Bovine Herpesvirus-1 (BHV-1), and bovine mammillitis virus, among others.
  • HSV-1 and HSV-2 have a wide host-cell range, multiply efficiently and rapidly destroy infected cells.
  • Others e.g., EBV, HHV6 have a narrow host-cell range or, in the case of HCMV, replicate slowly.
  • gancyclovir foscarnet
  • cidofivir Three drugs have been widely used for the treatment of HCMV infections: gancyclovir, foscarnet and cidofivir.
  • DRB analogs modified on the heterocyclic ring for example, the 2-substituted-5,6-dichloro-benzimidazole ribonucleosides such as 2,5,6- trichloro-l-( ⁇ -D-ribofuranosyl) benzimidazole (TCRB) and 2-bromo-5,6-dichloro-l-( ⁇ -D- ribofuranosyl) benzimidazole (BDCRB), as significant inhibitors of HCMV and this activity was well separated from its cytotoxicity. See for example, the United States Patent No. 5,248,672, issued to Townsend and Drach.
  • TCRB 2,5,6- trichloro-l-( ⁇ -D-ribofuranosyl) benzimidazole
  • BDCRB 2-bromo-5,6-dichloro-l-( ⁇ -D- ribofuranosyl) benzimidazole
  • the present invention provides benzimidazole nucleoside analogs wherein the sugar group is an arabinofuranosyl group or a derivative thereof.
  • One embodiment of the present invention pertains to a ⁇ -D-arabinofuranosyl compound of the structure:
  • Q is a substituted benzimidazole group attached at the benzimidazole 1 -position, and the 2- substituent of the benzimidazole is a group other than -H;
  • R 12 is -OH, -H, halo, -N 3 , or -X-R 13 , wherein X is -O- or -S- and R 13 is an alkyl of 1 to 8 carbon atoms; or a pharmaceutically acceptable salt, prodrug or derivative thereof; provided that: a) when R 10 is -H, R ⁇ is not -H, and the compound is not a ⁇ -compound; and b) the compound is not 2,5,6-trichloro-l-( ⁇ -D-arabinofuranosyl) benzimidazole.
  • the compound is a ⁇ -D-arabinofuranosylbenzimidazole compound having the structure:
  • R 2 , R 4 , R 5 , R 6 , and R 7 are independently selected from the group consisting of:
  • R 8 is -H or an alkyl group of 1-8 carbon atoms
  • R 12 is -OH, -H, halo, -N 3 , or -X-R 13 , wherein X is -O- or -S- and R 13 is an alkyl group of 1 to 8 carbon atoms; or a pharmaceutically acceptable salt, prodrug or derivative thereof; provided that: a) when each of R 4 , R 5 , R 6 , and R 7 is a hydrogen, R 2 is a group other than hydrogen; b) when R 10 is -H, R n is not -H, and the compound is
  • This invention further provides methods of preventing or inhibiting viral replication and/or infection in a cell by contacting the cell with an effective amount of a compound or composition as described herein.
  • a cell includes a plurality of cells, including mixtures thereof.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • composition is intended to mean a combination of active agent and another compound or composition, inert or active, such as an adjuvant.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting
  • compositions also can include stabilizers and preservatives.
  • stabilizers and preservatives For examples of carriers, stabilizers and adjuvants, see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975).
  • “Pharmaceutically acceptable salt, prodrug or derivative” as used herein, relate to any pharmaceutically acceptable salt, ester, ether, salt of an ester, solvate, such as ethanolate, or other derivative of a compound of the present invention which, upon administration to a recipient, is capable of providing (directly or indirectly) a compound of this invention or an active metabolite or residue thereof.
  • Particularly favored derivatives and prodrugs are those that increase the bioavailability of the compounds of this invention when such compounds are administered to a mammal (e.g. , by allowing an orally administered compound to be more readily absorbed into the blood) or which enhance delivery of the parent compound to a biological compartment (e.g., the brain or lymphatic system).
  • salts of the compounds of the present invention may be derived from inorganic or organic acids and bases.
  • acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and benzenesulfonic acids.
  • acids such as oxalic
  • bases include alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and compounds of formula NW 4 + , wherein W is C,_ 4 alkyl.
  • salts include: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylproprionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and unde
  • “In vivo” use of a material is defined as introduction of the material into a living human, mammal, or vertebrate.
  • “Ex vivo” use of a compound is defined as using a compound for treatment of a biological material outside a living human, mammal, or vertebrate, where that treated biological material is intended for use inside a living human, mammal, or vertebrate.
  • removal of blood from a human, and introduction of a compound into that blood is defined as an ex vivo use of that compound if the blood is intended for reintroduction into that human or another human.
  • Reintroduction of the human blood into that human or another human would be in vivo use of the blood, as opposed to the ex vivo use of the compound. If the compound is still present in the blood when it is reintroduced into the human, then the compound, in addition to its ex vivo use, is also introduced in vivo.
  • salts of the compounds of the present invention will be pharmaceutically acceptable.
  • salts of acids and bases which are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • effective amount includes “prophylactically effective amount” as well as “therapeutically effective amount” and refers to an amount effective in preventing or treating a viral infection in a patient either as monotherapy or in combination with other agents. Effective amounts are easily determined by those of skill in the art and will vary with the cell, virus being effected and the purpose of the treatment. For example, when utilizing the drug in cell culture, it is important that the amount of drug not be cytotoxic to the cells.
  • a “prophylactically effective amount” is an amount which inhibits viral infection, reproduction and proliferation in a subject challenged with the virus without toxicity to the cells and subject being treated.
  • subject refers to a host such as a mammal, a mouse, bovine, rat or a human patient or a host cell that is virally infected.
  • a "cell” is intended to include, but not be limited to a mammalian cell, e.g. , a mouse cell, a bovine cell, a rat cell, a woodchuck cell, a simian cell, or a human cell.
  • terapéuticaally effective amount refers to the effective amount for "treatment,” that is, for alleviation of symptoms of a particular disorder in a patient or for the improvement of an ascertainable measurement associated with a particular disorder, for example, a reduction of viral titer in the host.
  • treatment that is, for alleviation of symptoms of a particular disorder in a patient or for the improvement of an ascertainable measurement associated with a particular disorder, for example, a reduction of viral titer in the host.
  • One of skill in the art can determine when a host has been "treated” by noting a reduction in viral load or an alleviation in symptoms associated with viral infection.
  • a “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • the term "biologically acceptable carrier” refers to a carrier or adjuvant that may be administered to a host or patient, together with a compound of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver an effective amount of the antiviral compound.
  • suitable carriers include liquid phase carriers, such as sterile or aqueous solutions, as well as those described below.
  • the compounds of the present invention comprise a benzimidazole group which is attached via the 1 -position to an arabinofuranosyl ring via the 1 '-position.
  • the sugar arabinofuranose is characterized by a 2' -OH and 4'-CH 2 OH on the same side of the furanosyl plane and a 3'-OH is on the opposite side of the furanosyl plane.
  • the benzimidazole ring is on the same side of the furanosyl plane as the 3' -OH, whereas in the case of 1- ⁇ -arabinofuranosyl benzimidazole, the benzimidazole ring is on the opposite side of the furanosyl plane as the 3'-OH.
  • arabinofuranosyl compounds can be ⁇ -D- ⁇ -L, ⁇ -D, or ⁇ -L.
  • ⁇ -D-arabinofuranosylbenzimidazoles having the general structure:
  • Q is a substituted benzimidazole group attached at the benzimidazole 1 -position, and the 2- substituent of the benzimidazole is a group other than -H;
  • the present invention includes the above compounds and their pharmaceutically acceptable salts, prodrugs and derivatives.
  • arabinofuranosyl prodrugs of the present invention include, for example, those with chemically protected hydroxyl groups (e.g., with O-acetyl groups), such as 2'-O-acetyl-arabinofuranosyl; 3'-0-acetyl- arabinofuranosyl; 5'-0-acetyl-arabinofuranosyl; 2',3'-di-O-acetyl-arabinofuranosyl and 2',3',5'-tri-0-acetyl-arabinofuranosyl.
  • derivatives of a compound as used herein means a chemically modified compound wherein the chemical modification takes place either at a functional group of the compound or on the aromatic ring.
  • Non-limiting examples of arabinofuranosyl derivatives of the present invention include N-acetyl, N-methyl, N-hydroxy groups at any of the available nitrogens in the compound. Additional derivatives may include those having a trifluoromethyl group on the benzimidazole ring or on the arabinofuranose ring.
  • ⁇ -D-arabinofuranosyl benzimidazole compounds have the following structure:
  • R 2 , R 4 , R 5 , R 6 , and R 7 are independently the same or different and are selected from the group consisting of: -H, -F, -Cl, -Br, -I, -NO 2 , -NH 2 , -NHR 8 , - N(R 8 ) 2 , -OR 8 , -SR 8 , and -CF 3 ; wherein R 8 is -H or an alkyl group of 1-8 carbon atoms; and R 10 , R", and R 12 are independently the same or different and are -H or a hydroxyl protecting group; with the proviso that: a) when each of R 4 , R 5 , R 6 , and R 7 is a hydrogen, R 2 is a group other than hydrogen; b) when R 10 is -H, R 11 is not -H, and the compound is not a ⁇ -compound; and c) the compound is not 2,5,6-trichloro-
  • alkyl as used herein include, but is not limited to, fully saturated, partially unsaturated, and fully unsaturated hydrocarbyl groups which may be aliphatic, alicyclic, or aromatic.
  • R 8 is selected from the group consisting of straight chain, branched or cyclic alkyl groups, including but not limited to, methyl, ethyl, 1 -propyl (i.e., n-propyl), 2-propyl (i.e., iso-propyl), cyclopropyl, 1 -butyl (i.e., n- butyl), 2-butyl (i.e., sec-butyl), 2-methylprop-l-yl, (i.e., iso-butyl) 2-methylprop-2-yl (i.e., tert-butyl), pentyl, isopentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, cyclo
  • the present invention includes not only the ⁇ -D-arabinofuranosylbenzimidazoles, but also their ⁇ -D- ⁇ -L, and ⁇ -L analogs.
  • R 2 can be an amino group (i.e., -NH 2 ), a substituted amino group (e.g., -NH 2 , -
  • NHR 8 , -N(R 8 ) 2 a cyclic amino group; a halo group (e.g., -F, -Cl, -Br, -I), a nitro group (i. e. , -NO 2 ), an oxy group (/ ' . e. , -OR 8 ), a sulfhydryl group (i. e. , -SH), a thioether group ( . e. ,
  • R 2 is preferably selected from the group consisting of: -H, -F, -Cl, -Br, -I,-NH 2 , -
  • R 2 is a Cl, -Br, -NH 2 , -NHR 8 , and -N(R 8 ) 2 group, most preferably, is a Cl, or -N(R 8 ) 2 group, wherein the R 8 group is an isopropyl group.
  • R 4 and R 7 are both -H.
  • R 5 and R 6 are independently the same or different and selected from the group consisting of: -H, -F, -Cl, -Br, and -I.
  • R 5 and R 6 are both chloro.
  • the benzimidazole group is a halobenzimidazole, such as halo-, dihalo-, trihalo-, tetrahalo-, and pentahalobenzimidazoles, including but not limited to, 5,6-dihalobenzimidazole (e.g., 5,6-dichlorobenzimidazole, 5,6-dibromobenzimidazole),
  • 2,5,6-trihalobenzimidazole e.g., 2,5,6-trichlorobenzimidazole, 2-bromo-5,6- dichlorobenzimidazole
  • 2,4,6-trihalobenzimidazole e.g., 2,4,6-trichlorobenzimidazole, 2- bromo-4,6-dichlorobenzimidazole
  • 2,4,5,6-tetrahalobenzimidazole e.g., 2,4,5,6- tetrachlorobenzimidazole, 2-bromo-4,5,6-trichlorobenzimidazole.
  • the benzimidazole is a 5,6-dichlorobenzimidazole with a chloro or isopropylamino substituent at the 2-position.
  • esters of the compounds of the present invention include carboxylic acid esters obtained by esterification of the 2'-, 3'- and/or 5 '-hydroxy groups, in which the carboxylic acid moiety is further substituted by a group selected from (1) straight or branched chain alkyl (for example, n-propyl, t-butyl, or n-butyl), alkoxyalkyl (for example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), aryl (for example, phenyl optionally substituted by, for example, halogen,
  • sulfonate esters such as alkylsulfonyl (e.g., methanesulfonyl) or aralkylsulfonyl; (3) amino acid esters (e.g., L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono- di- or triphosphate esters.
  • the phosphate esters may be further esterified by, for example, a C, .20 alcohol or reactive derivative thereof, or by a 2,3-di-(C 6 . 24 )acyl glycerol.
  • any alkyl moiety present advantageously-contains from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more particularly from 1 to 4 carbon atoms.
  • Any cycloalkyl moiety present in such esters advantageously contains from 3 to 6 carbon atoms.
  • Any aryl moiety present in such esters advantageously comprises a phenyl group.
  • substituents on the carboxyl moiety suitable for ester formation at the 2'-, 3'- and 5'- position include, but are not limited to, CH 3 , C 6 H 5 groups.
  • the present invention includes 2'-deoxy, 3'-deoxy, and 5'-deoxy arabinofuranosyl benzimidazoles.
  • each of the above-described compounds can exist in a D or L conformation and each of that conformation can be an ⁇ or a ⁇ anomer.
  • this invention contemplates following groups of compounds: ⁇ -D-arabinofuranosyl benzimidazole; ⁇ -L-arabinofuranosyl benzimidazole; ⁇ -D- arabinofuranosyl benzimidazole; ⁇ -L-arabinofuranosyl benzimidazole; ⁇ -D-2'deoxy- arabinofuranosyl benzimidazole; ⁇ -L-2'-deoxy-arabinofuranosyl benzimidazole; ⁇ -D- 3'deoxy-arabinofuranosyl benzimidazole; ⁇ -L-3'-deoxy-arabinofuranosyl benzimidazole; ⁇ - D-3'-deoxy-arabinofuranosyl benzimidazole; ⁇ - D-3'-deoxy
  • Some specific compounds provided by this invention include: l-( ⁇ -L-arabinofuranosyl)-2,5,6-trichloro-benzimidazole; l-( ⁇ -L-arabinofuranosyl)-2-bromo-5,6-dichloro-benzimidazole; l-( ⁇ -L-arabinofuranosyl)-2-methylamino-5,6-dichloro-benzimidazole; l-( ⁇ -L-arabinofuranosyl)-2-isopropylamino-5,6-dichloro-benzimidazole; l-( ⁇ -L-arabinofuranosyl)-2-cyclopropylamino-5,6-dichloro-benzimidazole; and l-( ⁇ -L-arabinofuranosyl)-2-cycloheptylamino-5,6-dichloro-benzimidazole.
  • the invention also includes pharmaceutically acceptable salts, prodrugs and derivatives of the above-described compounds.
  • Benzimidazoles compounds, and methods for their preparation are known in the art and described in detail below. See also U.S. Patent Nos. 5,248,672 and 5,360,795.
  • compositions containing the above noted compounds, prodrugs, salts, and derivatives thereof, alone or in combination with each other, and a carrier, are further provided.
  • the carrier is a solvent. This combination may be useful in the synthesis of further derivatives of the disclosed compounds.
  • the carrier is a pharmaceutically acceptable carrier which is useful in the prophylactic and therapeutic methods described below. It should be understood, although not always explicitly stated that the compositions can contain one or more of the above noted compounds, prodrugs, salts, and derivatives thereof or alternatively, additional art recognized compounds and/or yet to be discovered compounds.
  • Formulations within the scope of this invention include, but are not limited to, ointments, gels, pastes, creams, sprays, lotions, suspensions, solutions and emulsions of the active ingredient in aqueous or nonaqueous diluents, syrups, granulates or powders.
  • One aspect of the present invention provides methods for inhibiting viral replication and/or propagation by contacting the virus with an effective amount of one or more compounds and/or compositions of the present invention.
  • the contacting is conducted under suitable conditions to inhibit viral replication and/or propagation.
  • This invention also provides methods of preventing viral infection and/or propagation in a cell or tissue by contacting the cell or tissue with an effective amount of the compounds and/or compositions as defined above. The contacting is conducted under suitable conditions to such that viral infection and/or propagation are inhibited.
  • the present invention encompasses not only the treatment and prevention of viral infections, it also provides compositions and methods for treating and/or preventing pathologies associated with some viral infections. For example, restinosis has been correlated with prior HCMV infections in individuals.
  • suitable conditions includes in vitro, ex vivo or in vivo conditions.
  • In vitro use of a material is defined as a use of a material or compound outside a living human, mammal, or vertebrate, where neither the material nor compound is intended for reintroduction into a living human, mammal, or vertebrate.
  • an in vitro use would be the analysis of components of a blood sample using laboratory equipment or use of a compound in a cell culture to prevent or inhibit viral infection, proliferation or replication.
  • contacting may be effected by incubating virally infected cells with an effective amount of one or more compounds or compositions of the present invention.
  • the compounds or compositions can be added directly to the culture media or combined with a carrier prior to addition to the cells.
  • the invention provides a means to treat or prevent a viral infection by administering to an infected subject or a susceptible subject a composition of a pharmaceutically acceptable carrier or a biologically acceptable carrier and a therapeutically or prophylactically effective amount of one or more compounds as described herein.
  • an effective amount of the compound or compositions is adminstered to the subject.
  • Administration can be effected in one dose, continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the target virus, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician. Suitable dosage formulations and methods of administering the compounds can be found below.
  • the methods of this invention are useful to treat and/or prevent viral infection, e.g., virus of herpes and non-herpes origins.
  • viral infection e.g., virus of herpes and non-herpes origins.
  • Herpes viruses belong to a large family of viruses that affect humans and animals.
  • HSV herpes simplex viruses
  • B- virus herpes simplex viruses
  • varicella-zoster which causes chickenpox and shingles
  • Epstein-Barr virus EBV, which causes mononucleosis
  • lymphocrypto virus human herpesvirus 6 (HHV6); human herpesvirus 7 (HHV7) kaposi-associated herpes virus (KHV); human herpesvirus 8 (HHV8) and herpesvirus simiae, among others.
  • Human cytomegalovirus (HCMV) also a human herpes virus, is a leading opportunistic pathogen among immunosuppressed individuals. See, for example, Gallant J. E. et al, J.
  • Animal pathogens of herpesviral origin include infectious bovine rhinotracheitis virus, bovine mammillitis virus, and cercopithecine herpesvirus 1 (B-virus), among others.
  • the human viruses of non-herpes origin that are contemplated to be treatable with the compounds and according to the methods of the present invention include influenza viruses A, B and C; parainfiuenza viruses -1,2, 3 and 4; adenovirus; reovirus; respiratory syncytial virus; rhino virus; coxsackle virus; echo virus; rubeola virus; hepatitis viruses of the types B and C (HBV and HCV); and papovavirus.
  • the animal viruses of non-herpes origin include pseudorabies virus (PRV, of swine), equine rhinopneumonitis and coital exanthema viruses (varicellaviruses); lymphocryptovirus; Marek's disease virus (of fowl), among others.
  • PRV pseudorabies virus
  • variantaviruses equine rhinopneumonitis and coital exanthema viruses
  • lymphocryptovirus of fowl
  • a herpesvirus of economic importance in the cattle industry is Bovine Herpesvirus- 1 (BHV-1), which has been associated with a variety of clinical disease manifestations, including rhinotracheitis, vulvovaginitis, abortions, conjunctivitis, encephalitis and generalized systemic infections. Gibbs et al, "Bovine herpesviruses. I: Bovine herpesvirus- 1," Vet. Bull.
  • the herpesvirus Pseudorabies virus also called Aujeszky's disease virus (ADV)
  • PRV herpesvirus Pseudorabies virus
  • ADV Aujeszky's disease virus
  • PRV herpesvirus Pseudorabies virus
  • the pig is the natural host of ADV. Accordingly, compounds of the present invention can be presented for use in the form of veterinary formulations, which may be prepared, for example, by methods that are conventional in the art.
  • herpesviruses A variety of disease symptoms and a complex clinical course are caused by herpesviruses. In the case of a first infection in an adult human, the symptoms may be very severe. Herpesviruses can cause recurrent infections, and the disability associated with these recurrences is a significant health problem. In the case of EBV and HCMV, acute
  • Mononuclear cells are the major candidate as cells involved in the latent state of HCMV infection, and infectious mononucleosis may follow blood transfusions from seropositive to seronegative individuals. Seronegative individuals may also become infected via transplantation of cells or organs from seropositive donors.
  • HBV hepatitis B virus
  • infection is typically via contaminated blood or body fluids, and thus HBV infection is prevalent among intravenous drug abusers, homosexuals, and in countries with less developed health care systems where the risk of exposure to contaminated blood products is high. It has recently been estimated that throughout the world there are approximately 250 million people who are chronic carriers of HBV.
  • hepatitis C virus An additional member of the hepatitis virus family that can be treated as defined herein is hepatitis C virus (HCV).
  • Hepatitis C which is neither hepatitis A nor hepatitis B, forms 95 to 100% of post-transfusion hepatitis and 40 to 50% of sporadic hepatitis and easily becomes chronic, further changing at high rates to cancer of liver via chronic hepatitis or hepatic cirrhosis.
  • HCV hepatitis C virus
  • the method comprises: (a) providing a composition containing the compound of this invention to be tested; (b) providing an extracorporeal cell system capable of being infected by HCV; (c) providing a biological sample containing infective HCV; (d) incubating the compositions of (a) and (c) with the cell system of (b) under conditions that would, in the absence of (a), allow infection of HCV in the cell
  • Preferred cell systems as disclosed in U.S. Patent No. 5,679,342 include hepatocytes, macrophages, more preferably Kupffer macrophages, and B lymphocytes. Cell lines derived from organs of hepatocytic origin also are suitable for use in the assay described above, The Houghton method can also be used for prophylactic screening of the compounds of the present invention for their activity against HCV or other viruses in general.
  • Another method well known in the art for testing the antiviral activity of compounds against HCV is the helicase inhibition assay. See, for example, Lain et al, Nucleic Acids Resl, 69:1720-1726, (1991) and Kim et al., Biochem. Biophys, Res. Comm., 160-166, (1995).
  • interferon has been known as an agent having inhibitory effect on the proliferation of hepatitis C virus
  • problems such as its low rate in the effectiveness as little as 30 to 40%, the 60 to 70% recrudescence after discontinuance of the dosage thereof, the appearance of influenza-like symptoms, such as pyrexia, headache and vomiting, and of diverse side effects such as leukopenia, at the high rates. Accordingly, there exists currently no effective treatment or preventive with acceptable efficacy and toxicity profile.
  • compositions and methods of this invention also provide methods for treating, preventing or ameliorating the symptoms or disorders associated with the viral infection.
  • inclusion disease blindness, mononucleosis, restenosis (HCMV); chickenpox, shingles (varicella-zoster virus); infectious mononucleosis, glandular, fever, and Burkittis lymphoma (Epstein-Barr virus); cold sores (herpes simplex virus 1); genital herpes (herpes simplex virus 2); roseola infantum (human herpes virus 6, human herpes virus 7); and kaposi sarcoma (human herpes virus 8).
  • this invention also provides methods of ameliorating, preventing, or treating disorders or symptoms associated with viral infection, e.g., HCMV or HSV-1 infection, e.g., restenosis, opportunistic infections (such as retinal infections, gastrointestinal infections, pneumonia,
  • Restensosis is the narrowing of the blood vessels which can occur after injury to the vessel wall and is characterized by excessive proliferation of smooth muscle cells in the walls of the blood vessel treated. Restenosis can occur following a number of surgical techniques, for example, transplant surgery, vein grafting, coronary by-pass grafting and, most commonly, following angioplasty. Alternatives to the balloon catheter, such as pulsed lasers and rotary cutters, have been developed with a view to reducing or preventing restenosis following angioplasty, but have met with limited success. A number of drugs including anti-coagulants and vasodilators have also been tried with disappointing or equivocal results. Because restinosis appears to be associated with viral infections, the compounds of this invention can be used in methods to prevent or treat restenosis in a susceptible subject or patient.
  • a viral infection or when inhibiting viral replication or proliferation, wherein the virus is a hepatitis virus, such as a hepatitis B or hepatitis C
  • the virus is a hepatitis virus, such as a hepatitis B or hepatitis C
  • compounds specifically provided by the present invention and those disclosed hereinabove as within the scope of this invention, but also compounds such as unsubstituted arabinofuranosyl benzimidazoles, 1 -( ⁇ -D-arabino furanosyl)-2,5 ,6-trichlorobenzimidazole, and ⁇ -D-2'-deoxy-arabinofuranosyl benzimidazoles with the preferred substitutions disclosed above, can be used.
  • the same compounds, their salts, prodrugs or derivatives can also be used in preparing medicaments to be used in treating or preventing a viral infection or in inhibiting viral replication or propagation.
  • compositions When a subject such as a human patient is being treated, the compositions can be administered topically, orally, intranasally, parenterally or by inhalation therapy, and may take the form of pharmaceutically acceptable carriers such as tablets, lozenges, granules, capsules, pills, ampoules, suppositories or aerosol form. More particularly, a compound of the formula of the present invention also referred to herein as the active ingredient, may be administered for therapy by any suitable route including oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parental (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary. It will also be
  • a suitable dose for each of the above-named viral infections is in the range of about 0.1 to about 250 mg per kilogram body weight of the recipient per day, preferably in the range of about 1 to about 100 mg per kilogram body weight per day and most preferably in the range of about 5 to about 20 mg per kilogram body weight per day.
  • all weights of active ingredient are calculated as the parent compound of the formula of the present invention for salts or esters thereof, the weights would be increased proportionately.
  • the desired dose is preferably presented as two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
  • sub-doses may be administered in unit dosage forms, for example, containing about 10 to about 1000 mg, preferably about 20 to about 500 mg, and most preferably about 100 to about 400 mg of active ingredient per unit dosage form. It will be appreciated that appropriate dosages of the compounds and compositions of the invention may depend on the type and severity of the viral infection and can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the antiviral treatments of the present invention.
  • the active ingredient should be administered to achieve peak plasma concentrations of the active compound of from about 2 ⁇ M to about lOO ⁇ M, preferably about 5 ⁇ M to about 70 ⁇ M, most preferably about 1 to about 50 ⁇ M.
  • This may be achieved, for example, by the intravenous injection of about 0.1 to about 5% solution of the active ingredient, optionally in saline, or orally administered, for example, as a tablet, capsule or syrup containing about 0.1 to about 250 mg per kilogram of the active ingredient.
  • Desirable blood levels may be maintained by a continuous infusion to provide about 0.01 to about 5.0 mg/kg/hour or by intermittent infusions containing about 0.4 to about 15 mg per kilogram of the active ingredient.
  • the use of operative combinations is contemplated to provide therapeutic combinations requiring a lower total dosage of each component antiviral agent than may be required when each individual therapeutic compound or drug is used alone, thereby reducing adverse effects, e.g., cytotoxicity.
  • the active ingredient While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation comprising at least one active ingredient, as defined above, together with one or more pharmaceutically acceptable carriers therefor and optionally other therapeutic agents.
  • Each carrier must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, topical (including transdermal, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal) and pulmonary administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
  • the active ingredient may also be presented a bolus, electuary or paste.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g. , povidone, gelatin, hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross- linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying
  • Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • compositions for topical administration may be formulated as an ointment, cream, suspension, lotion, powder, solution, past, gel, spray, aerosol or oil.
  • a formulation may comprise a patch or a dressing such as a bandage or adhesive plaster impregnated with active ingredients and optionally one or more excipients or diluents.
  • the formulations are preferably applied as a topical ointment or cream containing the active ingredient in an amount of, for example, about 0.075 to about 20% w/w, preferably about 0.2 to about 25% w/w and most preferably about 0.5 to about 10% w/w.
  • the active ingredient may be employed with either a paraffmic or a water- miscible ointment base.
  • the active ingredients may be formulated in a cream with an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane- 1, 3 -diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • the oily phase of the emulsions of this invention may be constituted from known ingredients in an known manner. While this phase may comprise merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at lease one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is merely an emulsifier (otherwise known as an emulgent), it desirably comprises a mixture of at lease one emulsifier with a fat or an oil or with both a fat and an oil.
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabilizer. It is
  • Emulgents and emulsion stabilizers suitable for use in the formulation of the present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulphate.
  • the choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the active compound in most oils likely to be used in pharmaceutical emulsion formulations is very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for topical administration to the eye also include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the active ingredient.
  • the active ingredient is preferably present in such formulation in a concentration of about 0.5 to about 20%, advantageously about 0.5 to about 10%) particularly about 1.5% w/w.
  • Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient, such carriers as are known in the art to be appropriate.
  • Formulations suitable for nasal administration wherein the carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid
  • Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer include aqueous or oily solutions of the active ingredient.
  • Formulations suitable for parenteral administration include aqueous and non- aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily subdose, as herein above-recited, or an appropriate fraction thereof, of an active ingredient.
  • the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example, those suitable of oral administration may include such further agents as sweeteners, thickeners and flavoring agents.
  • the compounds of this invention also can be administered in combination with other known therapeutic agents for the inhibition of the replication or propagation of the above virus and associated conditions.
  • Combination therapies according to the present invention comprise the administration of at least one compound of the present invention and at least one other pharmaceutically active ingredient.
  • the compounds of the invention could be used in combination therapies to treat viral infections, such as HCMV and HSV-1 infections, in AIDS patients already receiving the antiviral drug zidovudine (AZT) and/or 3TC.
  • Combination therapies with AZT may provide the advantage of less toxicity over the combination of ganciclovir with AZT.
  • the combination of the compounds of this invention with AZT may produce less cytotoxicity (i.e.
  • the active ingredient(s) and pharmaceutically active agents may be administered simultaneously in either the same or different pharmaceutical formulations or sequentially in any order.
  • the amounts of the active ingredient(s) and pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • the combination therapy involves the administration of one compound according to the invention and one of the agents mentioned herein below.
  • the term "operative combination" is intended to include any chemically compatible combination of a compound of the present invention with other compounds of the present invention or other compounds outside the present invention (such as ganciclovir, AZT, and focarnet), as long as the combination does not eliminate the antiviral activity of the compound of the present invention.
  • agents that are effective for the treatment of viral infections or associated conditions are (1 -alpha, 2-beta, 3-alpha)-9-[2,3- bis(hydroxymethyl)cyclobutyl] guanine [(-)BHCG, SQ-34514], oxetanocin-G(3,4-bis- (hydroxymethyl)-2-oxetanosyl]guanine), acyclic nucleosides (e.g., acyclovir, valaciclovir, famciclovir, ganciclovir, penciclovir), acyclic nucleoside phosphonates (e.g., (S)-l-(3- hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC), ribonucleotide reductase inhibitors such as 2-acetylpyridine 5-[(2-chloroa nilino)thiocarbonyl] thiocarbonohydrazone, 3
  • inhibitors such as 7-chloro-5-(2-pyrryl)-3H-l,4-benzodiazepin-2-(H)one (Ro5-3335), 7- chloro- 1 ,3-dihydro-5-( 1 H-pyrrol-2-yl)-3H- 1 ,4-benzodiazepin-2-amine (Ro24-7429), interferons such as ( ⁇ -interferon, renal excretion inhibitors such as probenecid, nucleoside transport inhibitors such as dipyridamole; pentoxifylline, N-acetylcysteine (NAC), Procysteine, ( ⁇ -trichosanthin, phosphonoformic acid, as well as immunomodulators such as interleukin II or thymosin, granulocyte macrophage colony stimulating factors, erythropoetin, soluble CD 4 and genetically engineered derivatives thereof, or non- nucleoside reverse transcriptase inhibitors such as nevirapine (BI-RG
  • the inventors have provided certain new benzimidazole compounds with antiviral activity.
  • the compounds of this invention are useful as a positive control in screening for new drugs or agents having antiviral activity.
  • To practice the screen one performs the steps of: a) measuring anti-viral activity of a compound of this invention; b) measuring anti- viral activity of a potential anti-viral agent.
  • the potential agent is a candidate therapeutic if its viral activity equals or exceeds the anti- viral activity of the compound of this invention.
  • the candidate therapeutic also has little or no cytotoxicity.
  • the measuring anti-viral activity consists of: a) contacting a virus-infected cell with an agent or compound; and b) assaying the virus-infected cell for inhibition of viral proliferation whereby the extent of inhibition of viral proliferation indicates anti-viral activity.
  • the contacting can be in vtro, in vivo, or ex vivo.
  • the agent can be one or more of: a small molecule, a polynucleotide, a polypeptide, a polysaccharide, a glycopeptide, or a peptidonucleic acid.
  • the agent is a nucleic acid
  • the nucleic acid can be introduced into the virus-infected cell using a gene delivery vehicle such as a viral vector or liposome.
  • the agent when it is a nucleic acid, it can be contacted or introduced into the cell by methods well known in the art, which includes, but is not limited to, incorporating the nucleic acid into an expression or insertion vector.
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Examples of vectors are viruses, such as baculovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles used in the art.
  • nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens, e.g., TCR, CD3 or CD4.
  • liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • 2,3,5-tri-O-acetyl-glyco-furanosyl nucleosides can be prepared by a modified Vorbriiggen procedure from 1,2,3,5-tetra-O-acetyl-glycosides. See, for example, Vorbruggen, H. et al. , "Nucleoside Synthesis with Trimethylsilyl Triflate and Perchlorate as Catalyst," Chem. Ber., 114:1256-1268, (1981). The anomeric configuration is predominately trans, with respect to the 1' -heterocyclic moiety and 2'-O-acetyl group, in most cases. See, for example, Baker, B. R.
  • tetra-O-acetyl-L-arabinofuranose (compound 1) was prepared, in three steps in 61% yield after silica gel chromatography, from a procedure developed by Guthrie, R. D. and Smith, S. C, "An improved preparation of 1,2,3,5-tetra-O-acetyl- ⁇ -D-ribofuranose," Chem. Ind, 547-548, (1968) and generalized
  • the 2-chloro substituent of 1 -substituted benzimidazoles can be conveniently displaced by a variety of nucleophiles. See, for example, Harrison, D. and Ralph, J. T., "Nucleophilic substitution reaction of 2-chlorobenzimidazoles. Part 1. Formation of benzimidazolin-2-ones and 2-alkoxybenzimidazoles," J. Chem. Soc, 236-239, (1965). Following this approach, 5,6-dichloro-2-methylamino-l-( ⁇ -L- arabinofuranosyl)benzimidazole (compound 5) was prepared in one step (64% yield) by the direct treatment of compound 4 with a 2 M solution of methylamine in methanol at room temperature.
  • compound 1 (a protected L- arabinofuranose) was reacted with an appropriately substituted benzimidazole to result in a protected ⁇ -L-arabinofuranosyl benzimidazole (2), which was deprotected and the deprotected nucleoside was then isolated by using an appropriate eluent on flash column chromatography. It is within the ordinary skill to prepare a ⁇ -L-arabinofuranosyl benzimidazole according to this reaction scheme and isolate it from its ⁇ -anomer by selecting the appropriate eluent. Further, by substituting a D-arabinofuranose in the above reaction scheme, one can obtain the ⁇ -D- compounds of the present invention
  • the above ⁇ -L- and ⁇ -D- analogs of the ⁇ -L-arabinofuranosyl benzimidazoles of the present invention can be prepared from the methodology disclosed by the U.S. Patent 5,360,795. This patent describes in detail the preparation and isolation of ⁇ -D-arabinofuranosyl benzimidazole, the contents of which Patent are hereby incorporated by reference.
  • PCT publication WO 97/25337 discloses the preparation of 2'-deoxy- ⁇ -L- ribofuranosylbenzimidazole and PCT publication WO 94/08456 discloses the preparation of 2'-deoxy- ⁇ -D-ribofuranosylbenzimidazole. Since a 2'-deoxyribofuranosyl is equivalent to a 2'-deoxyarabinofuranosyl, one of skill in the art would readily understand that the above publications disclose the preparation of 2'-deoxy- ⁇ -L and ⁇ -D-arabinofuranosyl compounds. Analogous methods can be employed to prepare 2'-deoxy- ⁇ -L and ⁇ -D- arabinofuranosyl compounds. Additionally, Howell, H.G.
  • Stock HCMV was prepared by infecting HFF cells at a multiplicity of infection (m.o.i.) of ⁇ 0.01 plaque-forming units (p.f.u.) per cell as detailed previously. See, for example, Turk, S. R. et al, "Pyrrolo[2,3- ]pyrimidine Nucleosides as Inhibitors of Human Cytomegalovirus," Antimicrob. Agents Chemother., 31 :544-550, (1987). High titer HSV- 1 stocks were prepared by infecting KB cells (ATCC) at an m.o.i. of ⁇ 0.1 also as detailed previously. See, for example, Turk, S. R.
  • Virus titers were determined using monolayer cultures of HFF cells for HCMV and monolayer cultures of BSC-1 cells for HSV-1 as described earlier. See, for example, Prichard, M. N. et al, "A Microtiter Virus Yield Reduction Assay for the Evaluation of Antiviral Compounds against Human Cytomegalovirus and Herpes Simplex Virus,” J. Virol. Methods, 28:101-106, (1990).
  • HFF or BSC-1 cells were planted as described above in 96-well cluster dishes and incubated overnight at 37°C. The next day cultures were inoculated with HCMV or HSV-1 and serially diluted 1 :3 across the remaining eleven columns of the 96- well plate. After virus adsorption the inoculum was replaced with fresh medium and cultures were incubated for seven days for HCMV, two or three days for HSV-1. Plaques were enumerated under 20-fold magnification in wells having the dilution which gave 5 to 20 plaques per well. Virus titers were calculated according to the following formula: Titer
  • HFF cells in 24-well cluster dishes were infected with approximately 100 p.f.u. of HCMV per cm ⁇ cell sheet using the procedures detailed above. Following virus adsorption, compounds dissolved in growth medium were added to duplicate wells in four to eight selected concentrations. After incubation at 37°C for 7 to 10 days, cell sheets were fixed, stained with crystal violet and microscopic plaques enumerated as described above.
  • Drug effects were calculated as a percentage of reduction in number of plaques in the presence of each drug concentration compared to the number observed in the absence of drug.
  • HSV-1 An ELISA was employed to detect HSV-1. See, for example, Prichard, M. N. and Shipman, C, Jr., "A Three Dimensional Model to Analyze Drug-Drug Interactions," Antiviral Res., 14:181-206, (1990).
  • MEM(E) Three Dimensional Model to Analyze Drug-Drug Interactions
  • Antiviral Res. 14:181-206, (1990).
  • Ninety-six-well cluster dishes were planted with 10,000 BSC-1 cells per well in 200 ⁇ L per well of MEM(E) plus 10% calf serum. After overnight incubation at 37°C, selected drug concentrations in quadruplicate and HSV-1 at a concentration of 100 p.f.u./well were added.
  • Cytotoxicity produced in stationary HFF cells was determined by microscopic inspection of cells not affected by the virus used in plaque assays. See, for example, Turk, S. R. et al, "Pyrrolo[2,3-cT]pyrimidine Nucleosides as Inhibitors of Human Cytomegalovirus," Antimicrob. Agents Chemother., 31:544-550, (1987).
  • the effect of compounds during two population doublings of KB cells was determined by crystal violet staining and spectrophotometric quantitation of dye eluted from stained cells as described earlier. See, for example, Prichard, M. N.
  • test compound was added in quadruplicate at six to eight concentrations. Plates were incubated at 37°C for 48 hours in a CO 2 incubator, rinsed, fixed with 95% ethanol, and stained with 0.1% crystal violet. Acidified ethanol was added and plates read at 570 nm in a spectrophotometer designed to read 96-well ELISA assay plates.
  • Dose-response relationships were constructed by linearly regressing the percent inhibition of parameters derived in the preceding sections against log drug concentrations. Fifty-percent inhibitory concentrations (IC5Q'S) or ICgo's were calculated from the regression lines. Samples containing positive controls (acyclovir for HSV-1, ganciclovir for HCMV, and 2-acetylpyridine thiosemicarbazone for cytotoxicity) were used in all assays.

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Abstract

La présente invention a trait à des composés d'arabinofuranosyl benzimidazole, spécifiquement à un β-D-arabinofuranosylbenzimidazole représenté par la structure (I) et à ses analogues β-L, α-D et α-L. Dans la formule, R?2, R4, R5, R6 et R7¿ sont sélectionnés indépendamment dans le groupe constitué par: -H, halo, -NO¿2?, -NH2, -NHR?8, -N(R8)¿2, -OR8, -SR8, et -CF¿3; R?8 est -H ou un groupe alkyle de 1-8 atomes de carbone; R?10, R11 et R12¿ sont sélectionnés indépendamment dans le groupe comprenant -H, -OH et un groupe protecteur hydroxyle; l'invention concerne également des sels pharmaceutiquement acceptables de ces composés, à condition que: a) lorsque chaque R?4, R5, R6 et R7¿ est hydrogène, R2 est un groupe autre qu'hydrogène; b) lorsque R10 est -H, R11 n'est pas -H, et le composé n'est pas un composé β; et c) le composé n'est pas 2,5,6-trichloro-1-(β-D-arabinofuranosyl)benzimidazole. L'invention concerne des procédés d'utilisation des composés pour prévenir, inhiber ou traiter une réplication et/ou une propagation virale. De plus, l'invention concerne des procédés d'utilisation de ces composés pour préparer un médicament antiviral.
PCT/US1999/007308 1998-04-03 1999-04-02 Arabinofuranosyl benzimidazoles comme agents antiviraux WO1999051619A1 (fr)

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US6455506B1 (en) 1997-07-30 2002-09-24 Smithkline Beecham Corporation Lyxofuranosyl benzimidazoles as antiviral agents
US6770666B2 (en) 1999-12-27 2004-08-03 Japan Tobacco Inc. Fused-ring compounds and use thereof as drugs
US7112600B1 (en) 1999-12-27 2006-09-26 Japan Tobacco Inc. Fused-ring compounds and use thereof as drugs
US7285551B2 (en) 1999-12-27 2007-10-23 Japan Tobacco Inc. Fused-ring compounds and use thereof as drugs
US7511145B2 (en) 2003-08-01 2009-03-31 Genelabs Technologies, Inc. Bicyclic heteroaryl derivatives
EP2206715A1 (fr) 2004-02-24 2010-07-14 Japan Tobacco, Inc. Composé héterotétracycliques fusionnés et leur utilisation en tant qu'inhibiteurs de la polymérase du HCV
US7977331B1 (en) 2004-02-24 2011-07-12 Japan Tobacco Inc. Tetracyclic fused heterocyclic compound and use thereof as HCV polymerase inhibitor
US7659263B2 (en) 2004-11-12 2010-02-09 Japan Tobacco Inc. Thienopyrrole compound and use thereof as HCV polymerase inhibitor
CN115721606A (zh) * 2021-08-27 2023-03-03 博美利克斯技术公司 胶束、其制备方法以及包含其的药物组合物
US11998530B2 (en) 2021-08-27 2024-06-04 Biometrix Technology Inc. Micelle comprising benzimidazole-carbohydrate conjugate compound, preparation method thereof and use thereof as anticancer agent or antiviral agent comprising the same

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