METHODS AND COMPOSITIONS FOR POLYMERASE INHIBITION

专利摘要:
methods and compositions for inhibiting polymerase. The present invention relates to methods and compositions for inhibiting viral nucleic acid polymerases, such as RNA and DNA polymerases, and methods and compositions that are useful for treating viral infections in subjects. the methods comprise administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. the composition or method can optionally comprise one or more additional antiviral agents.
公开号:BR112013009029B1
申请号:R112013009029-4
申请日:2011-10-14
公开日:2021-06-29
发明作者:Shanta Bantia；Pravin L. Kotian；Yarlagadda S. Babu
申请人:Biocryst Pharmaceuticals, Inc.；
IPC主号:

专利说明:

This application claims priority to United States Provisional Application No. 61/393,522, filed October 15, 2010 and United States Provisional Application No. 61/492,054, filed June 1, 2011, both of which are incorporated here for reference.
Descriptions of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Background
The present invention relates to viral diseases that are responsible for both global pandemics and periodic annual epidemics such as influenza. Outbreaks can be characterized by potentiated virulence and can occur suddenly, resulting in serious mortalities. Importantly, viral diseases are not limited to humans. For example, influenza also affects livestock and poultry, which can have a significant impact on the food supply, in addition to increasing the risk of transmission to humans. Exemplary conditions relating to viral infection include, for example, influenza, smallpox, encephalitis, West Nile Virus disease, Yellow Fever, Dengue, hepatitis, human immunodeficiency, polio and Coxsackie.
The influenza A virus genome has an RNA-dependent RNA polymerase, which is a heterotrimetric complex of three subunits (PA, PB1 and PB2). RNA polymerase catalyzes the transcription and replication of viral RNA. Because virus transcription and replication depend on RNA polymerase activity, this enzyme has become of interest as a target for the development of new antiviral compounds, especially following the recent emergence of drug-resistant viruses. SUMMARY OF THE INVENTION
The invention provides methods and compositions for inhibiting viral nucleic acid polymerases, and methods and compositions that are useful for treating, suppressing and/or preventing viral infections in subjects.
The methods comprise administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof. , and a pharmaceutically acceptable carrier. The composition or method can optionally comprise one or more additional antiviral agents. The methods and compositions are useful for treating, suppressing and/or preventing viral infections in subjects that may arise from infection by one or more types of virus. Thus, the methods and compositions are useful for broad spectrum antiviral treatment, suppression and/or prevention.
The present invention is based, in part, on certain findings which are more fully described in the Examples section of the present application. For example, the present invention is based, in part, on the discovery that viral titer levels in cells were markedly reduced by treatment with a compound of formula I. Thus, the present invention also provides methods for reducing viral titer in a body fluid or cell comprising contacting said fluid or cell with a compound of formula I. The present invention is further based, in part, on the discovery that viral titer levels in cells for various viruses have been markedly reduced upon treatment with a compound of formula I, indicating broad spectrum antiviral activity for the compound of formula I against a variety of viral strains. Thus, the present invention also provides methods for reducing viral titer to various types, subtypes and/or strains of virus in a body fluid or cell comprised by contacting said fluid or cell with a compound of formula I.
In some embodiments, the present invention provides a method for inhibiting a viral RNA or DNA polymerase comprising contacting the polymerase with an effective inhibitory amount of the compound of formula I, or a pharmaceutically acceptable salt, solvate or hydrate thereof.
In some embodiments, the method is performed in vivo.
In some embodiments, the present invention provides a method of treating a subject suffering from a viral RNA infection which comprises administering to said subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt of the compound. same.
In some modalities, the bodily fluid is blood. In some modalities, the bodily fluid is plasma. In some modalities, the body fluid is blood serum.
In some embodiments, the subject is a mammal. In some modalities the subject is a human. In some modalities, the subject is a bird. In some modalities, the subject is a swine or pig.
These and other embodiments of the invention are further described in the following sections of the application, including the Detailed Description, Examples and Claims.
Still other objects and advantages of the invention will become apparent to those skilled in the art from the description herein, which is merely illustrative and not restrictive. Thus, other modalities will be recognized by a skilled artisan without deviating from the spirit and scope of the invention. SUMMARY OF FIGURES
FIG. 1 shows the phosphorylation of compound 1 in human hepatocellular carcinoma cells (Huh-7).
FIG. 2 shows 3H adenosine phosphorylation in Huh-7 cells.
FIG. 3 shows 3H compound 1 phosphorylation in Huh-7 cells.
FIG. 4 shows incorporation of genomic DNA and total RNA of compound 1 from 3H and 3H adenosine into Huh-7 cells after 24h.
FIG. 5 shows the effects of combination of compound 1 and peramivir (a neuraminidase inhibitor) on influenza in vitro.
FIG. 6 shows the effect of compound 1 (intramuscular) on weight loss in mice infected with influenza virus A/Victoria/3/75 H3N2.
FIG. 7 shows the effect of compound 1 (oral) on weight loss in mice infected with influenza virus A/Victoria/3/75 H3N2.
FIG. 8 shows the effect of compound 1 (intraperitoneal, intramuscular and oral) on the survival of mice infected with Ebola virus.
FIG. 9 shows the effect of compound 1 (intraperitoneal, intramuscular and oral) on weight loss in mice infected with Ebola virus.
FIG. 10 shows the effect of compound 1 (intramuscular and oral) on the survival of mice infected with Ebola virus.
FIG. 11 shows the effect of compound 1 (intramuscular and oral) on weight loss in mice infected with Ebola virus.
FIG. 12 shows the effect of compound 1 on the survival of hamsters infected with Yellow Fever virus.
FIG. 13 shows the effect of compound 1 on weight loss in hamsters infected with Yellow Fever virus.
FIG. 14 shows the oral pharmacokinetic curve of compound 1 dosed at 10 mg/kg as measured in rats. DETAILED DESCRIPTION
The invention provides methods and compositions for inhibiting viral nucleic acid polymerases, such as RNA and DNA polymerases, and methods and compositions that are useful for treating viral infections in subjects. The methods comprise administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. The composition or method can optionally comprise one or more additional antiviral agents. The methods and compositions are useful for treating, suppressing and/or preventing viral infections in subjects that may arise from infection with one or more types of virus. Thus, the methods and compositions are useful for broad spectrum antiviral treatment, suppression and/or prevention.
In particular, the present invention relates to methods of treating, suppressing and/or preventing diseases or conditions relating to viral infection comprising administering a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof.
The compounds of formula (I) are as follows:
or NH2.
Thus, in some embodiments of the compound of formula (I), A is NH 2 .
In some embodiments of the compound of formula (I), B is NH 2 .
In some embodiments of the compound of formula (I), A is OH.
In still some embodiments of the compound of formula (I), B is H.
In still some embodiments of the compound of formula (I), A is NH2 and B is H.
In still some embodiments of the compound of formula (I), A is OH and B is NH 2 .
In still some embodiments of the compound of formula (I), A is NH2 and B is NH2.
In still some embodiments of the compound of formula (I), A is OH and B is H.
The present invention is based, in part, on certain findings which are more fully described in the Examples section of the present application. For example, the present invention is based, in part, on the discovery that viral titer levels in cells have been markedly reduced by treatment with a compound of formula I. Thus, in some embodiments, the present invention provides methods for reducing the viral titer in a body fluid or cell comprised by contacting said fluid or cell with a compound of formula I. The present invention is further based, in part, on the discovery that viral titer levels in cells for various viruses have been markedly reduced by treatment with a compound of formula I, thus indicating broad spectrum antiviral activity for the compound of formula I against a variety of viral strains. Thus, the present invention also provides methods for reducing the viral titer to various types, subtypes and/or strains of virus in a body fluid or cell comprised by contacting said fluid or cell with a compound of formula I.
The compounds of the present invention are prepared in different forms, such as salts, hydrates, solvates or complexes, and the invention includes compositions and methods encompassing all variant forms of the compounds. In some embodiments, the compounds are prepared as salt hydrates. Abbreviations and Definitions
The abbreviation "PNP" refers to purine nucleoside phosphorylase.
The term "compound(s) of the invention" as used herein means a compound of formula I, and may include salts, tautomeric forms, hydrates and/or solvates thereof. Compounds of formula I can also include solvates or hydrates of salts thereof.
The term "solvate" as used herein means a compound of formula I, or a pharmaceutically acceptable salt thereof, wherein molecules of a suitable solvent are incorporated into the crystal lattice. A suitable solvent is physiologically tolerable in the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate".
A "pharmaceutical composition" refers to a mixture of one or more of the compounds described herein, or pharmaceutically acceptable salts, or hydrates thereof, with other chemical components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.
The term "pharmaceutically acceptable salt" is intended to include salts derived from inorganic and organic acids, including, for example, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric acids. , glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2-sulfonic and other acids. Pharmaceutically acceptable salt forms may also include forms where the ratio of molecules comprising the salt is not 1:1. For example, the salt may comprise more than one molecule of inorganic or organic acid per molecule of base, such as two molecules of hydrochloric acid per molecule of compound of formula (I). As another example, the salt may comprise less than one molecule of inorganic or organic acid per molecule of base, such as two molecules of compound of formula (I) per molecule of tartaric acid. Salts can also exist as solvates or hyd rats.
The term "acid" encompasses all pharmaceutically acceptable inorganic or organic acids. Inorganic acids include mineral acids such as hydrohalic acids such as hydrobromic and hydrochloric acids, sulfuric acids, phosphoric acids and nitric acids. Organic acids include all pharmaceutically acceptable aliphatic, alicyclic and aromatic carboxylic acids, dicarboxylic acids, tricarboxylic acids and fatty acids. Preferred acids are saturated or unsaturated, straight-chained or branched C1-C20 aliphatic carboxylic acids, which are optionally substituted by halogen or hydroxyl groups, or C6-C12 aromatic carboxylic acids. Examples of such acids are carbonic acid, formic acid, fumaric acid, acetic acid, propionic acid, iso-propionic acid, valeric acid, alpha-hydroxy acids such as glycolic and lactic acid, chloroacetic acid, benzoic acid, methanesulfonic acid and acid salicylic. Examples of dicarboxylic acids include oxalic acid, malic acid, succinic acid, tartaric acid and maleic acid. An example of a tricarboxylic acid is citric acid. Fatty acids include all pharmaceutically acceptable aliphatic or aromatic carboxylic acids having from 4 to 24 carbon atoms. Examples include butyric acid, isobutyric acid, sec-butyric acid, lauric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and phenylacetic acid. Other acids include gluconic acid, glucoheptonic acid and lactobionic acid.
As used herein the term "about" is used herein to mean roughly, roughly, around or in the region of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used here to modify a numerical value above and below the stated value by a 20 percent range plus or minus (higher or lower).
An "effective amount", "sufficient amount" or "therapeutically effective amount" as used herein is amount of a compound that is sufficient to effect beneficial and desired results, including clinical results. As such, the effective amount may be sufficient, for example, to reduce or ameliorate the severity and/or duration of the viral infection, or one or more symptoms thereof, prevent the advancement of the viral infection, prevent the recurrence, development or onset of one or more symptoms associated with viral infection, prevent or reduce the replication or multiplication of a virus, prevent or reduce the production and/or release of a viral particle, enhance or otherwise increase the prophylactic effect(s)( s) or therapeutic(s) of another therapy. An effective amount also includes that amount of the compound of formula I that substantially prevents or alleviates unwanted side effects.
As used herein and as well understood in the art, "treatment" is an approach to obtaining beneficial and desired results, including clinical results. Beneficial or desired clinical outcomes may include, but are not limited to, alleviating and ameliorating one or more symptoms or conditions, decreasing the extent of the disease, a stable (i.e., not worsening) state of the disease, preventing the spread of the disease, delay or delay of disease progression, improvement or palliation of disease state, and remission (partial or total case), whether detectable or not. "Treatment" can also mean prolonging survival compared to expected survival if you do not receive treatment.
The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which a compound is administered. Non-limiting examples of such pharmaceutical carriers include liquids such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Pharmaceutical carriers can also be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. Additionally, auxiliary agents, stabilizers, thickeners, lubricants and colorants can be used. Other examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin; incorporated herein by reference in its entirety.
The terms "animal," "subject" and "patient" as used herein include all members of the animal kingdom, including, but not limited to, mammals, animals (e.g., cats, dogs, horses, pigs, etc.) and humans. Description
The present invention provides methods and compositions for inhibiting viral nucleic acid polymerases, such as viral DNA and/or RNA polymerases, and methods and compositions that are useful for treating viral infections in subjects. The methods comprise administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, and a pharmaceutically acceptable carrier. The composition or method can optionally comprise one or more additional antiviral agents. The methods and compositions are useful for treating, suppressing and/or preventing viral infections in subjects that may arise from infection by one or more families, genus, subtypes, serotypes or strains of viruses.
The compounds of formula I are 9-deazaadenine derivatives, generally known as imucillins, the synthesis of which is described, for example, in WO 03/80620, and by Evans et al., in Tetrahedron2000, 56, 3053 and J. Org .Chem. 2001, 66(17), 5723 (each being incorporated herein by reference in its entirety). Syntheses of similar structures are discussed, for example, in U.S. Pat. 5,985,848; 6,066,722; 6,228,741 and PCT Publications WO 2003/080620 and 2008/030119 (each incorporated herein by reference in its entirety). Imucillin derivatives have been studied as PNP inhibitors ( Vide, Kicska et al., J. Biol. Chem. 2002, 277, 3219-3225, and Kicska et al., J. Biol. Chem. 2002, 277, 3226-3231 each being incorporated herein by reference in their entirety). Some imucillins have also been studied as inhibitors of 5'-methylthioadenosine phosphorylase (MTAP) or 5'-methylthioadenosine nucleosidase (MTAN). Such mechanisms have been implicated in the treatment of cancer and bacterial infections (See, WO 03/080620, incorporated herein by reference in its entirety).
The compounds of formula I can exhibit tautomeric properties. Thus, the present invention also encompasses decomposed tautomeric forms of formula I, and mixtures thereof. It will further be appreciated that certain compounds exist as pharmaceutically acceptable salts, solvates, and/or hydrates, each of which is within the embodiments of the invention.
In some embodiments, the formula I compound exists as a pharmaceutically acceptable salt. In some embodiments, the salt form is at a 1:1 ratio of acid and formula I compound. In some embodiments, the salt form is greater than about a 1:1 ratio of acid and formula I compound. In some embodiments, the salt form is about a 2:1 ratio of acid and formula I compound. In some embodiments, the salt form exists as a hydrate.
In some embodiments, the formula I compound exists as a hydrate or solvate.
The compounds of the description, therefore, are useful in treating and/or preventing viral infections in a host or subject. The methods of the invention can be used in the treatment and/or prevention of disease states and conditions caused by and/or related to such viral infections. Examples of such viral infections include, but are not limited to, adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza ( including human, avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease, Encephalitis Venezuelan equine, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae.
In some embodiments, compounds of the invention are used to treat or prevent a viral infection associated with a virus. In some embodiments, the viral infection comprises infection by one or more types of virus. In some embodiments, the viral infection comprises infection by one or more viruses selected from the group consisting of orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae viruses. In some embodiments, the viral infection comprises infection by one or more viruses selected from the group consisting of adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox , encephalitis, Yellow Fever, Dengue, influenza (including human, avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacarib and pachindae virus.
In some embodiments, the viral infection comprises infection by one or more viruses selected from the group consisting of adenovirus, Dengue, influenza A and influenza B (including human, avian and swine), junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, Rhinovirus, Rift Valley Fever, Severe Acute Respiratory Syndrome (SARS), Tacarib, Venezuelan Equine Encephalitis, West Nile Virus, and Yellow Fever Virus.
In some embodiments, the virus is Ebola, Marburg, Yellow Fever, influenza A, or influenza B. In some embodiments, the virus is Ebola. In some embodiments, the virus is Marburg. In some modalities, the virus is Yellow Fever. In some embodiments, the virus is influenza A or influenza B.
In some modalities, the virus is West Nile Virus or Dengue. In some modalities the virus is West Nile Virus. In some modalities, the virus is Dengue.
In some embodiments, compounds of the invention are used to inhibit the replication or possibility of infection of a virus. In some embodiments, compounds of the invention are used to inhibit the growth of a cell infected with a virus. Examples of such viruses include, but are not limited to, viruses of the family orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae. Specific examples of viruses include, but are not limited to, adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza (including human, avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae.
Thus, in some embodiments, the virus is selected from the group consisting of viruses of the family orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae. In some embodiments, the viral infection comprises a virus selected from the group consisting of hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza ( including human, avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease , Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacarib and pachindae virus.
In some embodiments, the viral infection comprises a virus selected from the group consisting of adenovirus, Dengue, influenza A and influenza B (including human, avian and swine), junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, rhinovirus , Rift Valley Fever, Severe Acute Respiratory Syndrome (SARS), Tacarib, Venezuelan Equine Encephalitis, West Nile Virus and Yellow Fever Virus.
In some embodiments, the virus is Ebola, Marburg, Yellow Fever, influenza A, or influenza B. In some embodiments, the virus is Ebola. In some embodiments, the virus is Marburg. In some modalities, the virus is Yellow Fever. In some embodiments, the virus is influenza A or influenza B.
In some modalities, the virus is West Nile Virus or Dengue. In some modalities the virus is West Nile Virus. In some modalities, the virus is Dengue.
In some embodiments, the present invention provides a method of inhibiting a viral RNA or DNA polymerase in a subject comprising administering to said subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate of the compound. same.
According to the Baltimore classification system, RNA polymerase viruses can be classified into groups such as, for example, double-stranded viruses, positive-sense single-stranded viruses, and negative-sense single-stranded viruses. Positive-sense single-strand families include, for example, coronaviridae, picornaviridae, togaviridae, flaviviridae, and the like. Negative sense single-strand families include, for example, paramyxoviridae, arenaviridae, bunyaviridae, orthomyxoviridae, filoviridae, and the like. Each of the virus families can further be classified into genus, species and serotype (or subtype). Other designations for taxonomic designations of viruses are established by classification guidelines in accordance with the International Committee on Taxonomy of Viruses.
In some embodiments, RNA polymerase is double-stranded. In some embodiments, the RNA polymerase is single-stranded. In some embodiments, RNA polymerase is single-stranded positive sense. In some embodiments, RNA polymerase is single-stranded negative sense.
In some embodiments, the methods of the present invention provide for broad-spectrum inhibition of viruses and/or RNA polymerases from a virus family, genus, subtype, strain, and/or serotype. In some embodiments, the methods provide for the treatment, suppression or prevention of broad-spectrum infection from one or more virus families, genus, subtypes, strains, or serotypes. In some modalities, the broad spectrum encompasses more than two virus families, genera, subtypes, strains, and/or serotypes.
In some embodiments, the present invention provides a method for inhibiting viral polymerases from one or more virus families, genera, subtypes, serotypes or strains. In some embodiments, the present invention provides a method for treating, suppressing and/or preventing a viral infection where the viral infection is a result of infection by one or more families, genera, subtypes, serotypes or strains of viruses.
In some embodiments, viral polymerases or viruses are from one or more virus genera. In some embodiments, the viral polymerases or viruses are from one or more virus species. In some embodiments, viral polymerases or viruses are selected from one or more subtypes or serotypes. In some embodiments, viral polymerases or viruses are selected from one or more strains.
In some embodiments, viral RNA polymerase is selected from the group consisting of polymerases from the families orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picomaviridae, and coronaviridae. In some embodiments, viral RNA polymerase is selected from the group consisting of polymerases from the families orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, and coronaviridae. In some embodiments, viral RNA polymerase comprises polymerase selected from the group consisting of viral polymerase from hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza (including human, avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Disease from Kyasanur Forest, Venezuelan Equine Encephalitis, Eastern Equine Encephalitis, Western Equine Encephalitis, Severe Acute Respiratory Syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae.
In some embodiments, viral RNA polymerase is selected from the group consisting of adenovirus, Dengue, influenza A and influenza B (including human, avian and swine), junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, rhinovirus, Rift Valley Fever, Severe Acute Respiratory Syndrome (SARS), Tacarib, Venezuelan Equine Encephalitis, West Nile Virus and Yellow Fever viral polymerase.
In some embodiments, the viral RNA polymerase is Ebola, Marburg, Yellow Fever, influenza A, or influenza B viral polymerase. In some embodiments, the viral RNA polymerase is Ebola viral polymerase. In some embodiments, the viral RNA polymerase is Marburg viral polymerase. In some embodiments, the viral RNA polymerase is Yellow Fever viral polymerase. In some embodiments, the viral RNA polymerase is influenza A viral polymerase or influenza B viral polymerase. In some embodiments, the viral polymerase is West Nile or Dengue Virus viral polymerase. In some embodiments the viral polymerase is West Nile Virus viral polymerase. In some embodiments, the viral polymerase is Dengue viral polymerase.
In some embodiments, viruses are selected from the group consisting of families orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae. In some embodiments, viruses are selected from the group consisting of hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza (including human, avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae.
In some embodiments, viruses are selected from the group consisting of adenovirus, Dengue, influenza A and influenza B (including human, avian and swine), junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, rhinovirus, Rift Valley Fever, Severe Acute Respiratory Syndrome (SARS), Tacarib, Venezuelan Equine Encephalitis, West Nile Virus and Yellow Fever Virus.
In some embodiments, the virus is Ebola, Marburg, Yellow Fever, influenza A, or influenza B. In some embodiments, the virus is Ebola. In some embodiments, the virus is Marburg. In some modalities, the virus is Yellow Fever. In some embodiments, the virus is influenza A or influenza B.
In some modalities, the virus is West Nile Virus or Dengue. In some modalities the virus is West Nile Virus. In some ways, the virus is Dengue.
The influenza A virus genome has an RNA-dependent RNA polymerase, which catalyzes the transcription and replication of viral RNA. Because virus transcription and replication depend on RNA polymerase activity, this enzyme has become of interest as a target for the development of new antiviral compounds following the recent emergence of drug-resistant viruses. Viruses can develop resistance to a drug upon treatment, thus reducing the drug's effectiveness and requiring the subject to be treated with another antiviral drug. A drug or treatment that exhibits simultaneous efficacy against a broad spectrum of viral strains would thus be helpful.
Additionally, the composition or method may further comprise one or more additional antiviral agents in combination with a compound of formula I. Examples of such antiviral agents include, but are not limited to, Cytovene, Ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT and Amantadine, Rimantadine and other anti-influenza agents; Acyclovir and related agents, Foscarnet and other anti-herpesvirus agents. Non-limiting examples of a neuraminidase inhibitor include laninamivir, oseltamivir, zanamivir and peramivir.
Compounds which relate to influenza polymerase inhibition are described, for example, in U.S. Patent Nos. 7,388.002; 7,560.434; and in U.S. Patent Application Nos. 12/440,697 (published as U.S. Patent Publication No. 20100129317); and 12/398,866 (published as United States Patent Publication No. 20090227524), each of which is incorporated herein by reference in its entirety. Currently, there is an influenza polymerase inhibitor in clinical trials known as T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide). T-705 has potent and broad-spectrum antiviral activity against multiple strains of influenza virus infection in vitro and in vivo (Kiso et al., PNAS 2010, 107, 882-887; incorporated herein by reference in its entirety). T-705 is characterized by a mechanism of action that is different from most anti-influenza viral drugs.
Another class of compounds used as antivirals are M2 inhibitors (Vide, Pielak, R., Schnell, J., &Chou, J. (2009) Proceedings of the National Academy of Sciences, 106 (18), 7379-7384 (incorporated herein by reference in its entirety.) Exemplary members of this class include amantadine rimantadine.
Thus, in some embodiments, the methods of the invention further comprise administering one or more additional antiviral agents.
In some embodiments, an additional antiviral agent is selected from the group consisting of Cytovene, Ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT and amantadine, rimantadine, T-705 and other antiinfluenza agents; Acyclovir, and related agents, Foscarnet and other anti-herpesvirus agents.
In some embodiments, an additional antiviral agent is an anti-influenza agent. In some embodiments, an additional antiviral agent is a neuraminidase inhibitor. In some embodiments, an additional antiviral agent is selected from the group consisting of laninamivir, oseltamivir, zanamivir, and peramivir. In some embodiments, an additional antiviral agent is paramivir. In some embodiments, an additional antiviral agent is laninamivir. In some embodiments, an additional antiviral agent is oseltamivir. In some embodiments, an additional antiviral agent is zanamivir.
In some embodiments, an additional antiviral agent is an M2 inhibitor. In some embodiments, an additional antiviral agent is selected from the group consisting of amantadine and rimantadine.
In some embodiments, the methods of the invention comprise administering two additional antiviral agents. In some embodiments, the additional antiviral agents are a neuraminidase inhibitor and an M2 inhibitor. In some embodiments, additional antiviral agents are selected from the groups consisting of 1) laninamivir, oseltamivir, zanamivir, and peramivir; and 2) amantadine and rimantadine. In some embodiments, additional antiviral agents are peramivir and amantadine. In some embodiments, additional antiviral agents are peramivir and rimantadine.
The present invention provides methods for inhibiting a viral RNA or DNA polymerase comprising contacting the polymerase with an effective inhibitory amount of the compound of formula I, or a pharmaceutically acceptable salt or solvate thereof.
In some embodiments, the present invention provides a method of treating a subject suffering from a viral infection comprising administering to said subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof.
In some embodiments, the present invention provides a method for suppressing a viral infection in a subject comprising administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof.
In some embodiments, the present invention provides a method of treating a subject suffering from a viral RNA infection which comprises administering to said subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt or solvate thereof.
In some embodiments, the viral infection comprises infection by one or more viruses.
In some embodiments, viral infections are infections selected from viruses of the families orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picomaviridae or coronaviridae, or any combination thereof. In some modalities, viral infections are infections selected from hepatitis viruses, immunodeficiency viruses, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza (including human , avian and swine), lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease, Venezuelan equine encephalitis , Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae, or any combination thereof.
In some embodiments, viral infection comprises infection by one or more viruses selected from the group consisting of adenovirus, Dengue, influenza A and influenza B (including human, avian and swine), junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, ri-novirus, Rift Valley Fever, Severe Acute Respiratory Syndrome (SARS), Tacarib, Venezuelan Equine Encephalitis, West Nile Virus, and Yellow Fever Virus.
In some embodiments, the virus is Ebola, Marburg, Yellow Fever, influenza A, or influenza B. In some embodiments, the virus is Ebola. In some embodiments, the virus is Marburg. In some modalities, the virus is Yellow Fever. In some embodiments, the virus is influenza A or influenza B.
In some modalities, the virus is West Nile Virus or Dengue. In some modalities the virus is West Nile Virus. In some modalities, the virus is Dengue.
In some modalities, viral infections are infections selected from influenza A, influenza B, PIV, RSV, Junin, Pichinde, Rift Valley Fever, Dengue, Measles, Yellow Fever and SARS-CoV viruses, or any combination of the same. In some embodiments, viral infections are infections selected from influenza A and B, subtypes thereof, strains thereof, or any combination d. In some modalities, viral infections are infections selected from Ebola, Marburg or Yellow Fever. In some modalities, the viral infection is E-ball. In some modalities, the viral infection is Marburg. In some modalities, the viral infection is Yellow Fever. In some modalities, the viral infection is West Nile Virus or Dengue. In some modalities the viral infection is West Nile Virus. In some modalities, the viral infection is Dengue.
In some embodiments, the description provides for the use of pharmaceutical compositions and/or medicaments comprised of the compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, in a method of treating a viral infection, and/or disease state, and/or condition caused by or relating to such viral infection.
In some embodiments, the method of treatment comprises the steps of: i) identifying a subject in need of such treatment; (ii) providing a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof; and (iii) administering said compound or composition in an amount therapeutically effective to treat viral infection in the subject or to inhibit viral DNA or RNA polymerase activity in a subject in need of such treatment.
In some modalities, treatment effectiveness results from inhibition of a viral DNA or RNA polymerase. In some embodiments, treatment efficiency results from inhibition of viral polymerases from one or more virus families.
In some embodiments, viral polymerases or viruses are from one or more virus genus. In some embodiments, the viral polymerases or viruses are from one or more virus species. In some embodiments, viral polymerases or viruses are selected from one or more subtypes, serotypes or strains.
In some embodiments, the method is performed in vivo.
In some embodiments, the subject is a mammal. In some modalities, the subject is a human. In some modalities, the subject is a bird. In some modalities, the subject is a swine or pig.
In some modalities, the bodily fluid is blood. In some modalities, the bodily fluid is plasma. In some modalities, the body fluid is blood serum.
In some embodiments, the compound or composition is administered intravenously, interperitoneally, intramuscularly, or orally.
In some embodiments, the compound or composition is administered intravenously.
In some embodiments, the compound or composition is administered intraperitoneally.
In some embodiments, the compound or composition is administered intramuscularly.
In some embodiments, the compound or composition is administered orally.
The methods comprise administering to the subject a therapeutically effective amount of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier . Pharmaceutically acceptable carriers are well known to those skilled in the art, and include, for example, adjuvants, diluents, excipients, fillers, lubricants and vehicles. Often, the pharmaceutically acceptable carrier is chemically inert towards active compounds and is non-toxic under the conditions of use. Examples of pharmaceutically acceptable carriers may include, for example, water or saline, polymers such as polyethylene glycol, carbohydrates and derivatives thereof, oils, fatty acids or alcohols. In some embodiments, the carrier is saline or water. In some embodiments, the carrier is saline. In some embodiments, the carrier is water.
In some embodiments, the method of preventing or suppressing the viral infection or disease state comprises the steps of: i) identifying a subject in need of such treatment; (ii) providing a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof, or a composition comprising a compound of formula I, or a pharmaceutically acceptable salt or hydrate thereof; and (iii) administering said compound or composition in a therapeutically effective amount to prevent or suppress the viral infection or disease state in the subject or to inhibit viral DNA or RNA polymerase activity in a subject in need of such treatment.
The compounds of the present invention are prepared in different forms, such as salts, hydrates, solvates, tautomers or complexes, and the invention includes methods encompassing all variant forms of the compounds.
In some embodiments, the methods of the invention comprise pharmaceutically acceptable salts of the compound of formula I. A compound of formula I may also be formulated as a pharmaceutically acceptable salt, e.g., acid addition salt, and complexes thereof. The preparation of such salts can facilitate pharmacological use by altering the physical characteristics of the agent without preventing its physiological effect. Examples of useful changes in physical properties include, but are not limited to, lowering the melting point to facilitate transmucosal administration and increasing solubility to facilitate the administration of higher drug concentrations.
Subjects of the invention are in vitro and in vivo systems, including, for example, isolated or cultured cells or tissues, in vitro non-cellular analysis systems, and animals (for example, an amphibian, a bird, a fish, a mammal, a marsupial, a human, a domestic animal, such as, for example, a suit, dog, monkey, mouse or rat; or a commercial animal such as, for example, a cow or pig).
The compounds of the invention can be formulated into pharmaceutical compositions for administration to subjects in a biologically compatible form suitable for in vivo administration. According to other aspects, the present invention provides a pharmaceutical composition comprising compounds of formula I in admixture with a pharmaceutically acceptable diluent and/or carrier. The pharmaceutically acceptable carrier must be "acceptable" in the sense that it is compatible with the other ingredients of the composition and not deleterious to the recipient thereof. The pharmaceutically acceptable carriers employed herein can be selected from various organic or inorganic materials that are used as materials for pharmaceutical formulations and that are incorporated as analgesic agents, buffers, binders, disintegrants, diluents, emulsifiers, excipients, extenders, glidants, solubilizers. - agents, stabilizers, suspending agents, tonicity agents, vehicles and viscosity-increasing agents. Pharmaceutical additives, such as antioxidants, flavors, colorings, flavor-improving agents, preservatives and sweeteners, can also be added. Examples of acceptable pharmaceutical carriers include carboxymethylcellulose, crystalline cellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate, sucrose, starch, talc and water, among others. In some embodiments, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the United States Pharmacopoeia or other pharmacopoeia generally recognized for use in animals and, more particularly, in humans.
Surfactants, such as, for example, detergents are also suitable for use in the formulations. Specific examples of surfactants include polyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetate and vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol, glycerol, sorbitol or polyoxyethylenated sorbitan esters; sodium lecithin or carboxymethylcellulose; or acrylic derivatives, such as methacrylates and others, anionic surfactants, such as alkaline stearates, in particular sodium, potassium or ammonium stearate; calcium stearate or triethanolamine stearate; alkyl sulfates, in particular sodium lauryl sulfate and sodium cetyl sulfate; sodium dodecylbenzenesulfonate or sodium dioctyl sulfosuccinate; or fatty acids, in particular those derived from coconut oil, cationic surfactants, such as water-soluble quaternary ammonium salts of the formula N+R'R"R'"R""Y', where the R radicals are radicals of optionally identical or different hydroxylated hydrocarbons and Y' is a strong acid anion, such as halide, sulfate and sulfonate anions; Cetyltrimethylammonium bromide is one of the cationic surfactants that can be used, amine salts of the formula N+R'R"R"', wherein the R radicals are optionally identical or different hydroxylated hydrocarbon radicals; octadecylamine hydrochloride is one of the cationic surfactants which can be used, nonionic surfactants such as optionally polyoxyethylenated sorbitan esters, in particular Polysorbate 80, or polyoxyethylenated alkyl ethers; polyethylene glycol stearate, polyoxyethylene castor oil derivatives, polyglycerol esters, polyoxyethylene fatty alcohols, polyoxyethylene fatty acids or copolymers of ethylene oxide and propylene oxide, amphoteric surfactants such as lauryl substituted betaine compounds.
When administered to a subject, compounds of formula I and pharmaceutically acceptable carriers can be sterile. In some embodiments, water is a carrier when the formula I compound is administered intravenously. In some embodiments, the carrier is saline when the formula I compound is administered intravenously. Aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol , propylene, glycol, polyethylene glycol 300, water, ethanol, polysorbate 20 and the like. The present compositions, if desired, may contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
The pharmaceutical formulations of the present invention are prepared by methods well known in the pharmaceutical arts. For example, the compounds of formula I are brought into association with a carrier and/or diluent, such as a suspension or solution. Optionally, one or more accessory ingredients (eg, buffers, flavoring agents, surface active agents and the like) are also added. The choice of carrier is determined by the solubility and chemical nature of the compounds, chosen route of administration, and standard pharmaceutical practice. In some embodiments, the formulation comprises a compound of formula I and water. In some embodiments, the formulation comprises a compound of formula I and saline.
Additionally, the compounds of the present invention are administered to a human or animal subject by known procedures including, without limitation, oral administration, sublingual or buccal administration, parenteral administration, transdermal administration, via inhalation or intranasally, vaginally, via rectally and intramuscularly. The compounds of the invention are administered parenterally, by epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymal, subcutaneous or sublingual injection, or by cath. to have. In some embodiments, the compound is administered to the subject via intramuscular delivery. In some embodiments, the compound is administered to the subject via intraperitoneal delivery. In some embodiments, the compound is administered to the subject via intravenous delivery. In some embodiments, the compound is administered orally.
For oral administration, a formulation of the compounds of the invention may be presented as capsules, tablets, powders, granules, or as a suspension or solution. Capsule formulations can be gelatin, gel capsules or solid. Tablets and capsule formulations may additionally contain one or more adjuvants, binders, diluents, disintegrants, excipients, fillers or lubricants, each of which are known in the art. Examples of such include carbohydrates such as lactose or sucrose, anhydrous dibasic calcium phosphate, corn starch, mannitol, xylitol, cellulose or derivatives thereof, microcrystalline cellulose, gelatin, stearates, silicon dioxide, talc, starch glycolate sodium, acacia, flavoring agents, preservatives, buffering agents, disintegrants and coloring agents. Orally administered compositions may contain one or more optional agents, such as, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as spearmint, Wintergreen oil, or cherry; coloring agents; and preserving agents to provide a pharmaceutically palatable preparation.
For parenteral administration (i.e., administration by injection via a route other than the alimentary canal), the compounds of the invention can be combined with a sterile aqueous solution that is isotonic with the subject's blood. Such a formulation is prepared by dissolving a solid active ingredient in water containing physiologically compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then making said sterile solution. The formulation can be presented in unit or multiple dosage containers, such as ampoules or sealed vials. The formulation can be delivered via any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, parenchymal, subcutaneous or sublingual or by through the catheter into the subject's body.
Parenteral administration includes both aqueous and non-aqueous based solutions. Examples of which include, for example, water, saline, alcoholic sugars or aqueous sugar solutions, alcoholics (such as ethyl alcohol, isopropanol, glycols), ethers, oils, glycerides, fatty acids and fatty acid esters. In some modalities, water is used for parenteral administration. In some embodiments, saline is used for parenteral administration. Parenteral injection oils include animal, vegetable, synthetic or petroleum based oils. Examples of solution sugars include sucrose, lactose, dextrose, mannose and the like. Examples of oils include mineral oil, petrolatum, soy, corn, cottonseed, peanut and the like. Examples of fatty acids and esters include oleic acid, myristic acid, stearic acid, isostearic acid and esters thereof.
For transdermal administration, the compounds of the invention are combined with skin penetration enhancers, such as propylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid, N-methylpyrrolidone and the like, which increases the skin permeability of the compounds of the invention. and allow compounds to penetrate through the skin and into the bloodstream. The enhancing compound/compositions can also additionally be combined with a polymeric substance, such as ethylcellulose, hydroxypropylcellulose, ethylene/vinylacetate, polyvinylpyrrolidone and the like, to provide the composition in gel form, which is dissolved in a solvent, such as hydrochloride of methylene, evaporated to the desired viscosity and then applied to the backing material to provide a bandage.
In some embodiments, the composition is in unit dosage form, such as a tablet, capsule, or single-dose vial. Suitable unit doses, i.e. therapeutically effective amounts, can be determined during clinical trials designed appropriately for each of the conditions for which administration of a chosen compound is indicated and will obviously vary depending on the desired clinical endpoint.
The present invention also provides articles of manufacture for treating and preventing disorders, such as viral disorders, in a subject. The articles of manufacture comprise a pharmaceutical composition of the compounds of formula I, optionally additionally containing at least one additional antiviral compound as described herein. Articles of manufacture are packaged with indications for various disorders that the pharmaceutical compositions are capable of treating and/or preventing. For example, articles of manufacture comprise a unit dose of a compound described herein that is capable of treating or preventing a certain disorder, and an indication that the unit dose is capable of treating or preventing a certain disorder, for example, a viral infection.
According to a method of the present invention, the compounds of formula I are administered to the subject (or are contacted with cells of the subject) in an amount effective to limit or prevent a decrease in the level of virus in the subject, particularly in cells of the subject. This amount is readily determined by the skilled artisan based on known procedures, including the analysis of established in vivo titration curves and the methods and analyzes described here. In some embodiments, a suitable amount of the compounds of the invention effective to limit or prevent an increase in the level of viral particles in the subject ranges from about 0.01 mg/kg/day to about 1000 mg/kg/day, and/or is an amount sufficient to obtain plasma levels ranging from about 300 ng/ml to about 1000 ng/ml or higher. In some embodiments, the amount of compounds from an invention ranges from about 5 mg/kg/day to about 1000 mg/kg/day. In some embodiments, from about 0.01 mg/kg/day to about 500 mg/kg/day is administered. In some embodiments, from about 0.01 mg/kg/day to about 300 mg/kg/day is administered. In some embodiments, from about 0.01 mg/kg/day to about 200 mg/kg/day is administered. In some embodiments, from about 0.05 mg/kg/day to about 100 mg/kg/day is administered. In some embodiments, from about 0.05 mg/kg/day to about 50 mg/kg/day is administered. In some embodiments, from about 0.05 mg/kg/day to about 30 mg/kg/day is administered. In some embodiments, from about 0.05 mg/kg/day to about 10 mg/kg/day is administered.
The precise dose to be employed in the compositions will also depend on the route of administration, and the severity of the infection or disorder, and should be decided in the judgment of the practitioner and the individual circumstances of the patient. However, effective dosage ranges suitable for intramuscular administration are generally from about 0.5 to about 1000 mg of the compound of formula I per kilogram of body weight. In specific embodiments, the dose via i.m. is about 500 to about 1000 mg/kg, about 300 to about 500 mg/kg, about 200 to about 300 mg/kg, about 100 to about 200 mg/kg, about 50 to about 100 mg/kg, about 10 to about 50 mg/kg, or about 5 to about 10 mg/kg (or the equivalent doses expressed per square meter of body surface area). Alternatively, a suitable dosage range for i.v. administration can be obtained using doses from about 5 to about 1000 mg, without adjustment for a patient's body weight or body surface area. Alternatively, a dosage range suitable for administration via i.p. it can be achieved using doses of about 5 to about 1000 mg, without adjustment for a patient's body weight or body surface area. Oral compositions may contain from about 10% to about 95% by weight of one or more compounds of formula I alone or in combination with another therapeutic agent. In some embodiments of the invention, dosage ranges suitable for oral administration, i.p. or i.m. are generally about 5 to about 1000 mg, preferably about 5 to about 500 mg of compound per kilogram of body weight or their equivalent doses expressed per square meter of body surface area. In some modalities the oral dose, i.p. or i.m. is about 5 to about 50 mg/kg, about 50 to about 80 mg/kg, about 80 to about 150 mg/kg, about 150 to about 250 mg/kg, about 250 to about 350 mg/kg, about 350 to about 450 mg/kg, about 450 to about 550 mg/kg, about 550 to about 700 mg/kg, about 700 to about 1000 mg/kg (or the equivalent doses expressed per square meter of body surface area). In some embodiments, the dosage range is suitable for oral administration, i.p. or i.m. administration is from about 5 to about 2000 mg, without adjustment for a patient's body weight or body surface area. Other efficient doses can be extrapolated from dose response curves derived from animal model or in vitro test systems. Such animal models and systems are well known in the art.
In certain aspects, an "efficient amount" of a compound in the context of a viral infection is an amount sufficient to reduce one or more of the following steps in the life cycle of a virus: the anchoring of the particular virus to a cell, the introduction of viral genetic information in a cell, the expression of viral proteins, the production of new virus particles and the release of virus particles from a cell by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, an efficient amount of a compound in the context of a viral infection reduces the replication, multiplication or spread of a virus by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least minus 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, an efficient amount of a compound in the context of a viral infection increases the survival rate of infected subjects by at least 5%, preferably by 10%, at least 15%, at least 20%, at least 25% , at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% , at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
Those skilled in the art will recognize, to ensure the use of no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be equivalents to be within the scope of the present invention.
The invention is further described by way of the following non-limiting examples. EXAMPLES
Example 1: Synthesis of (2S,3S,4R,5R)-2-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4- diol [compound 1 (formula I, where A = NH2 and B = H) as the HCl salt].
Step 1:
For a solution of 7-((2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-2-yl)-3H-pyrrolo[3,2-d]pyrimidin -4(5H)-one (1-1) [(prepared according to the procedure reported by Evans, Gary B.; Furneaux, Richard H.; Hutchison, Tracy L; Kezar, Hollis S.; Morris, Philip E., Jr.; Schramm, Come L.; Tyler, Peter C in Journal of Organic Chemistry (2001), 66(17), 5723-5730; incorporated herein by reference in its entirety) 115 g, 390 mmol] in water and methanol (1:1, 2.4 L) was added triethylamine (113 ml, 1.12 mol) at room temperature followed by (Boc)2O (227 g, 1.04 mol). The reaction mixture was stirred at room temperature overnight. The solid product was collected by filtration, washed with water, and dried in vacuo to furnish (2R,3R,4S,5S)-tert-butyl 3,4-dihydroxy-2-(hydroxymethyl)-5-(4- oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (1-2) (100%) as a white solid. 1H NMR (300 MHz, DMSO) δ 7.85 (s, 1H), 7.35 (s, 1H), 4.73 - 4.53 (m, 1H), 4.29 (s, 1H), 4.03 (s, 1H), 3.97 (s , 1H), 3.70 - 3.53 (m, 2H), 1.36 and 1.04 (s, 3H, 6H for rotamers). Step 2:
For a solution of (2R,3R,4S,5S)-tert-butyl 3,4-dihydroxy-2-(hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[ 3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (1-2) in pyridine (184mmol, 2.26mol) was added DMAP (0.79g, 6.46mmol) and acetic anhydride (107 ml, 1131 mmol) at room temperature. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with chloroform and washed with water, aqueous HCl, water, and saturated aqueous sodium bicarbonate. The organic layer was dried, filtered and concentrated in vacuo to provide (2R,3R, 4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5- dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyl diacetate (1-3) (150 g), which was pure enough to be used as such for next step. MS (ES+) 493.1 (M+1), 515.1 (M+Na); (ES’) 491.4 (M-1). Step 3:
For a solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3.2] -d]pyrimidin-7-yl)pyrrolidine-3,4-diyl diacetate (1-3) (150 g, 300 mmol) in acetonitrile (660 ml) was added benzyltriethylammonium chloride (137 g, 600 mmol), dimethylaniline ( 57 ml, 450 mmol), followed by POCl3 (164 ml, 1800 mmol) at room temperature. The reaction mixture was heated to 80°C for 1h. The reaction mixture was cooled to room temperature and concentrated to vacuum drying. The residue obtained was dissolved in chloroform and washed with saturated aqueous sodium bicarbonate, brine, dried, filtered and concentrated to dryness. The residue of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7- il) pyrrolidine-3,4-diyl diacetate (1-4) was used as such in the next step without purification. 1H NMR (300 MHz, DMSO) Ô 12.54 (s, 1H), 8.65 (s, 1H), 7.92 (s, 1H), 5.85 (m, 1H), 5.45 (m, 1H), 5.10 (m, 1H) , 4.49 (m, 2H), 4.07 (m, 1H), 2.07 - 1.99 (m, 9H), 1.19 (2 bs, 9H, rotamers). Step 4:
For a solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7-yl )pyrrolidine-3,4-diyl diacetate (1-4) (300mmol) in DMF (540ml) was added azide (97.5g, 1500mmol) and heated at 80°C overnight. The reaction mixture was concentrated in vacuo and the residue obtained was dissolved in chloroform. The chloroform layer was washed with water, dried, filtered and concentrated in vacuo. Purification by crystallization from (acetone: hexane = 1:2) gave (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrole [3,2-d ]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-5). 1H NMR (300 MHz, DMSO) δ 13.56 - 13.00 (bs, 1H), 9.86 (s, 1H), 7.95 (s, 1H), 5.78 (m, 1H), 5 .40 (m, 1H), 5.26 - 5.14 (m, 1H), 4.54 (m, 1H), 4.42 (m, 1H), 4.16 - 4.03 (m, 1H ), 2.06 (s, 3H), 2.02 (s, 6H), 1.14 (bs, 9H); MS (ES+) 540.0 (M+1); (ES') 515.9 (M-1). Step 5:
For a solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-i I)-1 -(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-5) (300mmol) in methanol (1L) was added Pd(OH)2 (30g). The reaction mixture was hydrogenated at (160 psi) overnight, and filtered to remove catalyst through celite. The filtrate was concentrated in vacuo to give (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)- 1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-6) (113g). 1H NMR (300 MHz, DMSO) δ 12.47 -11.92 (m, 1H), 8.84 - 8.03 (m, 3H), 7.90 - 7.68 (m, 1H), 5, 70 - 5.51 (m, 1H), 5.38 (m, 1H), 5.12 (m, 1H), 4.42 (m, 2H), 4.17 - 4.00 (m, 1H) , 2.07 (s, 3H), 2.05 (s, 3H), 2.00 (s, 3H), 1.14 (s, 9H); MS (ES+) 492.1 (M+1), (ES') 490.0 (M-1). Step 6:
For a solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl) )pyrrolidine-3,4-diyl diacetate (1-6) (111 g, 226 mmol) in methanol (500 ml) was added NaOMe (25% w/w in methanol, 4.88 g, 22.6 mmol) at the temperature environment. The reaction mixture was stirred at room temperature for 3h and concentrated in vacuo to provide (2S,3S,4R,5R)-tert-butyl 2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin -7-yl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate (1-7). 1H NMR (300 MHz, DMSO) δ 11.40 -10.73 (bs, 1H), 8.01 (s, 1H), 7.39 (2s, 1H), 6.90 (s, 2H), 4 .83 (m, 2H), 4.45 (m, 2H), 3.96 (s, 2H), 3.58 (m, 3H), 1.31 and 0.99(s, 3H, 6H, rotamers ); MS (ES+) 366.0 (M+1), 388.0 (M+Na); (ES) 363.8 (M-1). Step 7:
A solution of (2S,3S,4R,5R)-tert-butyl 2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxy- Aqueous 5-(hydroxymethyl)pyrrolidine-1-carboxylate (1-7) of aqueous HCI (160 ml of cone, HCI and 400 ml of water) was stirred at room temperature for 30 min and then concentrated in vacuo to dry. The residue obtained was dissolved in water, treated with activated charcoal and refluxed for 30 min. The solution was filtered through celite and concentrated to obtain a semi-solid product, which was recrystallized from water and ethanol to give (2S,3S,4R,5R)-2-(4-amino-5H-pyrrolo[3.2 - d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol (1) (50 g, total yield for 7 steps: 42.6%) as white crystal. 1H NMR (300 MHz, D2O) δ 8.41 (s, 1H), 8.02 (s, 1H), 4.99 (d,J = 9 Hz, 1H), 4.78 (m, 1H), 4.45 (dd, J = 3.1.5 Hz, 1H), 3.97 (m, 2H), 3.90 (m, 1H); MS (ES+) 266.2 (M+1), (ES') 264.0 (M-1); Analysis: Calculated for CIIH15N5O3•2HCl: C, 39.07; H, 5.07; N, 20.71; Cl, 20.97; Found: C, 39.09; H, 5.10; N, 20.49; Cl, 20.84. Example 2: Large scale synthesis of (2S,3S,4R,5R)-2-(4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3 ,4-diol [compound 1 (formula I, wherein A = NH 2 and B = H) as the HCl salt]. Step 1:
For a suspension of 7-((2S,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidin-2-yl)-3H-pyrrolo[3,2-d]pyrimidin -4(5H)-one (1 -1) [(prepared according to the procedure reported by Evans, Gary B.; Furneaux, Richard H.; Hutchison, Tracy L.; Kezar, Hollis S.; Morris, Philip E. ., Jr.; Schramm, Vern L.; Tyler, Peter C in Journal of Organic Chemistry (2001), 66(17), 5723-5730), 500.0 g, 1.474 mol, 1 eq)] in a water mixture : methanol (1:1, 10.4L) was added triethylamine (621 ml, 4.422 mol, 3.0 eq) at room temperature followed by (Boc)20 (987 g, 4.53 mol, 3.1 eq) ). The reaction mixture became clearly colored after the addition of (Boc) 20 with a slight increase in the internal temperature from 28°C to 33°C. The solution started showing some turbidity after 1 hour of stirring. The solution was stirred at room temperature overnight. The solid product was collected by filtration and washed with water (5.0L), dried under high vacuum at 50°C to give (2R,3R, 4S,5S)-tert-butyl3,4-dihydroxy-2-( hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (1-2) (482 g, 89% ). 1H NMR (300 MHz, DMSO) δ 11.92 (s, 2H), 7.81 (s, 1H), 7.32 (d, J= 22.7 Hz, 1H), 5.73 - 5.20 (m, 1H), 5.05 - 4.91 (m, 1H), 4.87 - 4.76 (m, 1H), 4.74 - 4.49 (m, 1H), 4.33 - 4 .17 (m, 1H), 4.09 - 3.86 (m, 2H), 3.64 - 3.48 (m, 2H), 1.39 - 1.00 (m, 9H); MS (ES+) 755.1 (2M+Na), (ES) 731.7 (2M-1); Analyze; Calculated for C16H22N4-6: C, 52.45; H, 6.05; N, 15.29; Found: C, 52.24; H, 6.02; N, 15.05. Step 2:
For a suspension of (2R,3R,4S,5S)-tert-butyl 3,4-dihydroxy-2-(hydroxymethyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[ 3,2-d]pyrimidin-7-yl)pyrrolidine-1-carboxylate (1-2) (482 g, 1.32 mol, 1.0 equiv.) in pyridine (740 ml, 9.21 mol, 7 equiv.) DMAP (3.22 g, 26.32 mmol, 0.2 equiv.) and acetic anhydride (435 mL, 4.61 mmol, 3.5 eq) was added at room temperature. The internal temperature started to rise with the addition of acetic anhydride, therefore cooling by an ice-water bath was performed. With complete addition of the anhydride, the temperature increased to 67°C, then decreased to room temperature. The ice-water bath was removed after the reaction reached 25°C. The suspension did not provide a clear solution, but a lighter suspension was observed. The reaction mixture was stirred at room temperature for 14h to yield an unclear solution. A stimulated aliquot showed no more starting material and two important points by TLC (9:1 chloroform: methanol), MS shows two important points in (493.0, M+1) for product and provided tetra-acetylated (M+ 1=535). The reaction mixture was diluted with 3.0L of chloroform, stirred for 10 minutes, then 2.0L of deionized water was added. A waxy white product was formed at the aqueous organic phase interface. This product remained in the aqueous phase after partitioning. The organic phase was separated and washed again with 2.0L of water. The combined water washes were back extracted with 1.0 µl of chloroform. The combined organic phases were washed with 2.0N aqueous HCI (2 x 1.0L), water (2 x 1.0L), saturated sodium bicarbonate (2 x 1.0L) and brine (2 x 1.0L). The organic layer was dried over MgSO4 filtered and concentrated to dry in vacuum and 50-55°C water bath. The vacuum was changed to a high vacuum oil pump until no distillate was observed to provide a dense sweetened product. The product was left in a high vacuum oil pump for 14h to minimize residual pyridine. A combination of solid foam that turned to a good white solid and a dense residue of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4 ,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine-3,4-diyl diacetate (1-3) was obtained (715, 110% yield). This percentage reflects the amount of tetra-acetylated compound. The product was pure enough to be used as is for the next step. An analytical sample was prepared for purification of the mixture using flash column chromatography [silica gel, eluting with 0-100% (9:1) ethyl acetate/methanol in hexane] to give (2R,3R,4S,5S) -2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-7-yl)pyrrolidine- 3,4-diyl diacetate (1-3) as a white solid; 1HRMN (300 MHz, DMSO) δ 12.13 (s, 1H, Interchangeable D2O), 11.98 (s, 1H, Interchangeable D2O), 7.82 (s, 1H), 7.29 (s, 1H), 5.76 (s, 1H), 5.37 (t, J = 4.5 Hz, 1H), 4.99 (s, 1H), 4.55 (dd, J = 11.3, 6.6 Hz , 1H), 4.34 (d, J = 8.3 Hz, 1H), 4.03 (q, J = 7.1 Hz, 1H), 2.01 (d, J = 12.6 Hz, 9H ), 1.23 (dd, J = 39.9, 32.8 Hz, 9H); MS (ES+) 493.0 (M+1); (ES’) 526.7 (M+C1); Analysis: Calculated for C22H28N4θ9:C, 53.65; H, 5.73; N, 11.38; Found: C, 53.18; H, 5.89; N, 11.10 Step 3:
For a solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-oxo-4,5-dihydro-3H-pyrrolo[3.2] -d]pyrimidin-7-yl)pyrrolidine-3,4-diyl diacetate (1-3) (622g, 1.26mol, 1.0eq) in acetonitrile (2.75L) was added benzyltriethylammonium chloride (575g , 2.5 mol, 2.0 eq), dimethylaniline (240 ml, 1.9 mol, 1.5 eq), followed by POCI3 (706 ml, 7.58 mol, 6.0 eq) at room temperature . A light yellow colored solution was obtained. The reaction mixture was slowly heated to 80°C and held at that temperature for 10 minutes. TLC in 9:1 chloroform:methanol shows the reaction is >98% complete. The homogeneous black solution was cooled to 50.0°C and concentrated in vacuo (70-73°C water bath) to remove the POCI3, the residue placed under the high vacuum of the oil pump until no distillate was observed. The residue was dissolved in 3.0L of chloroform and quickly washed carefully with saturated aqueous sodium bicarbonate until a neutral pH was obtained. The organic layer was washed separately with water (2L), brine (2L), dried over MgSO4, filtered and concentrated in vacuo to dryness (water bath 50-53°C). The black product of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7 -yl)pyrrolidine-3,4-diyl diacetate (1-4) was used as is in the next step without purification. Step 4:
For a solution of (2R,3R,4S,5S)-2-(acetoxymethyl)-1-(tert-butoxycarbonyl)-5-(4-chloro-5H-pyrrolo[3,2-d]pyrimidin-7 -yl)pyrrolidine-3,4-diyl diacetate (1-4) (622g, 1.26mol, 1eq) in DMF (1.5L) was added sodium azide (411g, 6.32mol, 5 equiv.) and heated with stirring at 60°C for 10 hours, at which time the reaction was complete (TLC in 9:1 chloroform:methanol and 1:1 hexane:ethyl acetate). The reaction was cooled to 25°C, poured onto ice (2L) and extracted with chloroform (2 x 1L). The chloroform layers were combined and washed with water (2 x 2L), brine (2L), dried, filtered and concentrated in vacuo (70-80°C water bath) to yield a black slurry. Purification of the slurry was achieved by column chromatography (987 g black slurry, 8x30 inch column, 1/2 full of silica gel, elution profile hexane:ethyl acetate; 9:1 (40.0L); 7 :3 (20.0L); 6:4 (20.0L); 1:1 (20L); 4:6 (20.0L) and 2:8 (20.0L); The appropriate fractions were pooled and concentrated in vacuo ( 50.0°C water bath) to provide (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl )-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-5) (407.05 g, 62.3% yield for two steps) as a reddish colored honey-like product An analytical sample was prepared for purification of the mixture by flash column chromatography [0-100% ethyl acetate in hexane] to give (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H- pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-5) as an orange solid.1HNMR (300 MHz, DMSO) δ 13.08 (d, J = 155.6 Hz, 1H, exchangeable D2O), 9.86 (s, 1H), 7.6 1 (d, J = 76.8 Hz, 1H), 5.78 (t, J = 4.5 Hz, 1H), 5.41 (t, J = 4.3 Hz, 1H), 5.21 (s, 1H), 4.55 (dd, J = 11.4 , 6.4 Hz, 1H), 4.41 (dd, J = 11.4, 3.9 Hz, 1H), 4.07 (d, J = 16.5 Hz, 1H), 2.06 (s, 3H), 2.01 (d, J = 9.9 Hz, 6H) ), 1.23 (dd, J = 39.8, 32.7 Hz, 9H); MS (ES+) 518.0 (M+1), 540 (M+23); (ES) 516.4 (M-1); Analysis: Calculated for C22H27N7O8: C, 51.06; H, 5.26; N, 18.95 Found: C, 50.97; H, 5.30; N, 18.62. Step 5:
(2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine -3,4-diyl diacetate (1-5) was reduced in three different batches as follows Batch 1: To a 2.0L Parr hydrogenator, addition of Teflon was added (2R,3R,4S,5S)-2-(acetoxymethyl) -5-(4-azido-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-5) (108, 01g, 300mmol in methanol, 800ml), Pd(OH)2 (21.6g, 20% w/w). Lot 2: To a 2.0L Parr hydrogenator, addition of Teflon was added (1-5) (140.70g, 271.9mmol in methanol, 1.0L), Pd(OH)2 (28.14g, 20% w/w). Lot 3: To a 2.0L Parr hydrogenator, added Teflon (1-5) (140.7 g, 271.9 mmol in methanol, 1.0L), Pd(OH)2 (28.14 g) , 20% w/w).
Reaction mixtures were hydrogenated at 150 psi for 15-18 hours. The reaction mixture was filtered to remove the catalyst through celite. The filtrate was concentrated in vacuo (60-70°C water bath) to constant weight to give a dark colored product (2R,3R, 4S,5S)-2-(acetoxymethyl)-5-(4-amino- 5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-6) (328.8 g, 89%). The product was pure enough to be used as is for the next step. An analytical sample was prepared for the mixture using flash column chromatography (0-10% methanol in chloroform). 1H NMR (300 MHz, DMSO) δ 11.06 (s, 1H), 8.12 (s, 1H), 7.49 (s, 1H), 6.94 (s, 2H), 5.86 (s, 1H), 5.44 (t, J = 4.2 Hz, 1H), 5.02 (s, 1H), 4.56 (dd, J = 11.3, 6.9 Hz, 1H), 4.40 (dd, J = 11.3, 4.2 Hz, 1H), 4.16 - 3.98 (m, 1H), 2.09 - 1.94 (m, 9H), 1.48-1.14 (m, 9H); MS (ES+) 492.1 (M+1); (ES) 526.4 (M+C1); Analysis: Calculated for C22H29N5O8’1.25H2O: C, 51.41; H, 6.18; N, 13.62; Found: C, 51.24; H, 5.92; N, 13.33. Step 6:
Lot 1. For (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert. -butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-6) (81.5g, 165.8mmol), anhydrous methanol (370ml) was added followed by the addition of NaOMe (sodium methoxide, 25% by weight solution in methanol, 4.49g, 20.76 mmol) at room temperature. The reaction mixture was stirred at room temperature until TLC (chloroform: methanol 9:1) showed that all of the starting material had reacted.
Lot 2. For (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert. -butoxycarbonyl)pyrrolidine-3,4-diyl diacetate (1-6) (117.8 g, 239.6 mmol), anhydrous methanol (530 ml) was added followed by the addition of NaOMe (sodium methoxide, 25% by weight) solution in methanol, 6.58g, 30.45mmol) at room temperature. The reaction mixture was stirred at room temperature until TLC (chloroform:methanol 9:1) showed that all starting material had reacted;
Lot 3. For (2R,3R,4S,5S)-2-(acetoxymethyl)-5-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-1-(tert-butoxycarbonyl) )pyrrolidine-3,4-diyl diacetate (1-6) (129.5 g, 263.5 mmol) was added anhydrous methanol (584 ml) followed by the addition of NaOMe (sodium methoxide, 25% by weight solution in methanol) , 6.99g, 32.35mmol) at room temperature. The reaction mixture was stirred at room temperature until TLC (chloroform:methanol 9:1) showed that all of the starting material had reacted (7-8 hours).
The above solutions were concentrated (65-75°C water bath) to give (2S,3S,4R,5R)-tert-butyl 2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin- 7-yl)-3,4-dihydroxy-5-(hydroxymethyl)pyrrolidine-1-carboxylate (1 -7) which was pure enough to be used as is for the next step. An analytical sample was prepared by purifying the mixture using flash column chromatography (0-10% methanol in chloroform). 1H NMR (300 MHz, DMSO) Ô 10.77 (s, 1H), 8.01 (s, 1H), 7.40 (s, 1H), 6.82 (s, 3H), 5.04 - 4.91 (m, 1H), 4.87 - 4.74 ( m, 1H), 4.56 - 4.35 (m, 2H), 4.04 - 3.90 (m, 2H), 3.72 - 3.63 (m, 1H), 3.59 - 3.41 (m, 1H), 1.15 (2s, 9H); MS (ES+) 366.1 (M+1); (ES’) 400.3 (M+C1); Analysis: Calculated for C16H23N5O5*0.25H2O: C, 51.33; H, 6.46; N, 18.71; Found: C, 51.04; H, 6.43; N, 18.48. Step 7:
(2S,3S,4R,5R)-tert-butyl 2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-3,4-dihydroxy-5-(hydroxymethyl) pyrrolidine-1-carboxylate (1-7) were treated as follows in three batches.
Lot 1. (1-7) was dissolved in aq. (118 ml cone, HCl and 293 ml water);
Lot 2. (1-7) was dissolved in aq. (169 ml conc. HCI and 421 ml water).
Lot 3. (1-7) was dissolved in aq. (186 ml conc. HCI and 468 ml water).
The reaction mixtures were stirred at room temperature for 30 min (strong evolution of CO2 gas) and then each batch was concentrated in vacuo to dryness (80-90°C). Lots 2 and 3 were pooled to provide 226g of wet light yellow product. Lot 1 provided 91.4 of a dark grayish product. Crystallization was carried out as follows: For the wet product of batches 2 and 3: 226 ml of water was added to the product then heated to 50°C at which point hot ethanol was slowly added until crystallization started. The mixture was held at 50°C for 10 minutes, then allowed to reach 25°C with vigorous stirring before filtration to provide light yellowish powder of (2S,3S,4R,5R)-2-(4 -amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-(hydroxymethyl)pyrrolidine-3,4-diol (1) (88g). Lot one was similarly purified to provide 33.0g of light gray colored product. The total yield is 121.0g after drying at 55°C in high vacuum. The mother liquor from the recrystallization of batches 1 and 2 was reprocessed to provide 15.0g of light yellowish powder product (1). 1H NMR (300 MHz, DMSO) δ 14.60 (s, 1H), 13.25 (s, 1H), 10.23 (s, 1H), 9.13 (s, 2H), 8.84 (s, 1H), 8.63 (s, 1H) , 8.11 (d, J = 3.1 Hz, 1H), 5.55 (s, 2H), 4.78 (d, J = 4.4 Hz, 1H), 4.44 (dd, J = 8.8, 5.0 Hz, 1H), 4.14 - 4.02 ( m, 1H), 3.73 (d, J = 5.1 Hz, 2H), 3.52 (s, 1H); 1H NMR (300 MHz, D2O) δ 8.33 (s, 1H), 7.94 (s, 1H), 4.90 (d, J = 8.9 Hz, 1H), 4.65 (s, 1H), 4.37 (dd, J = 4.8, 3.4 Hz, 1H), 3.89 (s, 1H), 3.88 (s, 1H), 3.81 (dd, J = 8.1, 4.5 Hz, 1H); MS (ES+) 266.3 (M+1); Optical rotation -52.69; (H2O,C=1.15); MP: 238°C; Analysis: Calculated for CnH^NsOa^HCI.O^N^O: C, 38.55; H, 5.15; Cl, 20.44; N, 20.69; Found: C, 38.67; H, 5.05; Cl, 20.45; N, 20.42. Example 3: Phosphorylation of Compound 1 (Formula I, where A = NH 2 and B = H) and DNA/RNA Incorporation Studies.
Human hepatocellular carcinoma cells (Huh-7) were incubated with 3H-compound 1 for 24h, followed by methanol extraction and HPLC analysis using SAX column and radioactive detector. FIG. 1 shows phosphorylation of compound 1 in Huh-7 cells, indicating efficient phosphorylation in cells.
Figures 2-4 show that compound 1 is phosphorylated but not incorporated into mammalian RNA or DNA (DP stands for diphosphate and TP stands for triphosphate). FIG. 2 shows adenosine phosphorylation in Huh-7 cells. FIG. 3 shows phosphorylation of compound 1 in Huh-7 cells. FIG. 4 shows the incorporation of genomic DNA and total RNA from compound 1 and adenosine into Huh-7 cells. Example 4: Effects of viral RNA polymerase inhibitor (formula I, where A = NH2 and B = H: compound 1) on measles virus replication in African green monkey kidney cells.
Materials and Methods: Vero 76 cells (African Green Monkey kidney cells) were obtained from American Type culture collection (ATCC, Manassas, VA). Cells were routinely passaged in minimal essential medium (MEM with 0.15% NaHCO3; Hyclone Laboratories, Logan, UT, USA) supplemented with 5% fetal bovine serum (FBS, Hyclone). When evaluating compounds, serum has been reduced to a final concentration of 2.5%, and gentamicin is added to test the medium to a final concentration of 50 pg/ml. The measles virus (MV), Chicago strain, was obtained from the Centers for Disease Control (Atlanta, GA). Antiviral Testing Procedures:
Cytopathic Effect Inhibition Analysis (Visual Analysis)
Cells were seeded into 96-well flat-bottomed tissue culture plates (Corning Glass Works, Corning, NY), 0.2 ml/well, at the appropriate cell concentration, and incubated overnight at 37°C for establish a cell monolayer. When the monolayer was established, the growth medium was decanted and various dilutions of test compound were added to each well (3 wells/dilution, 0.1 ml/well). Compound diluent medium was added to virus and cell control wells (0.1 ml/well). Virus, diluted in the test medium, was added to the compound test wells (3 wells/compound dilution) and the virus control wells (6 wells) at 0.1 ml/well. Virus (viral MOI = 0.001) was added approximately 5 min after compound. Virus-free test medium was added to all toxicity control wells (2 wells/dilution of each test compound) and to cell control wells (6 wells) at 0.1 ml/well. Plates were incubated at 37°C in a humidified incubator with 5% CO2, 95% atmospheric air until virus control wells had adequate cytopathic effect (CPE) readings (80-100% cell destruction). This was obtained from 4-11 days after exposure of the virus to the cells, depending on the virus. The cells were then examined microscopically for CPE, this being scored from 0 (normal cells) to 4 (maximum, 100%, CPE). Cells in the toxicity control wells were observed microscopically for morphological changes attributed to cytotoxicity. This cytotoxicity (cell destruction and/or morphology change) was also graded at 100% toxicity, 80% cytotoxicity, 60% cytotoxicity, 40% cytotoxicity, 20% cytotoxicity, and 0 (normal cells). The 50% efficient dose (EC50) and the 50% cytotoxic dose (IC50) were calculated by CPE regression analysis of the virus data and the toxicity control data, respectively. The selective index (SI) for each compound tested was calculated using the formula: SI = CC50 - EC50.
Neutral Red (NR) Absorption Analysis of CPE Inhibition
Red NR was chosen as the sham quantification method for evaluating antiviral drugs based on the findings of Smee et al (J. Virol. Methods2002, 106: 71-79; incorporated herein by reference in its entirety). This analysis was performed on the same CPE inhibition test plates described above to verify the inhibitory activity and cytotoxicity observed by visual observation. NR analysis was performed using a method modified from Cavenaugh et al. (Invest. New Drugs1990, 8:347-354; incorporated herein by reference in its entirety) as described by Barnard et al. (Antiviral Chem. Chemother. 2001, 12:220-231; incorporated herein by reference in its entirety). Briefly, medium was removed from each well of a plate scored for CPE from a CPE inhibition assay, 0.034% NR was added to each well of the plate and the plate incubated for 2h at 37°C in the dark. The NR solution was then removed from the wells. After sweetening (cells sometimes jammed from the plate causing an erroneous reduction of neutral red) and aspiration to dry, the remaining dye was extracted for 30 min at room temperature in the dark from the cells using buffered absolute ethanol. of Sorenson citrate. Absorptions at 540 nm/405 nm are read with a microplate reader (Opsys MRTM, Dynex Technologies, Chantilly, VA, USA). Absorption values were expressed as percentages of untreated controls and EC50, CC50 and SI values were calculated as described above.
Virus Yield Reduction Analysis
Virus yield reduction analyzes were performed using 50% infectious dose analysis of cell culture (CCID50) essentially as described above (Antimicrob. Agents Chemother. 1992, 3:1837-1842; incorporated herein by reference in its entirety). Briefly, supernatants from each well were serially diluted into triplicate wells of 96-well plates containing Vero 76 cells. Plates were incubated for 6 days and then checked for virus-induced CPE. Quantitation of virus yield titers was by the endpoint method of Reed and Muench (Am. J. Hyg. 1938, 27:493-498; incorporated herein by reference in its entirety). The EC90 value was calculated using linear regression to estimate the concentration needed to inhibit virus yield by 90% or a log 10 decrease in virus titer. Results and discussion
Measles virus was potentially inhibited by compound 1 (Table 1). EC50 values against measles virus were 0.6 and 1.4 μg/ml by visual analysis and NR analysis, respectively. The compound had no cytotoxicity in any of the NR and visual analyzes (IC50 >100). Therefore, the selective indices by both analyzes suggested that compound 1 was highly active against measles virus (MV). The potent inhibitory activity against MV was confirmed by a virus yield reduction analysis with an EC90=0.36 μg/ml, representing a drop of Iog10 in virus produced in infected cells. Conclusions
Compound 1 demonstrated selective and potent inhibitory activity. By virus yield reduction analysis, compound 1 was also a potent inhibitor of MV (EC90 = 0.37 µg/ml). Thus, compound 1 has been shown to be a potent inhibitor of many RNA viruses and suggests that compound 1 further warrants in vitro and in vivo evaluation as a broad-spectrum inhibitor of selected RNA viruses. Example 5: Effects of viral RNA polymerase inhibitor (formula I, where A = NH2 and B = H: compound 1) on replication of various RNA viruses. Materials and methods
Cells and Viruses
African green monkey kidney cell (MA-104) were obtained from Whitaker MA Bioproducts, Walkersville, MD, USA). All Vero cells (African Green Monkey Kidney Cells, Human Laryngeal Cell Carcinoma (A-549), and Madin-Darby Canine Kidney Cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) A-549 cells were cultured in Dulbecco's Minimal Essential Medium (DMEM) supplemented with 0.15% NaHCO3 (Hyclone Laboratories, Logan, UT, USA) and 10% fetal bovine serum (FBS, Hyclone) The remaining cells were routinely passaged through a minimal essential medium (MEM with 0.15% NaHCO3; Hyclone Laboratories, Logan, UT, USA) supplemented with 5% fetal bovine serum (FBS, Hyclone).
When evaluating compounds, serum has been reduced to a final concentration of 2.5%, and gentamicin is added to the test medium to a final concentration of 50 µg/ml. Test media for influenza analyzes consisted of serum-free MEM, 0.18% NaHCO3, 20 μg trypsin/ml, 2.0 μg EDTA/ml, and 50 μg gentamicin/ml.
For assessment of toxicity in actively growing cells, cytotoxicity was assessed by determining the total number of cells as reflected by an NR uptake analysis after a 3-day exposure to various concentrations of compound. To quantify cell growth at 72h in the presence or absence of drug, plates were seeded with 1 X 103 MDCK cells, and after 4h (allowed all cells to attach to plate wells) were exposed to select drug concentrations in MEM or MEM. After 72h, plates were treated as described above for NR analysis. Absorption values were expressed as percentage of untreated controls and CC50 values were calculated by regression analysis.
Dengue virus 2 (DV-2), New Guinea C strain, Respiratory syncytial virus (RSV) A2, Rhinovirus 2 (RV-2), HGP strain, Tacarib (TCV), TRVL 11573 strain, Venezuelan equine encephalitis virus ( VEE), and Yellow Fever virus (YFV), strain 17D were all acquired from the American Type Culture Collection (ATCC; Manassas, VA). All influenza viruses, Measles virus (MV), Chicago strain, SARS coronavirus (SARS-CoV), Urbani strain, and West Nile virus (WNV), isolated strain designated New York 1999 prototype 996625, were obtained from Centers for Disease Control (Atlanta, GA). The Punta Toro virus (PTV), strain Adames, was obtained from Dr. Dominique Pifat of the US Army Medical Research Institute for Infectious Diseases, Ft. Detrick (Frederick, MD). The Rift Valley Fever Virus Vaccine Strain (RVFV), MP-12, and the Junin Virus Vaccine Strain (JUNV), Candid 1, were kindly provided by Dr. Robert Tesh (World Reference Center for Emergency and Viruses and Arboviruses (World Reference Center for Emerging and Viruses and Arboviruses), University of Texas Medical Department, Galveston, TX). Pichinde virus (PICV), strain An 4763, was provided by Dr. David Gangemi (Clemson University, Clemson, South Carolina). Parainfluenza virus type 3 (PIV-3), strain 14702/5/95, was obtained from Jacquelin Boivin (St. Justin Hospital, Montreal, Canada). Adenovirus (AV-1) type 1, Chicago/95 strain, was isolated from tracheal lavage of a pediatric patient and was provided by M.F. Smaron (Department of Medicine, University of Chicago, Chicago IL). Antiviral Test Procedure
Cytopathic effect inhibition analysis (Visual analysis)
Cells were seeded into 96-well flat-bottomed tissue culture plates (Corning Glass Works, Corning, NY), 0.2 ml/well, at the appropriate cell concentration, and incubated overnight at 37°C for establish a cell monolayer. When the monolayer was established, the growth medium was decanted and the various dilutions of test compound were added to each well (3 wells/dilution, 0.1 ml/well). Diluent compound media was added to virus and cell control wells (0.1 ml/well). Virus, diluted in test medium, was added to compound test wells (3 wells/compound dilution) and to virus control wells (6 wells) at 0.1 ml/well. Virus (viral MOI = 0.001) was added approximately 5 min after compound. Virus-free test medium was added to all toxicity control wells (2 wells/dilution of each test compound) and to cell control wells (6 wells) at 0.1 ml/well. Plates were incubated at 37°C in a humidified incubator with 5% CO 2 , 95% atmospheric air until virus control wells had adequate cytopathic effect (CPE) readings (80-100% cell destruction). This was achieved 4-11 days after exposure of the virus to cells, depending on the virus. The cells were then examined microscopically for CPE, this being scored from 0 (normal cells) to 4 (maximum, 100%, CPE). Cells in the toxicity control wells were observed microscopically for morphological changes attributed to cytotoxicity. This cytotoxicity (cell destruction and/or morphology change) was also graded as 100% toxicity, 80% cytotoxicity, 60% cytotoxicity, 40% cytotoxicity, 20% cytotoxicity and 0 (normal cells). The 50% efficient dose (EC50) and the 50% cytotoxic dose (IC50) were calculated by CPE regression analysis of the virus data and the toxicity control data, respectively. The selective index (SI) for each compound tested was calculated using the formula: SI = CC50 - EC50.
Neutral Red (NR) Absorption Analysis of CPE Inhibition and Compound Cytotoxicity
NR red was chosen as the sham quantification method for antiviral drug evaluation based on the findings of Smee et al. (above). This analysis was performed on the same CPE inhibition test plates described above to verify the inhibitory activity and cytotoxicity observed by visual observation. NR analysis was performed using a method modified from Cavenaugh et al. (supra) as described by Barnard et al. (above). Briefly, medium was removed from each well of a plate scored for CPE from a CPE inhibition assay, 0.034% NR was added to each well of the plate and the plate incubated for 2h at 37°C in the dark . The NR solution was then removed from the wells. After sweetening (cells sometimes jammed from the plate causing erroneous reduction of neutral red) and aspiration to dry, the remaining dye was extracted for 30 min at room temperature in the dark from the cells using buffered absolute ethanol with Sorenson citrate buffer. Absorptions at 540 nm/405 nm are read with a microplate reader (Opsys MRTM, Dynex Technologies, Chantilly, VA, USA). Absorption values were expressed as percentages of untreated controls and 5 EC50, CC50 and SI values were calculated as described above. Table 1. Effects of a polymerase inhibitor (compound 1) on the replication of various viruses.

Other viruses that were found to be significantly inhibited by compound 1 (SI >10) were DV-2 (EC50 = 15, 13 μg/ml), JUNV (EC50 = 29, 16 μg/ml), YFV (EC50 = 8.3, 8.3 μg/ml) (Table 1). The following viruses were slightly inhibited by compound 1 (10<SI>3): PIV-3 (EC50 = 7.1, 10 μg/ml), SARS-CoV (EC50 = 14, 16 μg/ml), PICV ( EC50 = 61, 28 μg/ml), and RVFV (EC50 = 75, 64 μg/ml). Compound 1 was tested against a subset of influenza viral strains (Table 2), and exhibited a broad spectrum of anti-influenza activity against multiple strains. Table 2. Broad spectrum of Anti-influenza Activity of Compound 1.
Conclusions
Compound 1 demonstrated potent activity against all influenza viruses tested. Compound 1 has been shown to be a potent inhibitor of influenza virus replication and suggests that Compound 1 is effective as a broad-spectrum inhibitor of selected RNA viruses, including all influenza viruses. Example 6: In vitro antiviral activity of compound 1.
The antiviral activity of compound 1 was evaluated in vitro on various viruses for antiviral activity. EC50 values ranged from about 10 μg/ml to about >300 μg/ml against Marburg (filoviridae), Junin's Candid 1 (arenaviridae), Pichinde (arenaviridae), Chikungunya 181/25 (togaviridae) and NYCBH vaccine (poxviridae). Example 7: Synergistic antiviral activity of compound 1 and neuraminidase inhibitor in MDCK cells.
Canine Kidney Madin Darby (MDCK) cells were infected with the H3N2 influenza virus (A/Victoria/3/75) and treated with various combinations of compound 1 and peramivir for 72h. The cytopathic effect was determined using neutral red sham uptake analysis. Data are shown in table 3. Table 3: Percentage Inhibition of Cytopathic Effect in Influenza-infected Cells.

Experimental data were evaluated by three-dimensional analysis using Mac Synergy II® software (Prichard and Shipman, 1990; incorporated herein by reference in its entirety). The software calculates theoretical additive interactions from dose-response curves for individual drugs. The calculated additive surface, which represents the predicted additive interactions, is then subtracted from the experimental surface to reveal regions of larger (synergy) or smaller (antagonism) interactions than expected. The combination of peramivir and compound 1 in cell culture studies demonstrated a synergistic antiviral effect with a synergistic volume equal to 92 µM2 per % unit (FIG. 5). Example 8: Efficiency of Intramuscular (IM) Injection of Compound in Murine Influenza Model.
Balb/C mice between 6-8 weeks of age were adapted for H3N2 virus (A/Victoria/3/75). Doses of 0, 30, 100 and 300 mg/kg/d qd were provided by intramuscular (IM) injection for 5 days starting 1h before infection. N = 50 animals. All animals were followed for 16 days. End points included lethality, average days of death and weight loss. The effects are shown in Figure 6.
The results for compound 1 (IM) in mouse model influenza viruses are shown in table 4. Compound 1 provided via IM increases survival and weight loss in mice infected with influenza virus. Table 4: Compound 1 (IM) in mouse influenza model viruses - H3N2 A/Vic/3/75
*P<0.001 compared to infected vehicle group (log rank test) **P<0.001 compared to infected vehicle group (t test) Example 9: Efficiency of oral administration of compound 1 in murine influenza model.
Balb/C mice between 6-8 weeks of age were adapted for the H3N2 virus (A/Victoria/3/75). Doses of 0, 30, 100 and 300 mg/kg/d qd and 100 mg/kg/d bid were provided orally. N = 60 animals. All animals were followed for 16 days. End points included lethality, average days to death, and weight loss. The effects of orally administered compound 1 on weight loss in mice infected with influenza virus H3N2 A/Vic/3/75 are shown in Figure 7.
The results of oral administration of compound 1 in mouse model influenza viruses are also shown in table 5. Compound 1 given orally increases survival and weight loss in mice infected with influenza virus. Table 5: Compound 1 (Oral) in Mouse Influenza Model Virus - H3N2 A/Vic/3/75
*P<0.001 compared to infected vehicle group (log rank test) **P<0.001 compared to infected vehicle group (t test) Example 10: Pharmacokinetic studies in mice.
Female Balb/c mice (N = 30) were orally dosed with compound 1 at 100 mg/kg. Mice were bled through the retroorbital sine at t = 0.17, 0.5, 1.0, 3, 6 and 24h (5 mice each per time point), centrifuged and the plasma was stored at -80°C. Plasma drug levels were measured by LC/MS/MS analysis.
Mouse plasma levels for compound 1 after oral administration are shown in table 6. Table 6: Plasma levels of compound 1 in mice following oral administration
Example 11: Mouse Ebola Virus Prophylaxis Study.
Compound 1 was administered via i.p., i.m. and orally (300 mg/kg/day, BID) for 8-12 week old C57BI/6 mice (N = 10 per group, 4 groups - one saline and 3 drug-treated groups). Eight days of treatment starting 4 hours before infection. The mouse-adapted Ebola Virus challenge (Zaire) was administered intraperitoneally. Mortality and weight were monitored for 14 days post-infection.
The survival percentage of mice is shown in Figure 8. Saline-treated mice infected with Ebola virus all died by day 8. All mice treated intraperitoneally or intramuscularly with compound 1 survived to study endpoint (day 14). Eighty percent of mice treated orally with compound 1 survived the study endpoint (day 14).
The change in weight of mice is shown in Figure 9.
Saline-treated mice infected with Ebola virus exhibited overall weight loss by day 8 (all control mice were dead by day 8). Mice treated intraperitoneally or intramuscularly with compound 1 retained greater than 95% of initial weight on day 12. Mice treated orally with compound 1 retained greater than 80% of initial weight on day 12. All treated mice continued gaining weight around the 12th. Example 12: Mouse Ebola Virus Prophylaxis Study.
Compound 1 was administered i.m. and orally to 8-12 week old C57BI/6 mice. Study subjects were divided into 6 groups (N = 10 per group). Group 1 was a saline control, group 2 was dosed with 150 mg/kg of compound 1 (PO, BID); group 3 was dosed with 250 mg/kg of compound 1 (PO, BID); group 4 was dosed with 150 mg/kg of compound 1 (IM, BID). Group 5 was uninfected mice treated with saline (PO, BID), and group 6 was uninfected mice treated with 250 mg/kg of compound 1 (PO, BID). The treatment lasted nine days, starting 4 hours before the infection. The Mouse-adapted Ebola Virus challenge (Zaire) was administered intraperitoneally at 1,000 pfu). Mortality and weight were monitored for 14 days post-infection.
The survival percentage of mice is indicated in Figure 10. All mice infected with Ebola virus treated with saline died by day 8. All mice treated intramuscularly with compound 1 survived to study endpoint, indicating that the IM dosage of compound 1 was completely protective. Eighty percent or more of mice treated orally with compound 1 survived the study endpoint.
The weight change of mice is indicated in Figure 11. Saline-treated mice infected with Ebola virus exhibited overall weight loss by day 7 (all control mice were dead by day 8). Mice treated intramuscularly with compound 1 exhibited similar weight gain as the uninfected control group on day 11. Mice treated orally with compound 1 exhibited reversible weight loss, and retained more than 100% of initial weight on the 11th Example 13: Yellow Fever Virus (YFV) Time Window Golden Hamster Study.
Yellow Fever virus (Jimenez strain) was injected via IP to golden Syrian hamsters (99 g) at 20 CCID50 per hamster (-6.25 x LD50). The groups were divided as follows: 1) compound 1 was administered starting -4h (N = 15); 2) compound 1 administered starting at 1 dpi (days post-infection) (N = 10); 3) compound 1 administered starting at 2 dpi (N = 10); 4) compound 1 administered 3 dpi (N = 10); 5) compound 1 administered 4 dpi (N = 10); 6) ribavirin administered starting -4h (N = 10); 7) saline vehicle starting -4h (N = 16); 8) uninfected hamsters administered compound 1 starting -4h (N = 3); 9) uninfected hamsters administered saline vehicle starting -4h (N = 3); and 10) normal, untreated, uninfected controls (N = 3). The treatment dose was 100 mg/kg IP, BID for 7 days. The endpoint of the studies was 21-day mortality, weight measured on days 0, 3, 5, and 6; serum and liver virus titers (day 4, compound 1 in -4h, and vehicle in -4h), and ALT and AST on day 6.
The study showed improved survival for compound 1 with delayed treatment compared to placebo (Figure 12). Survival of hamsters infected with YFV and treated with compound 1 twice daily for 7 days starting at various times after challenge is indicated (***P<0.001, **P<0.1, compared to placebo). Survival rate was 100% for compound 1 starting pre-infection, and late treatment up to 3 days post-infection. The survival rate was 80% for the compound starting 4 days post-infection, indicating a significant increase over placebo in late treatment groups. In contrast, ribavarin provided 90% survival starting pre-infection and vehicle provided 12.5% survival starting pre-infection. Most deaths occurred within 10 days of infection. Surviving animals will be rechallenged with YFV 21 days post-infection.
The weight change of hamsters is shown in Figure 13. Hamsters infected with YFV and treated with compound 1 from pre-infection 4 days post-infection showed weight gain over placebo and ribavirin administered pre-infection. The percent weight change of hamsters infected with YFV and treated with compound 1 twice daily for 7 days starting at various times before and after virus challenge is shown. Example 14: Oral Bioavailability of Compound 1 in Rats.
Compound 1 was dosed at 10 mg/kg, PO in rats. The pharmacokinetic curve measuring the concentration of compound 1 in rat plasma within 6 hours is shown in Figure 14. Example 15: Marburg Virus Study for Compound 1.
Compound 1 was dosed via IM in 10-12 week old BALB/c mice challenged (interperitoneally) with 1000 pfu mouse-adapted MARV-Ravn. The study was divided into 10 groups (N=10 per group). Dosing regimens, routes and doses are shown in Table 7. Compound 1 was dissolved in 0.9% saline prior to administration, and health and weight were monitored for 14 days post-infection. Table 7: Study Design for Prophylaxis and Treatment with Compound 1 for Marburg Infection
*Day 0 of treatment started 4h before infection, except for group 6. Treatment for group 6 started 4h post-infection on day 0. PI = post-infection
The percentage survival for the 10 groups in this study for day 12 is included in Table 8. The survival rate for mice treated with vehicle alone (0.9% saline) was 60% on day 5 7 and 30% on days. days 8-12. Compound 1 has been shown to increase survival by at least 90% on day 7, and at least 80% on days 8-12 at all doses. Table 8: Percent Survival Rate for Prophylaxis and Treatment with Compound 1 for Marburg Infection

Although the invention has been described and illustrated in the above illustrative embodiments, it is understood that the present description has been made by way of example only, and that numerous changes to the details of implementation of the invention can be made without departing from the spirit and scope of the invention, which is limited only by the claims that follow. The features of the described embodiments may be combined and rearranged in various ways within the scope and spirit of the invention.

权利要求:
Claims (21)
[0001]
1. Use of a therapeutically effective amount of a compound of formula I:
[0002]
2. Use according to claim 1, characterized in that said viral infection comprises infection by one or more viruses.
[0003]
3. Use according to claim 1 or 2, characterized in that the viral infection comprises a virus selected from the group consisting of families orthomyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae ; or where the viral infection is selected from the group consisting of adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile Virus, smallpox, encephalitis, Yellow Fever, Dengue, influenza A, influenza B, lassa, lymphocytic choriomeningitis, junin, male, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese Encephalitis, Kyasanur Forest Disease, Venezuelan Equine Encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae virus; or where the viral infection is selected from the group consisting of adenovirus, Dengue, influenza A, influenza B, Junin, measles, parainfluenza, Pichinde, punta toro, respiratory syncytial, rhinovirus, Rift Valley Fever, SARS-CoV, tacarib, Venezuelan Equine Encephalitis, West Nile Virus and Yellow Fever Virus; or where the viral infection is selected from the group consisting of Ebola, Yellow Fever, Marburg, influenza A and influenza B viruses.
[0004]
4. Use according to claim 3, characterized in that the viral infection comprises a virus selected from the group consisting of families orthomyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae.
[0005]
5. Use according to claim 3, characterized in that the viral infection is West Nile virus.
[0006]
6. Use according to claim 3, characterized in that the viral infection is Dengue virus.
[0007]
7. Use according to claim 3, characterized in that the viral infection is Rift Valley Fever virus.
[0008]
8. Use according to claim 3, characterized in that the viral infection is Ebola virus.
[0009]
9. Use according to claim 3, characterized in that the viral infection is Marburg virus.
[0010]
10. Use according to claim 3, characterized in that the viral infection is yellow fever virus.
[0011]
11. Use according to claim 3, characterized in that the viral infection is influenza A or influenza B virus.
[0012]
12. Use of a therapeutically effective amount of a
[0013]
13. Use according to claim 12, characterized in that the second viral RNA polymerase is inhibited.
[0014]
14. Use according to claim 13, characterized in that the second viral RNA polymerase is selected from the group consisting of viral polymerases of orthomyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, and coronaviridae.
[0015]
15. Use according to claim 13 or 14, characterized in that the second RNA polymerase is selected from viral polymerases of influenza A, influenza B, parainfluenza, respiratory syncytial rhinovirus, Junin, Pichinde, Rift Valley Fever, Degue , measles, yellow fever, tacarib, Venezuelan equine encephalitis, West Nile Virus and SARS-CoV.
[0016]
16. Use according to any one of claims 12 to 15, characterized in that the viral RNA polymerase is selected from the group consisting of viral polymerases from rhinoviruses, polio, measles, Ebola, Coxsackie, West Nile Fever, Dengue , influenza A, influenza B, lassa, lymphocytic choriomeningitis, junin, machupo, guanarite, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese encephalitis, Kyasanur Forest Disease, Equine encephalitis Venezuelan, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pichinde virus/ or in which viral RNA polymerase is a polymerase selected from the group consisting of viral polymerases of dengue, influenza A, influenza B, Junin, measles, parainflyenza, Pichinde, Punta Toro, respiratory syncytial, Rift Valley Fever, West Nile fever and yellow fever/or in which RNA Viral polymerase is a polymerase selected from the group consisting of viral polymerases from Ebola, yellow fever, measles, influenza A and influenza B.
[0017]
17. Use according to any one of claims 1 to 16, 5 characterized in that it further comprises administration of an additional antiviral agent.
[0018]
18. Use according to claim 17, characterized in that the antiviral agent is selected from the group consisting of laninamivir, oseltamivir, zanamivir and peramivir.
[0019]
19. Use according to claim 17 or 18, characterized in that the additional antiviral agent is peramivir.
[0020]
20. Use according to any one of claims 1 to 19, characterized in that the administration is selected from the group consisting of intravenous, intraperitoneal, intramuscular and oral.
[0021]
21. Invention, in any form of its embodiments or in any applicable category of claim, for example, of product or process or use encompassed by the subject matter initially described, disclosed or illustrated in the patent application.

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法律状态:
2018-07-03| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2018-07-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-04-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-05-05| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-04-20| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-06-29| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 14/10/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US39352210P| true| 2010-10-15|2010-10-15|

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US61/393,522|2010-10-15|

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US201161492054P| true| 2011-06-01|2011-06-01|

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US61/492,054|2011-06-01|

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PCT/US2011/056421|WO2012051570A1|2010-10-15|2011-10-14|Methods and compositions for inhibition of polymerase|

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