Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/052—Imidazole radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/056—Triazole or tetrazole radicals

Definitions

• the field of the invention is improved specificity in treatment of diseases.
• nucleoside analogs can be employed to reduce viral replication by inhibiting the viral reverse transcriptase.
• nucleoside analogs are frequently associated with side effects, including anemia and/or neutropenia.
• prolonged exposure to nucleoside analogs favors development of resistance to the drug in some virus strains.
• cocktails of nucleoside analogs may be administered.
• cocktails of nucleoside analogs typically only postpone the onset of drug resistance.
• nucleoside analogs generally do not exhibit selectivity between viral replication and replication in rapidly dividing cells of a host organism, thereby exhibiting significant cytotoxicity towards the host.
• protease inhibitors may be employed to interrupt proper assembly of viral proteins.
• Protease inhibitors are highly specific towards viral proteases, thereby typically avoiding problems associated with limited selectivity between viral replication and replication in rapidly dividing host cells.
• side effects including nausea, diarrhea, diabetes, and kidney stones tend to occur.
• some protease inhibitors are only poorly soluble in aqueous solution, thereby reducing the potential amount that can be delivered to a patient.
• viral resistance to some protease inhibitors has been shown to occur after prolonged treatment.
• IFN-alpha may be employed to treat chronic hepatitis C.
• IFN-alpha tends to cause fever, headache, lethargy, loss of appetite, anxiety and depression, in lower dosages, and bone marrow suppression and low blood cell counts in higher dosages.
• compositions and methods known in the art for targeted treatment of hepatic diseases all or almost all of them suffer from one or more disadvantages. Therefore, there is still a need to provide improved compositions and methods for targeted treatment of diseases.
• the present invention is directed to methods and compositions for increasing selectivity of a drug.
• a drug is covalently modified by a blocking group.
• the blocking group is coupled to the drug via a nitrogen atom in the blocking group.
• the blocking group in the modified drug reduces metabolic conversion and sequestration (i.e., accumulation) of the drug in non-target cells, and the blocking group is enzymatically removed in the target cell.
• a metabolic conversion of a drug induces damage to a target cell
• a blocking group attached to the drug via a nitrogen atom in the blocking group prevents metabolic conversion, and that the blocking group is cleaved from the drug in a target cell. Consequently, it is contemplated that modifying a drug with a blocking group, and administering the drug to a system comprising target and non-target cells reduce cytotoxicity.
• a metabolic conversion of a drug in a non-target cell reduces the effective concentration of the drug
• a blocking group attached to the drug via a nitrogen atom in the blocking group prevents metabolic conversion, and that the blocking is cleaved from the drug at a target cell. Consequently, it is contemplated that modifying a drug with a blocking group may reduce a dosage. It is also contemplated that the drug is administered to a system comprising target and non-target cells.
• Contemplated drugs include a carboxamide group, and especially contemplated drugs are l-beta-D-ribofuranosyl-1.2,4-triazole-3 -carboxamide and 2-beta-D-ribofuranosyl- 4-thiazolecarboxamide, which may also be in their respective L-isomeric form.
• the blocking group is not restricted to a particular chemical nature, it is preferred that the blocking group comprises a nitrogen atom.
• Contemplated target cells are not limited to a particular cell type, and may or may not be infected with a virus, or they may be hyperproliferative. However, virus infected or hyperproliferative hepatocytes and neurons are especially preferred and non-target cells include erythrocytes.
• Figures 1A and IB are schematic illustrations of uptake and retention of an exemplary drug according to the inventive subject matter.
• Figure 2 is an exemplary synthetic scheme for the synthesis of Ribavirin.
• Figure 3 is an exemplary synthetic scheme for the synthesis of modified Ribavirin. Detailed Description
• the term "pharmacological effect” refers to any alteration of metabolism, replication, structure, or life span of a cell in a cell containing system, which is caused by a molecule that is added to the system.
• pharmacological effect For example, inhibition of an anabolic, catabolic, or polymerase-type reaction catalyzed by an enzyme is considered a pharmacological effect.
• depolymerization of tubulin by Kinl kinesins is considered a pharmacological effect under the scope of this definition.
• allosteric inhibition of an enzyme by a metabolite produced within the cell in a system is not regarded a pharmacological effect, because the allosteric inhibitor was not added to the system.
• target cell refers to a cell in which a drug is intended to exhibit a pharmacological effect.
• a virus-infected hepatocyte is considered a target cell for the drug Ribavirin.
• non-target cell encompasses all cells in a cell-containing system that are not target cells.
• non-target cells may metabolize those drugs at a significant rate, frequently leading to undesirable non-specific side effects.
• the inventors have discovered that such undesired metabolic conversion at (i.e., in, or on the surface of) non-target cells can be prevented by modifying a drug with a blocking group that is selectively removed in a target cell, thereby increasing the selectivity of a pharmacological effect of a drug, while concomitantly reducing the cytotoxicity and the dosage of the drug.
• the selectivity of the pharmacological effect of a drug is increased by a method in which in one step a drug is identified as having a desired pharmacological effect on a target cell.
• the drug is modified with a blocking group, wherein the blocking group is covalently attached to the drug via a nitrogen atom in the blocking group.
• the blocking group further reduces accumulation of the drug in a non-target cell, and is enzymatically removed from the drug in the target cell.
• the term "selectivity" of a drug with respect to a pharmacological effect refers to the propensity of a drug to preferentially exert the pharmacological effect on a target cell towards a treatment goal as opposed to exerting the pharmacological effect on a non-target cell towards an undesired side effect of the drug in the non-target cell.
• Ribavirin is known to have antiviral properties in hepatitis virus-infected hepatocytes (e.g., see review Marcellin, P. and Benhamou J.; Treatment of chronic viral hepatitis, Baillieres Clin Gastroenterol 1994 Jun;8(2):233-53). It is also known, that Ribavirin is readily phosphorylated in erythrocytes to the pharmacologically active form Ribavirin-phosphate at a significant rate (e.g., Homma, M. et al.
• FIG. 1A The reduction in selective accumulation of modified Ribavirin is illustrated in Figures 1A and IB.
• an erythrocyte (non-target cell) 100 is presented with Ribavirin (R). Ribavirin enters the erythrocyte, and is phosphorylated to the pharmacologically active Ribavirin-phosphate (R-P), which is retained in the erythrocyte.
• a hepatocyte (target cell) 110 is presented with Ribavirin (R). Ribavirin enters the hepatocyte, and is phosphorylated to the pharmacologically active Ribavirin-phosphate (R-P), which is retained in the hepatocyte.
• an erythrocyte (non-target cell) 101 is presented with modified Ribavirin (R*, l-beta-D-ribofuranosyl-l,2,4-triazole-3- carboxamidine).
• R* modified Ribavirin
• the modified Ribavirin enters the erythrocyte, however, it is not phosphorylated and can therefore exit the erythrocyte.
• a hepatocyte (target cell) 111 is presented with modified Ribavirin (R*).
• the modified Ribavirin enters the hepatocyte, and is enzymatically deaminated to Ribavirin, which is subsequently phosphorylated to the pharmacologically active phosphorylated Ribavirin (R-P), which is retained in the hepatocyte.
• drugs other than Ribavirin suitable for the inventive concept presented herein.
• appropriate drugs include drugs that are metabolized, activated and/or inactivated in a cell other than a target cell
• drugs include nucleosides, nucleotides, nucleoside analogs and nucleotide analogs.
• an alternative drug comprises the nucleoside uracil, or the nucleoside analog 5'-fluoro uracil (5'-FU).
• blocking group refers to a chemical group that can be covalently attached to a drug, and when attached to the drug, blocks at least one metabolic conversion of the drug.
• the term "metabolic conversion" of a drug refers to any intra and/or extracellular chemical change of a drug that is brought about by the metabolism of a cell or cellular system, and particularly includes enzymatic degradation (e.g., oxidation, hydrolytic cleavage) and enzymatic modification (e.g., glycosylation, phosphorylation).
• enzymatic degradation e.g., oxidation, hydrolytic cleavage
• enzymatic modification e.g., glycosylation, phosphorylation
• R t and R 2 are independently hydrogen, linear or branched alkyl, alkenyl, alkynyl, aralkyl, aralkenyl, or aralkynyl, aryl, all of which may further comprise heteroatoms including nitrogen, oxygen, sulfur, or a halogen.
• alternative blocking groups are enzymatically removable from the drug, and particularly contemplated enzymes include liver specific aminohydrolases, including deaminases (e.g. , adenosine or cytosine deaminase), liver deamidases (e
• Contemplated blocking groups may be covalently bound to various positions of the drug molecule, and while it is generally preferred that contemplated drugs are modified on a carboxamide moiety, various positions other than a carboxamide group are also contemplated, especially carbonyl groups (e.g., a carboxylic acid, and a keton-type carbonyl). For example, each of the carbonyl groups in the ring portion of uracil or its analog 5'-FU may be modified by a blocking group.
• the blocking group may inactivate the drug, or prevent subsequent activation once the modified drug is presented to a non-target cell.
• the blocking group may inactivate the drug.
• the blocking group may be coupled to the drug at a position that may prevent metabolic activation.
• the blocking group may replace a functional group or substituent, or that the blocking group is attached to a functional group or substituent.
• the drug comprises a nucleophilic group (e.g., -O " )
• the blocking group comprises a secondary amine with a suitable leaving group
• the secondary amine may be attached to the nucleophilic group.
• the modification may comprise an organo-synthetic modification, an enzymatic modification, or a de-novo synthesis to produce the modified drug.
• the drug comprises an activated carbonyl function
• amidation of the carbonyl atom may be achieved in a single nucleophilic exchange reaction.
• appropriate drugs may also be enzymatically modified by introducing the blocking group into the drug in a reaction that employs the drug and the blocking group as enzymatic substrates.
• the enzyme for such modifications is derived from the target cell (e.g. , from an allogenic or xenogenic source, or from a recombinant source expressing the gene coding for the enzyme).
• the prevention of accumulation of the drug in the non-target cell may be achieved by at least one of various mechanisms, including reduction of uptake through drug-specific transporters, reduction of metabolic conversion into forms that will be retained (e.g., due to additional or new electrical charge, change in hydrophobicity, or change in recognition by an exporter), or, due to increased export from the non-target cell (e.g., due to an secretory signal in the blocking group) prevention of accumulation of the drug may be achieved.
• the drug is a nucleoside analog
• the non-target cell has a nucleoside transporter that selectively imports nucleosides without a lipophilic moiety; adding a lipophilic moiety as the blocking group to the drug may prevent accumulation of the drug.
• phosphorylation (and concomitant accumulation) of various nucleosides in erythrocytes can be prevented by converting a carboxamide group into a carboxamidine group (supra).
• Enzymatic removal may include enzymes from various classes, including hydrolases, transferases, lyases, and oxidoreductases, and particularly preferred subclasses are adenosine and cytosine deaminases, arginases, transaminases, and arylamidases.
• contemplated enzymes for the enzymatic removal of the blocking group may exclusively be expressed in the target cells, however, in alternative aspects of the inventive subject matter appropriate enzymes may also be expressed in cells other than the target cells, so long as the enzyme is not ubiquitously expressed in all cells in a cell containing system. It should further be appreciated that contemplated enzymes are natively expressed (i.e., are non-recombinant) in the respective target cells under normal and/or pathological conditions. For example, it is known that glutamine-pyruvate transaminase is constitutively expressed with relatively high selectivity in liver cells, and may therefore be a suitable enzyme for removal of a blocking group.
• cytosine deaminase is expressed in relatively high quantities in colon cancer cells, but not, or only in minor quantities in normal colon cells.
• the cytotoxicity of a drag to a non-target cell is reduced by a method in which in one step it is recognized that a metabolic conversion of a drug in a non-target cell induces damage to the non-target cell.
• cytotoxicity refers to an undesired pharmacological effect on a non-target cell, wherein the undesired pharmacological effect particularly includes inhibition of replication, energy-metabolism, or includes cell death.
• the drug is modified with a blocking group, wherein the blocking group is covalently coupled to the drug via a nitrogen atom in the blocking group, and wherein the blocking group reduces the metabolic conversion of the drug in the non-target cell, and is enzymatically cleaved from the drug in the target cell.
• the drag is admimstered to a system comprising the target cell and the non-target cell, wherein the blocking group is covalently coupled to the drug.
• the metabolic conversion includes phosphorylation of the drug to the corresponding drug- phosphate in an erythrocyte.
• Ribavirin phosphorylation of the antiviral drug Ribavirin in various cells produces pharmacologically active Ribavirin- 5'-monophosphate (e.g., Homma, M. et al. High-performance liquid chromatographic determination of Ribavirin in whole blood to assess disposition in erythrocytes; Antimicrob Agents Chemother 1999 Nov; 43(11): 2716-9), a compound involved in the inhibition of inosine monophosphate dehydrogenase (IMPDH).
• IMPDH inosine monophosphate dehydrogenase
• Ribavirin-5'- monophosphate has a pronounced cytotoxic effect on erythrocytes (De Franceschi, et al.; Hemolytic anemia induced by ribavirin therapy in patients with chronic hepatitis C virus infection: role of membrane oxidative damage, Hepatology 2000 Apr; 31(4): 997-1004), and it is consequently recognized that prevention or reduction of formation of Ribavirin-5'- monophosphate in erythrocytes will significantly reduce the cytotoxicity of Ribavirin.
• metabolic conversions of a drug in a non-target cell other than phosphorylation are also contemplated, including oxidation, reduction, hydrolytic cleavage of a covalent bond within the drug, addition or removal of pendent groups, and ring-opening reactions.
• the metabolic conversion may include various enzymatic detoxification or solubilization reactions known to occur in the liver (e.g. , glycosylation, cytochrome P 50 -mediated oxidation, etc.).
• metabolic conversions may include phosphatase or esterase activity.
• the conversion may be limited to a single type of non-target cell, but may also occur in more than one cell type.
• the non-target cell has a relatively high rate of nucleic acid synthesis, and the metabolic conversion is mediated by an enzyme involved in the nucleic acid synthesis, various types of fast growing cells may exhibit metabolic conversion.
• metabolic conversion may also be locally limited through accessibility of the drug to a particular set of cells or organs.
• non-target cells are cultured in vitro
• the non-target cells may be incubated with the corresponding radiolabeled drag, and that the metabolites of the radiolabeled drags may then be identified by various assays, including immunoassays, thin layer chromatography, or GC-MS.
• a tissue biopsy may provide a sufficient specimen to isolate and identify metabolites of the admimstered drug.
• the type of damage to the non-target cell may vary substantially, and may range from slowing the cellular metabolism in the non-target cell-to- cell death.
• the metabolic conversion produces an inhibitor of an enzyme located in the glycolytic pathway
• energy for the non-target cell may be provided at least in part through salvage pathways.
• the metabolic conversion of a drag into an enzyme inhibitor proceeds at a relatively slow rate
• up-regulation of expression of the enzyme affected by the inhibitor may almost completely compensate for the reduction in number of active sites.
• the metabolic conversion produces a radical species, lipid peroxidation may result in severe membrane damage and subsequent death of the cell.
• the damage resulting from the metabolic conversion of the drag may be caused directly or indirectly.
• the metabolic conversion produces an enzyme inhibitor blocking an enzyme
• the damage is considered direct.
• the metabolic conversion produces an intermediate, which after subsequent intracellular or exatracellar modification is further converted to an enzyme inhibitor, the damage is considered indirect.
• suitable drags will be administered in any appropriate pharmaceutical formulation, and under any appropriate protocol.
• administration may take place orally, parenterally (including subcutaneous injections, intravenous, intramuscularly, by intrasternal injection or infusion techniques), by inhalation spray, rectally, topically and so forth, and in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
• appropriate drugs can be administered orally as pharmacologically acceptable salts, or alternatively intravenously in physiological saline solution (e.g., buffered to a pH of about 7.2 to 7.5).
• buffers such as phosphates, bicarbonates or citrates can be used for this purpose.
• pro-drug forms of the drags are contemplated.
• One of ordinary skill in the art will recognize how to readily modify contemplated drugs to pro-drag forms to facilitate delivery of active compounds to a target site within the host organism or patient.
• One of ordinary skill in the art will also take advantage of favorable pharmacokinetic parameters of the pro-drag forms, where applicable, in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound.
• contemplated drugs may be administered alone or in combination with other pharmacologically active agents, which may be administered separately or together and when administered separately, administration may occur simultaneously or separately in any order.
• Contemplated pharmacologically active agents include anti- viral agents such as interferon (e.g., interferon ⁇ and ⁇ ), anti-fungal agents such as tolnaftate, FungizoneTM, LotriminTM, MycelexTM, Nystatin and Amphoteracin; anti- parasitics such as MintezolTM, NiclocideTM, VermoxTM, and FlagylTM; bowel agents such as lfnmodiumTM, LomotilTM and PhazymeTM; anti-tumor agents such as interferon ⁇ and ⁇ ,
• interferon e.g., interferon ⁇ and ⁇
• anti-fungal agents such as tolnaftate, FungizoneTM, LotriminTM, MycelexTM, Nystatin and Amphoteracin
• cytokines such as IL2, IL4, IL6, IL8, IL10, and IL12.
• a therapeutically effective amount will vary with the condition to be treated, its severity, the treatment regimen to be employed, the pharmacokinetics of the agent used and the patient (animal or human) being treated. It is further contemplated that various dosages are appropriate, including dosages between 0.5 mg/kg and 0.1 mg/kg and less, but also dosages between 0.5 and l.Omg/kg and more. While it is generally preferred that the system comprising the target cell and the non-target cell is a mammal (most preferably a human), various alternative systems are also appropriate, and particularly include in vitro cell and tissue culture.
• the drug the blocking group, the step of modifying the drug, the target cell and the non-target cell in contemplated methods of reduction of cytotoxicity of a drag to a non-target cell, the same considerations as described above apply.
• the dosage of a drag in a system is reduced by a method in which a drag is provided, wherein metabolic conversion of the drug in a non-target cell reduces the concentration of the drug in a system comprising the non-target cell and a target cell.
• the drug is modified with a blocking group, wherein the blocking group is covalently coupled to the drug via a nitrogen atom in the blocking group, and wherein the blocking group reduces the metabolic conversion of the drug in the non-target cell.
• the drug is administered to the system, wherein the blocking group is covalently coupled to the drag, and wherein the blocking group is enzymatically removed from the drag in the target cell.
• the drug is Ribavirin
• the target cell is a hepatocyte infected with a virus
• the non-target cell is an erythrocyte.
• Ribavirin is metabolically converted to Ribavirin-phosphate and that Ribavirin-phosphate is retained in the erythrocytes, thereby significantly lowering the concentration of Ribavirin.
• Ribavirin is known to be orally administered to a human as an antiviral drug in at least one single dose of about 600mg-1200mg.
• the initial concentration of Ribavirin in the system e.g., a human
• the initial concentration of Ribavirin in the system is between about l ⁇ M and several hundred ⁇ M, however, the Ribavirin concentration is typically reduced in the system by sequestration into erythrocytes within 24 hours to about 85% to 50% of the initial concentration due to phosphorylation of Ribavirin in the erythrocytes.
• Ribavirin to l-beta-D-ribofuranosyl-l,2,4-triazole-3 -carboxamidine significantly reduces the amount of phosphorylation (infra) of Ribavirin. Therefore, it is contemplated that all or almost all of the initial concentration of Ribavirin is available for the desired pharmacological effect in the target cells. Consequently, it is contemplated that modification of Ribavirin with a blocking group can be employed to reduce the dosage of Ribavirin by about 5wt%, preferably by about 10wt%, more preferably by 25wt% and most preferably by 50wt%.
• various dosages other than 600mg-1200mg are also contemplated, including dosages of 200mg-600mg, dosages of 20mg-200mg, and less.
• dosages of 200mg-600mg are also contemplated, including dosages of 200mg-600mg, dosages of 20mg-200mg, and less.
• Ribavirin is employed as an immunomodulatory drug
• lower dosages of about 100mg-300mg may be sufficient.
• dosages of 600mg-1800mg, and more are contemplated.
• the reduction of the dosage may vary considerably. For example, where the metabolic conversion is relatively rapid and takes place in a plurality of non-target cells, reductions of the dosage of between 25wt% and 80wt%, and more, are contemplated. On the other hand, where the metabolic conversion is relatively slow, reductions of the dosage of between 25wt% and 5wt%, and less, are contemplated.
• the blocking group With respect to the drag, the blocking group, the metabolic conversion, the step of modifying the drug, the system, the step of administering the drag, the target cell and the non-target cell in contemplated methods of reducing the dosage of a drug, the same considerations as described above apply.
• a mixture of methyl- l,2,4-triazole-3-carboxy late (25.4 g, 200 mmol) (1), 1,2,3,5- tefra-O-acetyl-y ⁇ -D-ribofuranose (63.66 g, 200 mmol) (2) and bis(p-nitrophenyl)phosphate (1 g) were placed in a RB flask (500 mL). The flask was placed in a pre-heated oil bath at 165-175 °C under water aspirator vacuum with stirring for 25 min. The acetic acid displaced was collected in an ice-cold trap that is placed between aspirator and the RB flask. The flask was removed from the oil bath and allowed to cool.
• Methyl- 1 -(2,3 ,5-tri-O-acetyl-yff-D-ribofuranosyl)- 1 ,2,4-triazole-3 -carboxylate (62 g, 161 mmol) (3) was placed in a steel bomb and treated with freshly prepared methanolic ammonia (350 mL, prepared by passing dry ammonia gas into dry methanol at 0 °C until saturation) at 0°C.
• the steel bomb was closed and stirred at room temperature for 18 h.
• the steel bomb was cooled to 0°C, opened and the content evaporated to dryness.
• the residue was treated with dry EtOH (100 mL) and evaporated to dryness.
• l- ⁇ -D-R ⁇ bofuranosyl-l,2,4-triazole-3- carboxamidine Hydrochloride (8) can be produced by an enzymatic reaction using a culture of a microorganism, intact cells of a microorganism, or a cell extract as an enzyme source (under non-proliferating conditions of the microorganism).
• 3-Cyano-l-(2,3,5-tri-O-acetyl- ?- D-ribofuranosyl)-l,2,4-triazole (7) can be produced by causing 3-cyano-l,2,4-triazole or salt thereof and a ribose donor to contact in the presence of an enzyme source based on the microorganism.
• compound 7 will be transformed into (8) by treating (7) with liquid ammonia solution.
• l,2,4-triazole-3-carboamidine hydrochloride can react with ribose donor in the presence of an enzyme to produce directly (8).
• Ribavirin has been shown to be phosphorylated in RBCs, and it has further been suggested that phosphorylated Ribavirin is a causative agent in he olytic anemia observed in long-term treatment or high dosages of Ribavirin in humans.
• the liver radioactivity concentration after oral dosing of modified Ribavirin was estimated to be approximately 50% higher than oral dosing of Ribavirin.
• the therapeutic ratio for modified Ribavirin is estimated to be about twelve times that of Ribavirin.
• modified Ribavirin can be administered in a dosage of about 65% of Ribavirin to achieve approximately the same efficacy as Ribavirin with substantially no hemolytic anemia; or that modified Ribavirin can be admimstered in the same dosage as Ribavirin to achieve higher efficacy as Ribavirin with substantially no hemolytic anemia. It is further contemplated that modified Ribavirin can also be administered in a dosage of only about 5%-50%, preferably 20%-50%, more preferably 10%- 15%, and most preferably 5-6% of the Ribavirin dosage to achieve the same therapeutic effect as Ribavirin.

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