Method of resolution and antiviral activity of 1,3-oxathiolane nucleoside enantiomers

Abstract

A process for the resolution of a racemic mixture of nucleoside enantiomers that includes the step of exposing the racemic mixture to an enzyme that preferentially catalyzes a reaction in one of the enantiomers. The nucleoside enantiomer (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane is an effective antiviral agent against HIV, HBV, and other viruses replicating in a similar manner.

We claim:  
     
         1 . A method for the resolution of a racemic mixture of nucleoside enantiomers, comprising the step of exposing the racemic mixture to an enzyme that preferentially catalyzes a reaction in one of the enantiomers.  
     
     
         2 . The method of  claim 1 , wherein the enzyme is selected from the group consisting of an esterase, a lipase, substillisin, α-chymotrypsin, and cytidine-deoxycytidine deaminase.  
     
     
         3 . The method of  claim 2 , wherein the esterase is pig liver esterase  
     
     
         4 . The method of  claim 2 , wherein the lipase is selected from the group consisting of porcine pancreatic lipase and Amano PS-800 lipase.  
     
     
         5 . The method of  claim 1 , wherein the nucleoside enantiomers are acylated at the C5′-hydroxyl position.  
     
     
         6 . The method of  claim 5 , wherein the enantiomers are acylated before resolution with a compound selected from the group consisting of alkyl carboxylic acids and substituted alkyl carboxylic acids.  
     
     
         7 . The method of  claim 6 , wherein the alkyl carboxylic acid is selected from the group consisting of acetic acid, propionic acid, butyric acid, pentanoic acid, 2-chloropropionic acid, 2-chlorobutyric acid, and 2-chloropentanoic acid.  
     
     
         8 . The method of  claim 1 , wherein the nucleoside enantiomers are passed through a column that includes the enzyme immobilized on a support.  
     
     
         9 . The method of  claim 1 , wherein the enantiomers are mixed with the enzyme in a solution.  
     
     
         10 . The method of  claim 1 , further comprising carrying out the enzymatic reaction in the presence of a non-ionic surfactant.  
     
     
         11 . The method of  claim 10 , wherein the non-ionic surfactant is Triton X-100.  
     
     
         12 . The method of  claim 1 , further comprising the step of exposing the product of resolution to a second enzyme that enhances the resolution.  
     
     
         13 . The method of  claim 1 , further comprising recrystallizing the product of resolution.  
     
     
         14 . The method of  claim 1 , further comprising treating the product of resolution with a chiral acid.  
     
     
         15 . The method of  claim 14 , wherein the chiral acid is selected from the group consisting of malic acid, mandelic acid, dibenzoyl tartaric acid, 3-bromocamphor-8-sulfonic acid, 10-camphorsulfonic acid, and di-p-toluoyltartaric acid.  
     
     
         16 . The method of  claim 1 , wherein the racemic mixture is selected from the group consisting of the 5′-O-ester and the unesterified (±)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.  
     
     
         17 . The method of  claim 1 , wherein the racemic mixture is selected from the group consisting of the 5′-O-ester and the unesterified 5′-O-ester of (±)-2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane.  
     
     
         18 . The compound (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.  
     
     
         19 . The compound (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane.  
     
     
         20 . A pharmaceutical composition consisting essentially of an effective amount to inhibit the replication of a virus in a human of a compound selected from the group consisting of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the monophosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the diphosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, and the triphosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.  
     
     
         21 . The composition of  claim 20 , wherein the pharmaceutically acceptable carrier is selected from the group consisting of oil, water, saline, phosphate, buffer, polyethylene glycol, glycerine, propylene glycol, and combinations thereof.  
     
     
         22 . The composition of  claim 20 , wherein the carrier comprises a controlled release formulation.  
     
     
         23 . The composition of  claim 20 , wherein the carrier comprises a liposomal suspension.  
     
     
         24 . The composition of  claim 20 , wherein the pharmaceutically acceptable carrier comprises a biodegradable implant.  
     
     
         25 . The composition of  claim 20  in a unit dosage form that delivers between 1 and 20 mg/kg bodyweight per dosage.  
     
     
         26 . The composition of  claim 20  that produces a serum concentration of compound of between approximately 0.2 and 20 μM.  
     
     
         27 . The composition of  claim 20  that produces a serum concentration of compound of between approximately 1.0 and 10 μM.  
     
     
         28 . The composition of  claim 20 , further comprising a compound selected from the group consisting of an antibacterial agent, antifungal agent, chemotherapeutic agent, and another antiviral agent.  
     
     
         29 . The composition of  claim 20  wherein the amount of composition is effective to inhibit human immunodeficiency virus.  
     
     
         30 . The composition of  claim 20  wherein the amount of the composition is effective to inhibit hepatitis B virus.  
     
     
         31 . A pharmaceutical composition consisting essentially of an effective amount to inhibit the replication of a virus in a human of a compound selected from the group consisting of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the monophosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the diphosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, and the triphosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.  
     
     
         32 . The composition of  claim 31 , wherein the pharmaceutically acceptable carrier is selected from the group consisting of oil, water, saline, phosphate, buffer, polyethylene glycol, glycerine, propylene glycol, and combinations thereof.  
     
     
         33 . The composition of  claim 31 , wherein the carrier comprises a controlled release formulation.  
     
     
         34 . The composition of  claim 31 , wherein the carrier comprises a liposomal suspension.  
     
     
         35 . The composition of  claim 31 , wherein the pharmaceutically acceptable carrier comprises a biodegradable implant.  
     
     
         36 . The composition of  claim 31 , in a unit dosage form that delivers between 0.1 and 100 mg/kg bodyweight per dosage.  
     
     
         37 . The composition of  claim 31  that produces a serum concentration of compound of between approximately 0.2 and 20 μM.  
     
     
         38 . The composition of  claim 31  that produces a serum concentration of compound of between approximately 1.0 and 10 μM.  
     
     
         39 . The composition of  claim 31 , further comprising a compound selected from the group consisting of an antibacterial agent, antifungal agent, chemotherapeutic agent, and another antiviral agent.  
     
     
         40 . The composition of  claim 31  wherein the amount of composition is effective to inhibit human immunodeficiency virus.  
     
     
         41 . The composition of  claim 31  wherein the amount of the composition is effective to inhibit hepatitis B virus.  
     
     
         42 . A method for inhibiting replication of HIV in cells comprising administering to a human an HIV inhibitory amount of a composition consisting essentially of a compound selected from the group consisting of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the monophosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the diphosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, and the triphosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.  
     
     
         43 . A method for inhihiting replication of HIV in cells comprising administering to a human an HIV inhibitory amount of a composition consisting essentially of a compound selected from the group consisting of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the monophosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the diphosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, and the triphosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.  
     
     
         44 . A method for inhibiting the replication of HBV in cells comprising administering to a human an HBV inhibitory amount of a composition consisting essentially of a compound selected from the group consisting of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the monophosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the diphosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, and the triphosphate ester of (−)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.  
     
     
         45 . A method for inhibiting replication of HBV in cells comprising administering to a human an HBV inhibitory amount of a composition consisting essentially of a compound selected from the group consisting of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the monophosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, the diphosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, and the triphosphate ester of (+)-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane, or a pharmaceutically acceptable salt thereof, in combination with a pharmaceutically acceptable carrier.