Isolated bis-linezolid, preparation thereof, and its use as a reference standard

Abstract

The present invention provides an isolated linezolid (1) impurity, bis-linezolid (4), preparation thereof and its use as a reference standard.

1 . Isolated bis-linezolid (4).  
   
   
       2 . The isolated bis-linezolid (4) of  claim 1 , characterized by data selected from:  1 H NMR (400 MHz, D 2 O+TFA) δ (ppm): 2.08 (s), 3.63 (m), 3.83 (t), 4.02 (s), 4.14 (m), 4.94 (m), 7.23 (d), 7.58 (m);  13 C NMR (400MHz, CDCl 3 ) δ (ppm):16.6 (q), 43-50 (s), 50.4 (s), 50.9 (s), 68.3 (s), 103.6, 104.0 (d), 111.1 (s) , 118.7 (s), 130-140 (s), 149-153 (d), 171.4 (s); FAB+m/z (MH + ): 616; and an IR spectrum at 1519, 1572, 1644, 1743, 2825, 2858, 2891, 2965 cm −1 .  
   
   
       3 . The isolated bis-linezolid (4) of  claim 2 , characterized by  1 H NMR (400 MHz, D 2 O+TFA) δ (ppm): 2.08 (s), 3.63 (m), 3.83 (t), 4.02 (s), 4.14 (m), 4.94 (m), 7.23 (d), 7.58 (m).  
   
   
       4 . The isolated bis-linezolid (4) of  claim 3 , characterized by  1 H NMR substantially as depicted in  FIG. 1 .  
   
   
       5 . The isolated bis-linezolid (4) of  claim 2 , characterized by  13 C NMR (400 MHz, CDCl 3 ) δ (ppm): 16.6 (q), 43-50 (s), 50.4 (s), 50.9 (s), 68.3 (s), 103.6, 104.0 (d), 111.1 (s), 118.7 (s), 130-140 (s), 149-153 (d), 171.4 (s).  
   
   
       6 . The isolated bis-linezolid (4) of  claim 5 , characterized by  13 C NMR substantially as depicted in  FIG. 2 .  
   
   
       7 . The isolated bis-linezolid (4) of  claim 2 , characterized by FAB+m/z (MH + ): 616.  
   
   
       8 . The isolated bis-linezolid (4) of  claim 2 , characterized by an IR spectrum at 1519, 1572, 1644, 1743, 2825, 2858, 2891, 2965 cm −1 .  
   
   
       9 . The isolated bis-linezolid (4) of  claim 8 , characterized by an IR spectrum substantially as depicted in  FIG. 3 .  
   
   
       10 . The isolated bis-linezolid (4) of  claim 1 , containing less than about 5% by weight of linezolid (1).  
   
   
       11 . The isolated bis-linezolid (4) of  claim 10 , containing less than about 2% by weight of linezolid (1).  
   
   
       12 . The isolated bis-linezolid (4) of  claim 11 , containing less than about 1% by weight of linezolid (1).  
   
   
       13 . A process for preparing the isolated bis-linezolid (4) of  claim 1 , comprising: 
 a) combining R-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl methyl azide (3) with an organic solvent and hydrogen gas in the presence of a catalyst to obtain crude (S)-N-(4-morpholinyl-3-fluorophenyl)-2-oxo-5-oxazolidinyl-methyl amine (2);    b) combining the crude product of step a) with an organic solvent to obtain a solution;    c) adding acetic anhydride to the solution and maintaining the reaction mixture for at least 12 hours to obtain crude linezolid (1);    d) combining the crude linezolid (1) of step c) with an organic solvent to obtain a mixture;    e) heating the mixture to reflux; and    f) recovering bis-linezolid (4).    
   
   
       14 . The process of  claim 13 , wherein the organic solvent in steps a), b) and d) is a C 1-4  alkyl ester or an aromatic hydrocarbon.  
   
   
       15 . The process of  claim 14 , wherein said organic solvent is selected from the group consisting of methyl acetate, ethyl acetate, butyl acetate, isobutyl acetate and toluene.  
   
   
       16 . The process of  claim 15 , wherein said organic solvent is ethyl acetate.  
   
   
       17 . The process of  claim 13 , wherein the catalyst in step a) is Pd/C.  
   
   
       18 . The process of  claim 13 , further comprising adding an amine prior to step c).  
   
   
       19 . The process of  claim 18 , wherein said amine is pyridine or a tertiary amine.  
   
   
       20 . The process of  claim 19 , wherein said amine is triethyl amine.  
   
   
       21 . Use of the isolated bis-linezolid (4) of  claim 1  as a reference standard.  
   
   
       22 . A method of determining the amount of bis-linezolid (4) in a sample of linezolid (1) comprising: 
 a) measuring by HPLC the area under a peak corresponding to bis-linezolid (4) in a reference standard comprising a known amount of bis-linezolid (4);    b) measuring by HPLC the area under a peak corresponding to bis-linezolid (4) in a sample comprising linezolid (1) and bis-linezolid (4); and    c) determining the amount of bis-linezolid (4) in the sample by comparing the area of step (a) to the area of step (b).    
   
   
       23 . A method of determining the amount of bis-linezolid (4) in a sample of linezolid (1) comprising: 
 a) subjecting a reference standard solution of bis-linezolid (4) comprising a known amount of bis-linezolid (4) to HPLC for at least 45 minutes and measuring by HPLC the area under a peak corresponding to bis-linezolid (4);    b) subjecting a sample solution comprising linezolid (1) and bis-linezolid (4) to HPLC for at least 45 minutes and measuring by HPLC the area under a peak corresponding to bis-linezolid (4); and    c) determining the amount of bis-linezolid (4) in the sample by comparing the area of step (a) to the area of step (b).    
   
   
       24 . A method for detecting bis-linezolid (4) comprising: 
 a) providing a preparation of crude linezolid (1) containing bis-linezolid (4);    b) subjecting the preparation of step a) to HPLC;    c) determining whether any material has an RRT of about 3.4 with respect to linezolid (1);    wherein the presence of material with an RRT of about 3.4 with respect to linezolid (1) indicates that the preparation contained bis-linezolid (4).    
   
   
       25 . The process of  claim 24 , wherein the HPLC in step b) is carried out for at least about 34 minutes.  
   
   
       26 . An HPLC method for assaying linezolid (1) comprising the steps: 
 a) combining a linezolid (1) sample with a mixture of acetonitrile/water diluent to obtain a solution;    b) injecting the solution of step a) into a Hypersil Gold 150×4.6 (or similar) column;    c) eluting the sample from the column at about 25 min using a mixture of about 0.01M K 2 HPO 4 : MeOH as an eluent at a gradient of about 80:20 to about 50:50; and    d) measuring the bis-linezolid (4) content in the relevant sample with a UV detector.    
   
   
       27 . The process of  claim 26 , wherein the bis-linezolid (4) at step d) is measured at a wavelength of 254 nm.