ABSTRACT
To explore the application potential of dual prodrug strategies in the development of anti-HCV agents, a variety of sofosbuvir derivatives with modifications at the C4 or N3 position of the uracil moiety were designed and synthesized. Some compounds exhibited potent anti-HCV activities, such as 4e and 8a-8c with similar EC50 values (0.20-0.22⯵M) comparative to that of sofosbuvir (EC50â¯=â¯0.18⯵M) in a genotype 1b based replicon Huh-7 cell line. Moreover, 8b displayed a good human plasma stability profile, and was easily metabolized in human liver microsomes expectantly. On the other hand, aiming to discover novel anti-HCV nucleosides, pyrazin-2(1H)-one nucleosides and their phosphoramidate prodrugs were investigated. Several active compounds were discovered, such as 25e (EC50â¯=â¯7.3⯵M) and S-29b (EC50â¯=â¯19.5⯵M). This kind of nucleosides were interesting and would open a new avenue for the development of antiviral agents.
Subject(s)
Antiviral Agents/pharmacology , Hepacivirus/drug effects , Prodrugs/pharmacology , Pyrazines/pharmacology , Sofosbuvir/analogs & derivatives , Sofosbuvir/pharmacology , Antiviral Agents/chemical synthesis , Blood/metabolism , Cell Line, Tumor , Drug Discovery , Drug Stability , Humans , Microsomes, Liver/metabolism , Prodrugs/chemical synthesis , Pyrazines/chemical synthesis , Sofosbuvir/chemical synthesisABSTRACT
Direct-acting antiviral agents (DAAs) represent the major advance in hepatitis C virus (HCV) infection treatment leading to extremely high eradication rates in HCV mono- and HIV/HCV co-infected patients. In this scenery, availability of Therapeutic Drug Monitoring (TDM) is of interest to assess plasma concentrations to prevent either therapeutic failure due to suboptimal medication adherence and drug-drug interactions or avoid adverse events. Aim of this study was to develop and validate an Ultra-Performance Liquid Chromatography Mass Spectrometry (UPLC-MS/MS) method for the simultaneous quantification of sofosbuvir, sofosbuvir metabolite (GS-331007), and daclatasvir in human plasma. A simple protein precipitation was applied by adding 200⯵L acetonitrile with internal standard 6,7-Dimethyl- 2,3-di(2-pyridyl) quinoxaline to 100⯵L plasma sample. Drug separation was performed on analytical C-18 Luna Omega column (50â¯mmâ¯×â¯2.1â¯mm I.D.) with particle size of 1.6⯵m. The mobile phase consisting of water containing 0.1% formic acid and acetonitrile at flow 0.4â¯mL/min and a gradient run time of 3.5â¯min. The injection volume was 10⯵L. Anti-HCV drugs were detected in positive electrospray ionization mode. The full scan mass spectral analyses of sofosbuvir, GS-331007, daclatasvir and quinaxoline showed protonated molecule ions and transitions m/z: 530.098â¯ââ¯243.02, 260.93â¯ââ¯112.94, 739.4â¯ââ¯339.27 and 313.03â¯ââ¯77.99 respectively. The linearity of standard curves was excellent (r2â¯>â¯0.99), the absolute recovery of anti-HCV drugs ranged between 95 and 98%, and both imprecision and inaccuracy were <15% according to FDA guidelines. The UPLC-MS/MS method was applied to 16 plasma samples of as many HIV/HCV co-infected patients treated with sofosbuvir and daclatasvir. While sofosbuvir was not detectable in all samples, the median plasma concentrations of daclatasvir and GS-331007 were 223.6⯱â¯319.56â¯ng/mL and 537.11⯱â¯242.09â¯ng/mL, respectively. In conclusion, we describe an UPLC-MS/MS method allowing the simultaneous quantification of sofosbuvir, GS-331007 and daclatasvir in plasma samples. The method was sensitive, specific, robust, and time-saving.