| Objective:To design and synthesis of a series of novel adefovir mono L-thioamino acid ester,mono bile acid ester derivatives with improved hepatotrophic property,bioavailability and enhanced antiviral activity by using adefovir as lead compound.Methods:?1?Synthesis of target compound:After activation by isobutyl chloroformate?IBCF?,N-Boc-L-amino acids reacted with hydrogen sulfide?H2S,gas?at-25?in the presence of N-methylmorpholine?NMM?to give N-Boc-L-amino monothioacids,which then reacted with 1,2-dibromoethane or 1,3-dibromopropane,using NaH as base,to give anticipated 2-bromoethyl ester or 3-bromo-1-propyl ester of N-Boc-L-amino monothioacids,whereas condensation of bile acid,like ursodesoxycholic acid and deoxycholic acid with 3-bromo-1-propanol in the presence of DCC/DMAP yielded the corresponding bile acid 3-bromo-1-propyl esters.The above prepared product was condensed with adefovir under the condition of N,N'-dicyclohexyl-4-morpholine-carboxamidine?DCMC?as acid scavenger by using"one-pot synthesis"method,and then was purified by column chromatography to obtain adefovir N-Boc-mono-L-thioamino acid ester,mono bile acid ester derivative.The obtained products were further treated with 85%phosphoric acid/chloroform system to obtain the target compounds 6a-6p;?2?In vitro stability of target compounds in artificial gastric juice,artificial intestinal fluid,rat blank plasma,and rat liver microsomes,and degradation pattern in rat liver microsomes were evaluated by using UPLC-MS/MS techniques;?3?In vitro antiviral activities of target compounds were evaluated by using HepG 2.2.15 cell line,and EC50,CC50 and SI of them were calculated;?4?Uptake mechanism for target compounds were evaluated in Na+/taurocholate cotransporting polypeptide?NTCP?transferred Hek-293 cell line.Results:?1?16 target compounds?6a-p?were obtained with total yield in the range of12.6-25.2%.The structures of synthesized target compounds were confirmed by1H-NMR,13C-NMR,ESI-MS and ESI-HRMS techniques;?2?The stability and metabolism in vitro results showed that the test compounds had good stability in artificial gastric juice,and their stability in rat blank plasma and rat liver microsome were better than that of adefovir dipivoxil,in above medium,their half-life were longer than adefovir dipivoxil.The metabolic pattern of compound 6d showed adefovir mono bile acid ester derivative was first formed by hydrolysis of target compound 6d by hepatic microsomes,and further hydrolyzed into adefovir.;?3?An antiviral test in vitro showed that the eight tested compounds?6d,6e,6f,6g,6i,6l,6m,6n?had anti-HBV activity,among them compounds 6d,6i,6l,6m,6n had more potent antiviral activity,their EC50 and SI(EC50 3.95-29.41?mol·L-1,SI 89.50-454.07)were comparable to that of the positive control adefovir dipivoxil(EC50 3.09?mol·L-1,SI 142.04).Compound 6l exhibit optimal antiviral property with EC50 3.95?mol·L-1,SI 454.07;?4?Uptake mechanism evaluation results in NTCP transferring Hek-293cell?NTCP-Hek-293 cell?showed that the uptake of target compounds?6d,6l,6m,6n?in HEK293 cells stably transfected with NTCP was significantly higher than that of untransfected HEK293 cells at the same concentration?P<0.05?;And the order of uptake rate was 6l>6m>6n>6d.However,the uptake rate of adefovir dipivoxil in above two cells lines did not show a significant positive linear relationship with the increase of the concentration.Conclusion:16 unreported target compounds were synthesized;In vitro stability studies indicated target compounds are relative stable in acidic conditions and release parent drug in basic and enzymatic conditions.Uptake mechanism study indicated that bile acid carrier such as NTCP might be at work in recognition as well as transport of the target compounds,which may represent transporting mechanism for above hepatotrophic prodrugs.Compound 6l has more potent antiviral activity,higher stability in vitro and transfected cells uptake,which warrants further development as a candidate. |
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