Design,Synthesis And Antiviral Activity Of Novel Nucleoside Analogues

Currently, infections account for about30%of annual mortalities worldwide, and60-65%of infections are caused by virus, which can lead to AIDS, hepatitis, herpes and other serious diseases. Therefore, development of the novel anti-viral drugs is necessary and urgent in the future study. Nucleoside/Nucleotide analogues play a crucial role in anti-viral therapy, but these drugs suffers from several clinical limitations, such as short half-life time, significant dose-related toxicity and drug resistance. In order to find novel nucleoside analogues to circumvent these limitation, various modifications of sugar or base on nueleosides have been extensively studies. Based on these studies, we designed two type of novel nucleoside analogues:one is the pegylation of zidovudine using different molecules of methoxy poly(ethylene glycol)(mPEG, Mw:750Da,2k,5k and10k Da), the other is novel base modifications of nucleosides.1. Study of PEGylated zidovudine.A series of methoxy poly(ethylene glycol)-succinyl-5'-O-zidovudine conjugates (mPEG-succinyl-AZT) with different molecular weight (Mw:750Da,2,5or10kDa) of mPEG were synthesized and characterized by fourier transform infrared (FTIR) spectroscopy,1H nuclear magnetic resonance (1H NMR) spectroscopy, and matrix-assisted laser desorption/Ionization time of flight mass (MALDI TOF MS) spectrometry analysis. All conjugates showed good stability in vitro release experiments, and good anti-HIV activity and low cytotoxicity in MT-4cells, in which, mPEG750-succinyl-AZT exhibited better inhibition to wild-type viruses (strains ⅢB and ROD) with EC50values of0.11and0.090μmol/L, respectively, and it showed no cytotoxicity up to110μmol/L. Oral pharmacokinetic study in rats showed the half-life time (T1/2) of all conjugates are prolonged compared to free AZT. Especially, mPEG750-succinyl-AZT displayed a～2.3-fold prolonged half-life and approximately 224%increased bioavailability of AZT.2. Study of1,2,4-thiadiazine base nucleoside analogues.Based on the base pair principle, we designed and synthesized a series of1,2,4-thiadiazine base nucleoside analogues. The compounds are characterized by mass spectrometry,1H nuclear magnetic resonance ('H NMR) spectroscopy. Some of them are also characterized by13C nuclear magnetic resonance (13C NMR). All of compounds are evaluated the in vitro anti-HBV activity.The in vitro anti-HBV activity of compounds are evaluated in HepG2.2.15cell lines, and the inhibitory of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are analyzed by IC50(The half maximal inhibitory concentration), and the toxicity are analyzed by CC50(The half concentration required to reduce the viability of mock-infected cells). For1,2,4-thiadiazine base nucleoside analogues, the inhibitory of HBeAg is stronger than inhibitory of HBsAg. The compound7f1showed higher anti-HBV activity than other compounds, especially for inhibitory of HBeAg (IC50was25.54μmol/L), which was equal to the corresponding inhibitory for Adefovir (IC50was25.16μmol/L). Also therapeutic index of7f1was significantly higher than other compounds (SI=4.01). And it is noteworthy that1,2,4-thiadiazine nucleoside analogues with the novel base structure retained the anti-HBV activity, although which are lower than Lamivudine and Adefovir, their special base structure provide a novel guide for the future modification of nuleosides.Otherwise, the anti-HIV activities of the compounds are evaluated by Rega Institute for Medical Research of Katholieke University, and the activity are under testing.3. Study of4-S-pyrimidine nucleoside analogues.Based on the phenylthio-(PhS-) modificaion on purine of leading compoud MCC-478, we designed a series of novel4-S-pyrimidine nucleoside analogues. The compounds are characterized by mass spectrometry,1H nuclear magnetic resonance (1H NMR) spectroscopy. Some of them are also characterized by13C nuclear magnetic resonance (13C NMR) and heteronuclear long range correlation. All of compounds are both evaluated the in vitro anti-HBV activity and in vitro anti-HIV activity.The in vitro anti-HBV activity of compounds are evaluated in HepG2.2.15cell lines, and the inhibitory of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) are analyzed by IC50(The half maximal inhibitory concentration), and the toxicity are analyzed by CC50(The half concentration required to reduce the viability of mock-infected cells). For4-S-pyrimidine nucleoside analogues, the inhibitory of HBsAg is stronger than inhibitory of HBeAg. In4-S-pyrimidine nucleoside analogues, propargyl thio-pyrimidine compounds (4d,5d,6d1,6d2,6d3) showed better inhibitory compared with other thio-pyrimidine, especially for compounds6d1,6d2and6d3, their IC50for HBsAg is12.01,14.55and14.98μmol/L, respectively, which are even lower than the control drug Lamivudine and Adefovir (IC50for HBsAg is27.20and26.92μmol/L, respectively). Therefore this series of propargyl thio-pyrimidine nucleoside might worth be further investigated and developed.Otherwise, the anti-HIV activities of the compounds are evaluated by Rega Institute for Medical Research of Katholieke University, and the activity are under testing.In summary, we designed and synthesized three different kinds of nucleoside modifications, and find the corresponding superior structure in each kind, which provided the new direction for deep investigation.