Separation Of Natural Inhibitor Of HIV-1Protease And Studies On Synthesis And Activity Of Benzofuran Derivatives

AIDS (AIDS) is one kind of degenerative diseases on immune and central nervous system, caused by the human immunodeficiency virus (HIV). HIV-1reverse transcriptase, protease and integrase are three key enzymes in HIV-1gene replication. Until now, the design of anti-HIV/AIDS drugs is mainly focused on those three key enzymes. Inhibitor of protease can prevent maternal protein splitting into functional proteins new HIV infecting cells and viral replicating, which makes AIDS treatment have a breakthrough.Now people have discovered many inhibitors including peptides and non-peptides. There have been some HIV protease inhibitors widely used in clinic:saquinavir, indinavir, ritonavir, etc. However, long-term use of these protease inhibitors has significant adverse effect and drug resistance. So it is important to discover novel HIV protease inhibitors.It is an important path to discover leading compound with new style of structure by extracting the active substance from the microbial fermentation broth. In our screening course for novel HIV-1protease inhibitors from microorganisms, Streptomyce I03A-04862(CGMCC4766) was picked up. Inhibiting activity in the fermentation broth was determined by high-throughput fluorometric assay HIV-1protease substrate model. The fermented material was fractionated by means of chromatography with diaion HP-20, ODS-A, Sephadex LH-20and preparative HPLC, activity tracked by high-throughput fluorometric assay HIV-1protease substrate model, obtained an active compound, which named4862F. The structure of this compound, with feature of N,N,N-trimethylated amino acid residues, one kind of pentapeptides, were elucidated primarily by spectra of ESI-MS、1H-NMR、13C-NMR、DEPT、1H-1H COSY、HSQC、 HMBC、TOCSY、and ROESY.Finally, there are five amino acids in this structure:two leucine, one valine, one dehydrogen histidine, one N,N,N-trimethylated tyrosine, whose sequence of connection is N,N,N-(trimethyl)-Tyr-Leu-Val-Leu-(-2H)-His. This compound has not been reported in domestic and overseas literatures.The half inhibition concentration IC50of4862F against HIV-1protease is15.26nM on high-throughput fluorescence detection HIV-1protease substrate model. Its half cytotoxic concentration CC50on MDCK cells by using CPE method is more than292.83μM. The activity of this compound is good, and the toxicity is little. It is promising to get better inhibitors of HIV-1protease with structure transformation. Protease inhibitors have become one of important methods of treating HCV and HIV. But until now both two types peptide protease inhibitors that in clinical trials are not very good with low oral absorption and high cost. So researchers have become more and more concerned about the non-peptide protease inhibitors.During the literature research, we found that benzofuran compounds, a class of highly bioactive compounds, were widely found in natural and non natural products, with anti-virus, anti-tumor and anti-osteoporosis activity. Recently, it is found that benzofuran-fused pyridine or pyrimidine heterocyclic could increase activity of anti-HIV virus, inhibit of HIV-1reverse transcriptase and HCV NS34A protease.In this study, as synthetic routes reported in references, two benzofuran compounds Kellin and Visnagin was used as raw materials, through hydrolysis and and Vielsmeier-Haack reaction and addition with2-amino-hexa-heterocyclic compounds, We obtained a total of26compounds, including four intermediate, three non-target product, and twenty target compounds. Structure of all products was confirmed by1H-NMR and ESI-MS analysis. In particular, structure of the target compounds was confirmed by’H-NMR,13C-NMR, DEPT,1H-1H COSY, HSQC, HMBC and ESI-MS analysis. Structure of all the products was retrieved by SciFinder Scholar CA Online database and Reaxys database. Twenty-one of them havn’t been reported, including20target compounds.The structure of target compounds is different from that reported inreference. Analysing of the reaction mechanism of the target compounds, it was possible that conjugated double bonds in mother ring were twice additioned with2-amino-hexa-heterocyclic compounds and the solvent methanol, while one molecular of water taken off. In the process of addition, the direction of methanol attack to7-double bond was non-selective, leading to target compounds being racemate, which can be confirmed by crystal X-ray diffraction stacked unit cell and the fact of the specific rotation close to zero.The toxicity on tumor cells and normal cells, and the activity of anti-osteoporosis, inhibitory on HCV NS34A protease, HIV-1protease, HIV-1reverse transcriptase, anti-HIV-1pseudo-typed-viral and anti-influenza virus were evaluated. K-10, K-11and K-14could inhibit HCV NS34A protease well, and K-6and K-18could inhibit HIV-1protease well. In advanced research, we will emphasize on studies on activity of inhibitory on HCV NS34A protease and HIV-1protease. Analysis of the structure-activity relationship will be significant in advanced research.