Design, Synthesis And Antiviral Activity Of Chalcone Derivatives Containing Purine (thio) Ether

As the earliest known plant viruse,tobacco mosaic virus（TMV）can cause serious plant diseases and huge economic losses to agricultural production.The relevant data indicated that the loss caused by TMV is up to hundreds of millions of dollars worldwide each year.At present,the varieties of antiviral agents that can completely controlling the virus are relatively scarce.Among them,as two commonly used antiviral agents,ningnamycin and ribavirin exhibited good antiviral activity,but they have some shortcomings,such as the high cost of field prevention and control of ningnamycin,and the risk of damage of ribavirin.Therefore,it is urgent to develop more novel,high-efficiency and low-risk antiviral agents.As two kinds of natural compounds,chalcone and purine have the potential biological activity,and their structure is easyily modified.It provides inspirations and ideas for the development of antiviral drugs.Based on this,in order to find the compounds containing chalcone and purine with higher antiviral activity,a series of new chalcone derivatives containing a purine（sulfur）ether moiety were designed,synthesized,and their antiviral activities were tested.Meanwhile,the preliminary antiviral mechanism was explored by relevant biological means.The research results of this work were summarized as the follows.1.Based on our previous research results,we used chalcone as the basic skeleton in this work,then introducing the purine（sulfur）ether,or adding diethyl malonate moieties nitromethane to the propyl-2-ene-1-one by Michael addition reaction,thirty chalcone derivatives containing a purine（sulfur）ether moiety were designed and synthesized,and their strcutures were characterized via the nuclear magnetic resonance（NMR）and high resolution mass spectrum（HRMS）.2.The anti-TMV activities in vivo of the target compounds were systematically evaluated by the conventional half-leaf method.The results showed that some target compounds have good anti-TMV activities.Of which,the compounds 5d,5f,5h,8a,8b,8c and 8f showed excellent inactive activity against TMV,and their inhibition rates are 89.5%,80.9%,84.0%,81.7%,82.7%,87.4%and 83.2%,respectively,which were better than that of ribavirin（69.8%）.The further activities results showed that the EC₅₀values of compounds 5d,8c and 8f are 65.8,69.2 and 64.1μg/mL,respectively,which were significantly better than that of ribavirin(EC₅₀=154.3μg/mL).3.In order to explore the initial interaction mechanism of the compounds with excellent anti-TMV inactive activity.We used microscale thermophoresis（MST）technology to measure the binding affinity between the compounds 5d,8c,8f and TMV coat protein（CP）.In addition,the compounds 5g(EC₅₀=190.2μg/mL)and 5k(EC₅₀=591.1μg/mL)with moderate and poor inactive activity were used as the reference.Compounds 5d,8c and 8f had stronger binding force with TMV CP,and the Kd values were 13.4,15.6 and 14.4μM,respectively.The compounds 5g and 5k had medium to weak binding force with TMV CP,with the Kd values were 33.2 and215.1μM,respectively（ribavirin:Kd=119.8μM）.These test results are consistent with the trend of inactive activity of the compounds against TMV.4.To further explore the binding modes between the compounds and TMV CP,we selected the compounds 5d,8c,8f,5g and 5k with excellent to poor anti-TMV inactive activity to dock with TMV CP.The results showed that compounds 5d,8c and 8f formed five hydrogen bonds（C=O---TYR139,2.38（?）;C=O---ASN73,2.21（?）;C=O---GLN257,3.08（?）;NH---TRP217,1.39（?）and NH---ASP219,2.34（?））,one hydrogen bond（NH---ASN73,2.70（?））and three hydrogen bonds（C=O---GLY137,2.48（?）;C=O---GLN257,2.95（?）;C=O---ASN73,2.27（?））with the amino-acid residues of TMV CP,respectively.The compounds 5g（NH---ARG134,2.58（?）;NH---TRP217,1.88（?））and 5k（NH---SER147,3.06（?）;NH---ARG261,2.46（?））both formed two hydrogen bonds with the amino-acid residues of TMV CP.Moreover,ribavirin formed three conventional hydrogen bonds with the amino-acid residues GLU222（NH,4.54（?））,ASP219（C=O,3.93（?））,and ASP266（-OH,5.50（?））of TMV CP.In addition,the comounds 5d,5g,5k,8c,8f and ribavirin all formed Pi-Alkyl with with the amino-acid residue LYS253 of TMV CP.These results indicated that the binding between the compounds and TMV CP may affect inactive ability of the compounds against TMV,and the binding modes of the compounds with TMV CP depend on the structure of the compound itself.5.The morphology changes of TMV particles after treatment with the compounds were observed by transmission electron microscopy（TEM）.The upshots manifested that the compound 5d can destroy the integrity of TMV particles,resulting in more severe rupture and breakage,which probably weakened the infectivity of TMV.In conclusion,thirty chalcone derivatives containing a purine（sulfur）ether moiety were designed,synthesized and their structures were characterized by NMR and HRMS.The results showed that compounds 5d,8c and 8f had good inactive activity against TMV,with the EC₅₀ values were 65.8,69.2 and 64.1μg/mL,respectively,which were significantly better than that of ribavirin（154.3μg/mL）.The antiviral mechanism of compounds 5d,8c and 8f was studied by MST,molecular docking and electron microscopy.The results revealed that the antiviral activity of compounds 5c,8c and 8f depended on their strong binding ability with TMV CP.