Design,Synthesis And Biological Activity Analysis Of Urea Cyanobacteria Inhibitors

The blooms of cyanobacteria affect human production and life.There are already some substances such as metallic salt CuSO4,herbicide diuron,etc.to inhibit algae.However,these substances also threaten other water plants and animals because of their low selectivity.Therefore,it is urgent to find a highly selective and environmentally friendly algaecide to effectively control blooms of cyanobacterial.In the early stage of the study,with Cy-FBP/SBPase as target,we obtain a series of compounds TADIs,which have good inhibitory effects on Cy-FBP/SBPase and cyanobacteria and TADI-7 is best inhibitor(IC50=1.1± 0.2 μM,EC50=5.8 μM).While,the mode of action of the compound TADI-7 on Cy-FBP/SBPase is not clear now.Exploration on the mode of action of TADI-7 can better explain its inhibition mechanism and it also has important guiding significance for the design of new inhibitors in the later stage.Diuron is a commercial herbicide that also has a high inhibitory effect on cyanobacteria(EC50=50.7 nM)but it lacks selectivity for other aquatic plants.Based on the exploration of TADI-7 and Cy-FBP/SBPase mode of action,combined with TADI-7 symmetry structure and diuron molecular skeleton,a series of urea compounds are designed,synthesized and analyzed for biological activity.The specific research content mainly includes the following aspects:1.Study on the mode of action of the TADI-7 and Cy-FBP/SBPase:Firstly,the fluorescent probe molecule TADI-NBD is synthesized.Then the binding of TADI-NBD to Cy-FBP/SBPase is studied by the endogenous fluorescence of Cy-FBP/SBPase.Finally,the binding of TADI-7 to the substrate cavity and the allosteric cavity is studied by the method of exogenous fluorescence of the TADI-NBD probe molecule.The results indicate that TADI-7 is incorporated in the FBP cavity,not the AMP cavity.The binding mode of the probe molecule TADI-NBD to Cy-FBP/SBPase is studied by using the mutant experiment method.The fluorescence enhancement of TADI-NBD binding to mutants ARG176,ARG178,TYR131,THR102 decreases to 0.84,0.79,0.80,0.76,respectively,in combination with wild-type Cy-FBP/SBPase.The probe molecules have strong interaction with the amino acid residues ARG176,ARG178,TYR131 and THR102 in the substrate cavity.The experimental results verify the correctness of the TADI-7 and Cy-FBP/SBPase binding modes predicted by the research group.2.Design,Synthesis and Biological Activity Analysis of Phenylurea:Based on the binding mode of TADI-7 and Cy-FBP/SBPase,combined with the symmetry characteristics of diuron and TADI-7,a total of 25 symmetric phenyl urea compounds(series Ⅰ)and asymmetric phenyl ureas are synthesized and characterized for their biological activity.Series Ⅰ compounds against Cy-FBP/SBPase is higher than diuron.The IC50 of Ⅰ-3 against Cy-FBP/SBPase reaches 20.1 μM,is diuron(IC50>20 mM)995 times.Compared with the symmetric series Ⅰ,the asymmetric series Ⅱ have a certain degree of inhibitory activity on Cy-FBP/SBPase,even though the most active Ⅱ-8 compound is about 10 times lower than 1-3.The inhibitory activity of the series Ⅰ against FACHB 905 is higher than that of TADI-7 on FACHB 905.The best inhibitory effect was Ⅰ-3,EC50=3.9 μM,which is 1.5 Times of TADI-7.Compared with the symmetric series Ⅰ,the asymmetric series Ⅱ compounds have a certain degree of inhibition on FACHB 905.It indicates that the symmetrical structure plays an important role in the phenylurea compounds.3.Design,synthesis and biological activity analysis of semicarbazone:1-3 has good activity,but poorly water solubility(0.025 mM dissolved in water).A total of 33 symmetrical semicarbazone compounds(series Ⅲ)and asymmetric semicarbazone compounds(series Ⅳ)are synthesized,and Ⅲ-4(0.3 mM)solubility is better than 1-3.The activity of the III series compound against Cy-FBP/SBPase is preferably III-4,IC50=19.4 μM,equivalent to Ⅰ-3(20.1 μM),which is 1030 times of diuron;Ⅲ-4 inhibition of FACHB 905 is 7.5 times that 1-3(3.9 μM)and 11.2 times that TADI-7(5.8 μM).The inhibitory activity of the asymmetric series Ⅳ compounds on Cy-FBP/SBPase and FACHB 905 is lower than that of Ⅲ-4.It indicates that the symmetrical structure plays an important role in the semicarbazone compound.4.Study on the inhibition mechanism of novel inhibitor Ⅲ-4 on FACHB 905:Analysis of the photosynthetic parameters rETRmax and Φe of FACHB 905 by Ⅲ-4 shows that it does not act in the photoreaction phase of photosynthesis;analysis of oxidative stress indicators SOD,GSH,MDA and ROS shows that III-4 enters the cyanobacterial cells and that causes excessive accumulation of reactive oxygen species ROS,which exceeds the threshold of oxidative stress detoxification.It is speculated that the inhibition of Ⅲ-4 may be caused by severe oxidative damage to the membrane of cells.The normal growth of cells eventually leads to cell damage and death.