Synthesis,Bioactivities And Structureactivity Relationship Of2?Phenylphthalazin-2-Iums

Phthalazine,a class of nitrogen-containing heterocycle,has gained more attention from medicinal chemists owing to its pharmacological potential.Phthalazine are formed by the condensation of a pyridazine ring with a benzene ring.There are many isomers such as quinoxaline,quinazoline,cinnoline and naphthyridines.Drugs containing phthalazines have a variety of pharmacological activities,especially antibacterial,anticancer,parasiticidal,antihypertensive,anti-inflammatory,analgesic and antidepressant activities.The design and synthesis of phthalazines with novel structure and higher activity has become a new research hotspot.Quaternary benzo[c]phenanthridine alkaloids（QBAs）are an important class of natural isoquinoline alkaloids.As the two most common QBAs,Sanguinarine（SA）and Chelerythrine（CH）have been considered as promising lead compounds for the development of novel QBA-like drugs because of their excellent bioactivity,distinctive structure,and low mammalian toxicity.We previously deigned two classes of structurally simple analogs of QBAs,that is,isoquinoline analogs andβ-carboline analogs by structural simulation.These analogues generally showed similar or even more potent bioactivity than SA and CH.Besides,the analogues also showed high safety for plant growth.Encouraged by the findings above,herein we further designed a class of novel phthalazine analogs.It was expected that the structural similarity between QBAs or their analogs can lead to the discovery of a novel and more potent scaffold for the development of new QBA-like drugs.In this study,a novel 2-phenylphthalazin-2-ium scaffold and a series of its derivatives were designed and synthesized,and their inhibition activities were explored in vitro on 12important plant pathogenic fungi.The most active compound was evaluated for the activity in vivo,and the action mechanism was investigated by scanning electron microscopy（SEM）and transmission electron microscopy（TEM）observations.The cytotoxicity of compounds of series 1 against two kinds of cancer cells and two kinds of normal cells and the antibacterial activity compounds of series 1 against two kinds of bacteria were evaluated.Additionally,the structure-activity relationships were deduced.The achievements of this study are as follows:1.By structural simulation,sixty-eight 2-phenylphthalazin-2-ium bromides were designed,synthesized,and identified by ¹H NMR,¹³C NMR,and MS analysis.2.The target Compounds were screened for inhibition activity on 12 strains of plant pathogenic fungi by the mycelium linear growth rate method at the concentration of 50μg/m L.For compounds of series 1,73.3%（308/420）of the trials gave good to excellent activity with inhibition rates of 50 to 100%.Eight strains of plant pathogenic fungi showed higher susceptibility to the compounds.3.Based on the initial screening results,30 target compounds were further measured for median effective concentrations（EC50）against 8 kinds of plant pathogens by the mycelium linear growth rate method.The results showed that 19 among them had excellent antifungal activity against almost all 8 plant pathogenic fungi,not only far superior to the natural model compounds,SA and CH,but also superior to the positive drugs,azoxystrobin for 6 out of the8 test fungi.Compounds 1-26（2’,4’-di Cl）and 1-27（2’,5’-di Cl）showed the highest total activity against all the fungi while F.solani showed the highest susceptibility to the 19 compounds above.4.As a representative compound,the most active compound 1-27 was further tested for the inhibition activity in vivo against C.gloeosporioides on apples.The results showed that the inhibition efficacy of 1-27 showed concentration dependency.The treatment with 25.0μg/m L 1-27 almost completely inhibited the growth of the fungi over the post-treatment 9th day.5.We carried out the analysis of the hyphal morphology and cell ultrastructure by using SEM and TEM.C.gloeosporioides was chosen as the test fungi.The most active compound1-27 against C.gloeosporioides was selected as the test compound.The test concentration was set to the EC50 of 1-27.The treatment with 1-27 for 72 h led to shrinkage and twisting of the mycelia.The cell membrane became wrinkled,contracted inward,and partially detached from the cell wall.The results above indicated that 1-27 was able to damage the integrity of the mycelial structure by affecting the structure of the cell membrane of mycelium.Thus,the damage effect on the cell membrane may be considered as the main reason for the inhibition action of 1-27 on the fungi.6.The antifungal structure-activity relationships showed that the presence of electron-donating groups such as halogen atoms,trifluoromethyl,nitro,and cyano on the C-ring significantly increases the activity for all or most of the fungi,whereas electron-donating groups such as Me,Et,and Me O cause a relatively small change in activity in most cases.Electron-donating group（-OCH2O-）on the A-ring will reduce the activity against in most cases.7.We evaluated the activities of target compounds against Staphylococcus aureus and Escherichia coli.The results indicated that compounds 1-7（4’-Cl）,1-10（4’-Br）,and 1-11（4’-I）showed higher activity than the natural model compounds,SA and CH.Compound 1-11showed the highest activity against S.aureus which was close to the positive drugs,ceftriaxone sodium.8.The cytotoxicity of target compounds on cancer cells（He La and MKN-45 cells）and normal cells（GFFC and PFKC cells）was evaluated by the MTT method.The results showed that 14 compounds showed higher cytotoxicity on MKN-45 cells than a standard anticancer drug cis-platinum（DDP）.All tested compounds showed higher cytotoxicity on He La cells than DDP.Compound 1-30（3’,5’-di Cl）and 1-27 showed higher cytotoxicity on Hela cells than DDP.Both 1-26 and 1-27 presented lower cytotoxicity on two normal cells than on MKN-45and Hela cells.Besides,DDP showed higher cytotoxicity on the normal cells compared with on the cancer cells.This result shows that compounds 1-26 and 1-27 are more selective for cancer cells than DDP.In summary,compared with the natural model compounds SA and CH,2-phenylphthalazin-2-ium bromides have some obvious advantages such as more potent activity,wider antifungal spectrum,more simple structure,easier synthesis and modification,and lower cost.Thus,2-phenylphthalazin-2-iums are very candidate compounds for the development of novel QBAs-like fungicides for plant protection.1-26 and 1-27 represent promising candidates for the development of novel antifungal agents.Compounds 1-26 and 1-27 were more selective for cancer cells than DDP,and expected to be the leading compound for the development of new anti-cancer drugs.There are still shortcomings in this study.The plant safety evaluation and field efficacy trials of candidate drugs need to be carried out,and the stability experiment of the compound is the next work.