Study On Design And Synthesis Of Novel Imidazole Nitronyl Nitroxide Radicals And Their Biological Activities

Imidazole nitronyl nitroxide radicals, containing both nitroxide radicals（N-O·）andnitrogen and oxygen dipolar ions (+N-O－), are a novel class of bifunctional nitronylnitroxide radicals. In this thesis,10novel nitroxyl nitroxide radicals were designed,synthesized, and characterized. Specifically, antitumor, anti-Coxsackie virus, andradioresistance properties were studied and some action mechanisms were explored. Thesestudies provide a theoretical basis and experimental data for the development of nitoxylradical drugs. This thesis includes the following chapters.Section1. In this thesis,10nitroxyl radicals were synthesized,5of which are newstructures. Their synthetic routes were also optimized, and the key steps were discussed.These compounds were further characterized in terms of their biological activities.Designed and synthesized compounds L-1and D-1, which included chiral skeleton configurations of pyrrolidine units with the chiral carbon atoms directly connected withthe nitroxides functional groups; Designed and synthesized compounds L-2，D-2；L-3，D-3， which included chiral skeleton configurations of pyrrolidine units with the chiralcarbon atoms connected with the nitroxide functional groups by phenoxyl bonds;Designed and synthesized compouds R-4/S-4, which included chiral skeletonconfigurations of1-phenylethylamine;Designed and synthesized two achiral compounds5F-1and Ph-1, which are derived from5-fluorouracil and4-hydroxybenzaldehyderespectively.The structures were analyzed by elemental analysis, IR, HRMS, EPR spectrum. ThePh-1crystal structure were determined by X-ray crystal diffraction. CD spectrumsdemonstrated that L-1/D-1、L-2/D-2、L-3/D-3and R-4/S-4were enantiomers.Section2. In vitro and in vivo antitumor experiments for the nitroxyl radicals(1) Screened two advantageous molecule structures5F-1and Ph-1by the MTT(Methyl Thiazolyl Tetrazolium Thiazolyl Blue Tetrazolium Bromide) assays. These radicalcompounds’ MTT assays were performed to investigate cell proliferation inhibitory ratesfor seven tumor cells. Experiments showed that the nitroxyl radicals5F-1and Ph-1hadmore significant inhibitory rates for breast cancer and gastric cancer cells. Thus, the breastcancer cell MDA-MB-231was chosen for the remaining in vitro experiments. Experimentgroups:5F-1and Ph-1, positive group: docetaxe (DOC). Cell cycle and cell apoptoticpercent were analyzed by flow cytometry (FCM). Cell ultrastructures were observed bytransmission electron microscope (TEM). Cell adhesion experiment, cells invasion assays,and cell migration assays were also designed.The results showed that5F-1and Ph-1had good cell proliferation inhibitory rates forthe breast cancer cell MDA-MB-231. In particular, Ph-1had the same or better antitumorbioactivities compared with docetaxe.(2) Anticancer effects of nitroxyl radicals5F-1and Ph-1in vivo. Established atumor-bearing nude mice model bearing human breast cancer (MDA-MB-231);Investigated the tumor growth inhibition rate of the nude mice under5F-1and Ph-1administration; TUNEL Assay for cell detecting apoptosis caused by5F-1and Ph-1; Microvessel density (MVD) effects of5F-1and Ph-1for tumor-bearing mouse tissues.The in vivo experimental results showed that the inhibitory effects of the Ph-1experimental groups were stronger than those of docetaxe positive groups, whereas5F-1experimental groups with medium dose (60mg/kg) had the same tumor growth inhibitoryrates as docetaxe with medium or low dose (40mg/kg，20mg/kg). Staining analysis assaysfound that5F-1and Ph-1could effectively induce apoptosis of MDA-MB-231humanbreast cancer cells. Microvessel density (MVD) results of5F-1and Ph-1fortumor-bearing mouse tissues showed that5F-1and Ph-1significantly reduced MVD andsubsequent formation of breast cancer (P<0.05), compared with the control groups.Section3. Studies on the antiviral activities of the nitroxyl radicals for CVB3.Developed the viral myocarditis model in vivo and in vitro with Coxsackie virus B3infecting primary cultured adult rat ventricular cardiomyocytes; Researched inhibitoryeffect of nitroxyl radicals L-1and L-2for CVB3in vitro and in vivo; Investigated theeffects of nitroxyl radicals L-1and L-2for scavenging ROS.Experiments concluded that the two radicals L-1and L-2could effectively scavengereactive oxygen species of the myocardial cells, improve survival rate of cells, and reduceviral titer. As a consequence, the mortality of mice infected with CVB3was reduced, therelease of cardiac enzymes AST, LDH, and CK of infected rats were effectively reduced,and the activities of SOD, MDA, CAT, and NO were regulated. Above all, these twonitroxyl radicals had a significant inhibitory effect on CVB3infection both in vitro and invivo, and could reduce acute and chronic myocardial necrosis caused by oxygen freeradicals to a certain degree.Section4. Studied the radioprotective activities in vitro for5nitroxyl radicals (L-3/D-3, Ph-1, and R-4/S-4), and3advantageous structures (L-3/D-3and Ph-1) were screenedout，where the L-3and D-3show better radioresistance than Ph-1. Radioproection ofthese advantageous structures (L-3and D-3) were studied in the integral animalexperiments. The results showed that L-3/D-3at high dose (7.5Gy γ) provedwell radioresistance effects.Experimental results showed that nitroxyl radicals L-3/D-3, Ph-1,and R-4/S-4had no cytotoxicity on C6cells of rat glioma when the drug doses were less than60μg/mL.Furthermore, L-3/D-3had better radioprotective effects for the radioresistance of C6cells.There was no significant difference between the L-3and D-3isomer at6.5Gy γradiant intensity in vitro, whereas at7.5Gy γ, L-3showed better radioprotective effectsthan the D-3isomer. Additionally, nitroxyl radicals’ radioprotection doses and the toxicdoses showed significant differences (P>0.05).