Design,Synthesis And Bioactive Studies Of Photo And Enzyme-Triggered Nitric Oxide Donors

Objective:Nitric Oxide（NO）is a short-lived free radical generated by nitric oxide synthase,which uses arginine as the substrate.NO plays important roles in many physiological and pathological processes and is the most widely studied signal molecule.Based on the above various biological effects of NO,important progress has been made to show the effects of NO on vascular relaxation,nerve signal transmission,immune system defense,cancer treatment,etc.However,studies on antibacterial and gastrointestinal diseases are relatively rare.The biosynthesis of NO is regulated by various factors,and its physiological function is closely related to its concentration,produce rate,and produce site.Although it has been shown that NO donors have the potential to treat many diseases,its clinical applications are limited because there is currently no effective way to achieve precise release of NO in temporal-spatial model,thus resulting in a series of toxic side effects.Two strategies using light and enzyme-controlled release can achieve precise drug delivery by controlling the position and intensity of exogenous light sources or the specific distribution of enzymes,and have attracted widespread attentions.Therefore,a class of light-controlled NO-hybrid drugs based on benzothienoazepine core were designed and synthesized in this dissertation.These NO-hybrids exhibit good antibacterial effects.This dissertation provides new methods and strategies for the development of new antibacterial agents.In addition,considering the ubiquitousα-glucosidase in the small intestine,a class ofα-glucosidase-responsive NO donors are designed and synthesized to achieve targeting release of NO in the small intestine,with the purpose of providing new basis for the treatment of gastrointestinal diseases.Methods:（1）Seven light-sensitive NO hybrids（NO-hybrids）were designed and synthesized based on benzothienoazepine,which was tagged with a nitroso group and further conjugated with quaternary ammoniums or phosphoniums.The photo-response,release kinetics,stability of these seven compounds were verified by UV-vis and fluorescence spectra.The fluorescence quantum yield were also studied.Griess reagent and electron paramagnetic resonance（EPR）spin-trapping method were used to verify the NO release upon photoirradiation.Minimum inhibitory（MIC） and bactericidal（MBC）concentrations were determined by the broth dilution method and agar plating method to evaluate the antibacterial activity of these compounds.The antibiofilm effects were evaluated against mature biofilm（48 h）.Scanning electron microscopy was used to study the changes in bacterial surface morphology.Finally,hemolytic assay on sheep red blood cells was conducted to evaluate the biocompatibility of these compounds.（2）The glucose group and the azolenium diolate（NONOate）were linked together through the self-immolative linkers（p-hydroxybenzyl alcohol）,which have the substituents of F,CH₃,NO₂,CH₃O on benzene ring.The synthesized NO donors showed different release rates in the presence ofα-glucosidase,and achieved targeting release of NO in the small intestine due to the wide and specific distribution ofα-glucosidase in the small intestine.The response of these compounds to the enzyme and the release kinetics were verified by UV-Vis spectroscopy.Griess method was used to detect the NO release amount.Results:（1）The results are consistent with our design concept.NO-hybrids have good photosensitivity.The results of UV experiments indicated that the release of NO follows pseudo-zero-order kinetic process,with the rates of 0.24mM min^-1,0.18mM min^-1,and 0.23mM min^-1 for 7a,7b and 7c respectively.The fluorescent experiments showed that NO-hybrids（7a-c）are essentially not fluorescent(Ф_7a=0.0022,Ф_7b=0.0033,Ф_7c=0.0013),while light-reduced NO release from 7a and 7b leads to fluorescent products 6a and 6b(Ф_6a=0.557,Ф_6b=0.564).And these compounds have good fluorescence stability,supported by negligible interference by other substances.It implies that the release of NO can also be detected by the fluorescent method.The fluorescent method indicates that the reaction rates as 0.23mM min^-1 and0.21mM min^-1 for 7a and 7b,which is consistent with the result of UV method.Griess reagent method and EPR method have been used to detect the NO release amount,showing about 40% of NO release from these NO-hybrids.EPR experiments confirmed that NO release is controlled by light.Antibacterial activity experiment indicated that NO-hybrid 7a with triphenylphosphine group and its NO-released product 6a have good effects against Gram-positive bacteria Staphylococcus aureus(ATCC12600^GFP),with its MIC and MBC as 30mg/m L and 60mg/m L.Anti-biofilm experiment indicated that 60mg/m L of 7a or 6a could significantly reduce the colony forming units（4-log CFUs）of Staphylococcus aureus after treatment for 8 hours.Once NO released by light,7a could further reduce CFUs,which is 2-log higher than that under no light or 6a under light.Scanning electron microscopy experiments show that 7a or 6a can deform or damage the bacterial cell membrane.Red blood cell hemolysis experiment shows that these compounds will only cause 1% red blood cell rupture at the concentrations between 300-600mg/m L,indicating that these compounds have good biocompatibility.（2）Compounds 4a-f have good enzyme responsiveness.The NO release of these compounds follows pseudo-first-order kinetic processes,with the rates of 0.019 min^-1,0.044 min^-1,0.038min^-1,0.017 min^-1,0.03 min^-1,and 0.013 min^-1 respectively.Griess reagent method indicate that the NO yield is between 80%-95% from these NO donors（4a-f）in the presence ofα-glucosidase.Conclusions:（1）A new type of light-triggered NO-hybrids were designed and synthesized,which have good photo-responsiveness.The NO release of these NO-hybrids follows pseudo-zero-order kinetic.The NO-hybrid（7a）with quaternary phosphoniums,its precursor（6a）,and NO all have good antibacterial activities.Therefore,NO-hybrid（7a）is such“two-in-one”hybrid,which could release active gas molecule NO and the conjugates of benzothienoazepine with quaternary phosphoniums,enabling potent activity against Gram-positive bacteria S.aureus biofilm.These studies could provide new strategies for the development of antibacterial agents.（2）The enzyme-triggered NO donors have good enzyme-responsiveness.The NO release of these compounds follows pseudo-first-order kinetic.The specific mechanism needs to be further studied,and a series of biological activity experiments will be carried out in the future.