| Bioorthogonal reaction is a type of chemical reaction that can be carried out in a living system.This reaction has extremely high biocompatibility and will not conflict or affect other substances in the living system.People can react predictably in the biological environment,and can easily use one or more of the bioorthogonal functional groups to interact with each other according to their demands.At present,we have summarized two major types of bioorthogonal reactions in the pharmaceutical applications:prodrug release strategies(decaging reaction)and in-situ compound synthesis strategies in vivo(coupling reaction).Prodrug design is an important field of bioorthogonal reaction in drug research and development,in which the "click and release" reaction strategy can solve the problems of some drugs usually encountered in the process of drug development,such as membrane permeability,toxicity,targeting or selectivity.The first part of our work is to explore new reaction types and pharmaceutical applications around bioorthogonal click and release reactions.At present,only a few bioorthogonal reaction types have rapid reaction kinetics,which is an important prerequisite for application in vivo.In addition,although the reaction rate between trans-cyclooctene and tetrazine is fast,some tetrazine derivatives are decomposed by mercaptan induction and are quickly cleared in vivo,with a short half-life.Trans-cyclooctene is also mediated by proteins containing transition metals and isomerization.Therefore,we hope to develop a new kind of bioorthogonal reaction with fast reaction rate,and design the N-oxide precursor structure of ether bond or carbamate bond.It can be clicked with the above precursor structure by B2pin2 and release the corresponding drugs.These types of reactions not only have rapid reaction kinetics and drug release rates,N-oxide structure can also improve the water solubility of compounds,and also has a good effect in cells,which can realize the controllable release and imaging of intracellular drugs.In the first category,N,N-dimethyl-4-(methyl oxide)phenylamine oxide synthesized by using 7-hydroxycoumarin N-oxide precursor(Compound 102)as template substrate to react with B2pin2.The second-order reaction rate constant is k2=7.14×102 M-1s-1,which is faster than most reaction types.In the second category,we initially explored N-dimethyl-4-(formamide)methyl)aniline oxide conversion experiments at 50 μM concentrations in PBS.Found that most of the 4-methyl-7-aminocoumarin can be released within an hour,and we found that the prodrugs at different DMSO ratios,the less product is generated with higher DMSO ratio at the same time.For further application of this reaction structure type to vivo,camptothecin tumor chemotherapeutic agents with poor water solubility and high cytotoxicity were selected as the original drug to explore the in vitro and intracellular effects of the 7-ethyl-10-hydroxy camptothecin(SN-38)as a tool drug.The conversion of fluorescence from blue to yellow can be observed by clicking and releasing experiments in vitro PBS of 7-ethyl-10-hydroxy camptothecin N-oxide prodrug(103),and its second-order reaction rate constant is k2=1.17×103 M-1s-1.Fast reaction rate and about 90%in vitro release rate ensure that we can further verify the release and therapeutic effect of 103 in cells.Through biological experiments on five kinds of cells,such as A549,HeLa,MCF-7,PC3 and KYSE520,it was found that the toxicity of 103 and B2pin2 to cells was smaller than that of SN-38,and the cell activity of the reaction group was between the original drug and the prodrug.The experimental results show that the prodrug strategy is feasible.Moreover,the results of intracellular fluorescence imaging showed that the strategy could be used for intracellular imaging,and the prodrug gradually released a small amount of prodrug over time,which explained the toxicity of prodrug in cells.On the basis of intracellular imaging experiments,further experiments showed that the prodrug 103 can be injected into mice through the tail vein and activated to release SN-38,and achieve fluorescence imaging of tumor cells in vivo.Another work mainly focuses on the application of palladium catalyzed bioorthogonal coupling of drugs in vivo.In situ synthetic drugs can reduce the damage of toxic drugs to the living system to a certain extent,improve membrane permeability,and even play a better therapeutic effect in vivo.cRGDfE-PdNP(composed of palladium nanoparticle functionalized fluorescent microspheres decorated with the cyclic-RGD cancer targeting functionality)are also catalytic,imaging and targeted,as well as excellent membrane permeability,which can be used as a tool for further application and exploration.PD173074 is a FGFR1 ATP competitive inhibitor with good cellular activity,but its own toxicity has been withdrawn from clinical phase II studies.We split the PD173074 to obtain iodinated or brominated fragments,respectively,and cRGDfE-PdNP catalytic coupling reaction with compound 124(boronate compound fragment)in PBS.The results showed that the coupling reaction of compound 123(iodinated compound fragment)was better,more target products can be produced.In summary,we have designed a new "click and release" strategy.The bioorthogonal prodrug design uses a click reaction pair between N-oxide and B2pin2.It’s feasibility has been in vitro,in cells,and it was verified in vivo.Fast reaction kinetics(～103 M-1s-1)and high release rate(>90%)can be widely used in other fields.In addition,in order to further expand the scope of the application of cRGDfE-PdNP catalyst in the in-situ synthesis of small molecule drugs,the feasibility of the catalyst in the in-situ synthesis of PD173074 was verified. |
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