Structure-Based Design And Synthesis Of The Lead Compounds Of Novel D1 Protease Inhibitors

D1 protease(CtpA),a kind of C-terminal processing protease,widely exists in the chloroplast of green plants.It cleaves 8～16 amino acid residues of the precursor D1 protein(pD1) to form mature D1 protein(mD1),and this process is necessary for assembling the manganese cluster in the oxygen-evolving center of PSâ…¡.Because of its necessary for the photosynthetic reaction in plants, low amount,high homology in organisms,D1 protease has been predicted to be an ideal herbicidal target.To meet the need of high-efficient and environment-friendly herbicides in modern agriculture and solve the problem of herbicidal resistance,it will possess great significance for rational design and synthesis of hit compounds targeting D1 protease.At present there is only one report concerning the D1 protease inhibitors,and there are no commercial inhibitors targeting D1 protease.In our previous work,the homology model of spinach D1 protease was built on the basis of the crystal structure of D1 protease from Scenedesmus obliquus. With the three-dimension structure of this spinach D1 protease model,virtual screening was performed by docking of small molecules from various compound libraries into the active site of the Dlprotease model and subsequent evaluating of the predicted bioactivities with scoring functions. Thus several types of hit compounds were identified.In this thesis,four series of compounds were rationally designed on the basis of the hit compounds for chemical synthesis,and total 90 compounds were synthesized.The molecular structures of all the target compounds were verified with ¹HNMR and MS,and some were also confirmed with ¹³CNMR and elemental analysis.With X-ray crystallography,the complex structures of isoxazolythiazolyl piperidine derivatives were determined. The herbicidal activities of the target compounds were tested and showed moderate and good herbicidal efficacies.In order to validate the hit,some compounds were also tested their in vitro inhibition effects against the native or recombinant D1 protease with HPLC and SPR methodologies.This dissertation may be summarized as followings.1.This dissertation first made an investigation of the strategy for the structure-based design of pesticide.A systematic review was made on the research of D1 protease as the herbicidal target and progress on the inhibitors targeting D1 protease.The research strategies and plans were proposed for this dissertation.2.By taking use of the isoxazolecarboxylic acid formed via 1,3-dipolar cycloaddition followed by oxidation and the subsequent amidation withε-lysinylurea units,totally 21 compounds(ILU 1～21) were synthesized.The herbicidal efficacies of these compounds were tested against the growth rates of two model plants rape and barnyard grass.The structure-activity relationship was preliminarily discussed.Among these compounds,when the urea moieties were same,the compound with 2,4-dichloro substituent on the phenyl of isoxazole had better herbicidal activity.While when the isoxazole moieties had the same substituent,the urea moiety containing morpholine possessed better herbicidal efficacies.Changing the substituent of urea moiety will influence the biological activity of compounds to a great extent.3.Totally 21 compounds(ITH 1～14),namely isoxazolylthiazolecarbohydrazine derivatives, were rational designed and synthesized.The synthesis was accomplished via the subsequent formation of isoxazole ring,thiazole ring,and carbohydrazine.The isoxazole moieties were formed via the condensation of P-diketone ester and hydroxylamine,while the isoxazolylthiazolecarboxylic esters were formed via amidation and thiolation with the Lawesson's reagent and subsequent condensation with 2-chloroacetoacetic ester(Hansztch reaction).The isoxazolylthiazolecarboxylic esters were subjected to react with hydrazines followed by acylation or aminocarbohydrazine,and finally the isoxazolylthiazolecarbohydrazine derivatives were obtained.Biological test showed that the title compounds with urea moieties possessed better activities.Some compounds show good biological selectivity.However,since the water solubilities of these compounds were relatively low and the redesign is necessary for these compounds to improve their bioavailability.4.With the use of 1,3-dipolar cycloaddition and Hansztch reaction as like as the procedures mentioned above,the 4-isoxazolylthiazolylpiperidines as intermediates were obtained.The piperidine moieties in these intermediates were subjected to react with acyl chlorides,isocyantes or isothiocyanate to afford two classes of target compounds,totally 43 compounds(ITP 1～43).The second class of compounds possesses an isoxazolocyclohexanothiazole rigid moiety,which was expected to endow these compounds with higher bioactivities,and the bioassays demonstrated this expectation.The structure-activity relationship shows that the ortho- and para- position of phenyl on the isoxazole ring are important and the fluorine atom located at the ortho-position will promote the bioactivity of compounds;The p-F phenyl possessing the urea motiey alsohas the same effect.The isoxazole and piperidine moieties should be important units of the compounds,and the rigid cyclohexanyl connecting the isoxazole and thiazole will increase the compounds inhibiting efficiency to the growth of rape.5.A series of 5-aminoimidazoline-2,4-dione derivatives were rationally designed and synthesized. The synthesis was accomplished via the coupling of triones with amine,followed by reduction of the imine group and acylations with acyl chlorides.Thus totally twelve 5-aminoimidazoline-dione derivatives(H1～12) were obtained.The hydantoin with N-acyl aryl group had the better biological activities than N-aryl substituented compounds.The substitutents of 5-NH will influence the herbicidal activities of compounds.In order to get higher bioactivity of compounds the 3th position of the ring and the 5-NH will be important positions to vary different substituents.6.On the basis of our previous work for the isolation,purification and expression of spinach D1 protease,the isoxazolylthiazolyl piperidine carboxamides,ITP 16,ITP 21,ITP 22,ITP 26,ITP 29, ITP 42,ITP 43,were measured their in vitro inhibition efficacies against D1 protease by the HPLC method.One of these compounds,ITP 21,was demonstrated to possess an inhibitor constant(K_i) 1.3μM.The result showed the compound possessed effective inhibition to CtpA with a competitive inhibition.On the other hand,the inhibiting efficacy ratio of the compound ITP 21 was tested with the competitive strategy by surface plasmon resonance technology,and it was shown that this compound possessed an in vitro inhibition efficacy 47%at the concentration of 0.1 mM.All the results from the in vivo and in vitro tests show that the compounds containing isoxazolylthiazolyl piperidine carboxamides may be the potential compounds targeting D1 protease directly.