Design And Synthesis Of Spirobenzoxazinepiperidione Derivatives As Chitin Synthase Inhibitors

Fungal infections are a relatively common problem in human health field and can be classified as superficial infections and life-threatening invasive fungal infections.Invasive fungal infections,which have high mortality and morbidity,threaten the lives of millions of people worldwide each year.Current used antifungal drugs have high toxicity,tend to develop drug resistance,a narrow fungal spectrum and so on,resulting in an urgent clinical need for new antifungal drugs to deal with the growing problem of fungal infections.Chitin is an essential component of the fungal cell wall.Blocking the synthesis of chitin leads a change in cellular osmotic stability,abnormal morphology and growth arrest.The synthesis of chitin requires multiple chitin synthases.Since there is no chitin biosynthesis in mammaliane cells,chitin synthases are considered to be superior targets for the development of novel antifungal drugs.Polyoxin B is used as a class of excellent chitin synthase inhibitors in agricultural sterilization,however,there is still a lack of drugs targeting chitin synthase for clinical application.At present,the exploration of chitin synthase inhibitors has moved from polyoxin analogs and their structural modifications to other classes of compounds in order to discover new lead compounds,including extracting from natural products and designing new compounds based on the Structure-based Drug Design（SBDD）,followed by the relevant activity screening.Benzoxazinones are significant azacyclic compounds,a pharmaceutically parent structure present abundantly in a variety of biologically active molecules.Based on the current progress of research on chitin synthase inhibitors,several classes of compounds that used spirobenzoxazinepiperidione as scaffold were designed,and examined for their chitin synthase inhibitory activity,in vitro antifungal activity,and antibacterial activity.Depending on the results of the above activity assays,the highly active compounds were chosen for sorbitol protection assays,substance combination assays to verify the concept of design.Three drug-resistant fungi strains were cultured and the high-activity compounds were checked for their activity against the drug-resistant fungi.The main work is summarized as follows:1.Synthesis of 6-methoxyspiro[benzo[d][1,3]oxazine-4,4’-piperidin]-2（1H）-one:p-methoxyaniline was used as raw material,and the key intermediate 6-methoxyspiro[benzo[d][1,3]oxazine-4,4’-piperidin-2-one was obtained by bromination,amino protection,lithium-halogen exchange,electrophilic addition of carbonyl groups,ester exchange,and finally removing the protecting group.2.Four substituted anilines were used as raw materials and condensed with four fragments of maleic anhydride,succinic anhydride,micellaric acid and tartaric acid,respectively,and the resulting intermediates were further condensed with6-methoxyspiro[benzo[d][1,3]oxazine-4,4’-piperidin]-2（1H）-one to give the target compounds 14a-d,17a-d,20a-d and 23a-d.For the mentioned target compounds experiments were carried out for evaluation of the chitin synthase inhibitory activity and antifungal activity.The results showed that the CHS inhibitory activity of the maleic anhydride compounds 17a-d were significantly better than those of the tartaric acid compounds 14a-d,succinic anhydride compounds 20a-d and micellaric acid compounds 23a-d.The IC₅₀ values of the compounds 17a-d were between 0.14 to 0.37m M,and the IC₅₀ value of compound 17a is the best(IC₅₀=0.14 m M).In the antifungal activity assay,compound 17a exhibited high antifungal activity against all four fungi,with the MIC values less than that of Polyoxin B.Similarly,compounds 17b-d also showed comparable fungal inhibitory activity to that of the reference drug Polyoxin B against four fungi.While the remaining compounds 14a-d,20a-d and 23a-d showed low inhibitory activity against the four fungi.3.The optimal linking fragment was obtained as maleic anhydride according to the above experiments.Twenty aromatic amines with different substituents were further selected to connect maleic anhydride,then condensed with intermediate6-methoxyspiro[benzo[d][1,3]oxazine-4,4’-piperidine]-2（1H）-one to giveα,β-unsaturated ketones 17e-x.4.The chitin synthase inhibitory activity and in vitro antifungal activity of compounds 17e-x have been tested,and the results showed that most of theα,β-unsaturated ketones showed medium or higher inhibition activity against chitin synthase.Among them,compounds 17h,17q and 17v showed comparable chitin synthase inhibitory activities with IC₅₀ below 0.12 m M.In an in vitro antifungal assay,the activity of compound 17j against Candida albicans was comparable to that of fluconazole against Candida albicans（MIC=16 ug/m L）.The antifungal activity of compounds 17q and 17v with strong chitin synthase inhibitory activity against the four fungi are equivalent to the reference drug Polyoxin B.The MIC values of compound17q against both Candida albicans and Aspergillus flavus were 64 ug/m L,which is the same as reference drug Polyoxin B.The MIC values of compound 17v against Aspergillus fumigatus,Aspergillus flavus and Cryptococcus neoformans were32ug/m L,64ug/m L and 64ug/m L,respectively,and the antifungal activity was the same as Polyoxin B.5.Sorbitol protection experiments:Sorbitol belongs to a class of permeability stabilizers that can maintain the survival of fungal cells with broken cell walls.Highly active compounds 17a,17q and 17v were selected and tested for their minimum inhibitory concentrations against fungi in sorbitol medium at permanent concentrations.The results revealed that the MIC values of all three compounds against fungi in sorbitol medium at constant concentrations were 8-fold higher than the values of the results without sorbitol,with MIC values reaching 256 ug/m L.It was observed that the inhibitory effects of the three compounds were achieved by causing the fungal cell wall to break,further confirming that the antifungal targets of compounds 17a,17q and 17v were on the fungal cell wall.6.Six compounds with good antifungal activity（17a,17b,17d,17j,17v,17w）were selected for drug combination tests with fluconazole,respectively.The results showed that the combination of compounds 17a,17d,17j and 17w with Fluconazole produced a synergistic effect and inhibited the growth of Candida albicans,Aspergillus fumigatus and Cryptococcus neoformans more effectively,indicating that these compounds were not acting on the same target as fluconazole,further confirming that the above compounds have different targets from Fluconazole.The results showed that the target of these compounds is defferent from the target of Fluconazole.The combination with Polymyxin B is being tested.7.Culture and treatment of drug-resistant fungi:Two drugs,Fluconazole and Micafungin,were chosen to culture Fluconazole-resistant Candida albicans,Micafungin-resistant Candida albicans and Fluconazole-resistant Cryptococcus neoformans,respectively.Compounds 17a,17q and 17w with high activity against Candida albicans and Cryptococcus neoformans were screened,and their minimum inhibitory concentrations against the three drug-resistant fungi were checked.The results showed that the MIC values of compounds 17a,17q and 17w did not change against the resistant fungi compared with those of the common non-resistant fungi.Combined with the resistance mechanisms of the three resistant fungi,it was also further verified that the inhibition targets of the compounds were not on the cell membrane ergosterol（Fluconazole target）and cell wallβ-1,3-glucan（Micafungin target）,which belonged to a new class of targets.In summary,88 compounds were synthesized in this paper,including 42unreported compounds and 36 target compounds.The target molecular structures were determined by modern spectroscopic means ¹H NMR,¹³C NMR,HRMS.Five types of activity assays were completed:chitin synthase inhibitory activity assay,in vitro antifungal and antibacterial activity assay,sorbitol protection assay,drugcombination assay and culturing drug-resistant fungi and testing their activity.It was demonstrated that the antifungal mechanism of the synthesized compounds was achieved by inhibiting the activity of chitin synthase on the cell wall and also showed better inhibition against Fluconazole or micafungin-resistant fungi.