Objective:
The incidence of invasive fungal infections ( IFIs ) is increasing in the past years. However due to the limitations of azole drug resistance in existing drugs, nephrotoxicity of polyenes, and oral toxicity of echinocandins, IFIs caused increasing number of deaths and seriously threatens human health. Consequently, the development of novel antifungal and drug-resistant fungus medications is essential. In this thesis, 2-phenylindole and 4,5-dihydroisoxazole were used as lead compounds to design and synthesize novel fungal efflux pump inhibitors for the development of novel anti-fungal drugs for clinical application.
Methods:
1. The modification of 2-phenylindole by introducing halogen, alkyl, nitro, methoxy and other groups to form a series of derivatives, and oxidative catalysis to form dimers to obtain indoline derivatives. The 4,5-dihydroisoxazole was modified after the R3 substituent was linked to alkyl, aryl, five-membered heterocyclic groups by carbonyl and the R4 substituent was modified by introducing aryl, long-chain alkane, halogen and other groups to obtain a series of derivatives. 1H-NMR, 13C-NMR, and HRMS were used to characterize the compounds' structures;
2. The antifungal activity and mechanism of indole derivatives were tested by conventional methods in vivo and in vitro;
3. Microdilution method was used to evaluate the inhibitory effect of the isoxazole derivatives against six pathogenic microorganism and drug-resistant Candida albicans;
4. The activity of isoxazole derivatives to reverse drug resistance gainst azoles was tested using a checkerboard microdilution method.
Results:
A total of 36 indole derivatives and 16 isoxazole derivatives were synthesized and their chemical structures were determined. Amongst, 4 indoline compounds and 5 isoxazole derivatives were new compounds. The results of erythrocyte hemolysis test showed that 8 compounds had hemolytic toxicity, and the rest had good biosafety.
The activity studies showed that most of the derivatives showed good inhibitory activity against six pathogenic microorganism; Amongst, compounds 3d, 3o, and 3r showed excellent inhibitory activity against drug-resistant Candida albicans with the MICs 8 times stronger than FLC ( MIC80 = 32 μg/mL ). The fungicidal tests showed that compound 3o could kill Candida albicans. According to experiments on mitochondrial membrane potential and reactive oxygen species (ROS), chemical 3o might cause mitochondrial damage and ROS generation, and this would have killed the strain. Additional research indicated that compound 3o could effectively block the development of fungal biofilm and hinder morphological transformation. Compounds 4o, 8f and 8j were found to have excellent synergistic antifungal activity in combination with fluconazole (FICI < 0.5).
The efflux tests of rhodamine 123 showed that compound 4o could inhibit the activity of efflux pump. The findings indicate that compound 4o successfully reduced the expression of efflux pump genes (MDR1、CDR1、CDR2). In the Galleria mellonella larvae infection models, compound 4o could significantly prolong the survival time of Galleria mellonella larvae.
Conclusion:
In this work, indole and isoxazole derivatives were designed and synthesized. A total of 52 derivatives were synthesized, of which 9 were new compounds. Compounds 3d, 3o and 3r (precursor compounds for new drug development), compounds 4o and 8j (alternative drugs for reversing drug resistance ) have in-depth development and research value. The overall structure-activity relationship found that the halogen substitution on the indole ring and the alkyl substitution on the aromatic ring in the indole compounds can enhance the antifungal effect of the compounds; In isoxazole derivatives, the R3 substituent with the halogen aromatic ring and halogen substituent in R4 can enhance the activity against sensitive strains. The results of this thesis would provide new basis for the future tudy on agents against drug-resistant Candida albicans.
谷歌
火狐
