Design,Synthesis,Biological Activity And Mechanism Of Small Molecular Compounds Of Fluconazole Resistant Candida Albicans

The situation of fungal resistance has become increasingly serious,especially Candida species,which have various degrees of resistance to various types of antifungal drugs,such as azole drugs,polyene drugs and echinocandins,and its resistance mechanism is complicated.The incidence of invasive fungal infections in the world has risen sharply.The growing fungal resistance is widely recognized as one of the leading causes of the increasing incidence and mortality of invasive fungal infections in immunodeficiency patients.It poses a serious threat to the health and safety of the general public.To build a healthy China,how to solve the problem of fungal resistance,it is urgent to develop new types of antifungal drug resistance drugs,and it is necessary to further explore the mechanism of resistance to opportunistic fungi in order to develop resistance.New fungal drugs provide new targets.Natural products such as berberine,forsythiaside A and baicalein have been found to be synergistic agents against Candida albicans..It has also been reported that calcineurin inhibitors,5?--reductase inhibitors and heat shock protein 90 inhibitors can also synergize with azole drugs against drug-resistant fungi.Although a large number of different types of compounds have been discovered and reported to have synergistic antifungal activity against drug-resistant fungi,few drugs have so far been marketed as potent synergists for resistant fungi,even in clinical studies.A large number of synergistic agents currently reported to have synergistic antifungal activity MIC are generally at 1.0?g/ml,and the synergist less than0.1?g/ml is not common.The synergist activity is not good enough,which may be one of the reasons.So far,natural products such as berberine,forsythiaside A,and baicalein have not formed a clear mechanism of action,but have not found the key biological macromolecules,which may be the second reason.In previous studies,we found that berberine had synergistic effect on FCZ against drug-resistant fungi,which had been transformed to get several kinds of compounds with simpler structure and activity comparable to berberine or slightly better than that of wavelet bases.On the basis of this study,we selected two small molecules of the best activity that found by our group as lead compound 1-1 and compound 1-2.A comprehensive structural modification was carried out to optimize the structure of the pepper base on the left side,the intermediate amide bond,the right aromatic ring and the soft chain.The activity data of the compound were obtained by the microdilution method and the chessboard microdilution method through the screening experiments in vitro.The data showed that there were 99compounds with synergistic resistance to Candida albicans.With 8?g/ml fluconazole,there are10 compounds such as F2,F6,F8,F13,F14,F21,G8,G9,G10 and G15,these MIC₈₀is less than 0.1?g/ml.F2,F6 and F8 compounds have be further confirmed the reliability of the activity data by checkerboard microdilution method.The structure-activity relationship of these compounds showed that piperonylic part of lead compound 1-1 is play an important fuction of biological activity,the amide bond is olso an essential part,the amide bond which is inversed amide bond can slightly improve its biological activity,biphenyl can further improve its biological activity,thiophene ring can remain quite active,in addition soft chain length of5-6 carbon was best.Therefore,we chose the best active compound F6?CZ-66?to study the mechanism of its resistance to Candida albicans,and the single use of FLC and F5?CZ-65?in the ROS test of resistant strain 103 could not induce the increase of ROS,F5?CZ-65?and FLC combined ROS increased about 3 times,F6?CZ-66?increased ROS alone about 10 times,F6?CZ-66?and FLC combined ROS increased about 26 times.The results showed that compound F6?CZ-66?alone or in combination with FLC significantly increased ROS levels,whereas compound F5?CZ-65?with similar structure did not increase ROS levels when used alone.In the rhodamine efflux experiment of resistant strain 103,rhodamine 6G efflux was enhanced after addition of glucose?2 mM?and increased over time.There was no significant difference between the efflux of rhodamine 6G and the control group in each drug group,indicating that compounds F5?CZ-65?,F6?CZ-66?,FLC,and combination drugs had no effect on the fluorescence protein elution of drug-resistant bacteria 103.CT-LINK reverse molecular docking targets were searched through the PDB database.After analysis of possible targets obtained in CT-LINK,only Mitogen-activated protein kinases,NADH-ubiquinone oxidoreductase chain 1 and Mothers against decapentaplegic homolog 3 were found in fungi.There are reports of existence.Combined with the relevant references already reported,this article speculates that F6?CZ-66?may act on the ROS-MAPK pathway.In addition,this subject selected the HSP90 inhibitor BIIB021 with a relatively clear target as lead compound2-1.The purine ring on the 2nd,6th and 9th place were respectively structurally modified,when we retain the lead compound 9-position substituents,in the 2nd and 6th for simple transformation,2 and 6 were respectively chlorine or amino.When used as a substituent,the effect on the activity of the compound is not significant.For example,the MIC₈₀ of compounds H1-H3 is between 4.0-8.0?g/ml.When the 9-position aromatic ring was replaced with a fat ring,the activity was slightly improved,and the six-membered fat ring was the best.When the aromatic ring was introduced at the 2-position,the activity disappeared.Therefore,retaining the small atomic group structure such as fluorine or amino group at the 2-position is necessary to retain the activity of the compound.Compounds H12 and H19 were the two compounds with the best activity obtained from the modification of lead compound 2-1,although their activity did not exceed that of compound F6 obtained from the modification of lead compounds 1-1 and 1-2,but their activity was small.Strontium is equivalent,and FICI is better than berberine.As a class of molecules with relatively clear targets,it is worth further structural transformation and optimization.