Design,Synthesis And Preliminary Antifungul Activity Evaluation Of Novel CYP51 Small Molecule Inhibitors

Sterol 14α-demethylase（CYP51）,a water-soluble protein lacking membrane binding in the N-terminal domain,is located in the endoplasmic reticulum and mitochondrial intima of eukaryotes and is involved in the core metabolic process of various endogenous substances and exogenous biological agents.CYP51 is an indispensable enzyme in the life process of cytochrome P450 enzyme eukaryote growth and reproduction.Reducing the level of natural CYP51 in lower eukaryotes can reduce the survival and reproduction of organisms,leading to cell death.The situation can be reversed by adding the amount of CYP51.At the same time,CYP51participates in the synthesis of sterols in the form of oxygenation in vivo,and sterol is a precursor of synthetic bioactive molecules,which is very important for the construction of cell membranes.The loss of enzyme activity can be directly lead to ergosterol synthetic blocked,cell membrane and the change of membrane permeability,sexual dysfunction,and eventually killing bacteria and fungi and microbial cells.Therefore,CYP51 is often used as the main target of antifungal agents to treat infectious diseases caused by microbial pathogens.In recent years,the abuse of broad-spectrum antibiotics,the extensive use of immunosuppressive agents in clinical practice,the extensive development of traumatic diagnosis and treatment measures,and the occurrence of immunodeficiency diseases have led to the emergence of infectious diseases in a state of leaping rise.At present,the extensive use of various synthetic antifungal drugs leads to the increasingly serious cross-resistance of microbial strains,and targeted antifungal drugs can act on the active sites of biological enzymes in the fungal body,avoiding the phenomenon of bacterial resistance and at the same time giving play to the antifungal activity.Therefore,we need to focus our drug development efforts on targeting small molecule inhibitors.By reviewing the literature on CYP51 small molecule inhibitors,our group found that the 1,2,4-triazole core group is an excellent chemical structure nucleus,including fluconazole,letrozole,voriconazole,etc.The commonly used typical CYP51 target antifungal drugs all have a 1,2,4-triazole structure.It can be seen that the 1,2,4-triazole core structure plays a considerable role in the antifungal activity of the CYP51 small molecule inhibitors.The main research content of this paper includes:1.Design of target compounds 6a-qIn the context of the chemical structure of CYP51 small molecule inhibitors and the crystal structure of inhibitors and protein complexes,4-（2-chlorophenyl）-5-（3,4-dichlorophenyl）-1,2,4-triazole-3-thione was designed and synthesized.The compound 6h was found that it has good antifungal activity,and its binding affinity to the active site of CYP51 receptor protein（PDB:6AY4）was examined by molecular docking experiments.The nitrogen atom of the mother nucleus and the target protein active site iron porphyrin center iron to form stable chelating key,while the chlorine atoms on the side chains of the two benzene rings play an important role in the formation of hydrogen bonds with amino acid residues during the binding process.It is the tight binding of the inhibitor to the target protein that verifies the compound 6h excellent antifungal activity.On the basis of the above studies and based on the principle of pharmacophore splicing,this paper expects to introduce substituted phenylpyridine hydroxyl and trifluoromethyl phenyl and other pharmacodynamic groups into the 5-site on the premise that 4-position of1,2,4-triazole ring is the introduction of halophenyl,and design and synthesize a series of derivatives with 1,2,4-triazol-3-thione as the parent nucleus.2.Synthesis and structural confirmation of target compounds 6a-qIn this paper,a series of 1,2,4-triazole-3-thione compounds were synthesized by using four steps of esterification,hydrazinolysis,condensation and cyclization step synthesis process using benzoic acid and phenyl isothiocyanate as raw materials.It was confirmed structurally correct by mass spectrometry（MS）,nuclear magnetic resonance spectroscopy（¹H-NMR）and nuclear magnetic resonance(¹³C-NMR)to synthesize 17 4-halophenyl-5-substituents.-1,2,4-triazole-3-thione compounds.3.Screening of in vitro antifungal activity of target compounds 6a-qIn this experiment,the agar diffusion method was used to preliminarily determine the antifungal activity of 17 target compounds against Candida albicans,Spore filamentous fungi and Fusarium oxysporum.The results showed that all compounds had a certain degree of antifungal activity.Further determination of compounds under test determined by the minimum inhibition concentration（MIC）values in the determination results.In the measurement results,the MIC values of compound 6h for the three strains were 12.5,12.5,6.25 g/mL,respectively,showing outstanding bacteriostatic activity,and the antifungal ability was comparable to the control drug ketoconazole.Compounds 6a,6d,6m,and 6p exhibited good inhibitory activities against the six strains,respectively,and compounds 6e,6f,6g,and 6k exhibited some antifungal activity against some strains.According to the preliminary analysis of the structure-activity relationship of the compounds,the ortho-halophenyl group at the N-4 position of the 1,2,4-triazole ring,the substituted benzyl group at the 5-position,and the metahalophenyl group and the pyridyl group all enhance the bacteriostatic strength of the compounds.