Design, Synthesis And Three Dimensional Quantitive Structure-Activity Relationship Of Novel Antifungal Compounds For Lanosterol 14a Demethylase Of Fungi

During the past two decades, the frequencies and type of life threathening fungal infection have increased dramatically among cancer patients, transplant recipients, patients with AIDS and patients receiving broad-spectrum antibiotic or parental nutrition. Azoles such as clotrimazole, ketoconazole, fluconazole and itraconazole are main antifungal drugs in clinic, which belongs to inhibitors of lanosterol 14a-demethylase (CYP51). Azole antifungal agents inhibit the CYP51 by a mechanism in which the heterocyclic nitrogen atom (N-3 of imidazole and N-4 of triazole) binds to the heme iron atom in the binding site of the enzyme. The resulting ergosterol depletion and the accumulation of precursor 14a methylated sterols interfere with the function of ergosterol as a membrane component. They disrupt the structure of the plasma membrane, leading to inhibition of fungal growth.CYP51 is a member of the cytochrome P450 superfamily and one of the key enzymes of sterol biosynthesis in different biological kingdoms, such as fungi, higher plants and mammals, with the same metabolic role. However, in fact, azole antifungal agents are generally toxic and are hampered in the treatment of deep-seated mycoses and life-threatening systemic infections because of their ability to coordinate with the heme of a lot of host cytochrome P450 enzymes, particularly mammalian CYP3A4. Cases of fatal hepatotoxicity have been reported. The azole ring has been demonstrated to be one of the most important pharmacophores for antifungal activity in extensive structure-activity studies, and both toxicity and activity of azole antifungal agents are mainly attributed to the coordination binding of the nitrogen atom of the azole ring to the iron atom of heme. So all of these findings urged us to search novel non-azole lead compounds with more structural specificity for the fungal enzyme to separate their activity from toxicity except for just only modified the structure of azoles.Because CYP51 represents an attractive target for antifungal therapy, Haitao Ji et al constructed the three-dimensional model of CYP51 from Candida albicans. Except for the site coordinating with the heme, the key regions in the active site ofCYP51 for ligand noncovalent binding can be divided into four subsites. The SI subsite was the hydrophilic hydrogen-bonding region. The S2 subsite was the hydrophobic region. The S3 subsite was the narrow hydrophobic cleft formed by the residues in the helix B-meanderl loop and the N terminus of helix I. The S4 subsite adjacent to the /?6-l/pi-4 sheet is another important hydrogen-bonding region in the active site.In this paper, on the base of the three-dimensional model of CYP51 from Candida albicans constucted in previous study, we designed de novo and synthesized a series lead compounds of N-alkylated 2-aminotetralins, which interacted with the amino acid residues without coordination binding with the prosthetic group heme in the active site of CYP51, avoiding the serious toxicity arising from the coordination binding with the prosthetic group heme, as found in azole antifungal agents. Research content of this paper:1. One hundred and nine novel tetralin compounds were designed and synthsized on the base of the three-dimensional model of CYP51, which interacted with the amino acid residues in the active site of CYP51. Their structures have been confirmed by 'H NMR, IR spectra and MS.2. A series of optical compounds were designed and synthsized by asymmetric synthesis in order to review the relationship of chirality with antifungal activities.3. These compounds in vitro antifungal activities were measured by means of the minimal inhibitory concentrations (MIC) using the serial dilution method in 96-well microtest plates. The MIC determination was performed according to the National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Test fungal strains were obtained from the ATCC or clinical isolates: Candida albicans, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, Trichophyton rubrum, Fonsecaea compacta, Aspergillus fumigatu, Microsporum gypseum and resistant Candida albicans . Fluconazol was taken as a control drug. The results showed that all of the lead molecules exhibited potent antifungal activities. Compared to fluconazole, A series of compounds exhibited a higher antifungal activity of Candida albicans, Aspergillus fumigatu and resistant Candida albicans.All of the results gave us the suggestion to further design and synthesize their derivatives.4. The test of the inhibition of CYP51 was carried out.5. Three-dimensional quantitive structure-activity ralationship(3D-QSAR) analyses with Comparative molecular field analysis(CoMFA) and Comparative molecular similarity indices analysis(CoMSIA). The lead molecules were aligned by rms Fit and a best grid was obtained. A maximum of Q2 and Optimal Number of Component (ONC) was obtained by Leave-One-Out PLS. The CoMFA and CoMSIA model was derived through none-cross-validation PLS. All of the values of Q2 are greater than 0.5, indicating that the models have a satisfactory predictive activity.Binding of lead compounds to the active site of CYP51 from Candida albicans of these compounds were performed. The mode of action of the lead compounds with the active site of CYP51 from Candida albicans is shown: The tetralin ring was loctated in the hydrophibic S2 subsite, which was not interaction with the heme iron atom. The 7-phenolic hydroxyl and methoxy groups of lead molecule formed H bonds with the residue Tyr69, Val509 of S4 subsite. At position 2 of the lead structure, long Hpophilic alkyl side chains were interacted to interact with the hydrophobic S3 subsit. No interactions were found between the lead molecule and the heme. The mode of action of lead compounds were represented, which is different from that of azoles.In a word, the affinity of the lead compound for CYP51 was mainly attributed to their nonbonded interaction with the apoprotein. 2-aminotetralins are a novel non-azole inhibitors of lanosterol 14o>demethylase of fungi. The present study affords the opportunity to develop novel antifungal agents with own intellectual property that specifically interact with the residues in the active site and avoid the serious toxicity arising from coordination binding with the heme of mammalian P450s. Innovation of this paper:1. A novel non-azole inhibitors of lanosterol 14a-demethylase of fungi wasdesigned and synthsized. It is a new type of the leading compounds which is different from azoles in the study of antifungal agents.2. A new idea was put out to design a novel non-azole compounds, which interacted with the amino acid residues in the active site of CYP51, avoiding binding with the prosthetic group heme.3. The mode of the action and the antifungal mechanism of the lead compounds were different from that of azoles.4. A series of optical compounds were designed and synthsized by asymmetric synthesis.5. The model of 3D-QSAR of the class of the compounds was represented first time.The work was supported by National Natural Science Foundation of China (Grant No. 30572257).