Computer-Aided Design And Synthesis Of Novel Antifungal Agents

During the past two decades, the life threatening infections caused by pathogenic fungi are becoming increasingly common, especially in those individuals with immunocompromised hosts, such as patients undergoing anticancer chemotherapy or organ transplants and patients with AIDS. Moreover, dermatomycoses, such as toenails and tinea pedis, are among the most widespread human superficial and cutaneous fungal infections. However, the current antifungal therapy can suffer from drug related toxicity, severer drug resistance, non-optimal pharmacokinetics and serious drug-drug interactions. Therefore, there is an emergent need to develop novel antifungal drugs with higher efficiency, broader-spectrum and lower toxicity. Lanosterol 14a-demethylase (P45014DM, CYP51) is the primary target of azole antifungal agents and it is of great importance to know the fungal CYP51 were built by homology modeling, the binding mode of the substrate and azole antifungal agents with CYP51 were explored by molecular docking, and important regions and residues in the active site were identified by MCSS and ET analysis. On the basis of the results from molecular modeling, a series of novel azole antifungal agents were designed and synthesized and most of them showed good antifungal activity with broad spectrum.I. Homology modeling of fungal CYP51s and the binding siteanalysis.1. Homology Modeling of Lanosterol 14α-Demethylase of Candida albicans and Aspergillus fumigatus and Insights into the Enzyme-Substrate InteractionsThe crystal structure of 14α-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) provides a good template for modeling the three dimensional structure of lanosterol 14α-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14α-demethylase from Candida albicans (CACYP51) and Aspergillus fumigatus (AFCYP51) were built on the basis of the crystal coordinates of MT14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandranplots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P45014DM. The overall structures of the resulting CACYP51 model and AFCYP51 model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues, the residues lining the substrate access channel, and residues in active site were identified from the model. To explore the binding mode of the substrate with the two models. 24(28)-methylene-24,25-dihydrolanosterol was docked into the active site of the two models and hydrophobic interaction and hydrogen-bonding were found to play an important role in substrate recognition and orientation. These results provided a basis for experiments to probe structure-function relationships in the P45014DM. Although CACYP51 and AFCYP51 shared similar core structural character, the active site of the two models were quite different, thus allowing the rational design of specific inhibitors to the target enzyme and the discovery of novel antifungal agents with broad spectrum. 2. MCSS functional maps for the active site of CACYP51The putative binding site in the CACYP51 was searched and identified by Insightll / Binding site analysis. To explore the key regions in the active site that are important for ligand binding, the MCSS program was employed to calculate energetically favorable position and the orientation of given functional groups in the active site. In general, the active site of CACYP51 can be divided into four subsites: SI subsite represented the core hydrophobic area, S2 subsite represented the narrow and hydrophobic cleft, S3 subsite represented the hydrophilic hydrogen bond binding site, and S4 subsite represented the hydrophobic hydrogen bond binding site. His310. Thr311 and Ser378 were three important hydrogen bonding residues in the active site. The results from MCSS calculation can provide important information for the design of novel antifungal agents.II. Design, synthesis and molecular modeling of novel azoleantifungal agents.1. Design and synthesis of novel azole antifungal agentsForty 1 -(1H-1,2,4-Triazolyl)-2-(2.4-Diflurophenyl)-3-(4-substituted-1 -piperazinyl) -2-propanols were designed and synthesized on basis of the properties of the active site of CACYP51. All the target compounds were reported firstly and their structures were determined by 'HNMR and IR. In vitro antifungal activity assay revealed that most of the compounds showed strong antifungal activity against eight common pathogenic fungi and the activities against deep fungi were higher than that against superficial fungi. Compounds Al, A5, A8, Cl, CIO, C12 and C17 had excellent potency against a broad range of fungal pathogen including Aspergillus fumigatus and further biological evaluation of these compounds is in progress.2. Optimization of the synthesis of key intermediate of azoles by orthogonal experimental design1 -[2-(2,4-Difluorophenyl)-2,3-Epoxypropyl]-l//-l ,2,4-Triazole Methanesulphonate is the key synthetic intermediate of the triazole antifungal agents and its synthetic process was optimized by orthogonal experimental design. Reaction temperate, time, solvent and the amount of NaOH were considered in the experiment. The yield of the new process was improved from 21.84% to 62.34%. The new method has several advantages such as cheap reactants, facile reaction condition, convenient operation and high yield.3. 3D-QSAR study of a series of novel triazole antifungal compounds Comparative molecular field analysis (CoMFA) and comparative molecularsimilarity indices analysis (CoMSIA) three dimensional structure-activity relationship (3D-QSAR) studies were conducted on a series of novel triazole antifungal compounds. For the CoMFA study, two different pharmacophoric conformations were compared and conformation generated from our previous homologous enzyme-inhibitor docking model got better result. Variation of grid spacing was used during the optimization of the CoMFA model. For the CoMSIA study, the influence ofthe combination of different field types was evaluated and the best combination was considered to be of steric, electrostatic, hydrophobic and H-bonding acceptor fields. Variation of grid spacing and attenuation factor was used to get the best CoMSIA model. The resulting CoMFA and CoMSIA models had a cross validated coeffiecient q2 of 0.718 and 0.655 respectively, which showed strong predictive ability on both test set and training set. The 3D contour maps of CoMFA and CoMSIA provided smooth and interpretable explanation of the structure-antifungal activity relationship for the compounds. Analysis of CoMFA and CoMSIA contour plots revealed that the substitutions on para-position of the phenyl were more important for the antifungal activity than that on ortho-position and meta-position. For the substitutions on the para-position, the steric and hydrophobic groups or the electro-rich groups as hydrogen bond acceptors were favored. For the substitutions on the ortho-position, hydrophobic and electro-rich groups (e.g. halogens) were favored. The substitutions on the meta-position of the phenyl were not important for the antifungal activity and it was suitable to leave this position unsubstituted. The results from the 3D-QSAR analysis will guide the design of novel antifungal compounds with relatively higher activity.4. Flexible molecular docking study of a series of novel triazole antifungal compoundsTo explore the binding mode of our synthesized azole antifungal compounds with the active site of CACYP51 and guide our further structural optimization, flexible molecular docking method (Insightll / Affinity) was used to investigate the interaction between them. Docking results revealed that the N4 atom of the triazole ring can be coordinated to the Fe atom of the heme group and the difluophenyl group was located into the hydrophobic pocket lined with Phel26, Leul39, Phel45, Ile304 and Met306 and form hydrophobic interaction with them. The substituted piperazinyl side chains attached to C3 were place into the ligand access channel 2 (FG loop) and the piperazinyl group can form hydrophobic interaction with Val509, Alal 1 7 and Leu376. The substitution groups linked to piperazinyl group can form various interactions with the active site of CACYP51, which resulted in their different antifungal activity. Forthe compounds with phenyl, benzyl and pyridine attached to piperazinyl group, the aromatic ring can form n-n with the side chain of Tyrl 18. The hydrogen bond acceptor groups on the para-position of the phenyl can form hydrogen bond interaction with Ser378 and the steric and hydrophobic groups on para-position of the phenyl can form good hydrophobic and van de waals interaction with Leul21, Phe228, Phe233. Phe380 and Met508. For the compounds with benzoyl group attached to piperazinyl group, the weak hydrogen bond between the carbonyl group and the backbone of Phe380 resulted in the deviation of the phenyl group from Tyrl 18 and the 7i-7i interaction between them was lost, which caused the relatively lower antifungal activity of these compounds. From the current flexible molecular docking studies, the important residues binding with azole antifungal agents were identified, which gave important information for the further drug optimization. 5. Evolutionary trace analysis of CYP51 familyMultiple sequence alignments were performed on the CYP51 family and thus evolutionary trace was constructed. The important functional residues of CYP51 family were identified by the ET analysis. From the ET results, the important residues confirmed by biological experiments, such as Tyr76, Phe83, Gly84 and Asp90 of ligand access channel 1 in MTCYP51, Leul72, Glyl75, Argl94 and Aspl95 of ligand access channel 2 in MTCYP51, and Cys470, Gly464 and Arg467 of Cys pocket in CACYP51, were identified successfully. Because few residues in the active site of CACYP51 have been confirmed important for the enzymatic activity and inhibitor binding by biological experiments, the trace residues identified by ET analysis are of great importance to study the structure-function relationship and also design specific inhibitors. The trace residues were mapped onto the active site of CACYP51 and most of the conserved residues were distributed around the heme group, which might be important for the structure and function of the enzyme. The class-specific residues were mainly distributed in the area that interacted with the 3-side chain attached of azole antifungal agents, which might be important for the affinity and specificity.III. Further structural optimization of azole antifungal agents.1. Design and synthesis of l-(l/M,2,4-triazoi-l-yl)-2-(2,4-diflurophenyI)-3-(N-methyl-N-substituted benzylamino)-2-PropanoIsFlexible molecular docking results revealed that the N-methyl group was more favorable for the compounds to interact with the active site of CACYP51 than piperazinyl group. Thus, 13 l-(l//-l,2,4-triazol-l-yl)-2-(2,4-diflurophenyl)-3-(N-methyl-N-substituted benzylamino)-2-Propanols were design and synthesized. The structures of the target compounds were determined by 'HNMR and IR. In vitro antifungal activity assay revealed that the antifungal activities of the target compounds were further improved when the piperazinyl groups was replaced by the N-methyl group, which was consistent with the results from molecular modeling. Furthermore, all the target cmpounds showed broad antifungal spectrum with excellent activities against both systemic pathogenic fungi and dermatophytes. Compounds Dl, D8 and D9 had excellent potency against a broad range of pathogenic fungi and needed further biological evaluation.2. Design and synthesis of l-(l/M,2,4-triazoI-l-yl)-2-(2,4-diflurophenyl)-3-(N-methyl-N-[4-substituted amide]benzylamino)-2-PropanoIs3D-QDAR and molecular docking results revealed that a hydrogen bond acceptor at the para-position of the phenyl group can form hydrogen bonding interaction with Ser378. We chose amide as a hydrogen bond acceptor and various substituted phenyl groups were selected to attach the amide in order to form additional hydrophobic residues in ligand access channel 2 (FG loop) such as Ala61, Leu87, Leu 88, Ile231 and Phe233. Thus 10 l-(lH-l,2,4-triazol-l-yl)-2-(2.4-diflurophenyl)-3- (N-methyl-N-[4-substituted amide]benzylamino)-2-Propanols were synthesized. The structures of the target compounds were determined by 'HNMR and IR. In vitro antifungal activity assay revealed that the antifungal activities of the amide compounds were further improved because of additional hydrogen bonding and hydrophobic interaction with the target enzyme. Most of the compounds showed very strong activity against eightpathogenic fungi. In particular, the MICgo value of compound E10 against was 0.008ug/mL, which was 32-fold more potent than fluconazole, 64-fold more potent than itraconazole and 250-fold more potent than terbinafine.3. Design and synthesis of l-(l//-l,2,4-triazol-l-yl)-2-(2,4-diflurophenyl)-3-(N-methyl-N-{2-substituted-3,4-dihydro-2//-l,2,4-triazol-3-one-4-yl}benzyla mino)-2-PropanolsTriazolone was selected to replace the amide group of the compounds E, because triazolone group can not only function as a hydrogen bond acceptor by its carbonyl but also adjust the phsico-chemical properties of the molecule and improve water solubility. Substituted benzyl, substituted 2-oxo-phenethyl and pentyl were chosen to attach the triazolone in order to form hydrophobic and/or van de waals interactions with residues in FG loop. 36 l-(l//-l,2,4-triazol-l-yl)-2-(2,4-diflurophenyl)-3-(N-methyl-N-{2-substituted-3,4-dihydro-2//-l,2,4-triazol-3-one-4-yl}benzylamino)-2 -Propanols were synthesized. The structures of the target compounds were determined by 'HNMR and IR. In vitro antifungal activity assay revealed that the target compounds showed best antifungal activity and many compounds had MICso values in the range of 0.001-0.002|ag/mL against Candida allbicans. Several compounds exhibited better activity than fluconazole, itraconazole, ketoconazole and terbinafine and needed further evaluation.4. Design and synthesis of N-[2-(2,4-Diflurophenyl)-2-hydroxy-3-(lH-l,2,4-Triazol-l-yl)propyl]-N'-(4-substituted phenyl) -3-(2i/,4//)-l,2,4-triazolonesBecause compounds containing triazolone showed very good antifungal activity, we attached the triazolone to the C3 atom directly and substituted phenyl groups were chosen to link the triazolone. Thus, three N-[2-(2,4-Diflurophenyl)-2-hydroxy-3-(lH -l,2.4-Triazol-l-yl)propyl]-N'-(4-substituted phenyl)-3-(2//,4//)-1.2.4-triazolones were synthesized. In vitro antifunga! activity assay revealed that the target compounds showed antifungal activity against the eight pathogenic fungi to some extent and compound 11 had better activity than fluconazole and terbinafine.