Design, Synthesis And Antifungal Activity Of Novel N-Myristoyltransferase Inhibitors

N-Myristoyltransferase(NMT) is a cytosolic monomeric enzyme, which catalyzes the transfer of myristate from myristoyl-CoA to the N-terminal glycine residue of a number of eucaryotic and viral proteins and forms N-myristoylation protein. Protein N-terminal myristoylation results in irreversible protein modification and enlarging lipophilicify, which initiates many kinds of reversible protein-cytomembrane interaction and protein-protein interaction. N-myristoylation protein has diverse biological effects and participates in many kinds of biology process such as signal transduction cascade connection and apoptosis and so on. Some N-myristoylated G-proteins in fungal cell are necessary for the growth of fungal ceils. Nmt has been a promising target enzyme for the development of novel fungicidal drugs having a broad antifungal spectrum. Thus, the search of selective inhibitors of fungal NMT will lead to the discovery of novel antifungal drug with new mechanism of action.In 2002, Sogabe et al. reported the crystal structure of CaNMT in complex with two benzofuran inhibitors, which lay a solid foundation for us to design selective inhibitors of fungal NMT. Granted by the National Natural Science Foundation of China (No. 30430750), we have performed systemic molecular modeling studies of CaNMT including the construction of predictive 3D-QSAR models, the detailed analysis of the properties and the distribution of important function residues in the active site of CaNMT and the clarification of the binding mode of current inhibitors. From these studies, we found that all the small molecule NMT inhibitors had diheterocycle core structure, such as benzofuran and benzothiazole, which was located into the core region of the active site and formed hydrophobic interaction with the surrounding residues lined with Tyr107, Phell 5, Tyrll9, Phe176, Tyr225, Tyr256, Tyr335 and Yyr354. Starting with this point, we designed and synthesized a series of diheterocycle compounds with indole and benzoxazole as core structures. The in vitro antifungal activities of the synthesized 18 new compounds were also determined.1. Synthesis and antifungai activities of benzoxazole NMT inhibitorsAn efficient synthetic route of benzoxazole NMT inhibitors was developed. Starting with 2-nitro-benzene-1, 3-diol, CICH₂C(=NH)OEt·HC1 was used to form the benzoxazole structure. Then, the C4-OH was protected by Boc and C2-side chain was synthesized. After the removal of Boc, the C4-side chain was added to give the title compounds. A total of nine new compounds were synthesized and their chemical structures were confirmed by ¹H NMR. In vitro antifungal activity revealed that most compounds showed good activity against Candida albicans with MIC range 0.25-16μg/mL. In particular, compound 13f is as potent as fluconazole. Most of the compounds showed moderate activity against Cryptococcus neoformans with MIC value of 16μg/mL. However, these compounds were inactive against Aspergillus fumigatus. Structure-activity relationship showed that the C2-side chain on benzoxazole ring had significant influence on the antifungal activity. Compounds with amide C2-side chain were inactive. On the other hand, compounds with ether C2-side chain showed good activity and the addition of fluorine atom the phenyl could lead to the increase of the antifungal activity.2. Synthesis and antifungal activity of indole NMT inhibitors Based on the test of various synthetic routes, an efficient route with mild condition and high yield for the synthesis of diversely substituted indole NMT inhibitors was developed. Using salicylaldehyde as the starting material, the hydroxyl was protected by benzyl bromide, then condensed with azidoacetate to form the indole ring. After the protection of indole NH by Boc, the benzyl group on hydroxyl was removed by hydrogenation. Then, the addition of C2 and C4 side chain obtained the title compounds. A total of nine new compounds were synthesized and their chemical structures were confirmed by ¹H NMR. In vitro antifungal activity revealed that most compounds were inactive against deep fungi and showed moderate activity against superficial fungi. Several compounds showed good activity against Microsporum gypseum with their MIC value of 2-16μg/mL, which was more potent than fluconaole and less active than itraconazole and tebinafine.