Design,Synthesis And Structure Activity Relationship Of The Inhibitors Of GPI-Anchored Protein Biosynthesis As Novel Antifungal Agents

Invasive fungal infections have become the most headache problem for the practising physician. Recently reports indicated the invasive candidiasis has become the third bloodstream infections in the world, and the incidence by country is much higher than that by urban. Improved diagnostics, new epidemiology studies and novel antifungals have revolutionized the field of medical mycology in the past few decades. Early antifungals have appeared in the 1950s, but their toxicity limited efficacy, such as nystatin and amphotericin B. Triazoleshe were effective against fungal pathogens and revolutionized medical mycology in the 1980s. Throughout the 1990s, the development of antifungals mainly concentrated on the transformation of triazoles and the reformulation of amphotericin B. The second-generation triazoles (such as voriconazole and posaconazole) and liposomal amphotericin B realized less toxic and improved pharmacokinetics and pharmacodynamics. In the 2000s, the echinocandins came up, the newest antifungals inhibited the synthesis of a key component of fungal cell wall (1,3-β-glucan). The safety of the echinocandins was remarkable, and the mechanism of new action was the inspiration for the development of antifungals. As fungi are metabolically similar to mammalian cells, it is difficult to find pathogen-specific targets. On the other hand, the clinical development is slow by challenges in timely diagnosis of rare or resistant fungi. Despite these problems, the development of antifungals still continues.Glycosylphosphatidylionsitol anchored protein (GPI) is a class of protein with C-terminal glycosylphosphatidylinositol anchor structure on the surface of eukaryotic membrane. It has been reported that there are many kinds of GPI anchored proteins, including enzymes, receptors, adhesion molecules and specific antigens. Now, it is found that GPI anchored proteins play an important function in the immune recognition, complement regulation, transmembrane signal transduction and so on.Structure and function of GPI anchored proteins are different between mammalian and fungi. It makes GPI anchor proteins a new target for novel antifungal drugs. Scientists in Japan found the small molecules, such as gepinacin, E1210 and 10b, through high-throughputscreening, and proved these molecules to be GPI-anchored protein inhibitors, which inhibit a critical acyltransferase Gwtlp in GPI biosynthesis.In this study, we analyzed the structure of gepinacin, E1210 and 10b. Several novel derivatives were designed, synthesized and evaluated for in vitro antifungal activity. Structure-activity relationship study on these compounds was discussed.We first carried out chemical modifications of gepinacin to increase the antifungal activity, 10 novel compounds were designed and synthesized. The optimal structure zj-g-5b-6b (MIC80=4.0 μg/ml) showed potent antifungal activity compared to gepinacin, but weaker potent than fluconazole (MICso= 0.5 μg/ml). We failed to find a potential lead compound during the next experiment.Then, the structural modifications of E1210 and 10b give us a clue that the lead compounds were divided into five parts:A ring, B ring, C ring, linker I and linker II, and we got 48 novel compounds. Replacement of 2-aminonicotinamide ring in E1210 with benzothiazole ring gives us zj-e-1b-11b, which exhibited good antifungal activity against Candida albicans with MIC80 values ranging from 0.125 to 4.0μg/ml. However, introduction other aromatic ring into compounds (zj-e-12b-18b, zj-e-40b-44b) reduced the activity. Though 2-aminonicotinamide group is not necessary to antifungal activity, the nitrogen atom must exist in A ring, which suggest there are hydrogen bonds between molecules and target protein binding mode. The amide group substitute for the isoxazole ring in E1210, and thiophene ring instead of benzene ring by the isostere principle in these compounds, which exhibited the higher activity with the MICso value of 0.125μg/mL. The experimental results showed the antifungal activity of the compounds was reduced or totally lost in either shorten or lengthen the carbon chain. The optimum length is two carbon atoms, which proved that the length of the molecule has influence on the antifungal activity. Linker II may be related to the properties, such as water solubility and lipid solubility. Data of zj-e-lb-3b (MIC8o= 0.125 μg/ml) suggest that fluoro substituted at the meta or ortho position on benzene ring was strongly favored the activity. The substitution at 2-position of thiophene ring showed potent activity, while the new type class of 3-position substituted compounds (zj-e-45b-48b) also showed good antifungal activity with the MICso value of 0.0625-0.5 μg/mL. This new discovery provides a new inspiration for the research and development of antifungal drugs in the future.All the structure of the title compounds were confirmed by 1H-NMR and LC-MS, and the structure-activity relationships were discussed in depth according to the antifungal activity, we provide an experimental basis for design and synthesis GPI-anchored protein inhibitors for future.