Mmunological Studies Of β-[1,2]Mannan-based Vaccines For Candida AlbicansⅡ.Synthesis And Antifungal Activity Of Novel Triazole Compounds

During the past few decades there has been a sharp increase in the number of immunocompromised patients for a variety of reasons that include widespread use of immunosuppressive medications and broad-spectrum antibacterial drugs, as well as the spread of human immunodeficiency virus (HIV) epidemic. Candida. albicans, which is considered as one of three major fungal infections strains, has been resulted in high incidence and mortality rate in immunocompromised patients. The blood infections caused by C. albicans has risen to the third among the blood infections in hospital. Meanwhile, the mucous infection which is also cause by C. albicans is the leading cause of death in HIV patients. The most common antifungal agents currently used in clinical for C. albicans are fluconazole, itraconazole, voriconazole and so on. However, with continuous and abundant use, those drugs are not effective enough due to severe drug resistance. This has led to an interest in vaccine development for Candidiasis, which provides a new prevention and treatment strategy.Research reveals that the cell wall (CW) of C. albicans consists of an internal skeletal layer and an external protein coat. This coat has a mosaic-like nature, containing about20different protein species covalently linked to the skeletal layer, Most of which are called glycosylphosphatidyinnositol (GPI) anchored proteins. Due to the advanced developement in genetic and biological engineering technologies, the functions of some GPI-CWPs, such as Alsl, Als3, Hwpl, Pag59and Sap9, which are involved in the primary interactions between C. albicans and the host, mediate adhesive steps or invasion of host cells, were clarified. These help us to test immunogenicity and modify the structure of the protein to discovery new antigen for developing antifungal vaccines candidates. Furthermore, the structure of the glycan component of C. albicans phosphomannan has been determined and compelling data has shown that the β-mannan portion of the C. albicans cell wall plays a decisive factor in pathogenicity, and further studies indicate that (1â†'2)-β-mannan oligomers have potential as the key epitope for protein conjugate vaccines. Although a lot of work was carried out for anti-candida albicans vaccines, we find that the major disadvantage in these researches is the low immunogenicity, which is limited to elicit immune response. Base on the studies above and in order to solve these problems, in our design, the ethanolamine phosphate ester, which is the natural connect unit for GPI-CWPs, was considered for connecting the GPI-CWPs peptide fragment with β-1,2-mannan to form the glycopeptides vaccines. Here five different peptides fragment on GPI-CWPs (Alsl, Als3, Hwp1, Pga59and Sap9), which are closely related to the growth, adhesion, invasion and virulence of C. albicans, were chosen. Compared with the research before, the fragment of peptide and the β-1,2-mannan are more representative and the connect unit is more reasonable, we have great confidence the desired taget compounds will have better immunogenicity.Starting from D-glucose, after several steps we got orthoester compound21. Based on the compound21, acceptor26and monosaccharide donor30,29can be easily synthesized according to the published literatures. Disaccharide31was obtained after glycosylation, followed by a deacetylation, oxidation, and reduction sequence to afford trisaccharide acceptor36. Reaction of the donor29with acceptor26, acceptor33and acceptor36afforded oligosacchrides37-39respectively. Compounds43-45were obtained by repeating the procedure above (deacetylation, oxidation, and reduction), followed by debenzylation, deprotection of TBDPS, phosphorylation and deprotection of Fmoc group in sequence to afford56-59. Reactions of peptide60with compounds56-59afforded61-63respectively. The desired compounds7-9were finally obtained after global deprotection. Finally, all synthesis of the glycopepetides (7-9), oligosaccharides (64-66), pepetide71were finally coupled to proteins (KLH/HSA) to afford the target glycopeptides-protein conjugates (1-6), glyco-protein conjugates (12-17) and peptide-protein conjugates (10-11).All of the glycopepetides (7-9) and synthesized KLH conjugates were injected to mice to test immunogenicity as antifungal vaccines candidates. The immune responses were evaluated by enzyme-linked immunosorbent assays (ELISAs) and the HSA conjugates were used as capture antigens to coat plates for the detection of antibodies specific for corresponding antigen. Now the immunological experiments were undergoing. Candidosis, cryptococcosis and aspergillosis are three major fungal infections in immunocompromised patients. The most common antifungal agents currently used in clinical are niftifine, caspofungin, fluconazole and so on. Among these antifungal drugs, triazole drugs are considered as an attractive option due to their generally broad antifungal spectrum, high potency, low toxicity and favorable pharmacokinetic characteristics. However, with continuous and extensive use, serious problems can occur. Fuconazole is not effective against invasive aspergillosis and has suffered severe drug resistance due to mutations. This situation compels chemists to design and synthesize new triazole derivatives in an effort to come up with new drugs which can be used in place of current drugs.TTS-12, which was extracted from tribulus terrestris, is a new steroid compound belongs to isospirostanol saponin. It has been shown the potent antibacterial activity against Candida albicans and filamentous fungus, whereas fluconazole almost shows no activity against filamentous fungus. So far, a considerable amount of work on structural modification of steroid saponin has been done. Studies indicate that the skeleton fragments of steroids compounds appear more frequently in the design of new pharmaceutical drugs for their inherent properties.In light of these promising results described above and on the basis of previous research, we have become interested in incorporation of these two fragments into a series of novel complex conjugates that aim to combine the properties of their individual components. Firstly, corresponding triazole fragment was introduced to the3-OH position of diosgenin, in order to see whether they have synergistic effect. Secondly, after opening the F ring of diosgenin was introduced triazole fragment to see whether opened F ring takes effect on the antifungal activity in vivo. In our design,1,2,3-triazole moieties are introduced as a connecting unit due to their stability to metabolic degradation, favorable ability to hydrogen bond and antifungal activities.With compound (1)1-(2,4-difluorophenyl)-2,3-epoxypropyl)-1H-1,2,4-triazol methanesulphonate as starting material, treated with various amines to afford1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-(N-substituted-amino)-2-propanols. Then reacted with propargyl bromide in acetonitrile, using potassium carbonate as acid binding agent, to give compound (3a-d)1-(1H-1,2,4-triazole-1-y1)-2-(2,4-difluoro phenyl)-3-(N-substituted amino-N-propargyl amino)-2-propanols. Treated diosgenin (4) with methyl sulfonyl chloride and triethylamine in DCM to afford compound (5), followed by reaction with NaN3in DMPU to give azide intermediate (6). Diosgenin (4) was treated with BF3·Et2O and Et3SiH afforded ring-opened compound (8), followed by a reaction sequence analogous to the one described above to give the desired azide compound (9). The taget compounds were finally obtained by treating intermediates (3a-d) with azide compounds (6) and (9) respectively in the presence of a Cu (I) catalyst ("Click Reaction"). All of the target compounds described above were first reported and characterized by1H NMR, ESI-MS and Q-TOF-MS spectroscopic analysis.All the synthesized target compounds (10a-d,11a-c,12a-c,13a-c) were tested for antifungal activity against eight various pathogenic fungi:Candida albicans SC5314, Candida albicans Y0109, Candida parapsilosis, Candida tropicalis, Trichophyton rubrum, Candida krusei,Cryptococcus neoformans and Aspergillus fumigatus. The in vitro minimal inhibitory concentrations (MICs) of the compounds were determined by the micro-broth dilution method in96-well micro-test plates. Fluconazole (FLC), itraconazole (ICZ) and voriconazole (VCZ) were served as the positive control drugs.The preliminary result showed that all of these title compounds were inactive against the tested strains of Tri. Rubrum and Asp. fumigatus. The MICso value for all these compounds was>64μg/mL. All the title compounds exhibited lower antifungal activity against C. tropicalis, C. krusei and Cry. neoformans strains compared with the positive drugs. The antifungal activity of compound10d was equal to fluconazole but higher than that of itraconazole against C. alb. SC514and C. alb. Y0109(with MIC80value of1μg/mL respectively) while compound10b exhibited comparable antifungal activity with itraconazole but less than that of fluconazole and voriconazole. Compounds12a-c and13a-c showed equal activity against both C. alb. strains, which is far below those of positive control drugs. Compounds10a-d and lla-c showed higher activity against both C. alb. strains than that of compounds12a-c and13a-c. The preliminary structure-activity relationship showed that the double bond on the disogenin didn’t take effect on the activity of conjugates while opening F ring of diosgenin resulted in decreasing the antifungal activity of conjugates.