Study On Manufacture And Antifungal Of Pseudoaspidinol B Modification Derivatives And Aspidinol B

Aspidinol B is the most active ingredients isolated from the Dryopteris Adanson.Pseudoaspidinol B and Aspidinol B are Phloroglucinols while being isomers to each other.It has been studied that the Phloroglucinols in the Dryopteris plants have good antifungal and antiviral activity against diseases such as hand tinea,foot tinea,tinea corporis,tinea unguium and other superficial fungal infections.Previously in our laboratory,studies focusing on methodology of extracting and isolating phloroglucinols from Dryopteris fragrans has been carried out extensively.We also have screened them in terms of their antifungal activity.The Aspidinol B isolated from 50% ethanol extract has promising effect against Trichophyton rubrum and Trichophyton mentagrophytes.As isomers to Aspidinol B,Pseudoaspidinol B is easier to synthesize comparing to its counterparts with equivalent curing ability.Surprisingly,few studied has been addressed on this.Therefore,for sake of optimizing the antifungal activity of Pseudoaspidinol B,this article mainly aim to develop its total synthesis and structure modification strategy.Here,the in vitro antifungal ability of Pseudoaspidinol B,its synthesis intermediate,it derivatives along with Aspidinol B were also characterized.Compounds with distinguishing activity was selected.The relationship between structure and activity of Pseudoaspidinol and Phloroglucinol was discussed qualitively,in order to consolidate further drug development of Phloroglucinols.Specific work content is as follows:1.Manufacture of Pseudoaspidinol B and Derivatives:With Formylphloroglucinol as the starting material,methyl phloroglucinol is acquired via reduction of Formaldehyl,Then,through Friedel-Crafts reaction,hydroxyl at 4 and 6 are protected.After reaction with methyl iodide,hydroxyl at 2 was methylated.Finally,with the facilitation of palladium carbon catalyst,benzyl group was ripped off,presenting Pseudoaspidinol B.For structure modification,acylation and introduction of halogen was applied to increase its lipophility,in order to enhance its membrane permeability and thus its absorption,activity and bioavailability2.Manufacture of Aspidinol B: Prepared by silica gel column chromatography and semi-preparative high performance liquid chromatography for the separation and purification of Aspidinol B.According to the spectral characteristics and physical and chemical properties of the Manufacture of Aspidinol B.3.Under the guidance of Filamentous Fungi Spores Liquid Dilution Antifungal Drug Sensitivity Test(M38-A2)recommended by Clinical and Laboratory Standards Institute(CLSI),the MIC of Pseudoaspidinol B,its synthesis intermediate,it derivatives and Aspidinol B against Trichophyton rubrum,Trichophyton mentagrophytes and Microsporum Canis was characterized.The result was then presented in the discussion of their structure-activity relationship.Results:1.Total synthesis route from Formylphloroglucinol to Pseudoaspidinol B was successful,effective and reproducible.The overall yield was 0.53%.The target compounds was identified and confirmed by 1H-NMR,13C-NMR,MS analysis.Using butyryl chloride and fluorine benzene sulfonyl chloride,structural modification on Pseudoaspidinol B was conducted.In the end,a Pseudoaspidinol B derivative with a butoxycarbonyl group at 6 position and one with a fluorophenylacetate at ortho position were obtained,with their overall yield 20% and 22% respectively.Structure of them were identified by 1H-NMR and MS.We therefore named them as Butyryl Pseudoaspidinol B and 2-fluorobenzenesulfonyl Pseudoaspidinol B.2.Prepared by silica gel column chromatography and semi-preparative high performance liquid chromatography for the separation and purification of Aspidinol B.The compounds were confirmed by1H-NMR,13C-NMR and MS.3.The MIC of the nine compounds against Trichophyton rubrum were: Aspidinol B(20.00μg/mL)>Pseudoaspidinol B(28.28μg/mL)> Formylphloroglucinol(40.00μg/m L),3-methyl-2,4,6-trihydroxybenzophenone(40.00μg/mL)>butyryl Pseudoaspidinol B(80.00μg/mL)> 2-fluorobenzenesulfonyl Pseudoaspidinol(>160.00μg/mL),2-Methyl phloroglucinol(>160.00μg/mL),3-methyl-4,6-dibenzyloxy-2-hydroxybenzophenone(>160.00μg/mL),3-methyl-4,6-dibenzyloxy-2-methoxybenzophenone(>160.00μg/mL).MIC of Trichophyton mentagrophytes were: Aspidinol B(11.89 μg/mL)>Pseudoaspidinol B(20.00μg/mL)> 3-methyl-2,4,6-trihydroxybenzophenone(28.28μg/mL)> Formylphloroglucinol(33.63 μg/mL)> butyryl Pseudoaspidinol B(80.00μg/mL)>2-fluorobenzenesulfonyl Pseudoaspidinol(> 160.00μg/mL),2-Methyl phloroglucinol(> 160.00μg/mL),3-methyl-4,6-dibenzyloxy-2-hydroxybenzophenon(> 160.00μg/m L),3-methyl-4,6-dibenzyloxy-2-methoxybenzophenone(> 160.00μg/m L).MIC of Microsporum Canis were: Aspidinol B(10μg/mL)> Pseudoaspidinol B(10μg/m L)>3-methyl-2,4,6-trihydroxybenzophenone(20μg/mL)>Formylphloroglucin ol(23.78 μg/mL)> butyryl Pseudoaspidinol B(160 μg/mL)> 2-fluorobenzenesulfonyl Pseudoaspidinol(>160μg/mL),2-Methyl phloroglucinol(>160μg/mL),3-methyl-4,6-dibenzyloxy-2-hydroxybenzophenon(>160μg/mL),3-methyl-4,6-dibenzyloxy-2-methoxybenzophenone(>160μg/mL).This research prepared Pseudoaspidinol B through chemical synthesis.Comparing approach of direct extraction from natural plant,it is less laborious,convenient and time saving,without any radical condition.Thus,it is a feasible and effective method of Pseudoaspidinol B preparation.Its antifungal activity against Trichophyton rubrum,Trichophyton mentagrophytes and Microsporum Canis are superior to its counterparts,considering on their frequent infection among patients.Although the lipophility and membrane permeability of Pseudoaspidinol B derivatives in this study are higher than Pseudoaspidinol B,their antifungal activity is not,suggesting the replaced hydroxyl at position 4 is indispensable group when inhibiting fungal activity.Further structure-function relationship discussion is required.Alternatively,effort can be focused on modifying methoxyl group at position 2 and butyryl group at position 4,which might demonstrated the essential core of fungal inhibition of Phloroglucinol,providing theoretical support for drug development on Dryopteris Adanson.