Synthesis,Antibacterial Activity And Mechanism Of Action Of Methyl Phloroglucinol Derivatives

Dryopteris fragrans was mainly used as a folk medicine at present.It has been found that the main active compounds of the Dryopteris fragrans had a significant effect on a variety of skin diseases caused by fungi.Various compounds of phlorglucinol were isolated from the plant by our team.Pharmacological studies showed that these compounds had strong antifungal activities,and the aspidinol exerted outstanding against skin fungal.The methyl phlorglucinol was the intermediate in the synthetic strategy of aspidinol,and it could be modified for a variety of complex compounds due to the unique structural feature.Our previous studies found that the hydroxyl group of methyl-phloroglucinol was the active group,and C-4 or C-6 was active site.Introducing different groups in the two positions could change the properties and bioactivity of the compounds.In this study,different functional groups were introduced in methyl-phloroglucinol,and got various new phloroglucinol compounds.Then the antimicrobial activity was observated by molecular modelling and Elisa.Finally,the difference of antibacterial and mechanism of the new phloroglucinol compounds was measured and it lays a foundation for the study of antifungal action and mechanism of phlorglucinols.The main purpose:(1)The synthesis method and route of methyl phloroglucinol derivatives were established by introducing butyroyl and allylamino on the basis of methyl phloroglucinol.(2)Through the screening of antifungal activity and the bactericidal dynamic curve,the antimicrobial activity and the onset time of phloroglucinols were investigated.(3)The antifungal target and mechanism of methylbenzene triphenol derivatives were screened via the molecular modeling and enzyme test.And it provides a clue to the study of the mechanism of m-triphenols.The main contents are as follows:(1)Synthesis of mesoglucinol derivatives:firstly,the synthesis of phloroglucinolformaldehydefromphloroglucinol.Secondly,methyl-phloroglucinol was synthesized through reduction reaction.Thirdly,butyloyl and allylamino were introduced by Friedel-gram reaction,and obtained the target compound.Finally,the structures of the compounds were confirmed by MS,~1H-NMR,~133 C-NMR.(2)According to the American Association of Clinical and Laboratory Standards(ACLSI)M38-A2 method,the(MIC)and the MFC of compounds against four kinds of dermatophytes were determined,respectively.The most active compounds and the most sensitive fungi were screened out.(3)The bactericidal curves of different concentrations of tested compounds against microsporum canis were determined by colony counting method,and the dynamic inhibition of tested compounds on tested fungi was investigated.(4)The molecular docking and molecular dynamics simulation used Discovery Studio2.5.5 and AMBER9.0.The binding between the tested drug and the target were compared through determining the-CDOCKER energy and the RMSD value,and the possible bacteriostatic mechanism was discussed.(5)The determinate method of ergosterol was established by UPLC,and the change of ergosterol content in microsporum canis was investigated.(6)The effects of different drug groups on the activities of CYP51,squalene epoxidase and?-1,3-glucan enzyme was investigated by ELISA,and further probe into its bacteriostatic mechanism.The results are as follows:(1)The synthesis test showed that methyl-phloroglucinol was synthesized successfully by Vilsmeier-Haack reaction,reduction reaction and Friede-gram reaction,and then modified with the structure of methyl-phloroglucinol.As a result,four derivatives were obtained,they are 2-methyl-4-butylol phloroglucinol(28.6%),2-methyl-4,6-two butylol phloroglucinol(47.2%)and an intermediate 1-(2-butene)-3-methyl-5-butylyl phloroglucinol(35.7%)and 1-(2-butene)-3-methyl phloroglucinol(81.2%).The optimum synthesis process is as follows:the molar ratio of raw material and the catalyst was 1:4,the amount of dichloromethane is 10 times,and the reaction time is 6 h at45 ~oC water bath temperature.Under these conditions,the yield of the target compounds was the highest.The purity of the compounds obtained by UPLC was higher than 90%,and the compounds were identified by mass spectrometry and nuclear magnetic resonance spectroscopy.(2)According to the M38-A2 of CLSI,the antibacterial activities of 6 phloroglucinol derivatives were screened to 4 kinds of common dermatophytes in varying degrees by using the microdilution.The results showed that 1-(2-butene)-3-methyl-5-butylyl phloroglucinol(g)was the strongest antibacterial activity of,the next was 2-methyl-4,6-two butylyl phloroglucinol(e),and then the phloroglucinol formaldehyde(b),methyl phloroglucinol(c),2-methyl-6-butylyl phloroglucinol(d)and 1-(2-butene)-3-methyl phloroglucinol(f)inhibitory effect was not obvious.It was worth noting that these compounds had a strong inhibition of Trichophyton mentagrophytes and Microsporum canis in Trichophyton and Microsporum gypseum.Time-bactericidal curves of 1-(2-butene)-3-methyl-5-butylylphloroglucinol(g)and 2-methyl-4,6-two butylyl phloroglucinol(e)against microsporozoa canis were investigated by colonycounting method.The results showed that the two derivativescould inhibit the growth of Microsporum canis for a certain periodof time,and when the drug concentration was greater than orequal to the MIC of the two drugs,the two derivatives couldinhibit the growth of microsporum canis.However,the differencewas that the compound g can kill microsporum canis faster thancompound e when the drug concentration is MIC and 2MIC.(3)The molecular simulation showed that the binding patterns of methyl-phloroglucinol and its derivatives were similar to those of CYP51,squalene epoxidase and?-1,3-glucan enzyme.All of them were bound by the?-?bond between benzene ring and TRP amino acid group and the hydrogen bond formed by H or O in compound molecule and O or H in amino acid,and the amino acid type of binding was similar.The binding effect of compound e and compound g was stronger than d,it might be related to the groups such as butyryl,allylamino in structure e and g.The results of molecular dynamics showed that the RMSD values of the three compounds remained between 1.0 and 1.5 for the three enzyme kinetic simulation within 30ns,indicating the rationality and stability of the molecular docking between the three compounds and the enzyme receptors.(4)The results of ergosterol determination showed that ergosterol had a good linear relationship in the range of 4-40?g/mL,the regression equation Y=6287X-361.06,R~2=0.9997,the precision and accuracy of the sample were good,and the sample within 24 hours was stabile.The ergosterol content in microsporum canis decreased significantly after drug treatment compared to blank group.Compared with the blank group,when the concentration of compound g was 1/2MIC,MIC,and 2 MIC,the decrease rate of ergosterol content was15.76%,34.28%,and 56.83%;when the concentration was 1/2 MIC,MIC,and 2MIC of compound e,the decrease rate of ergosterol content was 13.55%,26.17%and 55.59%.The terpinafine hydrochloride MIC and nitric acid MIC groups caused a decrease in ergosterol content of45.37%and 49.11%,respectively.(5)The results of enzyme linked immunosorbent assay(ELISA)showed that compound e and compound g could decrease?-1,3-glucan synthase,lanosterol 14?-demethylase,squalene epoxidase,and it was dose-dependent.In this study,established 2-methyl-6-butylol phloroglucinol,2-methyl-4,6-twobutylol phloroglucinol and an intermediate 1-(2-butene)-3-methyl-5-butylyl phloroglucinol,1-(2-butene)-3-methyl phloroglucinol synthesis route from Methylenetriphenol.This route was simple,mild compared with pseudo aspidinol,and the yield was high.The antibacterial activity study showed that compound 2-methyl-4,6-twobutylol phloroglucinol and 1-(2-butene)-3-methyl-5-butylyl phloroglucinol had good inhibitory effect on Microsporum canis.The molecular simulation test showed that the phenolic hydroxyl group,benzene ring and double bond structure of these compounds could be be related to the amino acid residues of CYP51,squalene epoxidase and?-1,3-glucan enzyme stably and reliably.The ELISA test results showed that the compounds had similar inhibitory activities against the three target enzymes.Both compounds can significantly reduce the activity of the three enzymes.In summary,the phenolic hydroxyl groups in the structure of the phloroglucinol was the active group.After introducing butyrate and allylamine into the compounds,the compounds 2-methyl-4,6-twobutylol phloroglucinol and 1-(2-butene)-3-methyl-5-butylyl phloroglucinol showed stronger antibacterial activity than other compounds.The effect of the compounds on ergosterol content test results showed that both compounds could control the synthesis of ergosterol by inhibiting the key enzymes squalene epoxidase and lanosterol-14-?-demethylase,eventually causing the rupture of fungal cell membranes,fungal apoptosis.In addition,both compounds have significant inhibitory effects on?-1,3-glucanase,indicating that both compounds could affect the activity of?-1,3-glucanase in the cell wall of fungi.It was further thought that the main component of the fungal cell wall,glucan,causes fungal apoptosis.