Design,Synthesis And Activity Evaluation Of Novel Phloroglucinol Derivatives

In the past ten years,China’s medical level has been developed rapidly,and the success rate of disease treatment has been higher and higher,but some diseases still have a long way to go.For example,the treatment of tumors and fungi are facing great challenges.So,finding new low-toxicity,high-efficiency,broad-spectrum antitumor and antifungal target drugs is imminent.Dryopteris fragrans(L.)Schott is a plant belonging to Dryopteris adanson genus of Dryopteridaceae family which is a perennial fern with a special fragrance,and also called geraniol dryopteris.It is mainly distributed in Europe,north America and temperate zone of Asia,and commonly existed in the hillsides and rock fractures above 1 km in northeast China.It is a folk medicine that mainly used to treat of a variety of skin diseases and showed good effect and was not easy to relapse.The main bioactive components of Dryopteris fragrans(L.)Schott are phloroglucinol,terpenoids,flavonoids,phenylpropanoids and volatile oils.Modern pharmacological studies have shown that many types of phloroglucinol compounds contained in Dryopteris fragrans(L.)Schottwhich have strong bactericidal,antiviral,tumor suppressing and insect repellent effects.So far,more than fifty phloroglucinol compounds were isolated from the Dryopteris fragrans(L.)Schott,including monocyclic,bicyclic,tricyclic,and polycyclic compounds.In recent years,the reports about natural phloroglucinol compounds have been increasing,and bioactive phloroglucinol have attracted more and more attention from researchers.In the early study,monocyclic phloroglucinol with obvious antifungal effects were isolated from Dryopteris fragrans(L.)Schott.At the same time,we have completed the research on the synthesis and antifungal activity screening of the pseudoaspidinol.Due to the extraction and separation process of pseudoaspidinol was complicated,and a lot of poisonous gas and waste liquid would produced.The synthetic route of pseudoaspidinol was optimized from the perspectives of economics and green chemistry in this study.Based on the study of monocyclic compounds,a series of phloroglucinol derivatives were designed and synthesized.The pharmacophore aminopyrimidine of tyrosine kinase inhibitors was introduced to design new bioactive compounds.The antifungal and antitumor activities of the designed and synthesized compounds were invetigated by the broth microdilution method and the MTT method,and the binding mode was explored by molecular docking.The results showed that most of the compounds have good antitumor and antifungal activity.The main research contents and results are as follows:1.The extraction and separation of the monocyclic compounds aspidinol and pseudoaspidinol which are isomers equivalent in activity from Dryopteris fragrans(L.)Schot have lower yield,higher cost and longer time.In order to obtain a large amount of pseudoaspidinol for subsequent research,we have optimized the synthetic route of it.The optimized synthetic route is as follows:the pseudoaspidinol was synthesized by using phloroglucinol as raw material through Vilsmeier-Haack,Wolf-Kesinar-Huang-Minlon reduction,Friedel-Crafts,methylation reaction and hydrogen reduction.2.The 1,3,5-trimethoxybenzene was obtained by using phloroglucinol as raw material and dimethyl carbonate as methylation reagent through ether formation reaction.The product was acylated with the acyl chloride under the catalysis of aluminum trichloride,and the A series phloroglucinol derivatives were obtained.The 1,3,5-trimethoxybenzaldehyde was reduced by hydrazine hydrate and potassium hydroxide.The reduced product underwent a Friedel-crafts acylation reaction with the acyl chloride again,and the class B phloroglucinol derivative was obtained.The 1,3,5-trimethoxybenzaldehyde was catalyzed by hydrogen oxide to get 1,3,5-trimethoxybenzoic acid,and the C series phloroglucinol derivatives were obtained through the hydrolysis reaction,acylation reaction and urethane condensation.Finally,twent-two target compounds were designed and synthesized,and were characterized by MS,~1H-NMR and ~1C-NMR.At the same time,the reaction conditions of the target compounds A4,A5,B,C1 were optimized and screened to ensure,the experiment was carried out safely and efficiently.3.Evaluation of antifungal activity of phloroglucinol derivatives A,A1-A4,B,B1-B4 against Trichophyton rubrum and Trichophyton mentagrophytes were performed by CLSI+M38-A broth microdilution method.The results showed that A4 has the best antifungal effect on the two types of dermatophytes,and with the MFC value of 19.87μg/mL and 16.45μg/mL.4.The MTT method was used to detect the antitumor activity of twenty-two target compounds.The results showed that the twenty-one derivatives had obvious inhibitory effects on A-549,MCF-7,HepG2 and Hela cancer cells.The derivatives A1,A4,B4,C1,C2 and C3 had better antitumor effects.Among them,the effect of the derivative C2 was the best.And the inhibitory effect on MCF-7 was more significant than other tumor cells.5.In order to further explore the antitumor mechanism of C2,the Annexin V-FITC/PI double staining method was used to determine the effects of compound C2 and afatinib on MCF-7 cell apoptosis.The results showed that C2 could promoted MCF-7 apoptosis at low concentrations with apoptosis rate of 13.05%.It can be preliminarily determined that the designed and synthesized derivatives could be similar to the antitumor mechanism of afatinib.6.The twenty-one target compounds and afatinib were docked with the target proteins by MOE2014 molecular docking software.The binding force between the test compounds and the target proteins were used to predict the possible mechanism of action of the compound.The results showed that these derivatives have roughly adopted the same action points and docking mode with the target proteins.According to the docking scoring table,the compound of C2 has the highest scoring score and strongest binding energy than other compounds.This docking result was consistent with the experimental results of the antitumor activity of the target compounds.In summary,in this experiment,the route of the total synthesis of pseudoaspidinol was optimized.Pseudoaspidinol and its intermediate phloroglucinol were used as lead compounds,and the pharmacophore aminopyrimidine and acyl groups were introduced to synthesize twenty-one target compounds and three intermediates(2,4,6-trimethoxybenzoicacid,2,4,6-trimethoxybenzamide,2,4,6-trimethoxybenzoyl isocyanate).The experimental conditions were optimized to improve and ensure the safety and feasibility of the experiment.Finally,the compounds were tested for antifungal and antitumor activity.The results showed that the compound of A4 had the strongest inhibitory effect on two types of dermatophytes,and compound C2 had the most obvious inhibitory effect on four types of tumor cells.The compound of A4 and C2 provided lead compounds for the development of new antifungal and antitumor agents.