Virtual Screening,Synthesis Of Derivatives And Preliminary Application Research Of Antitumor Active Ingredient From Dalbergia Benthamii Prain

The research of anti-tumor drugs has become a challenging and significant topic in life science and pharmaceutical chemistry.With the development of scientific and technological method,access to get new medicines is also diversified.But the main source of the new chemical entity is still from medicinal plants.It is a systematic problem to find novel active compounds efficiently from traditional Chinese medicine and reasonable modification and activity research on them.Dalbergia benthamii Prain belongs to Dalbergia genus,which is a traditional Chinese herbal medicine commonly used by the Zhuang people in Guangxi.And the extracts of the plant with different polar solvents have been proved to have potent antitumor activity in our study.Therefore,screening out the anti-tumor active components,structural modification and anti-tumor activity research are of great significance.The main research aspects were as follow:（1）The antitumor activity of the extracts of different solvents of Dalbergia benthamii Prain was used by MTS method,indicating that the chloroform extracts were exhibited the best inhibitory effect against Hela,with the IC₅₀of 13.17±0.32μg/m L.The chemical component analysis of the extracts were determined by liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry.There were 51 compounds identified from the ethanol extracts,including some active ingredients,such as sanguinarinea,piperine,cephalotaxine,etc.The volatilel oils were extracted by SFE-CO₂,and the difference oil composition was analyzed by GC-MS.A total of 16compounds were analyzed and identified,such as elemicin,Medicarpin,ethyl linoleate,etc.（2）For discovering lead compounds for antitumor activity,97 chemical compounds from Dalbergia benthamii Prain were selected and all related antitumor targets and pathways were predicted and analyzed based on computer virtual screening methods.Robustic acid was found to have the greatest correlation with anti-tumor targets,referring to 116 related tumor targets.Therefore,robustic acid may be related to antitumor activity of Dalbergia benthamii Prain.（3）Robustic acid was isolated from chloroform extracts by Silica gel column sheet gradation and recrystallization,with yield of 3.5%.Through reverse virtual filtering technology and pharmacological experimental verification,robustic acid was proved to have certain anti-tumor effect,with IC₅₀valve of 48.72±1.06 and 28.99±0.81μg/m L to He La and HL-60,respectively.Multiple related anti-tumor targets were related to robustic acid,including Topo I inhibitory activity,antiangiogenic effects,and strong scavenging of free radicals.（4）Based on robustic acid and Topo I,five series of compounds were designed and synthesized by computer-assisted drug design and principle of hybridization.They are including 4-acyloxy derivatives of robustic acid（2a-2j）,alkyl and benzyl derivatives of robustic acid（4a-4l）,robustic acid amine derivatives（6a-6c,7a-7c）,1,2,3,4-tetrazole derivatives of robustic acid（10a-10f,11a-11f）and 1,2,3-triazole derivatives of robustic acid（14a-14e）.45 compounds in total were synthesized,and all the target compounds have confirmed by¹H-NMR,¹³C-NMR,MS and X-ray 44 compounds of them have been unreported.Anti-tumor activities of all compounds in vitro was used by MTS methods.The results were shown that they had some anti-tumor activity,and parts of them were better than robustic acid.Compound 2i was showed the strongest inhibitory activity to HL-60,with the IC₅₀valve of 16.38±0.27μM,Compound 4g was showed the strongest inhibitory activity to He La,with the IC₅₀valve of 30.71±1.36μM.Further studies of pharmacological mechanisms have revealed that this two compound have good inhibitory effect to DNA Topo I at the concentration of 0.1 m M.And they can aslo inhibit tumor growth by inducing apoptosis and regulating cell cycle of tumor cells.That may be one of the mechanisms of the anti-cancer effect of the two compounds.（5）Robustic acid derivatives have linear pyran coumarin structure,which conforms to the characteristics of fluorescent probe,Therefore,their ion recognition capability was used by UV spectrum and fluorescence spectrophotometry method.Compound 4i was shown a single selectivity colorimetric recognition to F^-at emission peak of 421 nm.Fluorescence intensity was gradually increasing by the addition of F^-.Compoud 7b was showed good selectivity to Fe³⁺at emission peak of 437 nm,and fluorescence intensity was gradually increasing by the addition of Fe³⁺.Compouds（10a,11a）aslo were exhibiting perfect Fe³⁺selectivity,and fluorescence intensity was gradually decreasing at fluorescence emission peak of 437 nm and 442 nm respectively.In a word,67 chemical compositions have been identified from Dalbergia benthamii Prain,and the chloroform extracts were exhibited obvious anti-tumor activities.Robustic acid was screened as lead compounds for multi-target anti-cancer drugs through methods of Virtual screening-HPLC analysis-Active verification based on CADD technology.45 robustic acid derivatives were designed and synthesized targeting at Topo I by molecular docking,some of which have high anti-tumor activity.The fluorescence property studies of these compounds have shown that some compounds were highly sensitive and selective to some metal cations or negative ions.