Design, Synthesis And Activity Characterization Of Ligustrazine Series Compounds (3)

Traditional Chinese medicine(TCM)therapy emphasizes on global effect caused by all effective compounds,which means that the efficacies were the results of all effective compounds acting on multi-targets and synergies.Based on the classic recipe,to discover multi-effective and low toxicity novel drugs are currently pursued by many researchers.Our design idea is enlightened by ’combination principle’ in drug discovery and ’compatibility principle’ in TCM prescription.Both of the two principles are aim to unite the different pharmacodynamic components to be a whole and to maximize the pharmacological effects.The attempt to apply structure combination to discover more efficient and multi-effective leading compounds from TCM formulations is focused.With the application of structure combination,we had chosen several effective ingredients from ’Chuanxiong-Tianma’ and ’Chuanxiong-Danshen’ to synthesize a series of ligustrazine derivatives with the treatment of migraine and stroke.Similarly,to discover antitumor leading compound,we synthesized several novel tetramethylpyrazine derivatives which were composed of the main active components from Huazhenghuisheng Pian and Shiquandabu Wan.Pharmacological evaluation showed that target compounds exhibited low toxic and multi effective,and more efficient than materials;the effect was comparable with positive control drug.The design idea and results of the experiment reflect that traditional Chinese medicine treating diseases via multi-components,multi-targets and synergies.More importantly,we had discovered more efficient and low toxicity leading compounds by this way.Part of the research have been applied for patents,both national and PCT patents.To further discover leading compound with multi-effect and low toxicity,a series of ligustrazine derivatives were designed and synthesized using several effective ingredients of’Chuanxiong-Danshen’ and ’Chuanxiong-Danggui’ as starting materials.108 ligustrazine derivatives(of which 91 was first synthesized in our laboratory)were synthesized,such as ligustrazine-triterpenoids derivatives,ligustrazine-steroid derivatives and ligustrazine-phenolic acid derivatives.Moverover,108 ligustrazine derivatives and 14 starting materials’ activities and toxicities were systematically evaluated on 9 cell models,for example:neuron research model(CoCl2-induced damage in differentiated PC 12 cells),human cancer cell model(HT－29,Hela,HepG2,Bel7402 and MCF-7 cell lines),as well as normal tissue cells model(MDCK,3T3 and Ehy926 cell lines).Structure-activity relationship(SAR)analysis of these ligustrazine derivatives was also revealed.These findings may provide a new framework for the design of new ligustrazine derivatives as antitumor agents or neuroprotective drugs.Results:(a)Anti-tumor effects of 49 ligustrazine derivatives were screened on five human cancer cell lines;and compounds 4,13,26 had significant cytotoxic activity with IC50<10μM.Some leading compounds’ antitumor effects were even better than the positive control drug-cisplatin(DDP,IC50≈5.5μM).All of the 3 agents’ anti-tumor activity was significantly higher than synthetic material(IC50>20μM).SAR analysis found that ligustrazine-triterpenoids derivatives and ligustrazine-steroid derivatives had different SAR rules.For example,ligustrazine-steroid derivatives’antitumor effect was strengthened with the ligustrazine moiety increase,and approximately followed a tendency in activity 3:1>2:1>1:1.While ligustrazine-triterpenoids derivatives’ antitumor SAR rules were contrary with ligustrazine-steroid derivatives’,1:1 was better than 2:1.In vitro toxic tests showed compound 4,13 were high cytotoxicity on normal cells;while compound 26 could only modest inhibit growth of Ehy926.There were low effects on MDCK and 3T3 cells with IC5o>80μM.Moreover,it was completely inactive on the growth of differentiated PC 12 cells at the concentration of 30μM.Current study displayed that compound 26’s cytotoxicity was related to cell division.Cell division was strong(such as cancer cells),the cytotoxicity effect was obvious,whereas cell division was weak(such as MDCK,3T3 cells)also reduced its lethal effect;so it was no effect on non-dividing cells(NGF-induced PC 12 cells).Compound 26 exhibited selective efficacy and low toxicity.Further mechanism reseach for the anti-cancer activity of three promising compounds 4,13 and 26 in HepG2 cell line were studied by flow cytometric analysis.All of three compounds significantly induced apoptosis in HepG2 cell at low concentrations(<8μM).In addition,compound 26 also induced Gl/S cell cycle arrest,blocked the cells from the G1 phase into the S phase.And HepG2 cell proliferation was relatively inhibited.Antitumor mechanisms’ study displayed that compound 26 exerted its antitumor activity by both inducing apoptosis and cell cycle arrest,which was similar with traditional Chinese medicine’s efficacies acting on multi-targets and synergies.In view of this,we optimized the synthetic process to prepare compound 26 about 5.0g(purity>95%).The acute toxic test showed that LD5S value of 26 exceeded 900 mg/kg via hypodermic injection in mice.Then anticancer efficacy of 26 was evaluated in vivo using murine sarcoma H22 model.Reduction of the tumor weight demonstrated that 26(30mg/kg,hypodermic injection)had promising inhibition effects and a 60.27%inhibitory rate in H22 mice.Moreover,compared with cisplatin,compound 26 did not cause severe toxicity damage,such as the inhibition of liver and spleen index,severe weight loss and other toxic symptoms.Experimental results revealed that 26 had a broad antitumor spectrum activities and low toxicity to normal cells in vitro.And in vivo tests also exhibited that compound 26 had promising antitumor effect with low toxicity.Cumulatively,this study highlights the attempt to apply structure combination to discover more effective,low toxic anti-tumor lead compounds by conjugating anti-tumor ingredients from TCM formulations.Results(b):Protective effect of 60 ligustrazine-phenolic acid derivatives against CoCl2-induced neurotoxicity in differentiated PC 12 cells was evaluated.Compound 78 displayed promising protective effect on the proliferation of the injured PC 12 cells(EC50=0.0796 μM),and it was more active than TMP(EC50=64.459μM).Combining hematoxylin and eosin staining(HE),we found that pretreatment with 78 could dramatically alleviate morphological manifestations of cell damage and lead to a pronounced increase in neurite-bearing cells compared to model groups.It revealed a dose-effect relationship.All of three concentrations of 78 significantly inhibited apoptosis in differentiated PC 12 cell induced by C0C12.Both MTT assay and HE staining revealed that 78 had promising neuroprotective effect and it was worth for further research.Structure-activity relationship analysis of 60 ligustrazine-phenolic acid derivatives displayed that bis-ligustrazine derivatives’(2:1)neuroprotective activities were better than that of the other substituendum-ligustrazine componds(1:1 or 3:1 or 4:1);ether-joined derivatives’protective effects were better than those of the ester ones;the trans double bond group may be favorable for the protective activity;compounds with-OH at the para-position and-OCH3 at ortho-position of the benzene moiety seemed to be more active than those with substituents at other positions in the same series.The above-mentioned 108 TMP derivatives(of which 91 was first synthesized in our laboratory)and 14 precursor materials were biological evaluated in vitro and in vivo.Finally one anti-tumor leading compound(26)and one neuroprotective leading compound(78)were gained.Current study suggest that the attempt to apply structure combination and modem pharmacological screening technology to discover multi-effective and low toxic anti-tumor lead compounds from TCM formulations is viable.This paper enriched the leading compounds’ designing idea that was conjunctive using ’combination principle’ in drug discovery and Compatibility principle’in TCM prescription.This dissertation is an interesting attempt for innovative drug’s discovery.