The Synthesis Of 1,3-Diyne Natural Productsd And Anti-depressant Activity Research

Rationales:Traditional Chinese medicine is an important source of lead compounds,and there are numerous natural products in the plant with different structures and various bioactivities.It is considered to be an effective way of finding innovative lead compounds.In our group previous work,(2Z,8E,10E)-pentadecane-2,8,10-trien-4,6-diyn-1-ol(RB-2)was isolated from classical antidepressant prescription xiaoyaosan through separation method under the guidance of actitivity.The results of preliminary pharmacological activity test showed that RB-2 can inhibit monoamine neurotransmitter reuptake of rat brain synaptosomes at 30 μM level.Meanwhile,the compound at this concentration is without obvious cytotoxicity.But,so far,the effect of polyacetyl compounds on depression has not been reported.Therefore,RB-2 is a leading compound of antipressants drugs with novel skeleton containing 1,3-diyne function groups.However,there is still two question needed to be answered.Does the RB-2 have anti-deressive activity in the organism;what is the relationship between the chemical space of 1,3-diyne skeleton and anti-depessive activity.what kind of mechanism of exhibiting their anti-depressive activity through.We may lay a good theoretical basis for the new skeleton of antidepressant innovative drug research.However,due to the low content of the RB-2 in the plant,further studies on the antidepressant activity of this compound are limited.Objective: The total synthesis of RB-2 and its analogues bupleurynol;The derivants were prepared by structural modification of RB-2;the neuron protection activity of RB-2 and it’s derivatives were detected;the relationship between 1,3-diyne compound and nerve cell protective activity was summarized;we further investigated the protective mechanism of 1,3-diyne compound on CORT-induced PC12 injury,meanwhile we explore the activity of compound F07 on GABA receptor;Finally,the antipressant activity of compound F07 was evaluated by animal model.Methods: 1.Based on the RB-2 and bupleurynol structure,retrosynthetic analysis for total syntheses RB-2 and bupleurynol was carried out.Using Scifinder software for RB-2 synthesis route design,the basic skeleton of RB-2 and bupleurynol was constructed through the sonogashira cross-coupling reaction,cadiot-chodkiewitz coupling reaction and a one-pot J-K olefination.Eventually,the synthesis of 1,3-diyne natural product RB-2 and bupleurynol was completed.2.On the basis of the above RB-2 and bupleurynol synthesis route,the derivatives of RB-2 were designed and synthesized.Firstly,the left part of diyne in the RB-2 structure was replaced with other terminal alkynes and a series of RB-2 analogues were synthesized.The cell based biological test indicated that compound F07 has significantly neuroprotective activity.The essential functional groups of compound F07 were further modified.3.The neuronal protective activity of RB-2 and its derivatives was determined by using CORT-induced PC12 cells as a model.The structure-activity relationship between 1,3-diyne compounds and neuronal protective activity was summarized based on the results of screening of neuronal protective activity.The relationship between the chemical space and the antidepressant activity of these skeletons was explored as well.4.Compound F07 as the representative was employed to study nerve cell protection mechanism of 1,3-diyne compounds.The expression of apoptosis-related proteins was studied through western blot test.The patch-clamp technique was employed to study the effect on GABAA receptor of compound F07.5.Pharmacodynamics study of in vivo antidepressant activity of compound F07.To evaluate the antidepressant activity of compound F07 in mice by forced swimming depression model.Result: 1.The basic skeleton of RB-2 and bupleurynol was constructed by Sonogashira cross-coupling reaction,cadiot-chodkiewitz coupling and a one-pot Julia-Kecieneski alkenylation.Total syntheses of RB-2 and bupleurynol have been achieved in 12 steps with 26% and 22% yield,respectively.2.Sixteen RB-2 analogs were synthesized based on the RB-2 structure.The nerve-cell protective activity of RB-2 and its analogues were screened with CORT induced PC12 cell model.Five analogues with PC12 cell protective activity were obtained,and the structure-activity relationship between the 1,3-diyne compounds and protective activity of neuronal cell was summarized.3.In the study of the mechanism of the nerve-cell protective effect of compound F07,the expression of pro-apoptotic protein bax,caspase-3 and cytochrome c was up-regulated in PC12 cell,and the anti-apoptotic protein was Bcl-2 protein was down-regulated in CORT-induced model group.While,the expression of anti-apoptotic Bcl-2 protein was up-regulated in thegroup treated with compound 4g,the expression of pro-apoptotic protein Bax,Caspase-3,cytochrome c was down-regulated at the same time.The results showed that the neuroprotective activity of 1,3-diyne compounds was mainly through the regulation of expression of apoptosis-related proteins in neurons.4.The effect of compound F07 on the GABAA was determined through the regulation of current mediated by GABAA receptor in rat hippocampal neurons.The results showed that compound F07 could increase the amplitude of the minimal inhibitory postsynaptic curreents significantly and at 10 μM level shorten their initial time to produce agonistic activity against GABAA receptor.5.In the evalution of the in vivo antidepressant activity of the compound F07,the administration group was divided into three groups in dosage of high,middle and low.The dosage was 0.0125 mg/kg,0.025 mg/kg,0.05 mg/kg.The forced swimming test results in depressed mice showed the three dose group could shorten the immobile time significantly(P < 0.01).The results showed that F07 has significant antidepressant activity in animal level.Conclusion: Total synthesis of 1,3-diyne natural product RB-2 and bupleurynol were completed;the synthesis of sixteen RB-2 analogues was completed and their neuroprotective activity was determined.The structure-activity relationship between 1,3-diyne compounds and neuronal protective activity was summarized.Further study on the mechanism of neuronal protective activity of 1,3-diyne compounds,and the results showed that compound F07 mainly through activation of GABAA receptor and regulation of apoptosis related protein level.Finally,the mouce forced swimming test proved that the compound F07 has significant antidepressant activity.