Design,Synthesis,Biological Evaluation Of Novel Phthalides And Study On The Synthesis Of Amino Polyphenols

In the First half of this thesis,it is about the design,synthesis and inhibiting activities on platelet aggregation of phthalide analogues.It also concerns about the study on cell protection of some selected targets and the preliminary mechanism study of this action.Thrombosis is an important cause of cardiovascular and cerebrovascular diseases,platelets play a major role in thrombosis.Therefore,the research and development for new antiplatelet agents is still one of the focuses for medicinal chemists in various countries.Butylphthalide(NBP),a new chemical entity developed in our own country,has multiple pharmacological effects including antiplatelet aggregation and neuro-protection,and is used clinically for the treatment of stroke.However,NBP bears some drawback properties such as poor water-soluble oil,inadequate pharmacokinetics and low oral bioavailability,which inspired us to design and synthesize some novel analogues.In order to improve the physical and chemical properties,and to increase the bio-activites of butylphthalide as a lead,here we design and synthesize total 141 targets consisting of four series considering the structures of its main metabolites with the help of computer-aided rational drug design.The targets included 16 of phthalide-3-carboxylic acids in Series I,76 of phthalide-3-formamides in Series Ⅱ,35 of phthalide-3-acetamides in Series Ⅲ and 14 of phthalide-3-propanamides in Series Ⅳ,amongst 124 compounds had never been reported in literatures.The structures of all targets were confirmed by ESI-MS,1H NMR and 13C NMR spectral data.The individual compounds were evaluated for inhibition of platelet aggregation in rabbit platelet rich plasma(PRP)in response to adenosine diphosphate(ADP,10 μM)or arachidonic acid(AA,1 mM)using Born’s turbidimetric method.NBP and aspirin(Asp)were used as positive controls.The pharmacological results showed that some of targets displayed inhibitory activities against platelet aggregation in response to ADP or AA,3-acid-typed compounds(Series I)showed the same or better inhibitory activities than NBP against platelet aggregation in response to ADP or AA;In the case of the 3-allkylamides(Series Ⅱ～Ⅳ),T-3c、T-3d、T3g、T-3m、T-4g、T-6b、T-6c、T-6d、T-6k、T-9o and T-10h showed better inhibitory activities than NBP(IC50=4.18±0.46 mM)against platelet aggregation in response to ADP,while T-3h、T-3i、T-6k and T-9o showed inhibitory activities close or equal to NBP(IC50=1.26±0.02 mM)in response to AA.Preliminary structure-activity relationships(SARs)were concluded that:(1)3-carboxylic acids acted well in most cases compared with their 3-alkylcarbamide analogues(Series Ⅱ-Ⅳ)although T-3c,T-3i,T-6k and T-9o still exhibited good activity;The better total atom length of the side chain was between 2～5 atomic units because too long distance would decrease the inhibitory activity;(2)The substituents on the B-ring had a great influence on the activity,the activity was better when with EWGs than that with EDGs whereas-NO2 or-CN could decrease the activity;Substituents on 5-or 6-position would be suitable especially when-Br was introduced.(3)In the case of Series Ⅱ-Ⅳ of alkylamides,amides with ammonia(CONH2)or with the-OH or-NH2 group on the terminal carbon was essential for the activity,whereas further substituted-OH or-NH2 would lead to the decrease of activity.In-depth antiplatelet activity evaluation,four 3-carboxylic acids and one amide(T-2,T-5,T-6-1,T-9,and T-3c)with better in vitro activities were selected to further assay in vivo.The activity of selected targets were performed by adopting tail breaking method compared with butylphthalide or aspirin as the positive controls.After being administered orally once daily for 7 days in mice,it was shown that bleeding time of all the target administration groups were significantly prolonged compared with the control group,it gave comparatively equal bleeding time with positive NBP.The cell protective activity of 19 selected targets was evaluated on African green monkey kidney epithelial cells.The preliminary screening results presented that selected targets showed certain anti H2O2-induced cell injury activity,which was equivalent to the positive NBP.Activity of the targets with-Br/Cl on B ring was better than that of the positive.The mechanism of target T-5 on antioxidant activity was further studied.Pretreated with 50,100 μM of T-5 for 2 h could obviously reduce the oxidative damage of Vero cells induced by H2O2 and increase the cell viability.In addition,T-5 could protect the integrity of mitochondrial membrane and improve the apoptosis induced by H2O2 in Vero cells.T-5 could also reduce the cell ROS,decrease the intracellular MDA content,increase the activity of CAT and the level of GSH,and inhibit the oxidative damage of Vero cells induced by H2O2.Further studies had shown that T5 regulates the expression of Nrf2/HO-l in intracellular antioxidant stress pathway.It could induce antioxidant activity by up regulating the expression of Nrf2 and HO-1.In the Second Half of this thesis,it is about the methodology study on the concise and efficient synthesis of amino polyphenols starting from a multi-functionalized carbasugar derivative---(2R,3S,4S,5S’)-5-hydroxy-2,3,4-tris(phenylmethoxy)-5-[(phenylmethoxy)methyl]-cyclohexanone(cyclohexanone 1).Amino-polyphenols are widely distributed in nature or widely synthesized active molecules.It is pretty difficult to synthesize this kind of relative simple but rather unstable structures due to the multiple amino and/or phenolic hydroxyl groups on their aromatic rings.Because of their potential antioxidation,antitumor and other biological activities,novel synthetic study is still a hot field in the research of synthetic chemistry and medicinal chemistry.Multi-functionalized cyclohexanone 1 is commercially available and used as a key intermediate for the synthesis of Voglibose,a hypoglycemic drug.It is found in our process study of the drug that cyclohexanone 1 is prone to eliminate BnOH via retro-Michael reaction to give(4S,5S)-5-hydroxy-2,4-bis(phenylmethoxy)-5-[(phenylmethoxy)methyl]-2-encyclohexanone(cyclohexenone 2)in high yield.Both of the compounds can be used as the starting materials for our synthesis.Inspired by the easy formation of protected polyphenol 3 via dehydration and aromatization,it is pondered protected amino-polyphenols 4 could be synthesized if the imination with various amines before the aromatization,and the final target could be easily obtained after proper removal of protecting groups.After tuning the reaction conditions,it was eventually found that the ammonium,formed in situ by the addition of catalytic bronsted acid,could smoothly prompt the imination and aromatization to afford compound 4 in high to excellent yield.In our previous study,it was found the ketone 1 could be easily converted to cyclohexenone 2 by the elimination of BnOH under some basic conditions.It was then focused that compound 4 might be prepared in a "one-pot",way from ketone 1.The reaction underwent luckily under the above similar ammonium-catalyzed condition in over 80%yield.This tandem process might proceed via successive elimination of BnOH,imination with amines and then dehydration,or via the imination,elimination and then dehydration.After removal of all the Bn-protecting groups of compound 4 synthesized in the above alternative way using Pd/C-catalyzed hydrogenation,fifteen of the N-substituted amino polyphenols(5a～5o)were cleanly obtained in its salt form for the sake of stabilizing aminopolyphenol structure after filtration of the catalyst.These kind of compounds with multi electron-donating groups on the rings would be uneasily synthesized via other synthetic method.Hence,this is a concise process for the synthesis of amino-polyphenols from the carbasugar derivatives.Additionally,some of the targets(5b,5f,5h and 5n)were preliminarily evaluated the inhibitory activities on oral epidermoid carcinoma cell(KB)and human breast cancer cell(MCF-7)tumor cells using MTT assay.It showed certain inhibitory activity as expected.