Design And Synthesis Of Calixarene Compounds And Their Application In Drug Carrier And Anti-tumor Drug Research

Calixarenes,generated by the condensation reaction of para-tert-butyl phenol with formaldehyde,are an extremely versatile class of macromolecules due to their superior geometric shapes,excellent flexibility,improved conformational mobility,easily modification and no immune response in vivo.As the third generation of supramolecular compounds,calixarene could be functionalized by some groups with good water solubility or biological activity on the upper or lower rim to obtain the derivatives that can be used as the carrier for poorly water soluble drugs and anti tumor drugs.Based on the above research significance,calixarene-based derivatives have gradually become the focus of research in biomedical field.This paper is proposed to design and synthesize series of carboxylic acid,polyhydroxyamine and amide derivatives by functionally modification of the phenolic hydroxyl groups on the lower rim of tert-butyl calix[n]arene and tert-butyl dihomooxacalix[4]arene,investigate the drug loading and release behavior,evaluate antitumor activity via in vitro tumor cell toxic effects,reveal the activity relationship and discuss the possible mechanisms of actions.The Quantum calculation and molecular docking are also used to study their interaction mode.The results will contribute greatly to the research and development of more potential supramolecuar anti-tumor agents and drug delivery systems.The main research works in this dissertation are divided into the following five parts:1.Design and synthesis of functional calixarene derivativesThe p-tert-butyl calix[n]arenes were afforded by the condensation of p-tert butyl phenol with formaldehyde using the one-step synthesis method.As the raw materials,p-tert-butylcalix[n]arene(n = 4,6,8)were further derived through reverse F-C reaction,esterification and hydrolysis to afford the calixarene carboxylic acid derivatives,which were applied for the inclusion of paclitaxel and the construction of nano-carriers.On the basis of literatures reviews and principles of drug design,calixarene polyhydroxy amide derivatives were designed as anti-tumor drugs by changing the cavity size and introducing hydroxyl amino groups with different chains length.Thirteen novel Calix[n]arene polyhydroxy amine derivatives 3a-3m were obtained by esterification and ammonia hydrolysis with p-tert-butyl calix[n]arene(n = 4,6,8).Structures of all the compounds were further successfully identified by IR,1H NMR,13C NMR and HR-MS analysis.The single crystal of 3a was developed and detected by single crystal diffraction.Taking the calix[4]arene ethanolamine derivative 3a as the lead compound and using the principles of drug design,all the dihomooxacalix[4]arene-based amide derivatives were successfully designed by replacing one CH2 group with the CH2OCH2 group on the bridge,followed by introducing the active groups on the lower rim of the benzene rings.By controlling the reaction conditions,mono,1,3-disubstituted acids and esters intermediates were afforded through esterification and hydrolysis,which further reacted with ethanol amine,aromatic amine,or amino methyl pyridine to obtain CA series,DA series and DA2 series dihomooxacalixarene[4]amide derivatives.EA series of 2,3-ethylene-bridged dihomooxacalix[4]arenes were selectively synthesized by direct O-alkylation of p-tert-butyldihomooxacalix[4]arene with excess of 1,2-dibromoethane in controlled basic systems..Noted that all their structures have been verified using by IR,1H NMR,13C NMR and HR-MS.The crystal of CA-11,EA-1,EA-2 and EA-3 were successfully obtained and their configuration were determined by single crystal diffraction.2.Study on the inclusion of calixarene carboxylic acid derivatives and drug loading behavior of nano-carriers for PaclitaxelPaclitaxel is one of the best antitumor drugs with definite therapeutic effect on breast cancer and ovarian cancer.However,its water solubility is very poor(less than 0.03 mg.mL-1).The inclusion and release of calix[8]arene carboxylic derivative C8OCA to the paclitaxel are explored by UV and fluorescence spectrometry methods,in which the influence of the molar ratio of the concentration,temperature are also discussed.The results indicate that the stable PTX-C8OCA inclusion complex could be formed under certain conditions.Two types of amphoteric calix[n]arene carboxylic acid C6HCA and C8OCA nanoparticles(NPs)were successfully prepared using the dialysis method.CnCA NPs had regular spherical shapes with an average diameter of 180-220 nm and possessed negative charges of greater than-30 mV.C6HCA and C8OCA NPs were stable in 4.5%bovine serum albumin solutions and buffers(pH 5-9)with a low critical aggregation concentration value of 5.7 mg·L-1 and 4.0 mg L-1,respectively.C6HCA NPs and C8OCA NPs exhibited good paclitaxel(PTX)loading capacity,with drug loading contents of 7.5%and 8.3%,respectively.The overall in vitro release behavior of PTX from the CnCANPs was sustained,and C8OCANPs had a slower release rate compared with C6HCANPs.These favorable properties of CnCA NPs make them promising nano-carriers for tumor targeted drug delivery.3.Preliminary evaluation of calix[n]arene polyhydroxyamine derivatives on antitumor activity in vitroCalixarene-based compounds are highly effective therapeutic agents against cancer.By performing cancer cell growth inhibition assays,the antitumor ability to induce cytotoxicity in six cancer cell lines(A549 cells,SKOV3 cells,SW1990 cells,HeLa cells,Raji cells,MDA-MB-231)were studied on the 3a-3m.The structure-activity relationship was also summarized.The results demonstrated that 3a-3m had different inhibitory activity,among of which,calix[4]arene polyhydroxyl amine derivatives 3a-3d had the best anti-tumor activity.3a-3d achieved IC50 values ranging from 1.6 ?M to 11.3 ?M.Among the different compounds,3a and 3b exerted the strongest cytotoxic effect in inhibiting the growth of SKOV3 cells.In relation to the underlying mechanisms of cytotoxic effects,cell cycle analysis revealed that the exposure of SKOV3 cells to 3a induced cell cycle arrest in the G0/G1 phase,suggesting a reduction in DNA synthesis.Immunofluorescent staining indicated that the protein expression levels of caspase-3 and p53 in cells significantly increased,whereas that of Bc1-2 was effectively suppressed.Meanwhile,no significant changes in Bax were observed in SKOV3 cells.In addition,the molecular docking of the target compound 3a was studied to provide the foundation for further design and structure optimization of the calixarene-based antitumor drugs.4.Anti-tumor activity evaluation based on the dihomooxacalix[4]arene amide derivativesBy performing cancer cell growth inhibition assays,the abilities of dihomooxacalix[4]arene amide derivatives to induce cytotoxicity in tumor cells were evaluated and the corresponding IC50 values were measured.In relation to the underlying mechanisms of cytotoxic effects,the key indicators including the cell cycle and apoptosis were observed by the flow cytometry analysis of CA-10 in MCF-7 cell.The results of subsequent cell cytotoxicity experiments have shown that they had varying degrees of inhibition on tumor cells,which were probably affected by the polarity and hydrophilicity adjacent to the acyl amino group at the lower rim of the rings.Derivatives CA-10 had the best inhibition on A549 cells,MCF-7 cells,HeLa cells and HepG2 cells with IC50 values ranging from 2.1?M to 9.6 ?M.The results of the flow cytometry revealed that CA-10 can induce the MCF-7 cell cycle arrest in G0/G1 phase.On the other hand,CA-10 simultaneously showed little damage to the normal cells.The antitumor mechanism of dihomooxacalix[4]arene amide derivatives will be further explored to lay the foundation on the research of calixarene-based antitumor agents in the future.5.Quantum chemical calculation and molecular docking of calixarene-based supramolecula compoundsThe suitability of carboxilic substituted calix[n]arenes(n = 6,8)as drug delivery agents for Paclitaxel(PTX)has been analyzed by means of DFT theoretical calculations.The calculation results showed that the electrostatic interaction between host guest molecules and the noncovalent interactions include hydrogen bonding as well as van der waals force,which made the carboxilic substituted calix[8]arene would be the potential paclitaxel carrier.VEGFR-2 signaling pathway mediated by VEGF plays an important role in the formation of tumor vessels.SurFlex-docking is employed to investigate the possible interaction mode between calixaren-based compounds and VEGFR2 enzyme.The molecular docking results indicated that compound 3a could be located in the region of the ATP binding site of VEGF-R2 and the van der waals force Fan was dominant.In addition,the possible interaction between the modified dihomooxacalix[4]arene derivatives and VEGF-R2 were also studied.The results showed that DA2-4 had the highest score value and could be well integrated with VEGF-R2 receptor,which would encourage us to conduct the enzyme activity experiments and find potential of calixarene antitumor targeted drugs in the future.