Design And Synthesis Of Urushiol-based Ishydroxamic Acid Hdac Inhibitors,Nano-micelles Preparation And Studies On Antitumor Activity

Lacquer is the secretion of lacquer tree,which belongs Chinese characteristic economic forest tree species,the main component of Lacquer is urushiol,which is a kind of alkyl phenolic compound with catechol structure,unsaturated urushiol has good antitumor activity,because of the difficulty of separation of urushiol monomer with different saturation,unstable structure and easy oxidation polymerization,so it limits the development and application of unsaturated urushiol as antitumor drug.HDAC is recognized as an important target for the treatment of cancer at home and abroad,and the discovery and synthesis of HDAC inhibitors have become a hot topic in the research of anti-tumor targeting drugs at home and abroad.However,the study on the structure modification targeting tumor targets and structure-activity relationship of unsaturated urushiol is lack at home and abroad.In this paper,triene urushiol monomers with purity of more than 90%were separated by Ag+coordination resin,and basing on the chemical structure of FDA-approved isohydroxamic acid HDAC inhibitor of SAHA,series of novel urushiol-isohydroxamic acid derivatives were designed,some compounds with good binding effect on HDAC2/HDAC8 were selected by molecular docking and dynamic simulation,and their chemical synthesis was carried out to obtain a series of urushiol-HDAC targeting inhibitor analogues.By in vitro evaluation of its inhibitory activity on HDAC2/HDAC8 and tumor cell inhibitory activity,the structure-activity relationship of synthesized compounds were preliminarily elucidated;and the antitumor mechanism was elucidated by detecting their effect on histone acetylation expression,apoptosis and cycle in tumor cells.Computer software was used to predict the ADMET properties of compounds,to speculate their pharmacokinetic properties and to predict their pharmacogenic potential.By synthesizing amphiphilic block copolymer m PEG-PBAE,the p H-responsive amphiphilic copolymer micelles loaded with urushiol derivatives were prepared,in order to obtain low toxicity and high efficiency nano-targeted antitumor drug preparations.These studies will provide theoretical and applied research basis for the development of unsaturated urushiol as a new HDAC inhibitor and antitumor targeting drug.The main contents and results are as follows:(1)Separation and purification of triene urushiol monomers from unsaturated urushiol by Ag+coordination resinThe Ag⁺coordination resin was prepared with 732-type ion exchange resin loaded with silver ions,the effect of silver nitrate concentration on the adsorption effect of the resin was investigated;the isothermal adsorption curve and adsorption kinetic curve of triene urushiol on the Ag⁺coordination resin were plotted;the stability difference of the formed complexes by different saturated urushiol and silver ions was studied,and the optimum adsorption and elution conditions for the separation and purification of triene urushiol by Ag⁺coordination resin were optimized.The results show that the resin treated with 1 mol/L silver nitrate solution has the best adsorption of triene urushiol in methanol;the isothermal adsorption of triene urushiol on Ag+coordination resin conforms to langmuir equation,which is monolayer adsorption with equation of q-1=0.1999C-1+0.0154;the adsorption kinetic curve conforms to Lagergren rate equation,and the fitting equation is ln51.04/(51.04-qt)=0.0165t.By measuring the stability constant of silver ion complexes with different saturated urushiol,it is shown that the stability constant of silver ion complexes with triene urushiol is obviously higher than that of silver ion complexes with monoene or diene urushiol.The maximum saturated adsorption capacity of Ag⁺coordination resin to triene urushiol is about 12 mg/g in the dynamic adsorption process,under the conditions of sample concentration of 10 mg/m L,10%ethyl acetate methanol solution as eluent,flow rate of 1.5 BV/h,the purity of separated triene urushiol can be increased from 61.24%to 90.58%,and the recovery rate can reach to 85.03%.(2)Design,molecular docking and dynamic simulation of urushiol derivatives as HDAC2/HDAC8 inhibitorsThree series of 30 novel urushiol derivatives were designed as potential HDAC inhibitors,with the introduction of isohydroxamic acid groups into the tail of the alkyl side chain and functional groups with different electron or site resistance into the fat chain or benzene ring of urushiol structure.The molecular docking and dynamic simulation situations of designed urushiol derivatives towards HDAC2/HDAC8 were studied.The binding affinity of the designed 30 compounds to HDAC2/HDAC8 was screened by glide docking scoring.10compounds showed good glide scores to HDAC2 from-7.65 to-8.47,and 10 compounds obtained better docking scores to HDAC 8 from-8.18 to-10.23.The forces and binding modes of the compounds with HDAC2/HDAC8 were studied by molecular docking,it is shown that Zn²⁺chelation,hydrogen bonding,and hydrophobic interactions contribute to the high binding affinity of these compounds to HDAC2/HDAC8.Additionally,the molecular dynamics simulations of complex systems formed by compounds with HDAC2/HDAC8 from the aspects of trajectory stability,hydrogen bonding dynamics,HDAC2/HDAC8 conformation flexibility,zinc ion coordination,binding free energy and energy composition,results showed that all complex systems had good stability,with low RMSD values,amino acid residues with low RMSF values,stable hydrogen bonds and Zn²⁺chelation,and low binding free energy values.The results of combining free energy composition analysis showed that van der Waals and electrostatic free energy provide the main contribution to the stability of these complex systems.(3)Chemical synthesis of series urushiol-based isohydroxamic acid derivatives11 urushiol derivatives which showed good molecular docking and dynamic simulation effects and stable binding with HDAC2/HDAC8 were selected to study their chemical synthesis methods.Using triene urushiol as raw material,by etherification of its o-diphenol hydroxyl group and blocking urushiol oxidation polymerization,methylene ether urushiol was obtained,and by Diels-Alder,hydrolysis and condensation reaction,the hydroxamic acid group was introduced at the tail of the side chain of triene urushiol,and by Friedel-Crafts,Schiemann,oxidation,reduction reaction and so on,the functional groups such as F,Cl,amino,sulfonamide,triazole,benzoamide,hydroxyl or nitro group were introduced into the benzene ring,and hydroxyl or carbonyl groups were introduced into the alkyl chain of triene urushiol.All the 11 urushiol-based isohydroxamic acid derivatives were successfully synthesized,they all have not been reported in the literature.And all the structures of synthesized target compounds were confirmed by ¹H NMR?¹³C NMR?ESI-MS and IR.(4)In vitro activity evaluation of urushiol-based isohydroxamic acid derivativesThe inhibitory activity of the 11 urushiol-ishydroxamic acid derivatives on HDAC2 and HDAC8 was tested by kit.The inhibitory activity of compounds on HDAC8 was better than that of HDAC2.And 6 compounds showed HDAC2 inhibitory IC₅₀ values between 82.84 and145.07 n M,6 compounds showed HDAC8 inhibitory IC₅₀ values between 16.22 and 24.62 n M,which all showed better inhibitory activity than positive drug SAHA.Computer software was used to predict the ADMET properties of the 11 compounds,indicating that they all had good pharmacokinetic properties and good proprietary properties.The MTT method was used to evaluate the inhibitory activity of compounds on the proliferation of four kinds tumor cells,results showed that the inhibitory effect on proliferation of MCF-7 cells was the best,the second was on Hela and HCT-116 cells,and the worst-performing was on A549 cells.And 6compounds showed Hela cell inhibitory IC₅₀ values between 2.47 and 24.27?M,6 compounds showed HCT-116 cell inhibitory IC₅₀ values between 12.24 and 25.91?M,and 7 compounds showed MCF-7 cell inhibitory IC₅₀ values between 5.22 and 13.58?M,which all showed better inhibitory activity than positive drug SAHA.The analysis of structure-activity relationship shows that when Cl-,F-,nitro,hydroxyl or formosulfonamide groups are introduced into benzene ring,and the introduction of hydroxyl or carbonyl groups in alkyl chain can all significantly increase the broad-spectrum,HDAC inhibition and anti-tumor biological activity.The order of inhibitory activity of substituents on benzene ring or alkyl chain is Cl->F->hydroxyl>carbonyl>formosulfamide>nitro>amino>triazoles>benzoylamine.West-blotting and flow cytometry were used to detect the effect of compounds on histone acetylation and apoptosis and cycle in tumor cells,to elucidate the anti-tumor mechanism of urushiol derivatives.The results showed that the compounds could significantly induce histone H3 and tubulin acetylation expression,the results of apoptosis and cycle test showed that the compound could obviously induce apoptosis of tumor cells,which mainly blocked the tumor cell cycle in the G1 and S stages.(5)Preparation and in vitro properties of urushiol derivatives / p H-responsive amphiphilic copolymer micellesUsing MPEG-NH₂(Mn=2000 or 5000)and hydrophobic amine monomers(laurylamine or tetradecylamine)with different molecular weights,four kinds of amphiphilic block copolymer poly ethylene glycol-b-poly?-amino ester(m PEG-PBAE)with different molecular weights were synthesized by a one-pot method,they were named m PEG₂₀₀₀-PBAE-C₁₂?m PEG₂₀₀₀-PBAE-C₁₄?m PEG₅₀₀₀-PBAE-C₁₂ and m PEG₅₀₀₀-PBAE-C₁₄ respectively.Their structure and molecular weight were characterized by IR?¹H NMR and GPC.The molecular weights of four copolymers were characterized as 7666,8920,10666 and 11920,respectively.The properties of CMC,particle size,zeta potential and DLC%of different copolymers were investigated,in which the m PEG5000-PBAE-C12 copolymer micelles have suitable particle size,the largest zeta potential value,the best stability,and the largest DLC%.Then by dialysis method urushiol derivative loaded m PEG₅₀₀₀-PBAE-C₁₂ copolymer micelles were prepared.The prescription of drug-loaded copolymer micelles was optimized by star-point design and response surface methodology.The prepared drug-loaded copolymer micelles exhibited a regular spherical structure with uniform size and good dispersion,having a mean 160.1 nm particle size,a DLC value of 23.45%,an EE value of 80.68%,a zeta potential of 33.4 MV and good stability.The p H responsivity and in vitro drug release of the drug-loaded micelles were investigated,the particle size increasing and the release rate of the drug-loaded micelles in buffer solution with p H value of 5.0 were significantly higher than that of buffer solution with p H values of 6.5 or 7.4.The cumulative drug release rate was 98.7%within 72 h under the condition of p H 5.0,which showed obvious p H-responsive drug release.The in vitro antitumor activities of drug-loaded micelles were evaluated by MTT method with HCT-116 and A549tumor cells.The half inhibitory concentration(IC₅₀)were 14.80 and 12.91?M respectively,the in vitro antitumor activity of urushiol derivative loaded copolymer micelles is obviously better than that of free urushiol derivatives.