Design,Synthesis And Structure-activity Relationship Of Novel TNBG Derivatives As Antitumor Agent

Tetrazanbigen(TNBG)is a new type of original lipotoxic antitumor compound with good in vitro and in vivo antitumor activities,which contains tetrahydroisoquinoline and quinoxaline structure,and has no cross resistance with other commonly used antitumor drugs.In vitro and in vivo experiments showed that TNBG could activate peroxisome proliferator activated receptor?(PPAR?)and SREBPs in tumor cells,enhanced lipid synthesis,inhibited lipid transport,and induced lipid accumulation in tumor cells,thus leading to"lipotoxicity"of tumor cells.However,the poor water solubility of TNBG limited its further application.ObjectiveIn this thesis,TNBG was selected as the lead compound for structural modification.We expected to discover candidates with good water solubility,good antitumor activity,and improved druggable properties for further study.Methods1.Four novel series of TNBG derivatives were designed and synthesized from TNBG in this thesis.Series I derivatives derived from the introduction of different sizes and electronegative groups into the A-ring or E-ring of TNBG skeleton to investigate their effects on the activity;Series II derivatives derived from the introduction of basic side chains into the C-ring of TNBG to improve water solubility and enhance the activity;Series III derivatives derived from the introduction of carboxyl,amide and basic groups into E-ring of TNBG;The introduction of basic chains into A-ring and C-ring,or C-ring and E-ring of TNBG to obtain series IV derivatives with two basic chains.The effects of the number and position of basic alkyl chains on antitumor activity of TNBG derivatives were investigated.2.In vitro screening of anticancer activities for TNBG derivativesHuman lung cancer cell line A549,colon cancer cell line LOVO,liver cancer cell line Hep G2 and QGY7701 were selected to measure the in vitro antitumor activities of the TNBG derivatives using CCK8 assay.Then the structure-activity relationship was established and discussed.3.In vivo pharmacodynamic study of TNBG derivativesThe growth inhibition of the TNBG derivative 16g on xenograft model bearing A549 cells was measured,and the tumor inhibition rate was calculated.4.Study of the preliminary mechanism of TNBG derivative 16gOil red O staining assay was used to detect the lipid accumulation in cancer cells;Flow cytometry was employed to detect the effects of 16g on cell cycle arrest and apoptosis of tumor cells;Luciferase reporter gene driven by peroxisome proliferator response element PPRE was used to detect the activation of PPAR?of Cos-7 cells;Western blotting technique was used to measure the effects of 16g on the expression of related proteins in cancer cells.5.Pharmacophore model and molecular docking of TNBG derivative16gThe pharmacophore model based on the molecular similarity was constructed by using the Discovery Studio software(2016),and further matched with TNBG derivatives to verify the rationality of the pharmacophore model.Then PPAR?-ligand complex was selected as docking model,and derivative 16g was docked to the protein to study the interaction mode between PPAR?and 16g.Results1.Substituted phenylethylamines were used as the starting material,and the key intermediates were synthesized through five steps reaction,which were cyclized to form series I derivatives.TNBG reacted with chloroalkyl chain through nucleophilic substitution reaction to give series II compounds.The ester derivatives of TNBG were hydrolyzed,acylated and reduced to obtain series III compounds,while series IV derivatives were synthesized by the reaction of the dihydroxy TNBG with chloroalkyls chain under the action of sodium hydride.All the TNBG derivatives were confirmed by NMR and high-resolution mass spectrometry,and the melting point and other physical constants were determined.The structures of some TNBG derivatives were confirmed by X-ray crystal diffraction.2.The in vitro antitumor activity suggested that some target compounds had strong antiproliferation activity on A549 and Hep G2.On the same cell line,the antitumor activity for TNBG derivatives follows the sequence:series IV>series II>series I or series III,which suggested that the introduction of basic side chains into C ring of TNBG are indeed helpful to the improvement of the activity,and the increase of the side chain number in ring A or E is helpful to the activity.In particular,the TNBG derivatives16d and 16g with three carbons side chain are most sensitive to Hep G2 and A549.The IC₅₀values of Hep G2 are 0.42 and 0.54?M respectively,and 0.33 and 0.47?M for A549,respectively,which are10–30 folds higher than that of TNBG and TNBG-5602.The water solubility of 16g is 31.4 mg/m L,which is more than 7850 folds than that of TNBG(4?g/m L).3.The in vivo study of A549 tumor xenograft model in nude mice showed that 16g could effectively inhibit the growth of tumor at the dose of10 mg/kg,and the tumor inhibition rate was 62%.4.Oil red O staining assay showed that derivative 16g exhibit a positive manner on human lung cancer cell line A549,which suggested that lipid accumulation in A549 tumor cells.5.The preliminary mechanism of action showed that TNBG derivative16g could induce apoptosis of in A549 cell line,which may be related to the activation of PPAR?,upregulation of the expression of PPAR?and PTEN protein,and inhibition of AKT phosphorylation.6.The pharmacophore model and molecular docking technologies were used to further verify the activities of compound 16g.The results showed that the introduction of two tertiary amine side chains into the TNBG skeleton was significantly helpful to improve the antitumor activity,which was consistent with the previous experimental results.Molecular docking showed that the derivative 16g could form strong hydrogen bond interactions with PPAR?,and result in the activation of PPAR?to exert biological activities.ConclusionIn this thesis,we optimized the structure of lead compound TNBG and synthesized 44 new compounds.We investigated the in vitro antitumor activities of TNBG derivatives and screened out the derivative 16g with improved water solubility and antitumor activity than that of TNBG.In vivo pharmacodynamics experiment showed that the derivative 16g could effectively inhibit the growth of tumor at the dose of 10 mg/kg,and the tumor inhibition rate was 62%.Oil red O staining assay showed that derivative 16g exhibited a positive manner in A549 cell line,suggesting that the lipid accumulation in tumor cells which has the"lipotoxic"effect on tumor cells.The preliminary mechanism of action study showed that derivative 16g could induce apoptosis of A549 cell line,which may be related to activation of PPAR?,upregulation of expression of PPAR?and PTEN protein,and inhibition of AKT phosphorylation.All these results indicate that 16g is a promising antitumor compound,which is worth of further study.