Design, Synthesis And Activity Evaluation Of LAT1-based TBA Antitumor Derivatives (1)

Backgroud:Cancer is one of the maj or diseases which threat human life and health seriously.The cytotoxic agents are still one of the main clinical treatments for the malignant tumor.However,these drugs usually have some severe side effects with poor patients compliance,therefore the discovery of the targeted anti-tumor drugs is of great significance.The L-Type Amino Acid Transporter-1(LAT1)is over expressed in various malignant tumor and posses potent amino acid transport capacity,so it is becoming one of the current research hotspots and an excellent target for the design of targeted anti-tumor drugs.Research purposes:The L-Type Amino Acid Transporter-1(LAT1)is over-expressed in various tumor cells and posses potent amino acid transport capacity.In current study,we decorated the anticancer lead compound TBA with LAT1 amine acid or oligopeptide substrates,in order to improve its antitumor activities,tumor targeting and screen out the effective and secure anti-tumor lead compounds.Research methods:On the basis of our previous studies,the synthesis route of TBA was redesigned.And we prepared a lot of TBA using the new method;A series of TBA amino acids or oligopeptide derivatives BH-X were designed and synthesized,their structures were confirmed using 1H-NMR,13C-NMR and high-resolution mass spectrometer(HRMS);The cytotoxicity of BH-X was evaluated on five cancer cell lines(HepG2,HT-29,Hela,BGC823 and A549)and normal cell line(MDCK)using the standard MTT assay.In addition,the structure-activity relationships of these derivatives were briefly discussed;The anti-tumor mechanism research of TBA,BH-12(TBA sarcosine derivative),BH-26(TBA alanine sarcosine dipeptide derivative)was performed by combination of staining observation(Giemsa,DAPI)and flow cytometric analysis;In this work,the logP values and polar surface area for BH-X were calculated using the Sybyl-X 2.0 software to estimate their druggability preliminarily.Research results:1 A new synthesis route and preparation process of TBA was established and about 20.0g(purity>95%)TBA was prepared using the new method;A novel synthesis route of turning hydroxyl to chloro-substituted was found in the synthesis process of the intermediate 2-(chloromethyl)-3,5,6-trimethylpyrazine.2 27 novel TBA amino acids or oligopeptide derivatives BH-X were designed and synthesized by using TBA as the scaffold;The cytotoxicity detection revealed that most of TBA amino acids derivatives(such as BH-02,BH-09,BH-10,BH-12 et al)and nearly all TBA oligopeptide derivatives showed significantly improved cytotoxicity compared to the starting material TBA.Among the candidates,BH-26 was the most active one,which exhibited perfect antiproliferative activities(mean IC50=2.31±0.78?M)on all tested cancer cell lines.For example,the IC50 values of BH-26 for HT-29,Hela,BGC-823(1.70±0.34?M,1.74±0.99?M,1.79±0.28?M)is much lower than those of the the standard anticancer drug cisplatin(DDP)(4.1±1.17?M,5.60±0.78?M,4.25±0.32?M).Meanwhile these compounds exhibited cytotoxic selective towards MDCK cells and showed high therapeutic index.3 The antitumor mechanism research suggested BH-12 and BH-26 have the similar antitumor mechanism with the starting material TBA.They all could induce HepG2 cell apoptosis and cause HepG2 cell nuclear condensation and fragmentation,chromatin condensation in a dose dependent manner.Further studies suggested that they induced apoptosis through depolarization of the mitochondrial membrane potential and increase of intracellular free Ca2+ concentration.4 The ClogP values calculated using the Sybyl-X 2.0 software showed that most of the derivatives with lower ClogP compared the starting material.Combineing with the result of cytotoxicity assay,we found that the derivatives,which showed better cytotoxicity than the starting material TBA,always possessed larger polar surface area and smaller CLogP.And we thought that the increased antitumor activity may be related to the improved membrane permeability and solubility of the derivatives.Conclusions:According to the cytotoxicity detection and anti-tumor mechanism research of the 27 novel TBA amino acids or oligopeptide derivatives,we found the introduction of LAT1 amine acid or oligopeptide substrates to TBA might contribute to enhance its antitumor activity,cytotoxic selective and solubility.And some pontent anti-tumor lead derivatives(such as BH-26)were found,further research and development of these compounds are currently underway.Structure-activity relationship analysis revealed that the compounds with different substituents seemed to exhibit different antiproliferative activities,such as TBA aliphatic amino acid derivatives showed better antitumor activity in vitro than the aromatic,heterocyclic amino acid derivatives;From the obtained results,it was also observed that the derivatives containing small molecule aliphatic amino acids seemed to be more active than those with larger molecular weight aliphatic amino acids substituents.In addition,it was observed the TBA oligopeptide derivatives seemed to be more active than TBA amine acid derivatives.In brief,LAT1 as a target for targeted antitumor drug design is feasible and significative.And our works paved a new way to develop targeted antitumor drugs and had important practical significance for the research and development of the cytotoxic agents.