Synthesis And Anticancer Activity Evaluation Of The Four Kinds Of Phenylboronic Acid Quaternary Ammonium Salts

Cancer is a serious disease that endangers human health.Chemotherapy is one of the most important treatments for cancer.But chemotherapy brings serious side effects to cancer patients.Therefore,the study of anticancer drugs with high efficiency and low toxic side effects has become a hot spot of cancer treatment.Because positively charged anticancer drugs can combines with negatively charged phosphate on the surface of DNA to prevent DNA formation,damage cell function and inhibit tumor growth.Therefore,The development of positively charged anticancer drugs has been favored by researchers.The positively charged quaternary ammonium liposomes can easily combines with the negatively charged tumor cell membrane through electrostatic binding,which damages the integrity of tumor cell membrane and has certain toxicity to tumor cells.In addition,quaternary ammonium liposomes have no buffering capacity at any pH value.Therefore,they are prone to generate greater cytotoxicity.In this paper,four phenylboronic acid quaternary ammonium salts using strong toxicity to tumor cells of quaternary ammonium salt liposomes and the potential targeting properties of phenoboric acid on tumor cells with high sialic acid expression were designed and synthesized.And their anticancer activities were evaluated.In this paper,four kinds of phenylboronic acid quaternary ammonium salts?PBA-DiC12MA,PBA-DiC14MA,PBA-DiC16MA and PBA-DiC18MA?with different hydrophobic chain lengths were constructed using hexamethylenediamine as starting material.One of amino group was selectively protected by?Boc?₂O.The free amino group wasreactedwith1-chloromethylnaphthaleneand2-methyl-Acylphenylboronic acid?PBA?.Then,the Boc protecting group was removed with trifluoroacetic acid?TFA?.The exposed amino group was reacted with alkane brominated of different lengths to obtain two carbon chain-substituted tertiary amine compounds.Finally,methylation reaction with methyl iodide gave phenylboronic acid quaternary ammonium salts.The structures of target products and some intermediates were characterized by ¹HNMR?¹³CNMR?gCOSY and HSQC.The average particle size,zeta potential and PDI value of four aqueous solutions of phenylboronic acid quaternary ammonium salts were determined by DLS.The results showed that the average particle size of the four kinds of phenylboronic acid quaternary ammonium salts aqueous solutions was200-400 nm.Zeta potential was distributed between+8-+20 mV.The PDI value was between 0.8-1.0,which mean the particle dispersion of the four kinds of phenylboronic acid quaternary ammonium salts solutions was poor.Biological activities of four kinds of phenylboronic acid quaternary ammonium salts were evaluated.1)The method of CCK8 was used to treat cancer cells:HepG2 cells of liver cancer,HeLa cells of cervical cancer,MDA-MB-231 cells of human breast cancer,U87 cells of human glioma,and HGC-27 cells of human chondrosarcoma.And normal cells:human embryonic renal epithelial cell Hek-293T,human umbilical vein endothelial cell HEUVEG,human normal liver cell L-O2 and other cells were evaluated for toxicity.The results showed that the four kinds of phenylboronic acid quaternary ammonium salts can inhibite the survival of cancer cells to a different extent.Especially to the HepG2 cells,four kinds of phenylboronic acid quaternary ammonium salts were showed lower semi-inhibitory concentrations?IC50?,which was 2.65?M,2.68?M,2.21?M,2.28?M in the order of PBA-DiC12MA,PBA-DiC14MA,PBA-DiC16MA,PBA-DiC18MA;However,the toxicity to normal cells is much weaker.The IC50 value of four kinds of phenylboronic acid quaternary ammonium salts on L-O2 cells is about 4 times higher than that on HepG2 cells,which indicating a relatively weak toxicity.2)The results of a single phenylboronic acid or sialic acid pre-incubation competition inhibition experiment showed that the IC50 value of the four kinds of phenylboronic acid quaternary ammonium salts to HepG2 cells increased about 2 times after the cells were incubated with 1 mM phenylboronic acid or sialic acid for 1 h.About doubling,phenylboronic acid or sialic acid pre-incubation reduces the binding ability between four kinds of phenylboronic acid quaternary ammonium salts and sialic acid receptors with high expression on the surface of liver cancer cells.That is,four kinds of phenylboronic acid quaternary ammonium salts have certain targeting properties.3)The positive charge of the four kinds of phenylboronic acid quaternary ammonium salts was blocked by the Mg²⁺concentration of 1 mM which was not toxic to the cells.This maybe the concentration of Mg²⁺was much higher than that of phenylboronic acid quaternary ammonium salts.Which was used to incubate cells,and the binding rate of Mg²⁺to tumor cells is much greater than the binding of quaternary ammonium phenylborate to tumor cells.The results showed that the toxicity of the four kinds of phenylboronic acid quaternary ammonium salts on cancer cells and normal cells was reduced after the coincubation of 1 mM Mg²⁺.That is,the positive charges of the four kinds of phenylboronic acid quaternary ammonium salts have certain toxicity to the cells.4)The morphology of cells incubated with four kinds of phenylboronic acid quaternary ammonium salts was photographed by scanning electron microscope.It can be known that the phenylboronic acid quaternary ammonium salts kills cancer cells by breaking cell membranes.The release experiment of lactate dehydrogenase?LDH?further showed that four kinds of phenylboronic acid quaternary ammonium salts could change the permeability of tumor cell membrane and induce the death of cancer cells.5)Hemolytic activity experiments showed that the hemolytic activity of human erythrocytes incubated with10?M phenylboronic acid quaternary ammonium salts was lower than20%.That is,the side effects of four kinds of phenylboronic acid quaternary ammonium salts to human erythrocytes were very small.6)Serum stability experiments showed that PBA-DiC12MA had the best stability,and the survival rate of cells was lower than 20%after incubation for 24 h with or without serum.PBA-DiC16MA and PBA-DiC18MA have the worst stability.In the presence of serum,PBA-DiC16MA and PBA-DiC18MA are almost completely inactivated after 4 hours.Studies have shown that the four electropositive four kinds of phenylboronic acid quaternary ammonium salts designed and synthesized have higher toxic activity and selectivity to cancer cells.However,they have less toxic side effects to normal cells.Therefore,the research results may have certain reference value for the development of quaternary ammonium salt as anticancer drugs.