Synthesis,Transmembrane Anionophoric Activity And Bioactivity Of Squaramide-based Anion Transporters

Anions are ubiquitous in biological systems.Their transport across cell membranes plays a crucial role in maintaining intracellular and extracellular ionic balance.Small-molecule organic compounds with transmembrane anion-transport activity are able to induce cell apoptosis by disrupting the balance of ion concentrations inside and outside tumor cells and therefore have been attracting considerable attentions in the field of new drug discovery.To date,various types of synthetic anion transporters have been reported,including prodigiosin,urea and thioureas,steroids,imidazolium salts and squaramides.Squaramides have rigid skeletons with aromatic four-membered rings and can serve as both hydrogen-bonding donors and acceptors.In this thesis,two types of squaramide-based anion transporers have been designed and synthesized.The first type is tris-(2-aminoethyl)amine-based tripodal squaramide conjugates 1 and 2,and the second type is aza-crown ether-squaramide conjugates 3-6,Compounds 1 and 2 were synthesized as below.First,the reaction of diethylsqu,arate with 3,5-bis(trifluoromethyl)phenylamine or 4-(trifluoromethyl)aniline gave squaramide-based intermediates 3-[[3,5-bis(trifluoromethyl)phenyl]amino]-4-ethoxy-3-cyclo butene-1,2-dione and 3-ethoxy-4-[[4-(trifluoromethyl)phenyl]amino]-3-cyclobutene-1,2-dione,respectively.Then,reaction of these two intermediates with ris-(2-aminoethyl)amine under the catalysis of triethylamine to give compounds 1 and 2.Compounds 3-6 were obtained by the following procedures.The reaction of 1,4,10,13-tetraoxa-7,16-diazacy clooctadecane with different equivalences of N-(2-bromoethyl)phthalimide gave bis(phthalimide)or mono(phthalimide)-substituted aza-crown ethers.Hydrazinolysis of bis(phthalimide)-disubstituted aza-crown ether and subsequent reaction with the above squaramide-based intermediates gave compounds 3 and 4,respectively.The mono(phthalimide)-substituted aza-crown ether was ethylated,hydrazinolized and then reacted with corresponding squaramide-based intermediate to give compounds 5 and 6,respectively.The structures of compounds 1-6 were fully characterized by use of ESI-MS,HR-ESI-MS,1H NMR and 13C NMR.Liposome models based on egg-yolk L-a-phosphatidylcholine(EYPC)were used to assess the transmembrane anion transport activity of compounds 1-6 by means of fluorescence and chloride ion selective electrode techniques.The results showed that compounds 1-6 exhibited potent anionophoric activity.Compounds 1 and 2 exhibited no significant selectivity for the tested monovalent anions(Cl-,Br-,I-and NO3-).In addition,compounds 1 and 2 exhibited a very low level of activity in the presence of sulfate anions,although compounds 1 and 2 were strong sulfate anion receptors.This may be because compounds 1 and 2 are strongly complexed with sulfate anions so that no free host compounds are available for the transportation of chloride anions.This result implies that a powerful receptor for a specific anion may not necessarily be its potent transmembrane transporter.Compounds 3-6 were capable of efficiently mediating transmembrane anion transport via an M+/Cl-symport mechanism.Among them,compounds 3 and 5 with 3,5-bis(trifluoromethyl)phenyl substituents have better anion transport activity than compounds 4 and 6 bearing 4-ffuoromethylphenyl substituents.It has been reported that cation/anion transport is able to trigger apoptosis by promoting the transport of chloride and sodium ions to cells.As compounds 3-6 act as effective cation/anion symporters,we used four solid tumor cell lines to study the antitumor activity and probable antitumor mechanism of compounds 3-6.These conjugates exhibit promising cytotoxicity toward the tested human cancer cell lines.Among them,compounds 3 and 5 are more active as both anion transporters and cytotoxic agents than their corresponding analogues 4 and 6.Mechanistic studies indicated that these compounds were able to alter the intracellular pH,disrupt the cellular homeostasis of chloride and sodium ions,and trigger cell death probably through an apoptotic pathway.The above preliminary studies clarified the correlations between anion binding ability and transmembrane anionophoric activity,transmembrane anion transport activity and antitumor activity of squaramide conjugates.These results are expected to provide some useful guidances for the rational design of effective anion transporters and antitumor agents that exert pharmacological effects their through transmembrane anion transport.