Effect Of Lipophilic Balance Modification On The Transmembrane Anionophoric Activity Of Functionalized Bis(choloyl) Conjugates

Synthetic anion transporters are designed to mediate the transmembrane transport of anions across phospholipid bilayer membranes,and are thought to have high potentials as chemotherapeutic agents for the treatment of channelopathies and cancers.Because of the amphiphilicity of phospholipid bilayer membranes,lipophilicity has become one of the major structural factors that affect the activity of synthetic anion transporters.In previous studies,we utilized cholic acid as a lipophilic scaffold to construct a family of bis(choloyl)conjugates that are capable of efficiently medicating the transmembrane transport of anions.In order to clarify how lipophilicity is correlated with the effectiveness of bis(choloyl)conjugates,in this dissertation we designed two series of functionalized bis(choloyl)conjugates.The first series is squaramido-finctionalized bis(choloyl)conjugates 1-6,the two squaramido groups of which serve as the additional functional groups and are expected to facilitate the transmembrane transport of anions.The second series is dimeric 3α-hydroxy-7α,12α-diamino-5β-cholan-24-oate conjugate 7 and its five derivatives 8-12.Here,the 7-,and 12-hydroxyl groups of the choloyl skeleton are transformed into amino substituents,which is expected to strengthen the anion-binding affinity and thereby lead to an enhancement in anion transport activity.In both series,alkyl chains of varying legth are introduced to gradually increase the lipophilicity of compounds 1-12.Compounds 1-6 were synthesized as follows.Acylation of cholic acid with L-lys(Boc)-OH and subsequent acylation with p-bis(aminomethy)benzene afforded a bis(choloyl)conjugate.Boc-deprotection by HCl gaved an amino-functionalized bis(choloyl)conjugate.Reaction of this conjugate with excess 3,4-diethoxy-cyclobut-3-ene-1,2-dione in the presence of Et3N,and subsequent reaction with alkylamines(from methyl to n-hexyl)afforded the desired compounds 1-6.Compounds 7-12 were synthesized as follows.First,3a-hydroxy-7a,12a-di[N-(t-butyloxycarbonyl)amino]-5β-cholan-24-oic acid was prepared from cholic acid using literature protocols,that is,via esterification,oxidation,oximation,hydrogenation,Boc-protection and hydrolysis reaction.Then,this modified cholic acid was acylated with p-bis(aminomethyl)benzene or p-bis(alkylaminomethyl)benzene(from methyl to n-pentyl)and Boc-deprotected with trifluoroacetic acid to gave the desired compounds 7-12.Compounds 1-12 were fully characterized on the basis of ESI MS(LR and HR)and NMR(1H and 13C)data.The anionophoric activities and ion selectivity of compounds 1-12 across liposomal membranes derived from egg-yolk phosphatidylcholine(EYPC),were studied by means of chloride ion selective electrode technique and pyranine assay.The data indicate that compounds 1-12 exhibit potent anionophoric activity,presumably via a carrier mechanism.Moreover,compound 1 acts mostly as a H+/Cl-symporter,whereas compounds 2-12 function mainly as anion exchangers.Both series exhibit moderate selectivity with respect to monoanionic ions.Detailed analysis of the effect of lipophilicity on the ion-transport activity indicates that the anionophoric activities of compounds 1-6 change with the lipophilicity in a concentration-dependent fashion.Specifically,at low concentrations lipophilicity has little effect on the ion transport activity.When the concentration increases,the effect of lipophilicity becomes apparent.For the second series,compounds 7-12 shows up to 25-fold higher transport effectiveness than compounds 1-6.It is clear that an optimum logP range exists for the transport effectiveness in term of both k2/K and EC50.This may be rationalized by the fact that enhancing the lipophilicity is favorable for the compounds to partition from aqueous phase into membranes and leads to an increase in the transport activity.However,increasing the lipophilicity may lower the mobility of the compounds within the lipid membranes and results in a decrease in the activity.The present results highlight the importance of high anionophoric activity in clarifying the effect of lipophilicity on anion transport effectiveness.Furthermore,lipophilic/hydrophilic balance plays a crucial role in the efficient transport of anions.Further study aimed at creating more effective anion transporters,guided by the present observations,will be conducted in our laboratory.