Synthesis, anionophoric activity and apoptosis-inducing bioactivity of benzimidazolyl-based transmembrane anion transporters

Highlights

• A series of 1,3-bis(benzimidazol-2-yl)benzene derivatives were synthesized.

• The anionophoric activity was greatly improved by adding electron-withdrawing groups.

• These derivatives exhibit potent anionophoric activity in liposomal models and cells.

• Most of these derivatives exhibit potent cytotoxicity toward the tested cancer cells.

• These derivatives induce cell death most probably via an apoptotic process.

Abstract

In this paper we show that a series of 1,3-bis(benzimidazol-2-yl)benzene (m-Bimbe) derivatives exhibit excellent performance as transmembrane anion transporters with anticancer activity. The transport efficiency of m-Bimbe and its derivatives has been firstly optimized by adding a strong electron-withdrawing nitro group at the 5-position of the central phenyl subunits to enhance the CH···anion interactions. Evidences for the interactions were obtained from ESI MS, spectrophotometric and ¹H NMR titrations. These compounds exhibit potent anionophoric activity in both liposomal models and live cells. In particular, the 5-nitrated derivatives having nitro or trifluoromethyl groups at the benzimidazoloyl subunits exhibit 2370- and 1721-fold enhanced anionophoric activity with the EC₅₀ values as low as 36 and 50 nM, respectively. These compounds can disturb the cellular homeostasis of chloride anions, modify the intracellular pH and induce the basification of acidic organelles. Most of this series of m-Bimbe derivatives exhibit potent cytotoxicity toward the tested human solid tumor cell lines, and the 5-nitrated derivative bearing trifluoromethyl groups at the benzimidazoloyl subunits is the most active with the IC₅₀ value in the low micromolar range. Mechanistic studies suggest that the transport of chloride anions across the cellular membranes plays a critical role in the cytotoxic effect and these compounds induce cell death probably via an apoptotic process.

Introduction

During the past decades, considerable interest has been attracted in identifying small-molecular anion transporters that are capable of efficiently mediating the transport of anions across lipid membranes [1,2]. By modulating the intracellular pH [3,4], or disrupting the cellular ionic homeostasis in cancer or bacteria cells [5,6], effective anion transporters are able to induce cell death and thereby may serve as a new therapy for cancers and bacterial infections [1,7]. For example, Gale et al. have shown that pyridine diamide-strapped calix [4]pyrroles are able to induce coupled chloride/sodium transport both in liposomal models and cells, and promote cell death by increasing the intracellular concentrations of chloride and sodium ions [8]. Recently, they have further shown that some squaramide-based anion transporters are able to disrupt the autophagy of cells and induce their apoptosis by perturbing the cellular chloride concentrations [9]. Talukdar et al. have shown that some bis(sulfonamide) compounds are able to facilitate the transport of chloride anions across cellular membranes and disrupt the ionic homeostasis to impose cell death [6]. Schmitzer et al. have described the strong antibacterial properties of some benzimidazolium-based anion transporters, which is considered as a result of their ability to insert into the cellular membranes and alter the membrane permeability for chloride anions [10]. Such chloride-mediated biological activity has driven forward the high potential applications of synthetic anion transporters in biomedical field [1,7].

On the other hand, some inherent disadvantages of the anion transporters reported to date are also apparent. In particular, the high molecular weights and lipophilicity do not meet the requirements for drug-likeness [11]. Therefore, small-molecular organic compounds that have high anion transport activity and meanwhile fall within the rules of thumb, such as the Lipinski's rule of five, are particularly attractive from the viewpoint of new drug discovery [1,2]. In the endeavor to optimize the efficiency of anion transporters, several strategies, such as lipophilicity [4,12], configuration [[13], [14], [15]] and flexibility [16], have been exploited. Because anions need to be desolvated and stabilized during crossing the hydrophobic phospholipid bilayer membranes [17], non-covalent interactions, such as electrostatic [18,19], hydrogen bonding [[20], [21], [22]] and anion–π interactions [[23], [24], [25]] have been successfully utilized to design powerful anion transporters. Among these interactions, hydrogen bonding formed from conventional OH and NH donors is a predominant one [[20], [21], [22]]. In some examples, anion transport activity has been maximized due to the strong hydrogen-bonding interactions of anions with pre-organized donors [21,22]. Recently, CH is recognized as a soft hydrogen-bond donor and therefore CH···anion interaction may serve as a promising alternative to achieve high binding affinity and efficient transmembrane anion transport [26,27].

In these aspects, because of the ability to recognize anions via hydrogen-bonding interactions [28], imidazolyl or benzimidazolyl-based compounds represent a class of attractive molecular scaffolds and have been demonstrated to exhibit promising anion transport properties with potential bioactivity [10,18,19,[29], [30], [31], [32], [33]]. In our recent studies we have shown that 1,3-bis(benzimidazol-2-yl)benzene (m-Bimbe, Fig. 1) exhibits efficient anion transport activity [34,35]. Preliminary study on the structure-activity relationship, by using m-Bimbe, 1,2-bis(benzimidazol-2-yl)benzene (o-Bimbe), 1,4-bis(benzimidazol-2-yl)benzene (p-Bimbe), 2-phenylbenzimidazole (Imbe) and 1,3-bis(N-methylbenzimidazol-2-yl)benzene (Me₂bimbe) (Fig. 1), indicates that the spatial orientation of the two benzimidazolyl subunits at the central phenyl group greatly affects the anion transport activity and the benzimidazoloyl-NH motifs are required for maintaining such activity [35]. More interestingly, the activity may be greatly improved by adding strong electron-withdrawing substituents, for example, trifluoromethyl and nitro groups onto the benzimidazolyl subunits (to give (CF₃)₂-Bimbe and (NO₂)₂-Bimbe, respectively, Fig. 1) [35]. The transport activity increases with the electron-withdrawing strength of the substituents in the order of NO₂ > CF₃ > F > Cl > Br > H [35]. These findings, together with the low molecular weight (310 Da), appropriate lipophilicity (clogP = 4.60) and ready availability (from commercial sources or the condensation of o-phenylenediamine with isophthalic acid or isophthalaldehyde), has highlighted the potentials of m-Bimbe as a lead compound for the development of potent, drug-like anion transporters.

The objectives of the work described herein are two-fold, that is, to further optimize the anionophoric activity of m-Bimbe and to explore the potentials of m-Bimbe derivatives as anti-cancer agents that function via a mechanism of transmembrane anion transport. The study by Tiburcio et al. has shown that m-Bimbe binds anions via the cooperative interactions with both benzimidazolyl-NH and –C₂H donor motifs [36]. Our study has indicated that the enhanced anion transport activity of m-Bimbe derivatives bearing strong electron-withdrawing substituents at the benzimidazolyl subunits, may be ascribed to the increase in the acidity of the benzimidazolyl-NH motifs caused by the strong electron-withdrawing groups [35]. Therefore, the first objective is to further optimize the anion-binding affinity and transport activity by increasing the acidity of –C₂H. This may be achieved by adding a strong electron-withdrawing substituent to the central phenyl subunit in m-Bimbe. Thus, we nitrated m-Bimbe, (CF₃)₂-Bimbe and (NO₂)₂-Bimbe at the 5-position of the central phenyl subunits to give NO₂-Bimbe, (NO₂)₃-Bimbe and NO₂(CF₃)₂-Bimbe, respectively (Fig. 1). For comparison, we also synthesized the corresponding analogues of (NO₂)₃-Bimbe and NO₂(CF₃)₂-Bimbe, that is, MeO(NO₂)₂-Bimbe and MeO(CF₃)₂-Bimbe having an electron-donating 5-methoxy group.

Secondly, we have shown in our recent study that m-Bimbe and its derivatives exhibit anion transport activity via a process of anion exchange with a minor level of proton/anion symport [34,35], a mechanism of action that is similar to that of prodigiosin, a natural chloride carrier [37]. Considering the strong correlation between the proton/chloride symport transport rates and the in vitro cytotoxic activity of prodigiosin and its analogues [38], we are concerned about (1) whether m-Bimbe and its derivatives depicted in Fig. 1 exhibit anti-proliferative activity toward human solid tumor cells, (2) how the cytotoxicity (if there is any) relates to the anion transport across liposomal and cellular membranes and (3) what the probable mechanism of action is.