2,6-Bis(benzimidazol-2-yl)pyridine as a potent transmembrane anion transporter

Article abstract of DOI:10.1016/j.bmcl.2016.03.114

2,6-Bis(benzimidazol-2-yl)pyridine was shown to exhibit potent anionophoric activity via a process of both Cl^-/NO₃^- antiport and H⁺/Cl^- symport. This is in sharp contrast to the finding that its corresponding N-methylated analog exhibited negligible activity and reveals the importance of the imidazolyl-NH fragments in the anion-transport process.

Full text of DOI:10.1016/j.bmcl.2016.03.114

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2,6-Bis(benzimidazol-2-yl)pyridine as a potent transmembrane
anion transporter
Chen-Chen Peng a, Zhi Li a, Li-Qun Deng a, Zhuo-Feng Ke b, Wen-Hua Chen a,
⇑
a Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, PR China
b School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
2,6-Bis(benzimidazol-2-yl)pyridine was shown to exhibit potent anionophoric activity via a process of
both ClÀ/NOÀ3 antiport and H+/ClÀ symport. This is in sharp contrast to the finding that its corresponding
N-methylated analog exhibited negligible activity and reveals the importance of the imidazolyl-NH
fragments in the anion-transport process.
Received 9 February 2016
Revised 28 March 2016
Accepted 31 March 2016
Available online xxxx
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Anionophore
Benzimidazole
Anionophoric activity
Ion selectivity
Anions play a central role in biology and transmembrane anion
transport across a cell membrane is crucial for maintaining cellular
functions.1 Uncontrolled transmembrane transport of anions may
lead to serious disorders, notably among which is cystic fibrosis
that originates from the defects in natural chloride ion channels.2
Therefore, during the past decades, increasing interest has been
attracted in identifying small-molecule organic compounds that
are capable of efficiently mediating the transport of anions, in
particular chloride across lipid bilayer membranes.3 By replacing
the missing anionophoric activity of defective anion channels, it
is thought that these compounds have high potentials as
chemotherapeutic agents for channelopathies and cancers.4 To
date, various non-peptidic structures have been reported to exhibit
promising anion transporting properties.3
Albeit successful to date, some of anion-transporting molecules
have potential disadvantages. In particular, their molecular
weights and lipophilicity may be too high to be drug-like.5 Accord-
ingly, it is highly challenging to develop small-molecule organic
compounds that have high anionophoric activity and meanwhile
fall within rules of thumb, such as Lipinski’s rule of five.6 To this
end, imidazoles and benzimidazoles are an attractive class of
molecular scaffolds for the creation of anion transporters,7 because
of their biocompatibility and ability to form complexes with
anions, in particular through hydrogen bonding.7b,8 While the
or (benz)imidazolium groups into the structures of transport
carriers enhances the anion affinity and thereby transport
activity. For example, Schmitzer et al. have reported a series of
imidazolium-based anion transporters,3c,7b–h some of which
exhibit potent antibacterial activity.7d
In the work reported herein, we sought to identify drug-like
anion transporters containing benzimidazolyl groups. As such,
we reason that 2,6-bis(benzimidazol-2-yl)pyridine (Bimpy,
Fig. 1), because of its appropriate lipophilicity (clogP = 4.6)9 and
the ability of the two benzimidazolyl groups to cooperatively act
on bound anions most probably through hydrogen bonding (vide
infra),10 would act as a potent anion transporter.11 To evaluate
the role of the imidazolyl NH fragments in the ion-transporting
process, Bimpy was methylated to afford 2,6-bis(N-methylbenzim-
idazol-2-yl)pyridine (Me2bimpy).12 Herein we describe the
detailed investigation into the anionophoric activity of Bimpy
and Me2bimpy by means of pyranine and lucigenin assays.
As discussed in literature, a precondition for anionophoric activ-
ity of a transporter is its ability to bind the anions that are to be
transported.13 Therefore, the potential of Bimpy as a receptor for
halides was studied virtually. It is clear from Figure 2 that Bimpy
is able to form complexes with halides through hydrogen-bonding
interaction,10 whereas no such interaction was observed between
Me2bimpy and halides (Fig. S2). To verify this, we measured the
association constants of Bimpy and Me2bimpy with halides and
nitrate by means of spectrophometric titrations (Figs. S3 and S4
and Table 1). The results indicate that Bimpy exhibits up to 22-fold
higher binding constants than Me2bimpy.
imidazole ring affords
a
wealth of biophysical-related
applications, it is reported that incorporation of (benz)imidazolyl
⇑
Corresponding author.
http://dx.doi.org/10.1016/j.bmcl.2016.03.114
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Peng, C.-C.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.114

2
C.-C. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Association constants (Ka’s, MÀ1) of Bimpy and Me2bimpy with tetrabutylammonium
Bimpy: R = H
Me2bimpy: R = Me
N
N
saltsa
Anion
N
NR
RN
Ka
b
Bimpy
Me2bimpy
RA
Figure 1. Structures of Bimpy and Me2bimpy.
ClÀ
BrÀ
IÀ
(8.71 3.44) Â 103
(3.04 0.36) Â 103
(1.57 0.06) Â 103
(4.90 1.19) Â 103
(4.00 1.08) Â 102
(5.03 1.65) Â 102
(7.07 5.23) Â 102
(7.23 3.32) Â 102
22
6.0
2.2
6.8
NOÀ3
The ionophoric activity of Bimpy and Me2bimpy was firstly con-
firmed by examining their ability to eliminate a pH differential
across liposomal membranes derived from egg-yolk -phos-
a
Measured by means of spectrophotometric titrations in a mixture of acetonitrile
L-a
and water (1:1, v/v) at room temperature.
phatidylcholine (EYPC).14 For this purpose, a pH sensitive dye,
pyranine is loaded within large unilamellar vesicles (100 nm diam-
eter, extrusion) and used as a fluorescence-responsive reporter of
pH changes within the vesicle interior. The data are shown in
Figures 3 and S5 and indicate that Bimpy is active in efficiently induc-
ing pH discharge, whereas Me2bimpy exhibits negligible activity.
Because pH discharge experiments cannot provide clear clues
for clarifying the transporting mechanism of action,15 we directly
detected the transport of chloride across EYPC bilayer membranes
by using a conventional fluorescence method based on a chloride-
sensitive fluorescent dye, lucigenin.16 It is known that the fluores-
cence of lucigenin is quantitatively quenched by chloride and
insensitive to nitrate, phosphate and sulfate. Thus, any change in
the fluorescence of lucigenin is evidence for the transport of
chloride.17 The data indicate that Bimpy is capable of promoting
chloride influx into the EYPC vesicles (Fig. 4a), whereas no chloride
transport was observed with Me2bimpy (data not shown). Interest-
ingly, the chloride transport by Bimpy was significantly suppressed
when the internal nitrate was replaced with highly hydrophilic sul-
fate (Fig. 4b), implying that ClÀ/NOÀ3 antiport is predominant in the
overall transport process.18 It should be noted that H+/ClÀ symport
cannot be excluded as the chloride influx was unaffected when
sulfate was present in both internal and external vesicles under
the condition of which ClÀ/SO42À antiport is not favored (Fig. S6).
RA denotes the relative affinity of Bimpy relative to Me2bimpy.
b
To gain further insight into the probable mechanism of action
and the ion selectivity, we carried out pH discharge experiments
with chloride salts of alkali metal ions (i.e., Li+, Na+, K+, Rb+ and
Cs+) and sodium salts of different anions (i.e., NOÀ3 , ClÀ, BrÀ and
IÀ), respectively.19 It is clear from Figure 5 that the discharge activ-
ity is essentially independent of alkali metal ions, excluding the
alkali metals in the ion permeation process. In contrast, the pH dis-
charge activity varies in the order of IÀ ꢀ NO3À > BrÀ > ClÀ. These
observations imply that the pH gradient decay correlates with
OHÀ/ClÀ antiport and H+/ClÀ symport.
In addition, preliminary study indicates that Bimpy displays
much lower pH discharge activity across lipid membranes derived
from POPC with 30% cholesterol (Fig. 6), indicative of a mobile car-
rier mechanism.20
Taken together, the above-mentioned observations reveal the
importance of the imidazolyl-NH fragments in the ion-transporting
process, and suggest that Bimpy mediates both ClÀ/NO3À antiport
and H+/ClÀ symport. This may be due to the ability of Bimpy to
bind anions to form complexes of appropriate lipophilicity that
are able to diffuse within the membranes. This was evidenced from
the above-discussed spectrophotometric titrations as well as the
Figure 2. Complexes of Bimpy with ClÀ, BrÀ and IÀ. Calculations were performed at the M06-2x/6-311++G⁄⁄/SMD (water) level of theory.
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C.-C. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 3. Discharge of a pH gradient across EYPC-based liposomal membranes in the presence of (a) Bimpy of varying concentrations in molar percent with respect to the
total concentration of EYPC lipid; and (b) 5 mol % of Bimpy and Me2bimpy. Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0); extravesicular
conditions: 25 mM HEPES (50 mM NaCl, pH 8.0). Ex 460 nm; em 510 nm. These and all the after-mentioned experiments were performed in triplicate, and the mean values
were taken.
Figure 4. Chloride influx into EYPC vesicles promoted by (a) Bimpy of varying concentrations in molar percent with respect to the total concentration of EYPC lipid and (b)
5 mol % of Bimpy. Intravesicular conditions for (a): 1 mM lucigenin in 25 mM phosphate buffer (225 mM NaNO3, pH 7.0) and for (b): 1 mM lucigenin in 25 mM phosphate
buffer (225 mM NaNO3 or Na2SO4, pH 7.0). Extravesicular conditions for both (a) and (b): 25 mM NaCl in 25 mM phosphate buffer (225 mM NaNO3, pH 7.0). Ex 455 nm; em
506 nm.
Figure 5. Discharge of a pH gradient by 3.0 mol % of Bimpy across EYPC-based liposomal membranes, under the measuring conditions of (a) internal vesicles: 0.1 mM
pyranine in 25 mM HEPES (50 mM MCl, pH 7.0) and external vesicles: 25 mM HEPES (50 mM MCl, pH 8.0) (M = Li, Na, K, Rb and Cs); and (b) internal vesicles: 0.1 mM
pyranine in 25 mM HEPES (50 mM NaX, pH 7.0) and external vesicles: 25 mM HEPES (50 mM NaX, pH 8.0) (X = NO3, Cl, Br and I). Ex 460 nm; em 510 nm. The experiment that
was conducted in NaCl media and in the absence of Bimpy was used as a control in both cases.
Please cite this article in press as: Peng, C.-C.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.114

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C.-C. Peng et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Supplementary data
Supplementary data (synthesis of Me2bimpy; experimental pro-
cedures for the measurement of ion transporting activity) associ-
ated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmcl.2016.03.114.
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Acknowledgment
This work was supported by the Program for New Century
Excellent Talents in University (NCET-11-0920).
Please cite this article in press as: Peng, C.-C.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.03.114