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

Article abstract of DOI:10.1016/j.ejmech.2018.04.036

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 fir

Full text of DOI:10.1016/j.ejmech.2018.04.036

Accepted Manuscript
Synthesis, anionophoric activity and apoptosis-inducing bioactivity of benzimidazolyl-
based transmembrane anion transporters
Xi-Hui Yu, Chen-Chen Peng, Xiao-Xiao Sun, Wen-Hua Chen
PII:
S0223-5234(18)30370-2
10.1016/j.ejmech.2018.04.036
EJMECH 10385
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 March 2018
Revised Date: 16 April 2018
Accepted Date: 18 April 2018
Please cite this article as: X.-H. Yu, C.-C. Peng, X.-X. Sun, W.-H. Chen, Synthesis, anionophoric
activity and apoptosis-inducing bioactivity of benzimidazolyl-based transmembrane anion transporters,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.036.
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ACCEPTED MANUSCRIPT
Graphical Abstract
A series of 1,3-bis(benzimidazol-2-yl)benzene derivatives were synthesized and found to
exhibit potent chloride-mediated apoptosis-inducing activity toward the tested cancer cells.

ACCEPTED MANUSCRIPT
Synthesis, anionophoric activity and apoptosis-inducing bioactivity of
benzimidazolyl-based transmembrane anion transporters
Xi-Hui Yu, Chen-Chen Peng, Xiao-Xiao Sun and Wen-Hua Chen*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences,
Southern Medical University, Guangzhou 510515, P. R. China. E-mail: whchen@smu.edu.cn
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
1
obtained from ESI MS, spectrophotometric and H 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 EC values as low as 36
50
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 IC value in the low micromolar range.
5
0
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.
Keywords: Anion transporter; Benzimidazole; Anionophoric activity; Cytotoxicity;
Apoptosis
1

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1
. 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
2

ACCEPTED MANUSCRIPT
lipophilicity [4, 12], configuration [13-15] 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 [20-22] and anion–π interactions [23-25] 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 [20-22]. 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, 29-33]. 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 (Me bimbe)
2
(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 (CF ) -Bimbe and (NO ) -Bimbe,
3
2
2 2
respectively, Fig. 1) [35]. The transport activity increases with the electron-withdrawing
strength of the substituents in the order of NO > CF > F > Cl > Br > H [35]. These findings,
2
3
together with the low molecular weight (310 Da), appropriate lipophilicity (clogP = 4.60) and
3

ACCEPTED MANUSCRIPT
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 –C H donor motifs [36]. Our study has indicated that the
2
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 –C H. This may be achieved by adding a strong
2
electron-withdrawing substituent to the central phenyl subunit in m-Bimbe. Thus, we nitrated
m-Bimbe, (CF ) -Bimbe and (NO ) -Bimbe at the 5-position of the central phenyl subunits to
3
2
2 2
give NO -Bimbe, (NO ) -Bimbe and NO (CF ) -Bimbe, respectively (Fig. 1). For
2
2 3
2
3 2
comparison, we also synthesized the corresponding analogues of (NO ) -Bimbe and
2
3
NO (CF ) -Bimbe, that is, MeO(NO ) -Bimbe and MeO(CF ) -Bimbe having an electron-
2
3 2
2 2
3 2
donating 5-methoxy group.
5
4
6
1
1
'
3
2'
N
N
7
5
'
'
R1
5
2
HN
NH
NH
H
HN
6
'
N N
3
'
4
3
6
4
'
1
R
N
N
R
m-Bimbe: R = H
2
o-Bimbe
F
Cl
Br
2
2
2
2
2
-Bimbe: R = F
-Bimbe: R = Cl
-Bimbe: R = Br
-Bimbe: R = CF
NH
H
HN
N
N
R
R
(
CF
NO
3
2
)
)
3
N
H
N
H
R1 = NO2
NO
(CF
NO
2
2
3
2
2
-Bimbe: R = H,
-Bimbe: R = CF , R = NO2
-Bimbe: R = NO , R = NO2
-Bimbe: R = NO , R = OMe
2
(
-Bimbe: R = NO
2
p-Bimbe
1
NO
2
3
2
2
3
)
)
)
)
3
1
(
2
1
N
MeO(NO
MeO(CF
N
N
1
-Bimbe: R = CF , R = OMe
3
N
N
N
H
Me
2
Bimbe
Imbe
Fig. 1. Structures of m-Bimbe and its derivatives.
4

ACCEPTED MANUSCRIPT
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.
2
. Results and discussion
2
.1 Chemistry
NO
2
NO
2
NO
2
NH
2
2
(i)
(ii)
N
N
N
N
N
+
HOOC
COOH
NH
NH
HN
NH
HN
O
2
N
C
NO
2
-Bimbe
(NO
2
)
3
-Bimbe
NO
2
NO
2
OH
NO
2
(ii)
(iii)
N
MeOOC
COOMe
iv)
OHC
CHO
OHC
CHO
NH
HN
(
F
3
CF
3
NO
2
(CF
3
)
2
-Bimbe
OMe
OMe
OMe
OMe
(v)
(vi)
HO
OH
N
N
MeOOC
COOMe
OHC
CHO
(vii)
NH
HN
NH
2
R1
R1
R1
R1 = NO
= CF
2
: MeO(NO -Bimbe
2
)
2
NH
2
R
1
3
: MeO(CF ) -Bimbe
3
2
Scheme 1. Synthesis of NO -Bimbe, (NO ) -Bimbe, NO (CF ) -Bimbe, MeO(NO ) -Bimbe and
2
2 3
2
3 2
2 2
MeO(CF ) -Bimbe. Reagents and conditions: (i) POCl , reflux; (ii) HNO /H SO , 0 °C to room
3
2
3
3
2
4
temperature; (iii) 4-trifluoromethyl-benzene-1,2-diamine, MeOH, room temperature; (iv) MeI, K CO ,
2
3
acetone, reflux; (v) LiAlH , anhydrous THF, reflux; (vi) PCC, CH Cl ; and (vii) DDQ and dioxane for
4
2
2
MeO(NO ) -Bimbe; MeOH for MeO(CF ) -Bimbe; room temperature.
2
2
3 2
The five new m-Bimbe derivatives, NO -Bimbe, (NO ) -Bimbe, NO (CF ) -Bimbe,
2
2 3
2
3 2
MeO(NO ) -Bimbe and MeO(CF ) -Bimbe, were synthesized according to the route shown in
2
2
3 2
Scheme 1. Thus, condensation of 5-nitro-isophthalic acid with benzene-1,2-diamine afforded
5

ACCEPTED MANUSCRIPT
NO -Bimbe, which was nitrated with HNO /H SO to give (NO ) -Bimbe [35]. Nitration of
2
3
2
4
2 3
benzene-1,3-dicarbaldehyde (to give 5-nitrobenzene-1,3-dicarbaldehyde) [39] and subsequent
condensation with 4-trifluoromethyl-benzene-1,2-diamine afforded NO (CF ) -Bimbe. 5-
2
3 2
Methoxyisophthalaldehyde was prepared starting from 5-methoxy-isophthalic acid dimethyl
ester [40-42], and reacted with 4-nitro-benzene-1,2-diamine or 4-trifluoromethyl-benzene-1,2-
diamine to give MeO(NO ) -Bimbe and MeO(CF ) -Bimbe, respectively. These compounds
2
2
3 2
1
13
were fully characterized on the basis of NMR ( H and C) and ESI MS (LR and HR) data
see supporting information).
.2 Anion recognition
(
2
In principle, the binding of the anions that are to be transported is required for a synthetic
anion transporter to exert its anion transport activity [43]. Therefore, the ability of NO2-
Bimbe, (NO ) -Bimbe, NO (CF ) -Bimbe, MeO(NO ) -Bimbe and MeO(CF ) -Bimbe as
2
3
2
3 2
2 2
3 2
receptors for chloride anions was firstly studied by means of ESI MS. ESI MS spectrometry
has been widely utilized as a rapid, sensitive and effective analytical technique for the
detection of non-covalent species, and the ESI MS results are assumed to reflect the
complexation phenomena in solution [44]. The negative ESI MS spectra of these compounds
+
–
with tetra(n-butyl)ammonium chloride (n-Bu N •Cl ) are shown in Fig. 2 and S20, and
4
+
–
indicate that they are able to form stable 1 : 1 complexes with n-Bu N •Cl in CH CN–H O
4
3
2
+
–
(
9/1, v/v). For example, in the ESI MS spectrum of NO (CF ) -Bimbe with n-Bu N •Cl (Fig.
2 3 2 4
–
2
, bottom), in addition to the ion peak at m/z 490.40 ([NO (CF ) -Bimbe – H] ), an abundant
2 3 2
ion peak at m/z 526.37 was observed. This ion peak is assigned to the 1 : 1 complex of
–
NO (CF ) -Bimbe with chloride anions ([NO (CF ) -Bimbe + Cl] ). The high relative
2
3 2
2
3 2
abundances of the complexes suggest that these compounds exhibit high affinity toward
chloride anions.
6

ACCEPTED MANUSCRIPT
To quantitatively assess the binding ability of NO -Bimbe, (NO ) -Bimbe, NO (CF ) -Bimbe,
2
2 3
2
3 2
MeO(NO ) -Bimbe and MeO(CF ) -Bimbe toward chloride anions, we measured their
2
2
3 2
+
–
association constants (K ’s) with n-Bu N •Cl in CH CN–H O (9/1, v/v) by means of
a
4
3
2
spectrophotometric titrations (Fig. S21, Tables 1 and S1). The results indicate that these
compounds exhibit high binding affinity toward chloride anions. As expected, the derivatives
having a 5-nitro group at the central aromatic ring tend to bind chloride anions more strongly
than the corresponding analogues having no 5-substituents or a 5-methoxy group, suggesting
that the addition of a 5-nitro group tends to increase the binding affinity. This improved
binding ability is considered as a likely consequence of the strong electron-withdrawing
properties of the 5-nitro group that increases the acidity of the –C H and thereby the binding
2
affinity with anions through enhanced CH···anion interactions.
Fig. 2. Negative ESI MS spectra of 4.1 mM of NO -Bimbe (top), (NO ) -Bimbe (middle) and NO (CF ) -
2
2 3
2
3 2
+
–
Bimbe (bottom) with n-Bu N •Cl (41 mM) in CH CN–H O (9/1, v/v).
4
3
2
The involvement of the –C H in the recognition of chloride anions most probably through
2
1
CH···anion interactions, was confirmed by H NMR titrations. As shown in Fig. 3 and S22,
+
–
upon the addition of n-Bu N •Cl , the –C H proton experiences large complexation-induced
4
2
downfield shift (CIS), a typical characteristic of hydrogen-bonding interactions [36]. Under
the same conditions, the –C Hs in NO -Bimbe, (NO ) -Bimbe and NO (CF ) -Bimbe exhibit
2
2
2 3
2
3 2
7

ACCEPTED MANUSCRIPT
larger downfield shifts than those in the corresponding analogues with no 5-substituents or a
5
-methoxy group at the central phenyl subunits, indicative of stronger interactions with
chloride anions. In addition, the -C H protons are also downfield shifted, which may be
4
(6)
ascribed to their intramolecular hydrogen-bonding interactions with the lone pair of the
benzimidazolyl nitrogen atoms, as reported by Tiburcio et al [36].
(a)
(b)
(c)
1
Fig. 3. H NMR (400 MHz, DMSO-d ) of 20 mM of (a) NO (CF ) -Bimbe, (b) (CF ) -Bimbe and (c)
6
2
3 2
3 2
+
–
MeO(CF ) -Bimbe before (bottom) and after (top) the addition of 30 equivalents of n-Bu N •Cl .
3
2
4
2
2
.3 Anion transport activity
.3.1 Anionophoric activity in liposomal models
Anionophoric activity. The ionophoric activity of NO -Bimbe, (NO ) -Bimbe, NO (CF ) -
2
2 3
2
3 2
Bimbe, MeO(NO ) -Bimbe and MeO(CF ) -Bimbe was firstly confirmed by examining their
2
2
3 2
ability to eliminate a pH differential across liposomal membranes derived from egg-yolk L-α-
phosphatidylcholine (EYPC) [20, 45]. The data are shown in Fig. 4a and S23, and indicate
8

ACCEPTED MANUSCRIPT
that these compounds are capable of efficiently discharging the pH differential in a
concentration-dependent fashion.
To gain quantitative insights into the transport efficiency of each compound, we calculated
the initial rate constants (k ’s, Table S2) from each pH discharge profile and analyzed their
in
relationship with the concentrations of each compound by using the Hill equation, k = k +
in
0
n
n
n
50
kmax[compound] /([compound] + EC ). This analysis gives the EC value of each
50
compound that is the effective transporter loading when 50% of the maximum rate (kmax) is
reached, and the Hill coefficient n value that defines the stoichiometry of the transport process
(
Fig. S24) [46]. The results, together with those of m-Bimbe, (NO ) -Bimbe and (CF ) -
2 2 3 2
Bimbe for comparison, are listed in Table 1.
It is clear from Table 1 that NO (CF ) -Bimbe, (NO ) -Bimbe and NO -Bimbe exhibit highly
2
3 2
2 3
2
−
3
efficient transport activity with the EC values being 2.73×10 ~0.49 mol% transporter/lipid
5
0
(or 0.036~6.50 µM under the assay conditions), 13~2370-fold lower than that obtained for m-
Bimbe (EC = 6.47 mol%, or 85.6 µM). NO -Bimbe, NO (CF ) -Bimbe and (NO ) -Bimbe
5
0
2
2
3 2
2 3
exhibit 13-, 26- and 3-fold higher activity than their corresponding analogues without a 5-
nitro group, that is, m-Bimbe, (CF ) -Bimbe and (NO ) -Bimbe, respectively. Replacement of
3
2
2 2
the 5-nitro group in NO (CF ) -Bimbe and (NO ) -Bimbe with a methoxy group (to give
2
3 2
2 3
MeO(CF ) -Bimbe and MeO(NO ) -Bimbe, respectively) led to 40- and 120-fold decrease in
3
2
2 2
the activity, respectively. These results unambiguously reveal the crucial role of an electron-
withdrawing 5-substituent in enhancing the transport efficiency. It is remarkable that
-3
NO (CF ) -Bimbe and (NO ) -Bimbe have very low EC values of 3.76×10 mol% (or 50
2
3 2
2 3
50
-3
nM) and 2.73×10 mol% (or 36 nM), respectively, suggesting that they are very active
transporters. More significantly, both NO (CF ) -Bimbe and (NO ) -Bimbe can still exhibit
2
3 2
2 3
-4
detectable pH discharge activity at the loadings as low as 4.48×10 mol% transporter/lipid (or
9

ACCEPTED MANUSCRIPT
5
.9 nM) (Fig. 4a and S23). Calcein leakage assay indicates that the activity is authentic, not
due to the disruption of the liposomal membranes by these compounds (Fig. S25) [47].
The high ionophoric activity of these compounds is considered as a likely consequence of the
enhanced binding affinity by the 5-nitro group. Because lipophilicity, widely measured by the
logarithm of n-octanol/water partition coefficient P (log P), is one of the major factors that
regulate the activity of an anion transporter [4, 12], we calculated the log P value (clogP) of
each compound (Table 1). The results indicate that the 5-nitro group does not significantly
change the clogP values of the related compound set (for example, m-Bimbe and NO2-
Bimbe), which rules out the possibility that the high activity is due to the alteration in the
lipophilicity [4, 12]. The finding that the EC values tend to decrease with the increase in the
5
0
binding affinity (Table 1), suggests that the transport activity may be largely regulated by the
binding ability of these compounds with chloride anions.
(a)
(b)
Fig. 4. (a) Discharge of a pH gradient across EYPC-based liposomal membranes by NO (CF ) -Bimbe of
2
3 2
varying concentrations. Intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH
.0); Extravesicular conditions: 25 mM HEPES (50 mM NaCl, pH 8.0). λEx 460 nm/λEm 510 nm. (b)
Chloride influx into EYPC vesicles promoted by NO (CF ) -Bimbe of varying concentrations.
7
2
3 2
Intravesicular conditions: 1 mM lucigenin in 25 mM phosphate buffer (225 mM NaNO , pH 7.0);
3
Extravesicular conditions: 25 mM NaCl in 25 mM phosphate buffer (225 mM NaNO , pH 7.0). λ 455
3
Ex
nm/λEm 506 nm. All the experiments were performed in triplicate, and the mean values were taken.
1
0

ACCEPTED MANUSCRIPT
(a)
(b)
-3
Fig. 5. Discharge of a pH gradient by NO (CF ) -Bimbe (1.95×10 mol%) across EYPC-based liposomal
2
3 2
membranes. (a) Measuring conditions for 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 or Cs). The
experiment that was conducted in NaCl media was used as a control. (b) Measuring conditions for 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 = NO , Cl, Br or I). Each profile was obtained by subtracting the background and
3
thereby represents the real increment in the presence of NO (CF ) -Bimbe.
2
3 2
a
Table 1. Association constants (K ’s) and kinetic parameters for pH discharge
a
d
Kinetic parameters
-1
b
c
Compound
K (M )
clogP
a
e
f
n
EC (mol%)
EC (µM)
RA
1
5
0
50
3
3
3
3
3
3
3
3
m-Bimbe
(2.48±0.82)×10
(3.94±1.19)×10
(2.22±0.79)×10
(8.18±0.96)×10
4.60
4.54
4.48
4.42
4.32
6.35
6.29
6.19
1.73±1.33
1.25±0.47
1.38±0.36
1.19±0.18
0.99±0.18
1.11±0.26
1.31±0.06
0.85±0.04
6.47
0.49
85.8
6.50
0.11
0.036
4.24
1.27
0.05
1.99
NO -Bimbe
13
2
-3
-3
(
NO ) -Bimbe
8.19×10
2.73×10
0.32
790
2370
20
2
2
(
NO ) -Bimbe
2
3
MeO(NO ) -Bimbe (1.56±0.46)×10
2
2
-2
-3
(
CF ) -Bimbe
(4.52±0.82)×10
9.59×10
3.76×10
0.15
67
3
2
NO (CF ) -Bimbe (7.53±0.91)×10
1721
43
2
3 2
MeO(CF ) -Bimbe (3.53±0.88)×10
3
2
a
b
c
d
e
The data for m-Bimbe, (NO ) -Bimbe and (CF ) -Bimbe have been reported previously [35].
2
2
3 2
Measured by means of spectrophotometric titrations in CH CN-H O (9/1, v/v).
3
2
Calculated using MarvinSketch (Version 6.1.0, Weighted Model, ChemAxon, MA).
See Fig. 4a and S23 for the detailed assay conditions.
The EC values in µM were calculated by multiplying the concentration of EYPC (1.33 mM) by the EC
50
50
in mol%.
f
RA denotes the transport efficiency of each compound relative to m-Bimbe.
1
1

ACCEPTED MANUSCRIPT
Probable mechanism of action The anion selectivity and the probable mechanism of action of
NO -Bimbe, (NO ) -Bimbe and NO (CF ) -Bimbe were investigated firstly by measuring the
2
2 3
2
3 2
pH discharge activity in the presence of different alkali metal ions or anions. The finding that
the activity is essentially independent of the alkali metal ions (Fig. 5a and S26), whereas
–
–
–
–
varies with the anions in the order of I > NO > Br > Cl (Fig. 5b and S27), suggests that
3
−
−
+
−
the pH gradient decay is a consequence of OH /X antiport and/or H /X symport [48]. The
anion-selective transport activity of NO -Bimbe, (NO ) -Bimbe and NO (CF ) -Bimbe was
2
2 3
2
3 2
confirmed by measuring their ability to mediate the influx of chloride anions into the EYPC-
derived liposomes, using a conventional fluorescence method based on a chloride-sensitive
fluorescent dye, lucigenin [49]. As shown in Fig. 4b and S28, these compounds exhibit
powerful chloride influx activity. Interestingly, the chloride influx was found to be
significantly suppressed when the vesicles entrapped with highly hydrophilic sulfate were
used (Fig. S29), suggesting that anion exchange is a predominant step in the permeation
+
−
process [49]. However, a minor level of H /Cl symport may be also involved, as the chloride
influx was slightly affected when sulfate was present in both internal and external vesicles,
−
2−
under the condition of which Cl /SO4 antiport is not favored (Fig. S30) [34, 35].
In addition, the finding that the pH discharge activity of NO -Bimbe, (NO ) -Bimbe and
2
2 3
NO (CF ) -Bimbe was significantly reduced in liposomes derived from POPC with 30%
2
3 2
cholesterol (Fig. S31), supports a mobile carrier mechanism. This is consistent with the Hill
coefficient n values being around 1.
The above-mentioned observations suggest that NO -Bimbe, (NO ) -Bimbe and NO (CF ) -
2
2 3
2
3 2
Bimbe function as anion-selective, highly effective carriers through a process of anion
exchange with a minor level of proton/anion symport.
1
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2
.3.2 In vitro anionophoric activity
The potent anionophoric activity of these compounds in liposomal models inspired us to study
their anionophoric activity in cells. For comparative purpose, we chose four compounds of
this set, that is, the parent compound m-Bimbe, NO (CF ) -Bimbe having a 5-nitro group at
2
3 2
the central phenyl subunit and trifluoromethyl groups at the benzimidazoloyl subunits, and the
corresponding analogues (CF ) -Bimbe and MeO(CF ) -Bimbe having no 5-substituents or a
3
2
3 2
5
-methoxy group at the central phenyl subunits. Firstly, we utilized the MQAE [N-
(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide] assay to measure the chloride entry
into cells in the presence of those four compounds [5, 6, 9]. MQAE is a cell-permeable,
chloride selective dye and its fluorescence is quenched by the anion. Thus, the fluorescence
quenching of MQAE upon the treatment of cells with m-Bimbe, (CF ) -Bimbe, MeO(CF ) -
3
2
3 2
Bimbe and NO (CF ) -Bimbe can be the direct evidence that these compounds are involved in
2
3 2
the transmembrane transport of chloride anions into the cells [5, 6, 9]. As shown in Fig. 6,
post incubation of either HeLa cervical cancer cells or MCF-7 breast cancer cells with these
compounds led to significant quenching of the MQAE fluorescence, which indicates the
influx of chloride anions in the intracellular matrix facilitated by these compounds.
Interestingly, this in vitro chloride transport activity of these compounds that is expressed as
the fluorescence quenching of MQAE parallels their anion transport efficiency in liposomal
models. It should be noted that at this stage we have no evidences for whether the compounds
themselves or the biological chloride channels/transporters activated by the compounds
mediate the influx of chloride anions into the HeLa or MCF-7 cells.
The in vitro anionophoric activity of m-Bimbe, (CF ) -Bimbe, MeO(CF ) -Bimbe and
3
2
3 2
NO (CF ) -Bimbe in HeLa cell lines, was further studied using vital staining with acridine
2
3 2
orange (AO), a cell-permeable dye [3,4]. When protonated and accumulated in acidic
compartments such as lysosomes, AO exhibits a characteristic orange fluorescence emission,
1
3

ACCEPTED MANUSCRIPT
whereas it emits green fluorescence when the acidic compartments are basified. As shown in
Fig. 7 and S32, when HeLa cells were stained with AO, granular orange fluorescence was
observed in the cytoplasm (Fig. 7a), suggesting that the orange fluorescence is due to acidic
organelles. Treatment of the HeLa cells with these compounds led to disappearance of most of
the orange emission for m-Bimbe and MeO(CF ) -Bimbe (Fig. 7b and 7c), and complete
3
2
disappearance of the orange emission for (CF ) -Bimbe and NO (CF ) -Bimbe (Fig. 7d and
3
2
2
3 2
7
e), respectively. This result suggests that these compounds, in particular NO (CF ) -Bimbe
2 3 2
and (CF ) -Bimbe are able to efficiently basify the acidic organelles. Notably, the basifying
3
2
ability in the order of NO (CF ) -Bimbe > (CF ) -Bimbe > MeO(CF ) -Bimbe > m-Bimbe,
2
3 2
3 2
3 2
parallels their ionophoric activity in liposomal models (Table 1). The proton/chloride symport
observed in the liposomal models, is thought to be responsible for the increase of the internal
–
–
pH although other mechanisms (e.g., Cl /HCO ) may also be involved [3, 4].
3
(a)
(b)
Fig. 6. Normalized fluorescence intensity of MQAE in (a) HeLa and (b) MCF-7 cells incubated with
MQAE (5 mM) for 3.5 h followed by the treatment with m-Bimbe (▼), (CF ) -Bimbe (▲), MeO(CF ) -
3
2
3 2
Bimbe (●) and NO (CF ) -Bimbe (■) of varying concentrations for 2 h. Fluorescence intensity was
2
3 2
recorded by the plate reader at λem = 460 nm (λex = 350 nm), and normalized with respect to the
fluorescence intensity of untreated cells. Each data point represents the mean intensity of three independent
experiments.
1
4

ACCEPTED MANUSCRIPT
(a)
(b)
(c)
(d)
(e)
Fig. 7. Acridine orange staining of HeLa cell lines. (a) Untreated cells (control); (b)-(e) Cells treated with
1
0 µM of m-Bimbe, MeO(CF ) -Bimbe, (CF ) -Bimbe and NO (CF ) -Bimbe for 3 h, respectively.
3 2 3 2 2 3 2
2
.4 Anti-proliferative activity
2
.4.1 Cytotoxicity
As these compounds are able to mediate the transport of chloride anions into live cells and
previous studies have shown that stimulated influx of chloride anions into cells induces cell
death [8, 9, 50], we tested the in vitro cytotoxicity of m-Bimbe and all the derivatives
described in Fig. 1 using a conventional MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay. Initially single-dose screening (50 µM) was carried out to
evaluate the inhibitory activity of each compound toward four solid tumor cell lines, including
HeLa, A549 lung adenocarcinoma, MCF-7 and HepG2 human liver cancer cell lines. The cell
viability was reported as a percentage of control cells (Fig. 8 and Table S3). The IC values
5
0
of those active compounds in the single-dose cytotoxicity tests at 50 µM, are listed in Fig.
S33-S42, Tables 2 and S4-S5. For comparison, the IC values of all the active compounds in
5
0
the single-dose screening toward LO2 human normal liver cells were also measured (Tables 2
and S6). Here the IC values represent the concentration of each compound resulting in 50%
5
0
1
5

ACCEPTED MANUSCRIPT
inhibition in cell growth. Doxorubicin was used as a positive control.
(a)
(b)
(c)
(d)
Fig. 8. Cell viability of m-Bimbe and its derivatives (50 µM) toward (a) HeLa, (b) A549, (c) MCF-7 and (d)
HepG2 cancer cell lines.
Table 2. Cytotoxicity (IC ) of m-Bimbe and its derivatives towards cancerous and normal cells
5
0
IC (µM)
5
0
Compound
HeLa
A549
MCF-7
> 100
HepG2
20.6±7.0
17.3±4.6
9.1±0.2
12.1±2.1
6.6±0.6
11.1±1.3
4.1±0.8
10.7±0.3
8.9±1.0
2.1±0.2
LO2
m-Bimbe
F2-Bimbe
8.5±0.7
11.6±0.5
10.7±1.5
21.1±0.1
8.0±1.5
1.2±0.1
1.9±0.3
8.4±1.2
1.0±0.2
0.5±0.1
38.5±4.0
> 100
19.9±1.0
20.8±3.7
13.7±1.5
17.8±1.2
10.9±0.6
44.9±4.5
3.7±0.2
> 100
Cl -Bimbe
2
23.7±3.7
21.9±0.9
8.9±0.6
13.6±4.2
3.9±0.7
8.1±1.9
18.5±6.1
0.6±0.1
25.9±2.8
9.7±4.2
12.8±0.9
1.2±0.3
9.7±0.5
11.9±0.3
17.1±0.6
0.8±0.1
Br -Bimbe
2
(
CF ) -Bimbe
3
2
(
NO ) -Bimbe
2 3
NO (CF ) -Bimbe
2
3 2
MeO(CF ) -Bimbe
10.1±1.4
26.3±2.0
0.11±0.03
3
2
MeO(NO ) -Bimbe
2
2
Doxorubicin
1
6

ACCEPTED MANUSCRIPT
It is evident that most of the compounds exhibit potent cytotoxicity toward the four cells, and
some of them are even comparable with doxorubicin. Though little selective cytotoxicity for
the cancer cells over the LO2 normal cells was observed, analysis of the cytotoxicity may
afford some preliminary structure-activity relationships, which may provide some useful
guidance for future rational design of m-Bimbe-based anion transporters.
Firstly, m-Bimbe shows much higher inhibitory activity than o-Bime, p-Bimbe, Imbe and
Me Bimbe. This result suggests that attaching two benzimidazolyl groups in the meta-position
2
of a phenyl group and keeping the benzimidazolyl NH motifs are required not only for anion
transport activity [35], but also for cytotoxicity. o-Bime, p-Bimbe, Imbe and Me Bimbe that
2
are inactive as anion transporters [35], are essentially non-cytotoxic. This finding implies that
a compound without anion transport activity exhibits a very low level of cytotoxicity (if there
is any).
Secondly, the effect of the substituents at the benzimidazolyl subunits on the inhibitory
activity varies with the substituents. For example, compared with m-Bimbe, F -Bimbe, Cl -
2
2
Bimbe, Br -Bimbe and (CF ) -Bimbe exhibit better or comparable activity. However, (NO ) -
2
3 2
2 2
Bimbe almost loses its cytotoxic effect. These results suggest that adding electron-
withdrawing substituents onto the benzimidazolyl subunits does not necessarily increase the
cytotoxicity, though such substituents are favorable to the anion transport activity [35].
Among all the substituents, trifluoromethyl group is the most favorable one for the
cytotoxicity.
Thirdly, the m-Bimbe derivatives having a 5-nitro group at the central phenyl subunit, such as
NO (CF ) -Bimbe and (NO ) -Bimbe are much more cytotoxic than their corresponding
2
3 2
2 3
analogues without a 5-nitro group, that is (CF ) -Bimbe and (NO ) -Bimbe, respectively.
3
2
2 2
However, it is unexpected that MeO(CF ) -Bimbe and MeO(NO ) -Bimbe with much lower
3
2
2 2
anion transport activity, also exhibit potent cytotoxicity. This result implies that other
1
7

ACCEPTED MANUSCRIPT
mechanisms besides anion transport may be also responsible for the cytotoxic effects [51].
Overall, the 5-nitrated m-Bimbe derivative having trifluoromethyl groups at the
benzimidazoloyl subunits, that is, NO (CF ) -Bimbe exhibits the most favorable cytotoxicity
2
3 2
(and anion transport activity), and therefore was used for further mechanistic study.
2
.4.2 Effect of chloride anions on cytotoxic effects
It is known that live cells involve a lot of complex ion homeostasis that are responsible for
antitumor activity [5, 6, 8]. As shown in Fig. 6-7 and Table 2, the entry of chloride anions into
live cells can be facilitated in the presence of NO (CF ) -Bimbe, (CF ) -Bimbe, MeO(CF ) -
2
3 2
3 2
3 2
Bimbe or m-Bimbe, and their in vitro anionophoric activity observed in the MQAE assay and
AO staining, parallels their cytotoxicity. These results imply that chloride transport across
cellular membranes may be responsible for the cytotoxic effects. To gain support for this, we
measured the cytotoxicity of NO (CF ) -Bimbe toward HeLa and MCF-7 cell lines in the
2
3 2
presence and the absence of chloride anions. As shown in Fig. 9, NO (CF ) -Bimbe was
2
3 2
found to be more cytotoxic toward both HeLa and MCF-7 cell lines in the presence of
chloride anions than in the absence of chloride anions. This result strongly suggests that
chloride transport plays a crucial role in the cytotoxic effects [5, 6, 8].
(a)
(b)
−
−
Fig. 9. Cell viability of (a) HeLa and (b) MCF-7 cells in the Cl -containing and Cl -free extracellular media
(
HBSS buffer) upon the dose-dependent treatment of NO (CF ) -Bimbe (2−10 µM) for 12 h (mean ± s.d., n
2 3 2
=
4, **P < 0.01). The differences in mean intensities are statistically significant (P < 0.01) according to
one-way analysis of variance (ANOVA).
1
8

ACCEPTED MANUSCRIPT
2
.4.3 Probable mechanism of action for cell death
Literature reports have shown that dysregulation of ion homeostasis, in particular via chloride
influx, can induce cell shrinkage and lead to apoptosis [6, 9, 50]. To gain insight into how
these compounds induce cell death, we used Hoechst 33342 staining to investigate the effect
of NO (CF ) -Bimbe on HeLa cell lines. Hoechst 33342 staining is able to differentiate
2
3 2
apoptosis from other cell death mechanisms through morphological observation [4, 6]. As
shown in Fig. 10, compared with the untreated cells, the cells treated with NO (CF ) -Bimbe
2
3 2
exhibit stronger blue fluorescence, indicative of chromatin condensation, fragmentation and
apoptotic bodies formation. The formation of “bean”-shaped nuclei was also observed. These
are typical features for cell death via an apoptotic process.
To further confirm the apoptotic mechanism of action, we used a JC-1 probe to observe the
loss of mitochondrial membrane potential in HeLa cells, a hallmark of apoptosis [6, 52]. JC-1
probe is a cell-permeable dye the fluorescence of which is sensitive to mitochondrial
membrane potential. As shown in Fig. 11, after the HeLa cells were treated with NO (CF ) -
2
3 2
Bimbe of varying concentrations and then with JC-1, the pixel intensity ratio of the red
fluorescence of JC-1 to the green fluorescence decreases significantly. This is indicative of
the decrease of the mitochondrial membrane potential, and is one of the characteristics for
apoptotic cell death in an early stage.
(a)
(b)
Fig. 10. Hoechst 33342 staining of HeLa cell lines. (a) Untreated (control) cells; (b) cells treated with
NO (CF ) -Bimbe (2.5 µM).
2
3 2
1
9

ACCEPTED MANUSCRIPT
(a)
(b)
(c)
(d)
Fig. 11. JC-1 staining of HeLa cell lines. (a) Untreated cells; (b)-(c) cells treated with NO (CF ) -Bimbe at
2
3 2
the concentrations of 5 µM and 10 µM, respectively; (d) Pixel ratio (red/green) for different concentrations
of NO (CF ) -Bimbe, analyzed by ImageJ. The error bars mean that the ratio of pixel intensities was
2
3 2
calculated from nine different horizons (mean ± s.d., n = 9, ***P < 0.001).
Taken together, the results obtained from Hoechst 33342 and JC-1 staining provide evidences
for the apoptosis-inducing activity of NO (CF ) -Bimbe.
2
3 2
3
. Conclusion
In conclusion, we have synthesized a series of m-Bimbe derivatives and investigated in details
their anion transport activity, anti-proliferative activity and probable mechanism of action.
Firstly, we have demonstrated that adding a strong electron-withdrawing nitro group at the 5-
position of the central phenyl subunits of m-Bimbe and its derivatives can greatly improve the
anion transport efficiency. This effect is considered due to the enhanced CH···anion
1
interactions, as evidenced from ESI MS, spectrophotometric and H NMR titrations. In vitro
anionophoric activity investigated by MQAE assay and AO vital staining indicates that these
compounds can disturb the homeostasis of chloride anions, modify the intracellular pH and
induce the basification of acidic organelles. We have measured the cytotoxicity of this series
2
0

ACCEPTED MANUSCRIPT
of m-Bimbe derivatives toward four solid tumor cell lines, using the conventional MTT assays.
The results indicate that most of the m-Bimbe derivatives exhibit potent cytotoxicity, and the
5
-nitrated derivative bearing trifluoromethyl groups at the benzimidazoloyl subunits is the
most active with the IC value in the low micromolar range. The findings that the entry of
5
0
chloride anions into live cells, evaluated by use of MQAE assay, parallels the anion transport
efficiency and the cytotoxicity, and these compounds exhibit higher cytotoxicity in the
presence of chloride anions, suggest that the transport of chloride anions across the cellular
membranes plays a critical role in the cytotoxic effects. Preliminary mechanistic study by
using Hoechst 33342 and JC-1 staining suggests that these compounds induce cell death most
probably via an apoptotic process. Hence, an m-Bimbe derivative that has both high anion
transport efficiency and biological activity (for example, disordering ion homeostasis) may be
developed as a potential apoptotic agent for tumor treatment. Further efforts are aimed at
clarifying the detailed mechanism of action of these compounds and creating structurally
optimized benzimidazole-based anion transporters with exceptionally high activity. The
outcomes will be reported in due course.
Supporting information
Experimental procedures and data for the synthesis, structural characterization, anion binding
affinity, transmembrane anion transport activity and biological activity of each compound.
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Research Highlights
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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.