Fluorinated bisbenzimidazoles: a new class of drug-like anion transporters with chloride-mediated, cell apoptosis-inducing activity

Article abstract of DOI:10.1039/c8ob03036g

Anion transporters have attracted substantial interest due to their ability to induce cell apoptosis by disrupting cellular anion homeostasis. In this paper we describe the synthesis, anion recognition, transmembrane anion transport and cell apoptosis-inducing activity of a series of fluorinated 1,3-bis(benzimidazol-2-yl)benzene derivatives. These compounds were synthesized from the condensation of 1,3-benzenedialdehyde or 5-fluoro-1,3-benzenedialdehyde with the corresponding 1,2-benzenediamines and fully characterized. They are able to form stable complexes with chloride anions, and exhibit potent liposomal and in vitro anionophoric activity. Their anion transport efficiency may be ameliorated by the total number of fluorine atoms, and the enhanced anionophoric activity was a likely consequence of the increased lipophilicity induced by fluorination. Most of these fluorinated bisbenzimidazoles exhibit potent cytotoxicity toward the selected cancer cells. Mechanistic investigations suggest that these compounds are able to trigger cell apoptosis probably by disrupting the homeostasis of chloride anions.

Full text of DOI:10.1039/c8ob03036g

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Fluorinated bisbenzimidazoles: a new class of drug-like anion
transporters with chloride-mediated, cell apoptosis-inducing
activity
Received 00th January 20xx,
Accepted 00th January 20xx
#
#
Xi-Hui Yu , Xiao-Qiao Hong and Wen-Hua Chen*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Anion transporters have attracted substantial interest due to their ability to induce cell apoptosis by disrupting cellular
anion homeostasis. In this paper we describe the synthesis, anion recognition, transmembrane anion transport and cell
apoptosis-inducing activity of a series of fluorinated 1,3-bis(benzimidazol-2-yl)benzene derivatives. These compounds
were synthesized from the condensation of 1,3-benzenedialdehyde or 5-fluoro-1,3-benzenedialdehyde with the
corresponding 1,2-benzenediamines and fully characterized. They are able to form stable complexes with chloride anions,
and exhibit potent liposomal and in vitro anionophoric activity. Their anion transport efficiency may be ameliorated by the
total number of fluorine atoms, and the enhanced anionophoric activity was a likely consequence of the increased
lipophilicity induced by fluorination. Most of these fluorinated bisbenzimidazoles exhibit potent cytotoxicity toward the
selected cancer cells. Mechanistic investigations suggest that these compounds are able to trigger cell apoptosis probably
by disrupting the homeostasis of chloride anions.
1
. Introduction
partitioning, and as a consequence it is able to boost transport rates
22-25
in a number of synthetic systems.
For example, Davis et al have
It is known that cellular anion homeostasis is essential for cells to
exert their biological function and imbalanced anion homeostasis
may result in cell deaths. Among the anionic species in living
systems, chloride anions are the most abundant ones in
reported that perfluorination of the acyl tail in monoacylglycerols
affords synthetic anion transporters with improved chloride
2
2
transport properties.
Gale et al have demonstrated that
1
fluorination of a series of structurally simple urea and thiourea
compounds leads to a significant increase in the transmembrane
extracellular fluid and play a critical role in regulating cell death.
Therefore, it is believed that one compound that is able to disturb
the homeostasis of chloride anions or modify the internal pH of
cells, may be developed as a chemotherapeutic agent for the
2
3
anion transport activity. They have further reported that some
ortho-phenylenediamine bis-ureas having fluorinated central phenyl
ring and/or peripheral phenyl groups, are highly effective anion
transporters and even outperform prodigiosin, the most active
2
,3
treatment of cancers.
During the past decades, this hypothesis
has been attracting substantial interest in identifying
transmembrane anion transporters, in particular for chloride anions.
2
4
anion transporter known to date. In addition, fluorination may
also lead to an alteration in the mechanism of action of ion
transport. For example, Gale et al have shown that calix[4]pyrrole
acts as a CsCl symporter, whereas the octafluoro analogue
3
-11
These endeavours have been further driven forward by the
findings that the transport properties of some anion transporters,
for example, prodigiosin and its derivatives, are linked to their
biological activity as anticancer agents.
diverse approaches have been adopted to optimize the anion
transport and potential biological activity of anion transporters.
2
5
12-20
functions as an anion exchanger. On the other hand, fluorination
is able to produce potent anion transporters by increasing the anion
binding affinity and/or the lipophilicity of the receptors. For
example, Gale et al have shown that fluorination of tripodal
trisureas and tristhioureas significantly improves the anion
transport efficiency both in vesicular models and in vitro, with the
best transporters depolarizing acidic compartments within GLC4
cells, reducing cell viability and inducing apoptosis in a range of
human cancer cell lines, whilst the non-fluorinated analogues have
As a consequence,
Fluorination represents a general strategy to ameliorate the
transport activity and cytotoxicity of small-molecular anion
transporters. Because of the electron-withdrawing properties of
fluorine atoms and the ability to increase the lipophilicity,
fluorination can enhance anion-binding affinity and/or membrane
3
2
1
1
6
a limited effect on cell viability. They have further shown that
even simple monoureas and thioureas containing fluorinated
indoles display promising anticancer activity towards A375
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
2
3
melanoma cells. Recently they have shown that fluorinated anion
transporters, such as squaramides having trifluoromethylphenyl
substituents, are able to trigger cell death via an apoptotic pathway
#
These authors contributed equally to this work.
†
Electronic Supplementary Information (ESI) available: Structural characterization
and experimental data for the anion recognition, anion transport and biological
activity of each compound. See DOI: 10.1039/x0xx00000x
2
6
and disturb the autophagy of cells. These findings have spurred
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great efforts to systematically study the effect of fluorination on the the benzimidazolyl NHs was observed, suggesting that both
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DOI: 10.1039/C8OB03036G
anion transport and biological activity of anion transporters.
benzimidazolyl NHs are simultaneously involved in the interactions
with chloride anions. These results suggest that chloride anions are
bound to the core 1,3-bis(benzimidazol-2-yl)benzene moiety
through the cooperative interactions with the benzimidazolyl NHs
To respond to this as well as to develop small-molecule, drug-like
anion transporters having both potent anion transport and
biological activity, in previous studies we have reported that 1,3-
bis(benzimidazol-2-yl)benzene (Bimbe 1, Fig. 1) functions as an
2
9,31
and the central aromatic H-2’s, as reported in literature.
anion transporter and the activity may be significantly improved by Molar ratio assays indicate that the changes in the chemical shifts
adding electron-withdrawing substituents onto the benzimidazolyl of the benzimidazolyl NHs or the central aromatic H-2’s with the
2
7,28,29
5
subunits and/or the central phenyl moiety.
For example, F - concentrations of each compound, follow a 1 : 1 binding mode with
2
Bimbe 3 (Fig. 1) having a fluorine atom at each terminal chloride anions (Fig. S48-S73). This 1 : 1 stoichiometry was further
benzimidazolyl subunit exhibits ca 10-fold higher anionophoric supported by means of ESI MS spectrometry. As shown in Fig. 2(b)
2
8
activity than Bimbe 1.
and Fig. S74, these compounds are able to form stable 1 : 1
complexes with chloride anions. For example, in the negative ESI
Inspired by these findings, in this study we utilized fluorination as
an alternative means to optimize the activity of Bimbe 1 with the
aim to obtain insightful structure-activity correlations, and
systematically studied the effect of fluorination on the transport
and biological activity. Specifically, we designed and synthesized
two series of fluorinated bis(benzimidazolyl) derivatives, that is,
-Bimbe 2-6 based on Bimbe 1 and
based on 5-fluoro-1,3-bis(benzimidazol-2-yl)benzene (FBimbe, 7)
Fn-Bimbe represents a Bimbe derivative having
totally n fluorine atoms at the m1,m2,…-positions of the terminal
5
,6
MS spectrum of F -FBimbe 12 mixed with TBACl (Fig. 2(b)), in
4
5
,6
–
addition to the ion peak at m/z = 399.66 ([ F -FBimbe – H] ), a new
4
ion peak at m/z = 435.59 was detected. This new ion peak is
5
,6
assignable to the complex of F4-FBimbe 12 with chloride anions
5
,6
–
([
F -FBimbe + Cl] ). Nonlinear fitting analysis of the relationship
4
m1,m2,…
m1,m2,…
between the chemical shift changes of the benzimidazolyl NHs (or
the central aromatic H-2’s) and the concentrations of each
compound, according to a 1 : 1 binding model, affords the
F
n
n
F -FBimbe 8-13
m1,m2,…
(Fig. 1). Here
association constants (K ’s) with chloride anions (Table 1 and Fig.
a
m1,m2,…
S48-S73). The association constants of compounds 1-13 with
benzimidazolyl subunits, whereas
Fn-FBimbe stands for an
chloride anions were also measured in CH CN/H O (9/1, v/v) by
3
2
FBimbe derivative having totally n fluorine atoms at the m1,m2,…-
positions of the terminal benzimidazolyl subunits. Most of these
compounds have drug-like features required by the Lipinski’s Rule
means of spectrophotometric titrations. As shown in Fig. S75,
addition of TBACl to compounds 1-13 led to a small change in the
absorbance, which is indicative of the complex formation. The
changes in the absorbance were analyzed against the
concentrations of each compound, according to a 1 : 1 model, gave
3
0
of Five,
including molecular masses of smaller than 500, two
hydrogen donor/acceptor groups and moderate lipophilicity. We
carried out a systematic investigation into their anion recognition,
anion transport, biological activity and the probable mechanism of
biological action.
the association constants of compounds 1-13 with chloride anions
1
(
Table S1). The association constants obtained from both the H
NMR and spectrophotometric titrations demonstrate that these
compounds show strong binding affinity toward chloride anions.
Notably, compounds 2-6 exhibit very similar binding affinity toward
chloride anions with compound 1, whereas compounds 7-12 show
2
. Results and discussion
Chemistry
The new fluorinated derivatives 2 and 4-13 were synthesized from higher affinity than the corresponding compounds 1-6. This result
the one-step reaction of 1,3-benzenedialdehyde or 5-fluoro-1,3- suggests that fluorination on the central phenyl subunit may have
benzenedialdehyde with the corresponding 1,2-benzenediamines more profound effect on the anion-binding affinity than on the
2
7-29
5
(Scheme 1).
Bimbe 1 and F -Bimbe 3 have been previously benzimidazolyl subunits.
2
2
8,31
reported by us and others.
was prepared from 1-fluoro-3,5-dimethyl-benzene in two steps
according to reported protocols.
fully characterized on the basis of melting points (m.p.), ESI MS (LR
and HR) and NMR ( H and C) data (see experimental section and
SI).
5-Fluoro-1,3-benzenedialdehyde
Liposomal anion transport
3
2
Ion transport efficiency. To evaluate how fluorination ameliorates
the ion transport efficiency of these compounds, we firstly
measured the EC50 value of each compound on liposomal models
formed from egg-yolk L-α-phosphatidylcholine (EYPC), using a
Compounds 2 and 4-13 were
1
13
3
3
conventional pyranine assay (Fig. S76-S77 and Table S2). Table 1
summaries the EC50 and the Hill coefficient n values that were
Anion recognition
It is known that fluorination may enhance the activity of an anion obtained from the Hill analysis of the relationship between the
transporter by increasing the hydrogen-bonding donor acidity and initial rate constants (k ’s) and the concentrations (mol%) of each
in
2
2-25
34
anion-binding affinity.
To evaluate how anion binding compound.
As a consequence, these derivatives, in particular
5
,6
4,5,6,7
contributes to the transport efficiency of these fluorinated
F4-FBimbe 12 and
F8-FBimbe 13 exhibit high pH discharge
compounds, we studied their anion recognition properties toward activity. This result suggests that fluorination of Bimbe 1 may serve
chloride anions as tetra(n-butyl)ammonium salt (TBACl) by means as a practical strategy to improve the ion transport activity. Calcein
1
of H NMR titrations in acetonitrile. As shown in Fig. 2a and Fig. S48- leakage assay (Fig. S78) clearly shows that these compounds do not
3
5
S73, addition of TBACl led to significant downfield shifts of the disrupt the liposomal membranes.
benzimidazolyl NHs and the central aromatic H-2’s, indicative of the
hydrogen-bonding interactions with chloride anions. Only one set of
2
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m1 ,m2,. ..Fn-Bimbe
m1,m2,...Fn-FBimbe
F
F
F
N
7
N
N
F
N
N
F
N
N
N
F
F
N
N
F
F
N
N
4
NH
HN
NH
HN
NH
HN
NH
HN
NH
HN
5
F
F
F
F
6
4F2-Bimbe 2
4F2-FBimbe 8
5F2-FBimbe 9
Bimbe 1
FBimbe 7
F
F
F
N
N
F
N
N
F
F
F
N
F
F
N
N
N
N
NH
HN
NH
HN
NH
F
F
F
F
F
HN
NH
HN
NH
HN
F
F
F
F
5F2-Bimbe 3
4,5F4-Bimbe 4
4,5F4
4,6F4-FBimbe 11
5,6F4-FBimbe 12
-FBimbe 10
F
F
F
F
F
F
N
F
N
N
N
F
N
NH
HN
NH
HN
NH
HN
F
4,6F4-Bimbe 5
5,6F4-Bimbe 6
F
F
F
F
F
F
F
4,5,6,7F8-FBimbe 13
Fig. 1. Structures of Bimbe 1 and the fluorinated derivatives 2-13.
R4
R5
R6
NH2
NH2
(i)
R4
R5
N
N
R4
R6
4F 2-Bimbe 2 (32%)
+
4,5
NH
HN
R5
F 2-Bimbe 4 (80%)
OHC
CHO
4,6
F -Bimbe 5 (38%)
R7
F
4
R6
R7
R7
5,6
F -Bimbe 6 (37%)
4
m1,m2,... Fn-Bimbe
F
F
(ii)~(iii)
FB imb e 7 (49%)
F2-FBimbe 8 (32%)
F2-FBimbe 9 (35%)
F4-FBimbe 10 (24%)
F4-FBimbe 11 (42%)
F4-FBimbe 12 (35%)
F8-FBimbe 13 (58%)
4
6
1
1%
H3C
CH3
OHC
CHO
(
Two steps)
R4
R4
(
i)
N
N
4
,5
R4
NH
HN
R5
R5
4,6
5,6
R5
R6
NH2
NH2
R6
R7
R7
R6
4,5,6,7
m1,m2,. ..Fn-FBimbe
R7
3
Scheme 1. Synthesis of fluorinated Bimbe derivatives 2 and 4-13. Reagents and conditions: (i) DMSO (or CH CN), room temperature-reflux;
4
5
6
7
(
ii) NBS, AIBN, CCl , reflux, 8 h; (iii) concentrated H SO , 110 °C, 1 d. See Fig. 1 for the specific substituents R , R , R and R .
4
2
4
(a)
(b)
1
5,6
−3
Fig. 2. (a) H NMR spectra (acetonitrile-d
(
3
, 400 MHz) of
4
F -FBimbe 12 (1.0×10 M) in the absence (bottom) and presence (top) of TBACl
−
3
5,6
−3
−3
7.08×10 M); (b) Negative ESI MS spectrum of F4-FBimbe 12 (1.0×10 M) in the presence of TBACl (7.08×10 M) in acetonitrile.
The data in Table 1 firstly demonstrate that the FBimbe derivatives previous finding that adding an electron-withdrawing group on the
2
9
tend to exhibit higher activity than the corresponding Bimbe central phenyl subunit is favourable to the anionophoric activity.
5
analogues. For example, F -FBimbe 9 is 6.9-fold more active than
2
Secondly, the activity of these fluorinated derivatives relies on the
total number of fluorine atoms on the benzimidazolyl and central
5
2
F -Bimbe 3. This suggests that fluorination of the central phenyl
subunits boosts the activity, which is in agreement with our
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phenyl subunits. Specifically, increase in the number of fluorine exhibit similar ion transport activity.
atoms is favourable to the activity. This is supported from the
View Article Online
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Fourthly, the activity of these fluorinated bisbenzimidazolyl
derivatives is largely regulated by the fluorination-induced
lipophilicity rather than the anion binding affinity. It is known that
fluorination boosts the activity of an anion transporter by increasing
4
,5,6,7
finding that the nona-fluorinated
F8-FBimbe 13 having one
fluorine atom on the central phenyl ring and four fluorine atoms on
each terminal benzimidazolyl subunit exhibits the highest activity,
3
07-fold more active than the non-fluorinated Bimbe 1. This result
the anion-binding affinity and/or lipophilicity of the transporter
suggests that high-degree fluorination of the 1,3-bis(benzimidazol-
2
enhance the transport efficiency.
22-25
molecule.
The data in Table 1 demonstrate that these
-yl)benzene core structure may serve as a practical strategy to
compounds exhibit similar association constants with chloride
anions, which rules out the possibility that anion-binding affinity is
2
3
Thirdly, the location of fluorine atoms on the terminal primarily responsible for the enhanced ion transport. Then we
benzimidazolyl subunits has limited effect on the activity. This is calculated the clogP values of these fluorinated compounds (Table 1)
demonstrated by the finding that the compounds having the same and analyzed the correlation between the clogP and EC50 values. As
total numbers, but different locations of fluorine atoms on the a consequence, it appears that the EC50 values decay exponentially
4
5
4
benzimidazolyl subunits, that is, F -Bimbe 2 vs F -Bimbe 3, F - with the clogP values (Fig. S79). This result demonstrates that the
2
2
2
5
4,5
4,6
5,6
FBimbe 8 vs F -FBimbe 9, F -Bimbe 4 vs F -Bimbe 5 vs F - transport efficiency of these compounds is primarily regulated by
2
4
4
2
4
,5
4,6
5,6
Bimbe 6, and F4-FBimbe 10 vs F4-FBimbe 11 vs F2-FBimbe 12, the lipophilicity within the tested range.
Table 1. Lipophilicity (clogP), association constants (K ’s) and anion transport efficiency (EC )
a
50
b
c
Anion binding
Ion transport (Pyranine Assay)
EC50 (mol%)
a
Compound
clogP
‒
1
d
d
K
a
(M )
RA
1
n
RA
2
3
3
3
3
3
3
3
4
3
3
3
3
3
Bimbe 1
4.60
4.88
4.88
5.17
5.17
5.17
4.74
5.02
5.02
5.31
5.31
5.31
5.88
(3.51±0.09)×10
(4.14±0.57)×10
(4.94±0.19)×10
(3.04±0.42)×10
(3.11±0.29)×10
(2.38±0.25)×10
(5.40±0.08)×10
(1.40±0.29)×10
(5.01±0.21)×10
(9.84±0.54)×10
(8.91±0.53)×10
(6.92±0.47)×10
(8.80±3.64)×10
1.0
1.2
1.4
0.9
0.9
0.7
1.5
4.0
1.4
2.8
2.5
2.0
2.5
2.06±0.06
1.14±0.07
0.91±0.25
0.98±0.06
1.04±0.22
1.03±0.18
2.00±0.04
1.14±0.02
1.08±0.10
1.07±0.08
0.93±0.04
1.00±0.04
0.92±0.07
9.67±0.89
3.16±0.23
3.61±0.09
0.62±0.07
0.21±0.06
0.31±0.05
8.63±0.14
1.66±0.22
0.52±0.08
1.0
3.0
2.7
15.6
46.0
31.6
1.1
4
5
F
F
2
-Bimbe 2
-Bimbe 3
2
4
,5
F
F
F
4
-Bimbe 4
-Bimbe 5
-Bimbe 6
4
5
,6
,6
4
4
FBimbe 7
4
5
F
F
F
F
F
2
2
4
4
4
-FBimbe 8
-FBimbe 9
-FBimbe 10
-FBimbe 11
-FBimbe 12
5.8
18.6
53.7
60.4
150
307
4
4
5
,5
,6
,6
0.18±0.01
0.16±0.02
-2
-2
(6.46±1.31)×10
(3.15±0.09)×10
4
,5,6,7
8
F -FBimbe 13
a
b
Calculated using MarvinSketch (Version 6.1.0, Weighted Model, ChemAxon, MA).
1
Measured by means of H NMR titrations in CD
3
CN. See Fig. S75 and Table S1 for the association constants measured in CH
3
CN/H
2
O (9/1,
v/v) by means of spectrophotometric titrations.
c
Measured under the intravesicular conditions: 0.1 mM pyranine in 25 mM HEPES (50 mM NaCl, pH 7.0) and extravesicular conditions: 25
mM HEPES (50 mM NaCl, pH 8.0). λ 460 nm/λem 510 nm.
ex
d
RA
1
and RA
2
are the relative binding affinity and ion transport efficiency of each compound relative to Bimbe 1, respectively.
(a) (b)
4
,5,6,7
Fig. 3. Relative chloride efflux out of EYPC liposomes enhanced by (a) each compound (1 mol%) and (b)
8
F -FBimbe 13 of varying
concentrations. Intravesicular conditions: 500 mM NaCl in 25 mM HEPES buffer (pH 7.0); Extravesicular conditions: 500 mM NaNO in 25
3
mM HEPES buffer (pH 7.0).
4
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(a)
(b)
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+
+
+
+
+
4,5,6,7
Fig. 4. (a) Relative chloride efflux out of EYPC liposomes containing Li , Na , K , Rb or Cs , enhanced by
F8-FBimbe 13 (0.125 mol%),
under the intravesicular conditions: 500 mM MCl in 25 mM HEPES buffer (pH 7.0) and extravesicular conditions: 500 mM MNO in 25 mM
3
4
,5,6,7
HEPES buffer (pH 7.0) (M = Li, Na, K, Rb or Cs). (b) Relative chloride efflux out of EYPC liposomes, enhanced by
mol%), under the intravesicular conditions: 500 mM NaCl in 25 mM HEPES buffer (pH 7.0) and extravesicular conditions: 500 mM NaNO3,
00 mM NaHCO or 250 mM Na SO in 25 mM HEPES buffer (pH 7.0).
F8-FBimbe 13 (0.125
5
3
2
4
(a)
(b)
4
,5,6,7
Fig. 5. (a) Relative chloride efflux out of EYPC (■) and EYPC/Chol (7/3, ●) liposomes, promoted by
F8-FBimbe 13 (0.125 mol%), under
the intravesicular conditions: 500 mM NaCl in 25 mM HEPES buffer (pH 7.0) and extravesicular conditions: 500 mM NaNO in 25 mM HEPES
3
4
,5,6,7
buffer (pH 7.0). (b) Chloride transport across a bulky nitrobenzene membrane, promoted by
chloride ion selective electrode in the receiving aqueous phase of U-tube.
F8-FBimbe 13 (1.0 mM) and detected by a
Anion selective transport. To assess the anion-selective transport highly hydrophilic sulfate anions and be recovered by carbonate
activity of these compounds, we measured their ability to facilitate anions, strongly suggesting that anion exchange is the primary
3
6,40
the efflux of chloride anions out of the EYPC liposomes by means of mode of action.
3
6
chloride ion selective electrode techniques (Fig. 3 and S80). The
concentration-dependent chloride efflux indicates that these
compounds are capable of mediating the transport of chloride
anions across lipid bilayers. Similar as observed in pH discharge
assays, the FBimbe derivatives 7-13 tend to exhibit higher chloride
efflux activity than the corresponding Bimbe analogues 1-6. It is
noteworthy that the EC50, 260 s values are much higher than those
obtained from the pH discharge experiments (Table S3), suggesting
that pyranine assay is a much more sensitive approach for probing
4,5,6,7
Probable mechanism of action As
active, we measured its chloride efflux out of 30% cholesterol
Chol)-containing EYPC vesicles to probe the probable mechanism
of action (Fig. 5a and Fig. S83). The observed reduction in the
F8-FBime 13 is the most
(
4,5,6,7
chloride efflux implies that
F8-FBime 13 may function as a
23
mobile carrier. To further confirm this probable mechanism, we
23
carried out a U-tube assay (Fig. 5b). In this experiment, a bulky
nitrobenzene membrane was used to separate a sodium chloride
solution as the resource phase from a sodium nitrate solution as the
receiving phase. The transport of chloride anions through the
3
7,38
the ion transport properties of an anion transporter.
To gain further insights into the anion selectivity and the probable nitrobenzene phase was monitored by using a chloride ion selective
4
,5,6,7
mechanism of action, we measured the chloride efflux activity of electrode. Compared with the blank, addition of
F8-FBime 13
these fluorinated compounds in the presence of different alkali led to a higher concentration of chloride anions in the receiving
metal ions or anions (Fig. 4 and Fig. S81-S82). The chloride efflux phase. This result in combination with the reduced chloride efflux
4
,5,6,7
activity is independent on the alkali metal ions, but varies with the observed by the cholesterol assay, suggests that
F8-FBime 13
−
−
tested anions. These results indicate that Cl /NO antiport and/or mediates the transmembrane transport of anions most probably via
3
+
−
39
H /Cl symport may be involved during the chloride efflux. The a mobile carrier mechanism.
chloride efflux activity was found to be significantly suppressed by
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Taken together, the above observations suggest that these in acidic compartments such as lysosomes, and green fluorescence
View Article Online
4-19,29,41
1
DOI: 10.1039/C8OB03036G
fluorinated compounds function as highly effective anion-selective when the acidic compartments are basified.
carriers primarily through a process of anion exchange.
As shown in
Fig. 6a, when HeLa cells were stained with AO, scattered orange
fluorescence was observed in the cytoplasm, suggesting that AO is
In vitro anion transport
located in acidic organelles. Treatment of the HeLa cells with
5
Inspired by the potent transport activity of these compounds on
FBimbe 7 and F -FBimbe 9 led to partial reduction in the orange
2
5
5
,6
4,5,6,7
liposomal models, we chose four compounds, that is, FBimbe 7, F -
2
emission (Fig. 6b and c), and with F4-FBime 12 and
F8-FBime
5
,6
4,5,6,7
F8-FBimbe 13 to investigate their
FBimbe 9, F4-FBimbe 12 and
anionophoric activity in live cells.
13 led to complete loss of the orange emission (Fig. 6d and e). This
result suggests that these compounds are able to basify the acidic
–
organelles, in the same order observed in liposomal models. The Cl
Acridine orange staining Firstly, we carried out vital staining on
HeLa cervical cancer cells by using cell-permeable acridine orange
–
/
HCO
3
antiport observed in the liposomal experiments may be
1
4-19, 29
responsible for the increase of the internal pH.
(AO). It is known that AO emits characteristic orange fluorescence
(a) (b)
(c)
(d)
(e)
5
Fig. 6. Acridine orange staining of HeLa cells. (a) Untreated cells (control); (b)-(e) cells treated with 10 μM of (b) FBimbe 7, (c) F -FBimbe 9,
2
5
,6
4,5,6,7
(
d) F4-FBimbe 12 and (e)
F8-FBimbe 13, respectively, for 1.5 h.
intensity of three independent experiments.
MQAE assay Similar to pH discharge experiments, AO staining does
not provide any evidence for whether these compounds are able to
facilitate the influx of anions into cells. To clarify this, we
5
5,6
investigated the ability of FBimbe 7, F -FBimbe 9, F4-FBimbe 12
2
4
,5,6,7
and
HeLa cells by using
ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE).
F8-FBimbe 13 to mediate the entry of chloride anions into
a
fluorescent assay based on N-
(
1
4-19,29
Because MQAE is a cell-permeable, chloride selective dye, its
fluorescence quenching upon the treatment of the HeLa cells with
FBimbe 7, F -FBimbe 9, F4-FBimbe 12 or
5
5,6
4,5,6,7
2
F8-FBimbe 13 may
serve as a direct evidence that these compounds are involved in the
influx of chloride anions into the HeLa cells. As shown in Fig. 7, post
5
5,6
incubation of the HeLa cells with FBimbe 7, F -FBimbe 9, F -
FBimbe 12 and
2
4
Fig. 7. Normalized fluorescent intensity of MQAE in HeLa cells
4
,5,6,7
F8-FBimbe 13 led to significant quenching of the
incubated with MQAE (5 mM) for 3.5 h followed by the treatment
5
5,6
MQAE fluorescence. This result clearly indicates that these
compounds are able to mediate the influx of chloride anions into
the intracellular matrix.
with FBimbe 7 (), F2-FBimbe 9 (●), F4-FBimbe 12 (▲) and
4
,5,6,7
F
8
-FBimbe 13 (▼) of varying concentrations for
2 h.
Fluorescent intensity was recorded by the plate reader at λem = 460
nm (λex = 350 nm), and normalized with respect to the fluorescent Biological activity
intensity of untreated cells. Each data point represents the mean
Cytotoxicity. It is well recognized that facilitated transport of
6
| J. Name., 2012, 00, 1-3
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ARTICLE
2
0,26,29
chloride anions across cells may induce cell death.
The ability Specifically, fluorination at the 4- or 7-position of the
View Article Online
DOI: 10.1039/C8OB03036G
of these fluorinated derivatives to mediate the transport of chloride benzimidazolyl subunits is not favorable to the cytotoxicity. Thus,
anions into live cells inspired us to test their in vitro cytotoxicity the compounds having significant anion transport, such as F -
toward four solid tumor cells, including HeLa, A549 lung FBimbe 12 and
5
,6
4
4
,5,6,7
F8-FBimbe 13 exhibit potent cytotoxicity. In
adenocarcinoma, MCF-7 breast and HepG2 human liver cancer cells. addition, no discrimination between cancerous and normal cells is
Initially, we screened the anti-proliferative activity of all the observed.
compounds at the concentration of 50 μM and then measured their
IC50 values, by using a conventional MTT [3-(4,5-dimethylthiazol-2-
Probable mechanism of biological action
Effect of chloride anions on the cytotoxicity It is reported that
dysregulation of ion homeostasis, in particular via chloride influx
into cells, can induce cell shrinkage and lead to apoptosis.
yl)-2,5-diphenyltetrazolium bromide] assay. Here the IC50 value
represents the concentration of each compound resulting in 50%
inhibition in cell growth. The cell viability expressed as a percentage
of control cells is shown in Fig. 8 and Table S4, and the IC50 values
are reported in Fig. S85-S96, Table 2 and Tables S5-S6. For
comparison, the IC50 values of all the compounds toward LO2
human normal liver cells were also measured (Fig. S85-S96, Table 2
and Table S7). Doxorubicin was used as a positive control.
2
0,26,42,43
As shown above, these fluorinated compounds are able to facilitate
the entry of chloride anions into live cells, and their cytotoxicity
parallels their in vitro anionophoric activity. These results imply that
chloride transport across cellular membranes may be responsible
for the cytotoxic effects. To gain support for this, we measured the
5
,6
4,5,6,7
cytotoxicity of F4-FBimbe 12 and
F8-FBimbe 13 toward HeLa
The data in Table 2 demonstrate that in general, the FBimbe series
tend to exhibit higher cytotoxicity than the corresponding Bimbe
series, which suggests that fluorination of the central phenyl rings is
favourable not only to the anion transport but also to the cytotoxic
effect. Interestingly, the location of fluorine atoms on the
benzimidazolyl subunits has an obvious impact on the cytotoxicity.
cells in the presence and absence of chloride anions. As shown in
5,6
4,5,6,7
Fig. 9a-b, both F4-FBimbe 12 and
F8-FBimbe 13 were more
cytotoxic in the presence of chloride anions than in the absence of
chloride anions. This result strongly suggests that chloride transport
may be one of the major factors that are responsible for the
cytotoxic effects.
(a)
(b)
(c)
(d)
Fig. 8. Cell viability of Bimbe 1 and the fluorinated derivatives 2-13 (50 μM) toward (a) HeLa, (b) A549, (c) MCF-7 and (d) HepG2 cancer cells.
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Table 2. Anionophoric activity (EC50, μM) and cytotoxicity (IC50, μM) of Bimbe 1 and the fluorinated derivatives 2-13
View Article Online
b
DOI: 10.1039/C8OB03036G
IC50 (μM)
a
Compound
EC50 (μM)
HeLa
12.5±1.2
> 50
6.2±0.3
> 50
A549
18.8±1.6
> 50
22.2±4.0
> 50
36.6±4.7
6.2±0.2
21.6±1.1
> 50
5.8±0.7
4.5±0.8
8.7±0.1
3.2±0.6
4.0±0.4
0.46±0.04
MCF-7
> 50
> 50
> 50
> 50
HepG-2
12.4±0.7
> 50
20.5±0.8
> 50
LO2
Bimbe 1
17.0±1.60
5.55±0.41
6.34±0.16
1.08±0.11
0.38±0.10
0.54±0.09
15.2±0.20
2.91±0.39
0.91±0.14
0.32±0.01
0.28±0.04
0.11±0.02
(5.54±0.16)×10
/
16.8±0.9
> 50
18.8±0.3
> 50
4
5
F
F
2
-Bimbe 2
-Bimbe 3
2
4
,5
F
F
F
4
-Bimbe 4
-Bimbe 5
-Bimbe 6
4
5
,6
,6
4
4
> 50
> 50
> 50
> 50
> 50
> 50
9.8±0.8
28.2±1.6
> 50
10.6±0.9
44.0±4.3
> 50
6.3±0.1
2.6±0.6
2.5±0.3
30.2±3.1
26.1±4.1
> 50
8.3±0.6
6.3±1.3
> 50
4.1±0.3
3.4±0.5
0.14±0.01
FBimbe 7
15.1±4.0
47.3±4.4
5.6±0.2
6.5±1.3
> 50
7.6±2.2
3.8±0.1
0.16±0.06
-4
37.8±2.3
> 50
21.0±1.5
15.7±0.8
> 50
4.6±0.8
11.4±0.6
0.15±0.01
4
5
F
F
F
F
F
2
2
4
4
4
-FBimbe 8
-FBimbe 9
-FBimbe 10
-FBimbe 11
-FBimbe 12
4
4
5
,5
,6
,6
4
,5,6,7
-2
F
8
-FBimbe 13
Doxorubicin
a
b
Calculated by multiplying the EYPC concentrations (1.76×10 M) of EYPC by the EC50 values (in mol%) of each compound listed in Table 1.
>
50 μM means that the cell viability tested at 50 μM was > 50% (Fig. 8).
4
5
Effect of necrotic, apoptotic and autophagic inhibitors on the mitochondrial membrane potential. As shown in Fig. 11 and Fig.
5
,6
4,5,6,7
cytotoxicity According to the morphological characteristics and S100, treatment of the HeLa cells with F4-FBimbe 12 or
8
F -
biochemical markers, cell death may be divided into several FBimbe 13 of varying concentrations and then with JC-1 led to a
44
categories, mainly including necrosis, apoptosis and autophagy.
significant change in the fluorescence of JC-1 from red to green.
To clarify which one is responsible for the cell death induced by This is indicative of a decrease in the mitochondrial membrane
5
,6
these fluorinated compounds, we measured the cytotoxicity of F - potential and is one of the characteristics for apoptotic cell death at
4
4,5,6,7
16,17
FBimbe 12 and
F8-FBimbe 13 toward HeLa cells in the presence an early stage.
of a necrotic inhibitor Nec-1, an autophagic inhibitor 3-MA or an
apoptotic inhibitor Z-VAD-FMK (Fig. 9c and Fig. S97-S99). As a result,
the cell viability was not recovered after the administration of Nec-
3
. Conclusions
In conclusion, we have successfully synthesized a series of
fluorinated bisbenzimidazolyl derivatives and systematically studied
their anion recognition, transmembrane anion transport,
1
or 3-MA, ruling out the possibility that the cell death proceeds via
a process of necrosis or autophagy. Though it is reported that
synthetic anion transporters are able to disrupt the autophagy of
1
cytotoxicity and probable mechanism of biological action. H NMR
2
6
cells,
the present finding suggests that the formation and
titrations and ESI MS spectrometric detections indicate that these
compounds are able to form stable complexes with chloride anions.
These compounds exhibit potent anion transport in liposomal
models, and the anionophoric activity may be regulated by the total
number of fluorine atoms. MQAE assay and AO vital staining
indicate that these compounds are able to disturb the homeostasis
of chloride anions, modify the intracellular pH and induce the
basification of acidic lysosomes. MTT assays indicate that most of
these fluorinated bisbenzimidazolyl derivatives exhibit potent
cytotoxicity toward the selected four solid tumor cells, and the
derivatives having one fluorine atom on the central phenyl ring and
two or four fluorine atoms on each terminal benzimidazolyl subunit
have the IC50 values in the low micromolar range. The higher
cytotoxicity in the presence of chloride anions suggests that the
transport of chloride anions across the cellular membranes plays a
critical role in the cytotoxic effects. Mechanistic study based on
necrotic, apoptotic or autophagic inhibitors, Hoechst 33342 and JC-
1 staining suggests that these compounds trigger cell death most
probably via an apoptotic process. The present findings together
with the drug-like features of these fluorinated compounds strongly
suggest that they are exploitable as apoptotic agents for cancers.
Further investigations into the probable mechanism of action are
accumulation of autophagosomes are not crucial for the cytotoxic
effects of these fluorinated bisbenzimidazolyl derivatives. However,
administration of Z-VAD-FMK led to significant recovery in the cell
5
,6
viability. This result suggests that the cell death triggered by F -
4
4
,5,6,7
FBimbe 12 and
F8-FBimbe 13 may proceed via an apoptotic
pathway. This probable mechanism of apoptosis was further
confirmed by means of Hoechst 33342 and JC-1 staining of HeLa
cells.
Hoechst 33342 staining It is known that Hoechst 33342 staining
may be used to differentiate apoptosis from other cell death
1
6,19
mechanisms through morphological observation.
Fig. 10, compared with the untreated cells, the cells treated with
FBimbe 7, F2-FBimbe 9, F4-FBimbe 12 or
As shown in
5
5,6
4,5,6,7
F8-FBimbe 13
exhibit stronger blue fluorescence, indicative of chromatin
condensation, fragmentation and apoptotic bodies formation. The
formation of “bean”-shaped nuclei was also observed. These are
1
6,17,19
typical features for cell death via an apoptotic process.
JC-1 staining The reduction of mitochondrial membrane potential is
1
6,17
a hallmark of apoptosis,
and this may be monitored by a cell-
permeable dye JC-1 the fluorescence of which is sensitive to
8
| J. Name., 2012, 00, 1-3
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ARTICLE
presently under progress, with the aim to develop potential anti- disordering the ion homeostasis of cancer cells.
cancer chemotherapeutic agents that exert their activity by
View Article Online
DOI: 10.1039/C8OB03036G
(a)
(b)
(c)
Fig. 9. (a, b) Viability of HeLa cells in the presence and absence of chloride anions in extracellular media (HBSS buffer) upon the dose-
5
,6
4,5,6,7
dependent treatment of
F
4
-FBimbe 12 (a, 18 h) and
8
F -FBimbe 13 (b, 24 h) (mean ± s.d., n = 4, **P < 0.01, Independent-Sample T
Test). (c) Viability of HeLa cells incubated with Nec-1 (25 μM), 3-MA (2.0 mM) or Z-VAD-FMK (25 μM) for 2 h, followed by the treatment
4
,5,6,7
with
8
F -FBimbe 13 (10 μM) for 24 h.
(a)
(b)
(c)
(d)
(e)
5
5,6
Fig. 10. Hoechst 33342 staining of HeLa cells. (a) Untreated cells; (b)-(e) cells treated with 10 μM of (b) FBimbe 7, (c) F
2
-FBimbe 9, (d)
4
F -
4
,5,6,7
FBimbe 12 and (e)
8
F -FBimbe 13 for 24 h, respectively.
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Journal Name
(a)
(b)
(c)
View Article Online
DOI: 10.1039/C8OB03036G
(d)
(e)
(f)
4
,5,6,7
Fig. 11. JC-1 staining of HeLa cells. (a) Untreated (control) cells; (b)-(e) cells treated with
2
F
8
-FBimbe 13 at the concentrations of 1.25 μM,
4
,5,6,7
.5 μM, 5 μM and 10 μM, respectively; (f) Pixel ratio (red/green) for different concentrations of
8
F -FBimbe 13, analysed by ImageJ
(
mean ± s.d., n = 9, ***P < 0.001, Independent-Sample T Test).
4
. Experimental
from commercial sources and used without further purification.
Generals. A Bruker Avance AV 400 NMR spectrometer was used to Synthesis of 5-fluoro-benzene-1,3-dicarbaldehyde
1
13
measure the H and C NMR spectra, and the data were reported
relative to the deuterium solvents. Waters UPLC/Quattro Premier
XE and Bruker maXis 4G ESI-Q-TOF mass spectrometers were used
to measure the LR and HR ESI-MS spectra, respectively. Melting
points (mp) were measured on an X-5 micro melting point
apparatus. Analytical thin-layer chromatography (TLC) plates (silica
gel, GF254) were detected by use of iodine and UV (254 or 365 nm).
EYPC vesicles were prepared by extrusion through nuclepore track-
etched polycarbonate membranes (100 nm, Whatman, Florham
Park, New Jersey, USA) on an Avanti’s Mini-Extruder (Avanti Polar
A solution of 1-fluoro-3,5-dimethyl-benzene (1.3 mL, 10.8 mmol),
NBS (11.4 g, 64.7 mmol) and AIBN (1.10 g, 6.70 mmol) in CCl (70
4
mL) was refluxed for 8 h. The mixture was filtered and the filtrate
was concentrated under reduced pressure. The residue was
dissolved in concentrated sulfuric acid (30 mL). The resulting
mixture was heated at 110 °C for 1 d, and then added drop wise
into iced water (50 mL). The mixture was extracted with ethyl ether
(
100 mL×3). The ether layer was washed subsequently with
saturated Na CO solution (100 mL×3) and saline (100 mL×3), and
2
3
dried over anhydrous sodium sulfate. Purification was accomplished
by re-crystallization from petroleum ether (b. p. 60-90 °C) to afford
Lipids, Inc., Alabaster, Alabama, USA). Chloride efflux was measured
TM
by using a Mettler-Toledo PerfectIon
chloride ion selective
5
-fluoro-benzene-1,3-dicarbaldehyde (181 mg, 11%) having
electrode assembled with a Mettler-Toledo Seven Compact S220
ionometer. Ultraviolet–visible and Fluorescence spectra were
measured on a Perkin Elmer Lambda 25 spectrophotometer and
Perkin Elmer LS55 spectrofluorimeter, respectively. MTT-based
cytotoxicity assay and MQAE assays were measured on a Tecan
Infinite M1000 PRO microplate reader. Acridine orange, Hoechst
‒
1
negative ESI-MS: m/z 151.62 ([M‒H] ); H NMR (CDCl , 400 MHz) δ
3
1
0.09 (s, 2H), 8.20 (t, J = 1.2 Hz, 1H), 7.84 (dd, J = 1.2 and 7.8 Hz, 2H)
13
and C NMR (CDCl , 100 MHz) δ 189.6 (×2), 163.5 (d, J = 252.0 Hz),
3
1
1
39.0 (d, J = 5.9 Hz), 127.0 (×2), 120.9 (d, J = 22.4 Hz). The H and
1
3
C NMR data were in agreement with the reported ones in
3
2
literature.
3
3342 and JC-1 staining experiments were carried out on a Carl
4
Zeiss Axio Observer A1 microscope.
2
Synthesis of F -Bimbe 2
EYPC and pyranine were purchased from Sigma Chemical Co. (St A solution of 1,3-benzenedialdehyde (51 mg, 0.38 mmol) and 3-
Louis, USA). Calcein, cholesterol, acridine orange and Hoechst fluoro-1,2-benzenediamine (115 mg, 0.92 mmol) in DMSO (1.5 mL)
3
3342 were purchased from J&K Chemical Co. (Beijing, China). MTT was stirred at room temperature. The reaction was monitored with
was supplied by Amresco (Ohio, USA). MQAE and 3-MA were TLC (CHCl /CH OH, 20/1, v/v). After 48 h, the reaction solution was
supplied by Aladdin (Shanghai, China). JC-1 mitochondrial added to water (200 mL). The resulting precipitates were collected
membrane potential detection kit was supplied by Genview (Florida, through filtration and purified by preparative TLC (CHCl /CH OH,
-Bimbe 2 (42 mg, 32%) having mp 271.1-272.3
C; H NMR (DMSO-d , 400 MHz) δ 9.02 (s, 1H), 8.41 (d, J = 7.6 Hz,
3
3
3
3
4
USA). Nec-1 and Z-VAD-FMK were purchased from Selleck (Houston, 15/1, v/v) to give F
2
5
o
1
USA) and Apexbio (Houston, USA), respectively. Bimbe 1 and F -
6
2
Bimbe 3 were prepared according to the protocols reported by us 2H), 7.76 (t, J = 8.0 Hz, 1H), 7.45 (d, J = 7.6 Hz, 2H), 7.23 (m, 2H),
2
8,31
13
and others.
All the other chemicals and reagents were obtained 7.04 (m, 2H); C NMR (DMSO-d
6
, 100 MHz) δ 151.8, 130.9, 130.1,
1
0 | J. Name., 2012, 00, 1-3
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1
28.6, 125.6, 123.4 (d, J = 5.7 Hz), 107.6 (d, J = 16.7 Hz); ESI-MS: m/z purified with preparative TLC (CHCl
3
/CH
3
OH, 15/1, v/v) to give
View Article Online
+
+
o
1
DOI: 10.1039/C8OB03036G
347.80 ([M+H] ), 369.54 ([M+Na] ) and HR-ESI-MS for C H F N
0 13 2 4
FBimbe 7 (54 mg, 49%) having mp 272.9-273.5 C; H NMR (DMSO-
d , 400 MHz) δ 13.57 (br, 1H), 9.03 (s, 1H), 8.19 (d, J = 9.6 Hz, 2H),
6
2
+
([M+H] ) Calcd: 347.1103; Found: 347.1100.
1
3
4
,5
7.73 (d, J = 4.8 Hz, 2H), 7.61 (d, J = 6.0 Hz, 2H), 7.27 (br, 4H);
C
Synthesis of
4
F -Bimbe 4
NMR (DMSO-d , 100 MHz) δ 163.1 (d, J = 242.0 Hz), 150.0 (×2),
6
A solution of 1,3-benzenedialdehyde (51 mg, 0.38 mmol) and 3,4-
difluoro-1,2-benzenediamine (132 mg, 0.92 mmol) in DMSO (3.0 mL)
was heated at 80 ºC. The reaction was monitored with TLC
1
43.9, 135.5, 133.5 (d, J = 9.0 Hz), 123.5, 122.5, 121.3, 119.5, 114.5
+
(d, J = 23.8 Hz), 112.2; ESI-MS: m/z 329.91 ([M+H] ), 351.86
+
+
([M+Na] ) and HR-ESI-MS for C H FN ([M+H] ) Calcd: 329.1197;
20 14 4
(
CHCl /CH OH, 20/1, v/v). After 36 h, the reaction solution was
3 3
Found: 329.1195.
added to water (200 mL). The resulting precipitates were collected
4
4
,5
Synthesis of F
2
-FBimbe 8
through filtration and washed with methanol thrice to afford F -
4
o
1
Bimbe 4 (114 mg, 80%) having mp > 300 C; H NMR (DMSO-d , 400 A solution of 5-fluoro-benzene-1,3-dicarbaldehyde (53 mg, 0.35
6
MHz) δ 13.56 (br, 2H), 9.06 (s, 1H), 8.32 (dd, J = 7.6 and 1.2 Hz, 2H), mmol) and 3-fluoro-1,2-benzenediamine (91 mg, 0.72 mmol) in
1
3
7.79 (t, J = 7.6 Hz, 1H), 7.41 (d, 2H, J = 5.2 Hz), 7.30 (m, 2H);
C
CH CN (3 mL) was refluxed. The reaction was monitored with TLC
3
NMR (DMSO-d , 100 MHz) δ 153.1, 145.9 (d, J = 233.2 Hz), 145.8 (d, (CHCl /CH OH, 20/1, v/v). After 48 h, the reaction solution was
6
3
3
J = 233.5 Hz), 130.6, 130.2, 128.7, 125.5, 112.3 (d, J = 20.7 Hz); ESI- concentrated under reduced pressure. The obtained residue was
+
+
MS: m/z 383.64 ([M+H] ), 405.58 ([M+Na] ) and HR-ESI-MS for purified with chromatography on a silica gel column, eluted with a
+
4
C H F N ([M+H] ) Calcd: 383.0914; Found: 383.0913.
mixture of CHCl /CH OH (125/1, v/v) to give F -FBimbe 8 (41 mg,
2
0
11
4
4
3
3
2
o
1
4
,6
32%) having mp 291.8-292.7 C; H NMR (DMSO-d
.05 (s, 1H), 8.24 (d, J = 9.6 Hz, 2H), 7.74 (d, J = 8.0 Hz, 2H), 7.28-
.23 (m, 2H), 7.09-7.05 (m, 2H); C NMR (DMSO-d , 100 MHz) δ
63.1 (d, J = 242.2 Hz), 150.7, 150.6, 133.1 (d, J = 8.9 Hz), 123.7 (d, J
6.3 Hz), 121.6, 115.1 (d, J = 23.9 Hz), 107.8 (d, J = 16.7 Hz); ESI-MS:
6
, 400 MHz) δ
Synthesis of F4-Bimbe 5
9
7
1
=
1
3
A solution of 1,3-benzenedialdehyde (51 mg, 0.38 mmol) and 3,5-
difluoro-1,2-benzenediamine (134 mg, 0.93 mmol) in DMSO (3 mL)
was stirred at room temperature. The reaction was monitored with
6
+
+
TLC (CHCl /CH OH, 20/1, v/v). After 48 h, the reaction solution was
3
3
m/z 365.82 ([M+H] ), 387.77 ([M+Na] ) and HR-ESI-MS for
C H F N ([M+H] ) Calcd: 365.1009; Found: 365.1008.
+
added to water (200 mL). The resulting precipitates were collected
through filtration and purified by chromatography on a silica-gel
column, eluted with a mixture of CHCl /CH OH (110/1, v/v) to
2
0 12 3 4
5
Synthesis of F -FBimbe 9
2
3
3
4
,6
o
1
afford F -Bimbe 5 (56 mg, 38%) having mp > 300 C; H NMR A solution of 5-fluoro-benzene-1,3-dicarbaldehyde (31 mg, 0.21
4
(
DMSO-d , 400 MHz) δ 13.58 (br, 2H), 8.29 (d, J = 7.6 Hz, 2H), 7.77 (t, mmol) and 4-fluoro-1,2-benzenediamine (84 mg, 0.67 mmol) in
6
13
J = 8.0 Hz, 1H), 7.26 (d, J = 7.6 Hz, 2H), 7.10 (t, J = 10.4 Hz, 2H); C DMSO (3 mL) was stirred at room temperature. The reaction was
NMR (DMSO-d , 100 MHz) δ 158.7 (dd, J = 236.8, 10.8 Hz), 152.9 (dd, monitored with TLC (CHCl /CH OH, 20/1, v/v). After 48 h, the
6
3
3
J = 251.4 and 16.3 Hz), 152.3, 137.7 (dd, J = 15.3 and 11.1 Hz), 130.6, reaction solution was added to water (200 mL). The resulting
1
2
30.2, 129.4 (d, J = 15.7 Hz), 128.5, 125.1, 97.9 (dd, J = 28.5 and precipitates were collected through filtration, and purified by
–
3.2 Hz), 95.0 (d, J = 25.7 Hz); negative ESI-MS: m/z 381.62 ([M–H] ) chromatography on a silica gel column, eluted with a mixture of
–
5
and negative HR-ESI-MS for C H F N ([M–H] ) Calcd: 381.0769; CHCl /CH OH (110/1, v/v) to give F -FBimbe 9 (27 mg, 35%) having
Found: 381.0776.
2
0
9
4
4
3
3
2
o
1
mp 288.6-289.3 C; H NMR (DMSO-d , 400 MHz) δ 13.53 (br, 2H),
6
5
,6
8.89 (s, 1H), 8.06 (dd, J = 9.2 and 0.8 Hz, 2H), 7.67 (br, 2H), 7.46 (br,
Synthesis of
4
F -Bimbe 6
1
3
2
H), 7.14 (dd, J = 10.0 and 2.0 Hz, 2H); C NMR (DMSO-d , 100 MHz)
6
A solution of 1,3-benzenedialdehyde (51 mg, 0.38 mmol) and 4,5-
difluoro-1,2-benzenediamine (130 mg, 0.90 mmol) in DMSO (6 mL)
was stirred at room temperature. The reaction was monitored with
δ 163.1 (d, J = 242.1 Hz), 159.3 (d, J = 234.5 Hz), 151.3, 149.9 (d, J =
3
.2 Hz), 133.3 (d, J = 8.9 Hz), 123.1, 121.2, 114.5 (d, J = 23.8 Hz),
+
111.3 (d, J = 25.0 Hz); ESI-MS: m/z 365.56 ([M+H] ), 387.49
+
+
TLC (CHCl /CH OH, 20/1, v/v). After 48 h, the reaction solution was
3
3
([M+Na] ) and HR-ESI-MS for C H F N ([M+H] ) Calcd: 365.1009;
20 12 3 4
added to water (200 mL). The resulting precipitates were collected
Found: 365.1005.
5
,6
through filtration and washed with methanol thrice to give F -
4
4,5
o
1
Synthesis of
4
F -FBimbe 10
Bimbe 6 (53 mg, 37%) having mp > 300 C; H NMR (DMSO-d , 400
6
MHz) δ 8.99 (s, 1H), 8.25 (dd, J = 7.6 and 1.2 Hz, 2H), 7.70-7.66 (m, A solution of 5-fluoro-benzene-1,3-dicarbaldehyde (50 mg, 0.33
1
3
4
1
1
H), 7.52 (t, J = 7.6 Hz, 1H); C NMR (DMSO-d , 100 MHz) δ 153.0, mmol) and 3,4-difluoro-1,2-benzenediamine (104 mg, 0.72 mmol) in
6
47.4 (d, J = 237.6 and 15.7 Hz), 130.8, 130.1, 128.1, 125.1, 103.4, DMSO (2 mL) was heated at 40 ºC. The reaction was monitored with
03.0; negative ESI-MS: m/z 381.68 ([M–H] ) and HR-ESI-MS for TLC (CHCl /CH OH, 20/1, v/v). After 24 h, the reaction mixture was
C H F N ([M–H] ) Calcd: 381.0769; Found: 381.0773.
–
3
3
–
added to water (200 mL) and extracted with EtOAc (100 mL×3). The
organic layer was concentrated under reduced pressure to give
2
0 9 4 4
Synthesis of FBimbe 7
4
,5
o
1
F
4
-FBimbe 10 (32 mg, 24%) having mp > 300 C; H NMR (DMSO-
A solution of 5-fluoro-benzene-1,3-dicarbaldehyde (51 mg, 0.33
d , 400 MHz) δ 13.61 (br, 2H), 8.86 (s, 1H), 8.06 (d, J = 9.6 Hz, 2H),
6
1
3
mmol) and o-phenylenediamine (88 mg, 0.81 mmol) in CH
mL) was refluxed. The reaction was monitored with TLC
CHCl /CH OH, 20/1, v/v). After 48 h, the reaction solution was
3
CN (3.0
7
.39 (br, 2H), 7.26-7.33 (m, 2H); C NMR (100 MHz, DMSO-d ) δ
6
162.9 (d, J = 242.4 Hz), 151.8, 145.9 (d, J = 227.3 Hz), 145.8 (d, J =
(
3
3
228.3 Hz), 134.1, 133.4, 132.7 (d, J = 8.8 Hz), 121.5, 115.2 (d, J =
+
+
concentrated under reduced pressure. The obtained residue was
23.8 Hz), 107.9; ESI-MS: m/z 401.53 ([M+H] ), 423.54 ([M+Na] ) and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Organic & Biomolecular Chemistry
Page 12 of 13
ARTICLE
Journal Name
+
HR-ESI-MS for
C
20
H
10
F
5
N
4
([M+H] ) Calcd: 401.0820; Found: Conflicts of interest
There are no conflicts to declare.
Acknowledgements
View Article Online
DOI: 10.1039/C8OB03036G
4
01.0817.
4
,6
Synthesis of
4
F -FBimbe 11
A solution of 5-fluoro-benzene-1,3-dicarbaldehyde (53 mg, 0.35
mmol) and 3,5-difluoro-1,2-benzenediamine (150 mg, 1.04 mmol) in
DMSO (10 mL) was stirred at room temperature. The reaction was
monitored with TLC (CHCl /CH OH, 20/1, v/v). After 48 h, the
This work was financially supported by the National Natural Science
Foundation of China (No. 21877057).
Notes and references
3
3
1
F. Lang, M. Ritter, N. Gamper, S. Huber, S. Fillon, V. Tanneur, A.
Lepple-Wienhues, I. Szabo and E. Gulbins, Cell. Physiol.
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reaction mixture was added to water (200 mL). The resulting
precipitates were collected through filtration. The precipitates were
partitioned in methanol, filtered and washed with methanol to
4
,6
o
1
2
3
I. Alfonso and R. Quesada, Chem. Sci., 2013, 4, 3009–3019.
P. A. Gale, J. T. Davis and R. Quesada, Chem. Soc. Rev., 2017, 46,
afford F -FBimbe 11 (59 mg, 42%) having mp > 300 C; H NMR
4
(
2
1
DMSO-d , 400 MHz) δ 13.64 (br, 2H), 8.86 (s, 1H), 8.07 (d, J = 9.6 Hz,
H), 7.28 (d, J = 7.2 Hz, 2H), 7.14-7.08 (m, 2H); C NMR (DMSO-d6,
00 MHz) δ 162.9 (d, J = 242.5 Hz), 158.8 (d, J = 235.4 Hz), 158.7 (d,
6
1
3
2497−2519.
4
5
N. Busschaert, C. Caltagirone, W. Van Rossom and P. A. Gale,
Chem. Rev., 2015, 115, 8038–8155.
Z. Li and W.-H. Chen, Mini-Rev. Med. Chem., 2017, 17,
J = 235.4 Hz), 151.2, 132.8 (d, J = 8.8 Hz), 121.4, 115.0 (d, J = 24.0
+
Hz), 98.5, 98.0; ESI-MS: m/z 401.53 ([M+H] ) and HR-ESI-MS for
+
1398−1405.
C H F N ([M+H] ) Calcd: 401.0820; Found: 401.0815.
2
0 10 5 4
6
7
D. S. Kim and J. L. Sessler, Chem. Soc. Rev., 2015, 44, 532−546.
N. Busschaert and A. P. Gale, Angew. Chem. Int. Ed., 2013, 52,
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G. W. Gokel and S. Negin, Acc. Chem. Res., 2013, 46, 2824–
2833.
5
,6
Synthesis of F4-FBimbe 12
A solution of 5-fluoro-benzene-1,3-dicarbaldehyde (53 mg, 0.35
mmol) and 4,5-difluoro-1,2-benzenediamine (105 mg, 0.73 mmol) in
DMSO (3 mL) was heated at 85 ºC. The reaction was monitored with
TLC (CHCl /CH OH, 20/1, v/v). After 48 h, the reaction solution was
8
9
P. A. Gale, R. Pérez-Tomás and R. Quesada, Acc. Chem. Res.,
2013, 46, 2801–2813.
3
3
added to water (200 mL). The resulting precipitates were collected
5
,6
through filtration, and washed with methanol thrice to give F - 10 F. De Riccardis, I. Izzo, D. Montesarchio and P. Tecilla, Acc.
4
o
1
FBimbe 12 (49 mg, 35%) having mp > 300 C; H NMR (DMSO-d ,
Chem. Res., 2013, 46, 2781–2790.
6
4
00 MHz) δ 13.38 (br, 2H), 8.79 (s, 1H), 7.98 (d, J = 9.2 Hz, 2H), 7.65 11 C. J. E. Haynes and P. A. Gale, Chem. Commun., 2011, 47,
1
3
(br, 4H); C NMR (DMSO-d , 100 MHz) δ 163.0 (d, J = 242.1 Hz),
8203–8209.
6
151.74, 151.73, 146.65 (dd, J = 238.8 Hz and 22.6 Hz), 139.5, 132.9 12 J. L. Sessler, L. R. Eller, W.-S. Cho, S. Nicolaou, A. Aguilar, J. T.
(
d, J = 8.9 Hz), 121.0, 114.5 (d, J = 24.0 Hz), 107.0, 106.3, 100.4, 99.8;
Lee, V. M. Lynch and D. J. Magda, Angew. Chem. Int. Ed., 2005,
44, 5989–5992.
13 A. M. Rodilla, L. Korrodi-Gregorio, E. Hernando, P. Manuel-
Manresa, R. Quesada, R. Pérez-Tomás and V. Soto-Cerrato,
Biochem. Pharm., 2017, 126, 23–33.
–
negative ESI-MS: m/z 399.51 ([M–H] ) and negative HR-ESI-MS for
C H F N ([M–H] ) Calcd: 399.0675; Found: 399.0678.
–
2
0 8 5 4
4
,5,6,7
Synthesis of
8
F -FBimbe 13
A solution of 3,4,5,6-tetrafluorobenzene-1,2-diamine (98 mg, 0.54
mmol) and 5-fluoro-1,3-benzenedialdehyde (42 mg, 0.27 mmol) in
DMSO (3 mL) was heated at 50 °C for 48 h. Then the reaction
mixture was added to water (200 mL). The formed precipitates
were collected through filtration and washed thoroughly with
1
4
E. Hernando, V. Soto-Cerrato, S. Cortes-Arroyo, R. Pérez-Tomás
and R. Quesada, Org. Biomol. Chem., 2014, 12, 1771–1778.
V. Soto-Cerrato, P. Manuel-Manresa, E. Hernando, S. Calabuig-
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15
4
,5,6,7
F8-FBimbe 13 (74 mg, 58%) having mp 267.0-
methanol to give
o
1
2
67.8 C; H NMR (DMSO-d , 400 MHz) δ 8.81 (s, 1H), 8.05 (d, J = 9.2
6
1
Hz, 2H); H NMR (TFA-d, 400 MHz) δ 9.07 (s, 1H), 8.32 (d, J = 7.6 Hz,
1
6
7
N. Busschaert, M. Wenzel, M. E. Light, P. Iglesias-Hernandez, R.
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1
3
2
1
1
H); C NMR (TFA-d, 100 MHz) δ 166.1 (d, J = 240.8 Hz), 151.4,
43.6 (dddd, J = 256.7, 17.2, 15.6 and 4.2 Hz), 137.7 (ddd, J = 255.3,
5.6 and 4.2 Hz), 127.6 (d, J = 8.5 Hz), 126.7 (×2), 123.8 (d, J = 25.2
1
4136–14148.
1
P. I. Hernandez, D. Moreno, A. A. Javier, T. Torroba, R. Pérez-
Tomás and R. Quesada, Chem. Commun., 2012, 48, 1556–1558.
Hz), 120.0 (ddd, J = 16.8, 5.1 and 2.2 Hz); negative ESI-MS: m/z
–
+
4
71.37 ([M–H] ) and HR-ESI-MS for C H N F ([M+H ]) Calcd:
20 6 4 9
18 T. Saha, A. Gautam, A. Mukherjee, M. Lahiri and P. Talukdar, J.
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73.04433; Found: 473.04366.
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9
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20
Literature protocols were used to carry out the experiments for
2
9,31
46
33
anion recognition,
vesicle formation,
pH discharge,
3
6
23
37
chloride efflux, U-tube assay, calcein leakage, acridine orange
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4-19,29,41
14-19,29
16,17
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JC-1 staining
MQAE assay,
and MTT-based cytotoxicity assay.
Hoechst 33342 staining,
1
6,17
16,17
1
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
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