Structure-activity relationships in tripodal transmembrane anion transporters: The effect of fluorination

Article abstract of DOI:10.1021/ja205884y

A series of easy-to-make fluorinated tripodal anion transporters containing urea and thiourea groups have been prepared and their anion transport properties studied. Vesicle anion transport assays using ion-selective electrodes show that this class of com

Full text of DOI:10.1021/ja205884y

ARTICLE
pubs.acs.org/JACS
StructureÀActivity Relationships in Tripodal Transmembrane Anion
Transporters: The Effect of Fluorination
Nathalie Busschaert,† Marco Wenzel,† Mark E. Light,† Paulina Iglesias-Hernꢀandez,‡ Ricardo Pꢀerez-Tomꢀas,‡
and Philip A. Gale*,†
†Chemistry, University of Southampton, Southampton, SO17 1BJ, U.K.
‡Department of Pathology and Experimental Therapeutics, Cancer Cell Biology Research Group, University of Barcelona,
Barcelona, Spain
S Supporting Information
b
ABSTRACT: A series of easy-to-make fluorinated tripodal
anion transporters containing urea and thiourea groups have
been prepared and their anion transport properties studied.
Vesicle anion transport assays using ion-selective electrodes
show that this class of compound is capable of transporting
chloride through a lipid bilayer via a variety of mechanisms,
including chloride/H+ cotransport and chloride/nitrate, chloride/
bicarbonate, and to a lesser extent an unusual chloride/sulfate
antiport process. Calculations indicate that increasing the degree of
fluorination of the tripodal transmembrane transporters increases
the lipophilicity of the transporter and this is shown to be the major
contributing factor in the superior transport activity of the
fluorinated compounds, with a maximum transport rate achieved
for clog P = 8. The most active transporter 5 contained a urea functionality appended with a 3,5-bis(trifluoromethyl)phenyl group and was
able to mediate transmembrane chloride transport at receptor to lipid ratios as low as 1:250000. Proton NMR titration and single crystal
X-ray diffraction revealed the ability of the tripodal receptors to bind different anions with varying affinities in a 1:1 or 2:1 stoichiometry in
solution and in the solid state. We also provide evidence that the most potent anion transporters are able to induce apoptosis in human
cancer cells by using a selection of in vitro viability and fluorescence assays.
’ INTRODUCTION
antiporters, and these properties have been linked to their biological
activity as anticancer agents.7,9,10 This lack of natural anionophores
has led to the design of a variety of synthetic molecules that can act
as transmembrane transporters for anions, including compounds
based on peptide fragments,11 anionÀπ slides,12 steroids,13 calix-
pyrroles14 and calixarenes,15 and other scaffolds.4
We have recently become interested in the transmembrane
anion transport mediated by small, structurally simple molecules,16
including the tripodal tris-urea 1 and tris-thiourea 6 based on the
tris(2-aminoethyl)amine (tren) scaffold (Chart 1).17 Reinhoudt and
co-workers were the first to show that tren-based tris-amides are
effective receptors for anions such as chloride.18 More recently, tren-
based tris-ureas have been shown to effectively bind oxo-anions, such
as sulfate and phosphate, by Ghosh,19 Custelcean,20 and others.21
The transmembrane transport abilities of tren-based anion receptors
have also been investigated. D. K. Smith et al. showed that tris-amides
based on tren are capable of HCl transport,22 while J. T. Davis et al.
showed that tren-based receptors containing catechol moieties are
capable of transporting chloride across a lipid bilayer.23 Tren-based
Transmembrane transport of anions across lipid bilayers is
an important biological process that is normally regulated by
specialized proteins embedded within the biological membrane
(ion channels). Malfunctioning transport proteins can lead to a
variety of pathologies, so-called ‘channelopathies’,1 including
the commonly inherited disease cystic fibrosis,2 the renal dis-
ease Bartter’s syndrome and some forms of myotonia (muscle
stiffness).3 This has led in recent years to an interest in the
development of small molecules that can act as potential future
therapeutic substitutes for these malfunctioning proteins.4 Further-
more, transport of anions in living cells is also essential in regulating
membrane potentials, cell volume and intracellular pH.5 Since the
alteration of pH regulation is an early event in apoptosis, it has been
suggested that compounds that can change internal pH regulation,
e.g. through HCl transport or through chloride/bicarbonate trans-
port, can offer a possible approach for anticancer therapy.6,7
Although there are several examples of nonprotein natural
products that can function as cation transporters, such as
valinomycin,8 only few examples of natural anion transporters can
be found. The most notable examples are the prodigiosins, which
have been shown to function as HCl transporters and as anion
Received: June 29, 2011
Published: August 16, 2011
r
2011 American Chemical Society
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Chart 1. Structures of Ureas 1À5 and Thioureas 6À10
Table 1. Association Constants Ka (MÀ1) for the Binding of
Compounds 1À10 to Various Anions in DMSO-d6 Contain-
ing 0.5% Water at 298 K, Following the Most Upfield
(Thio)urea NH (errors are within 15%).a
2À
À
À
À
ClÀ
SO4
H2PO4
HCO3
NO3
Urea-Based Compounds
b,d
b,e
1
2
3
4
5
882 b
575
166
405
517
>104 b
>104
>104 f
>104
>104
443 c
452 c
>104 f
243 c
À
À
e
365
>104 f,g
156
À
e,f
À
e
À
h
i
e
À
À
À
Thiourea-Based Compounds
b,i
b,e
6
191 b
179
128
156
>104 b
>104
>104
256 c
227 c
130
À
À
i
e
7
À
À
i
e
e
e
8
À
À
receptors containing sulfonamide functionalities have been investi-
gated by B. D. Smith and co-workers for their ability to transport
phospholipid head groups through a lipid bilayer, i.e. for their flippase
activity.24 In our previous studies on compounds 1 and 6, we found
that this type of receptor is capable of transporting chloride via
chloride/nitrate or chloride/bicarbonate antiport mechanisms. How-
ever, for practical applications of these anion transporters in medic-
inal and biological settings, it is necessary that high ion fluxes
can be achieved at low transporter concentrations, without compro-
mising other ADMET (absorption, distribution, metabolism, excre-
tion, and toxicity) properties of the compound. A common strategy
in the pharmaceutical arena to improve the physiological pro-
perties of potential new drugs is by investigating fluorinated analo-
gues. Fluorinated compounds are often less toxic and show higher
metabolic stability than their unfluorinated analogues.25 Further-
more, fluorination of aromatic compounds results in increased
lipophilicity and hydrogen bond acidity, which may subsequently
lead to stronger anion binding properties.26 Both characteristics are
expected to be favorable for the transport of anions across a lipid
bilayer.
Herein we report the transmembrane transport abilities of
tren-based tris-ureas 1À5 and tris-thioureas 6À10 (Chart 1), to
provide insight into the relationship between the structure, anion
affinity, lipophilicity and anion transport ability of the fluorinated
and unfluorinated tripodal receptors 1À10. The anion binding
properties in solution and the solid state are investigated and
linked to anion transport abilities obtained through vesicles
tests using ion-selective electrodes or fluorescence assays. The
results show a significant increase of anion transport in vesicles
for the fluorinated compounds compared to the unfluorinated
analogues. We demonstrate that this effect is mainly due to an
increase in lipophilicity, rather than to variations in anion bind-
ing affinity. We also provide initial results showing that the
fluorinated compounds that display the highest anion transport
activity in vesicles, possess anticancer properties, presumably
by inducing apoptosis in cancer cells through anion transport.
d
h
i
9
À
À
À
À
À
d
d
h
i
10
À
À
À
À
a Anions added as TBA salts, except HCO3À which was added as a TEA
salt. Fitted to 1:1 model. b Previously published data.17 c Data for
DMSO-d6/10% water, data for DMSO-d6/0.5% water could not be
fitted. d Data could not be fitted to any model. e No significant shift of
NH peaks, no binding. f Data for neat DMSO-d6 by Ghosh et al.,19 values
for DMSO-d6/0.5% water are expected to be lower. g 2:1 model. h New
peaks due to deprotonation of bound H2PO4À and subsequent binding
of HPO42À. i Peak broadening.
iso(thio)cyanate in dry dichloromethane. Synthetic details and
characterization of the compounds are described in the Support-
ing Information.
Anion Binding in Solution. The ability of receptors 1À10 to
bind anions in solution was investigated using 1H NMR titration
techniques in DMSO-d6 containing 0.5% water (with the anions
added either as tetrabutylammonium (TBA) or tetraethylammo-
nium (TEA) salts). The binding studies were performed for
various anions relevant in biological systems and in transmem-
brane transport assays. Where possible, the change in chemical
shift of the (thio)urea NH signals was fitted to a 1:1 or 1:2
binding model using the WinEQNMR2 computer program,27
and the results are summarized in Table 1. Previously reported
stability constants are included for comparison.17,19 Stacked
NMR plots, fitted curves, and Job plots can be found in the
Supporting Information (Figures S15ÀS63).
In agreement with previously reported tripodal tris-ureas,17,19
all compounds show strong 1:1 binding with tetrabutylammo-
nium sulfate (Ka > 104 MÀ1) and only limited interaction with
tetrabutylammonium nitrate. Titration experiments with tetra-
butylammonium chloride showed 1:1 complexation processes
occurring in solution with association constants of moderate
strength (Ka ≈ 102 MÀ1). The only receptor that did not fit to a
1:1 model was compound 10, and Job plot analysis with
tetrabutylammonium chloride confirmed a mixture of 1:1 and
2:1 receptor to chloride stoichiometries (Supporting Informa-
tion, Figure S22). In all cases the binding constants obtained
for the more acidic thioureas are lower than for the equivalent
ureas. Surprisingly, the obtained stability constants decrease with
the addition of increasingly electron-withdrawing substitu-
ents. This trend is opposite to what is normally expected, since
electron-withdrawing groups, such as fluorines,26 increase the hydro-
gen bond acidity of a receptor and hence show stronger hydrogen
’ RESULTS AND DISCUSSION
Synthesis. We have previously reported the synthesis and
properties of compounds 1 and 6.17 The synthesis and anion
binding properties of receptor 3 were reported by Ghosh et al.19
All other receptors (2, 4, 5, and 7À10) are novel and could be
readily synthesized in good yields by the reaction of tris(2-
aminoethyl)amine with three equivalents of the appropriate
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bond interactions with anions.28 A possible explanation for the
reverse trend observed here may be due to solvent effects. The
electron-withdrawing effect of the substituents not only increases the
binding to anions, but it will also increase the binding to other
hydrogen bond acceptors such as DMSO or water. Therefore,
fluorine substitution might lead to increased competition from the
solvent and hence lead to lower binding constants with anions in
competitive solvents.28
same NMR spectra as obtained by the addition of excess dihydrogen
phosphate (Figure 1). A control experiment where tetrabutylammo-
nium hydroxide was titrated in the absence of dihydrogen phosphate
did not result in the emergence of new downfield peaks, indicating
that deprotonation of bound dihydrogen phosphate is responsible
for the new peaks, rather than deprotonation of the receptor. It is
clear that some of the tripodal tris(thio)ureas can sufficiently change
the pKa of dihydrogen phosphate through multiple hydrogen bond
formation to allow deprotonation of the bound anion by free
dihydrogen phosphate in solution. It is possible that similar processes
can account for the complex binding curves observed for the other
tripodal tris-ureas and tris-thioureas 1À10 with both tetraethy-
lammonium bicarbonate and tetrabutylammonium dihydrogen
phosphate.
The interactions of 1À10 with tetraethylammonium bicarbo-
nate and tetrabutylammonium dihydrogen phosphate proved to
be less straightforward to interpret. Data analysis of the NMR
titrations of thioureas 6À10 with tetraethylammonium bicarbo-
nate was greatly hindered by peak broadening, which can be
indicative of a proton transfer from the ligand to bicarbonate or
from bound bicarbonate to free bicarbonate in solution. How-
ever, proton transfer is not the only process present, since the
addition of aliquots of strong base (tetrabutylammonium hydro-
xide) did not yield the same spectra as did the addition of
bicarbonate (compare Supporting Information Figures S52, S55,
and S56 with Figures S44, S46, and S48). Although significant
peak broadening also occurred upon the addition of bicarbonate
to the urea compounds 1À5, the peaks could still be followed
throughout the titration. However, the data could either not be
fitted to any model or the 1:1 fit gave larger errors (between 10%
and 15% error). This might be due to multiple equilibria in the
system—deprotonation events and mixed stoichiometries being
the most likely processes to occur—but Job plot analysis
indicated that 1:1 stoichiometry is dominant in DMSO-d6/
0.5% water solutions. Similar problems were encountered during
NMR titrations with dihydrogen phosphate. When the NMR
binding studies were performed in DMSO-d6/0.5% water, the
data could not be fitted to a 1:1 or 2:1 binding model (Supporting
Information), even though strong 1:1 complex formations
have been observed for 1, 3, and 6 in neat DMSO.19,29 This
problem did not occur when the titrations were repeated in
DMSO-d6 containing 10% water, yielding the stability constants
shown in Table 1. However, when considering the binding
constants obtained for both NH functions separately, it appears
that the interaction of the ligands with dihydrogen phos-
phate is still complex in this highly competitive solvent mixture
(e.g., compound 1, Ka for aromatic urea NH is 6363 MÀ1 and Ka
for alkyl urea NH is 443 MÀ1 in DMSO-d6 containing 10%
water). The large difference in binding constants between the
two urea NHs was also observed by Ghosh and co-workers for
the interaction of 3 with dihydrogen phosphate and was attrib-
uted to desolvation of DMSO from the cavity, or to conforma-
tional changes in the receptor.19 In the case of compounds 5, 9,
and 10 the NMR titration with tetrabutylammonium dihydrogen
phosphate showed another unexpected result where a combination
of fast and slow exchange processes were present in both DMSO-d6/
0.5% water and DMSO-d6/10% water mixtures. Up to the addition
of one equivalent of dihydrogen phosphate a downfield shift of the
NH groups was observed, while the addition of more aliquots of
anion resulted in the appearance of new peaks further downfield.
This behavior has been observed previously for receptors that bind
anions through multiple hydrogen bonds and has been explained by
the deprotonation of bound dihydrogen phosphate and the subse-
quent formation of a monohydrogen phosphate complex.30 To test
whether the same process occurs in the tripodal systems, we
conducted NMR experiments in which one equivalent of dihydrogen
phosphate was added to 5, 9, or 10, followed by the addition of
aliquots of tetrabutylammonium hydroxide. This resulted in the
In summary, we found that tripodal receptors 1À10 can
bind anions in DMSO/water solutions according to the trend
SO42À > H2PO4À > ClÀ > HCO3À . NO3À (e.g., compound 2,
Table 1 and Supporting Information Table S1), with various
À
deprotonation events occurring upon interaction with H2PO4
or HCO3À. We have also shown that, in competitive DMSO/
water mixtures, the compounds with the most acidic NH protons
have the lowest association constants—resulting in higher bind-
ing constants for the ureas compared to those of thioureas and for
those of the unfluorinated compounds compared to those of the
fluorinated compounds.
Anion Binding in Solid State. Binding properties in the
solid state were studied by single-crystal X-ray diffraction. Crystal
structures of anion complexes with tripodal tris-ureas have
been reported previously by Ghosh,19 Custelcean,20 and others.21
Here we provide a comprehensive overview of the structural
features in solid-state complexes of compounds 1À10 with various
anions obtained by our group and others. The focus will lie on the
complexes of 7 and 8 and on how the structural information can
be related to the transport properties of these compounds. Method
of crystallization, tables of hydrogen bonds, data collection
and refinement details, and thermal ellipsoid plots can be found
for each structure in the Supporting Information, CIF files are also
provided.31
X-ray analysis of single crystals of compounds 2, 7, and 9
revealed a preference of the tripodal (thio)ureas to form cage-like
structures. In the structure of urea 2, the cage is stabilized by two
intramolecular hydrogen bonds between an oxygen atom of
one arm and the NH functions of another arm (N O distances
3 3 3
2.911(2) Å and 2.967(2) Å with NÀH O angles of 156°
3 3 3
and 148°). The other two oxygen atoms are involved in intermolecular
hydrogen bonds with neighboring molecules, allowing crystal packing
(N O distances 2.843(2)À3.091(2) Å and NÀH O angles
3 3 3
3 3 3
between 146°À159°). A similar observation was made by Ghosh et al.
for free receptor 3.19 In the case of thioureas 7 and 9, two sulfur atoms
are involved in intramolecular hydrogen bonds (N S distances
3 3 3
3.267(4)À3.521(2) Å and NÀH S angles 137°À168°), whilethe
3 3 3
remaining sulfur atom is involved in crystal packing via inter-
molecular hydrogen bonds in the range 3.370(4)À3.502(4) Å
and with NÀH S angles 141°À162° (Figure 2a).
3 3 3
The tetrabutylammonium chloride complexes of receptors
2, 7, and 8 crystallized as 1:1 complexes, reflecting the stoichi-
ometry observed in solution. Six hydrogen bonds, one from each
NH group, stabilize the complex (Figure 2b). All NÀH Cl
3 3 3
angles are larger than 145°, and N Cl distances vary from
3 3 3
3.200(2) Å to 3.600(2) Å.
We were able to grow single crystals of tetrabutylammonium
sulfate complexes of compounds 7 and 8. Even though the anion
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1
Figure 1. (a) H NMR titration with compound 5 in DMSO-d6/0.5% water with tetrabutylammonium dihydrogen phosphate; (i) free receptor;
(ii) one equivalent of tetrabutylammonium dihydrogen phosphate; (iii) 2 equivalents of tetrabutylammonium dihydrogen phosphate; (iv) 3 equivalents
of tetrabutylammonium dihydrogen phosphate. (b) 1H NMR titration with compound 5 in DMSO-d6/0.5% water with tetrabutylammonium
dihydrogen phosphate and TBA hydroxide; (i) free receptor; (ii) one equivalent of tetrabutylammonium dihydrogen phosphate; (iii) one equivalent of
tetrabutylammonium dihydrogen phosphate + one equivalent of tetrabutylammonium OH; (iv) one equivalent of tetrabutylammonium dihydrogen
phosphate +1.5 equiv of tetrabutylammonium OH.
binding studies in solution showed a clear 1:1 stoichiometry
with sulfate, the crystal structure obtained for (7)2⊃SO4 revealed
a 2:1 complex in the solid state (Figure 2c). The disordered
sulfate ion is encapsulated between two ligands and is stabilized
via 14 hydrogen bonds to the six thiourea groups (Figure 2c).
Eleven of these hydrogen bonds can be classified as moderate
with N O distances <3.2 Å (N O distances from 2.840(4)
complex of 8 with sulfate did show the 1:1 stoichiometry
observed in solution (Figure 2d). Six strong hydrogen bonds
between the sulfate oxygens and the thiourea NH functions
are responsible for complex formation (N O distances 2.757-
3 3 3
(4)À2.883(4) Å and NÀH O angles 149°À169°). In this
3 3 3
structure, one of the TBA counterions is held in close contact to the
sulfate complex via two CÀH O interactions between oxygen
3 3 3
3 3 3
3 3 3
Å to 3.091(4) Å), while three of them are weaker (N
O
O
atoms in sulfate and CH2 groups adjacent to the positively charged
3 3 3
3 3 3
distances from 3.210(4) Å to 3.295(4) Å).32 All NÀH
nitrogen in TBA (C O distances 3.326(5) Å (CÀH O angle
3 3 3
3 3 3
angles are larger than 145°. Similar 2:1 complexes with sulfate
have been reported before for other tren-based tris-urea
receptors.19À21 On the other hand, the crystal structure of the
172°) and 3.334(4) Å (CÀH O angle 171°)), similar to
3 3 3
previously reported contact ion-pair interactions between tetra-
butylammonium and tripodal sulfate complexes.33
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behavior was observed in crystals grown from 5 with TEAHCO3.
All of these complexes ((1)2⊃CO3, (5)2⊃CO3 and previously
reported19 (3)2⊃CO3) form 2:1 complexes with CO3 via
2À
twelve hydrogen bonds or more, with NÀH O angles from
3 3 3
142° to 174° and the majority of the N O distances in the
3 3 3
range of 2.829(2)À3.392(2) Å, but some weaker hydrogen
bonds are also present (Figure 2e). Attempts to crystallize a
bicarbonate complex of receptor 8 failed, presumably because
bicarbonate functioned as a Brønsted base and deprotonated one
of the thiourea NH groups instead of forming an anion complex.
Thus, the crystal structure obtained was that of a dimer of
deprotonated 8 (Figure 2f), stabilized by intermolecular hydro-
gen bonds between the remaining NH functions and the sulfur
atom of the deprotonated thiourea group (five hydrogen bonds
per sulfur atom, N S distances from 3.274(4) Å to 3.451(5) Å
3 3 3
and NÀH S angles from 140° to 170°).
3 3 3
As was observed in solution, the solid-state structures for
dihydrogen phosphate (with 7, 8, and 10) proved to be morÀe
varied than for other anions. The complex of 8 with H2PO4
crystallized as the same 2 + 2 dimer observed for the analogous
urea 3.19 This structure consists of two H2PO4 ions that are
À
hydrogen bonded to each other. The phosphate dimer is further
encapsulated by two ligand molecules, stabilized by hydrogen
bonding to the (thio)urea NH functions and by anionÀπ
interactions. On the other hand, the solid-state complexes
formed by 7 and 10 with TBAH2PO4 showed the same depro-
tonation of bound dihydrogen phosphate as observed in solution
during NMR titrations and crystallized as 2:1 complexes with
2À
HPO4
in the presence of excess TBAH2PO4. Complex
(7)2⊃HPO4 is stabilized by 14 hydrogen bonds, where each
NH in the two ligands contributes to one hydrogen bond (except
N7ÀH7, which is bifurcated). Each oxygen in the phosphate
anion accepts three or four hydrogen bonds. N O distances
3 3 3
vary between 2.800 (4) Å and 3.253(4) Å and NÀH O angles
3 3 3
are in the range 144°À165°. Complex (10)2⊃HPO4 is stabilized
by 12 hydrogen bonds, one from each NH in the six thioureas
and three for each oxygen in phosphate. N O distances vary
3 3 3
between 2.829(6) Å and 3.047(6) Å and NÀH O angles
3 3 3
range from 149° to 169° (Figure 2g).
Even though no binding could be observed for nitrate in
solution under the conditions of the NMR titration experiments,
we were able to obtain a nitrate complex in the solid state
(8⊃NO3). The structure shows a nitrate ion bound inside the
cavity of the ligand via six hydrogen bonds (N O distances
3 3 3
from 2.884(6) to 3.366(7) and NÀH O angles from 129° to
3 3 3
166°) in a 1:1 stoichiometry. However, closer inspection reveals
a 2 + 2 dimer, where two nitrate:ligand complexes are held
together via anionÀπ interactions. The distance between one
oxygen atom of the nitrate ion and the centroid of an electron-
poor aromatic ring of the second ligand is 3.013(4) Å (distance
between nitrate N and the nearest aromatic carbon is 3.105(7) Å).
These short distances imply a significant interaction,34 justifying
the claim of a 2 + 2 stoichiometry (Figure 2h).
Figure 2. X-ray crystal structures of 5, 7, and 8 with a variety of anionic
guests. Ligands are represented as wireframes, and bound anions are
shown in a spacefilling view (0.6 times van der Waals radius). Solvent
molecules, counterions, and noninteracting hydrogens are omitted for
clarity: C (gray), H (white), N (blue), S (yellow), O (red), F (green), Cl
(dark green), P (orange). Hydrogen bonds are represented by dashed
lines. (a) Free ligand 7; (b) 7⊃Cl; (c) (7)2⊃SO4 (disordered sulfate);
Figure 3 shows spacefilling models of the chloride complex of
receptor 2 and the carbonate complex of compound 5. This
representation shows clearly that only a small fraction of the
bound anion is accessible to the environment, because the anion
is completely encapsulated by one or two tripodal receptors. This
would imply that the hydrophilic anions can be almost comple-
tely screened from the hydrophobic membrane by the lipophilic
ligands—especially in the cases of 2:1 complexes. Such structures
would be ideal for transporting anions across lipid bilayers.
(d) 8⊃SO4 TBA; (e) (5)2⊃CO3; (f) (8-H)2; (g) (7)2⊃HPO4
3
(disordered hydrogen phosphate); (h) (8⊃NO3)2.
Solid-state bicarbonate interactions were studied for com-
pounds 1, 5, and 8. We have previously reported that crystal-
lization of 1 in the presence of TEAHCO3 resulted in a 2:1
complex of 1 with CO32À (rather than with HCO3À).17 Similar
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Figure 3. Spacefilling representations of the X-ray structures of
(a) 2⊃Cl and (b) (5)2⊃CO3. Ligands are shown in grayscale and
anions in color: C (gray), O (red), Cl (green).
Transport Studies. We wished to assess the ability of com-
pounds 1À10 to transport ions across a lipid bilayer and hence
assess the effect of fluorination on the transport ability of this
class of compound. Ion-selective electrode (ISE) or fluorescence
assays were used to monitor ion transport. In a typical assay, we
prepared a series of unilamellar 1-palmitoyl-2-oleoylphosphatidyl-
choline (POPC) vesicles of defined size (200 nm in diameter).
The vesicles were loaded with a buffered sodium chloride
solution (489 mM in 5 mM phosphate buffer at pH 7.2) and
suspended in an isotonic sodium nitrate solution according to
literature procedures.35 Putative transporters 1À10 were added
as a solution in a small amount of DMSO and the resultant
transport of chloride out of the vesicles was monitored using a
chloride selective electrode. At the end of the experiment, the
vesicles were lysed by addition of detergent, and the final reading
was used to calibrate the ISE to 100% chloride release (Figure 4).
It is evident from Figure 4 that fluorination has a profound
impact on the transport abilities of these systems. In all cases, the
fluorinated compounds show a significantly faster release of
chloride than the nonfluorinated analogues.
Evidence for Anion Antiport Mechanism. Transport of ions
by simple synthetic molecules usually only occurs via passive
transport mechanisms in which the charge balance across the
membrane is maintained. This can be achieved by either a
symport mechanism, where both an anion and a cation are
transported, or by an antiport mechanism, where two anions are
transported across the membrane in opposite directions.4 The
chloride efflux shown in Figure 4 could be the result of Na+/ClÀ
Figure 4. Chloride efflux promoted by 1À10 (2% molar carrier to lipid)
from unilamellar POPC vesicles loaded with 489 mM NaCl buffered to
pH 7.2 with 5 mM sodium phosphate salts. The vesicles were dispersed
in 489 mM NaNO3 buffered to pH 7.2 with 5 mM sodium phosphate
salts. At the end of the experiment, detergent was added to lyse the
vesicles and calibrate the ISE to 100% chloride efflux. Each point
represents the average of three trials. DMSO was used as control. (a)
Urea compounds 1À5. (b) Thiourea compounds 6À10.
or H+/ClÀ symport or ClÀ/NO3 antiport. We have shown
À
chloride/bicarbonate antiporters, because this is a more biologi-
cally relevant process37 that has been linked to a number of
pathologies.2,38 This was combined with the aforementioned
sulfate assay, as shown in Figure 5. Two minutes after the
addition of the transporters, NaHCO3 was added to the external
Na2SO4 solution, which led to a marked acceleration of chloride
efflux (Figure 5). However, the transport rates are still slower
than those obtained for chloride/nitrate exchange (Figure 4).
Even though the experimental conditions are different (40 mM
NaHCO3 compared to 489 mM NaNO3), this might be explained
by the reduced lipophilic nature of bicarbonate compared to nitrate
(ΔGhydr(HCO3À) À335 kJ molÀ1).36 In general, the chloride efflux
mediated by the tripodal recÀeptors depends on the external anion
previously that receptors 1 and 6 function predominantly via
anion antiport mechanisms (ClÀ/NO3 or ClÀ/HCO3 anti-
port) using a variety of ISE vesicle studies and 13C NMR assays.17
To test whether the same mechanism operates for the fluorinated
analogues, vesicles containing a NaCl solution were prepared and
suspended in a solution of Na2SO4. Transport was initiated by
the addition of receptor in a DMSO solution at a 2 mol %
receptor to lipid concentration (Figure 5). With a double
negative charge, sulfate is a much more hydrophilic anion than
nitrate (ΔGhydr(SO42À) À1080 kJ molÀ1; ΔGhydr(NO3À) À300
À
À
kJ mol )
À1 36 and is consequently considerably more challenging
to transport across a hydrophobic membrane. As shown in
Figure 5, the majority of the compounds release only a limited
amount of chloride under these conditions. This strong
dependence on the external anion suggests that the receptors
transport chloride predominantly via an exchange mechanism.
We also tested the compounds for their ability to function as
À
and follows the trend NO3 > HCO3 . SO42À. This trend
suggests that an antiport mechanism is predominant and also
shows the importance of lipophilicity in the transport abilities of
simple molecules. The highest transport rates are obtained for the
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prepared POPC vesicles loaded with NaCl (489 mM) and 1 mM
of the pH-sensitive dye 8-hydroxy-1,3,6-pyrenetrisulfonate
(HPTS).39 The vesicles were suspended in a Na2SO4 solution
and upon addition of the carrier the fluorescence was measured.
At the end of the experiment the vesicles were lysed with
detergent. The result for compound 8 showed an initial rise in
the internal pH followed by a decrease (Supporting Information,
Figure S96). Similar behavior has been observed previously by
our group for 1 and 617 and by Berezin and Davis23 and was
explained by an initial, small amount of HCl transport, creating a
pH gradient across the membrane that could be compensated
by slow transport of HNO3. In this experiment this might
indicate that the initial HCl cotransport is compensated by back
transport of protons leading to a lower internal pH. The same
experiment was repeated for the other tripodal compounds
1À10, all of which showed an increase in internal pH, indicative
of a small amount of HCl cotransport (Supporting Information,
Figure S96). In most cases the rise in pH was not compensated
and remained stable at higher pH, with the exception of
compound 5 where the initial rise in intravesicular pH was
reversed, and a decrease in pH was observed, presumably due
to the back transport of protons into the vesicles.
Even though the high hydrophilicity of sulfate and the HPTS
test suggest otherwise, there are still several reasons to believe
that the activity observed for 8 is partly due to ClÀ/SO4
2À
antiport. First, the high binding constant for sulfate—combined
with a lipophilic receptor that can easily screen the sulfate from
the lipid bilayer—might compensate for the high hydrophilicity
of sulfate. Second, the other related receptors all function
predominanÀtly as antiporters, as shown by the ClÀ/NO3À and
ClÀ/HCO3 assays. A final argument is that the HPTS test
indicated that the initial HCl efflux can be compensated by a
back-transport mechanism. One possibility for this back trans-
port could be the influx HSO4À, which would formally result in a
ClÀ/SO42À exchange mechanism. To provide further evidence
to support this theory, we prepared a series of liposomes loaded
with NaCl and lucigenin that were suspended in a NaCl solution
(100 mM). The lack of a chloride and pH gradient rules out HCl
cotransport. Lucigenin was used to monitor the internal chloride
concentration, since the fluorescence of this dye is quenched by
halide ions.40 The experiment was initiated by the addition of
various anions (Na2SO4, NaNO3, or NaCl) followed by the
addition of a methanol solution of 8. At the end of the experiment
the vesicles were lysed with detergent. The result shown in
Figure 6 shows a significant increase in fluorescence upon the
addition of 8 when sulfate was originally added, indicating that
chloride is transported out of the vesicle. When a NaNO3
solution was added instead of Na2SO4, a much faster increase
in fluorescence was observed, as would be expected for an anti-
port process of a more lipophilic anion (Figure 6). As a control,
the experiment was repeated with the addition NaCl instead of
Na2SO4, and in this case the addition of compound 8 did not
result in an increase in fluorescence (a small decrease in fluo-
rescence was observed, as expected for a small influx of chloride
to compensate for differences in ionic strength). The variation of
the change in fluorescence in the presence of these anions
suggests that the nature of the externally added anion is
important and that a change in ionic strength alone is not enough
to transport chloride out of the vesicle. It is therefore plausible
that the increase in fluorescence seen for external sulfate is due to
chloride/sulfate antiport mediated by compound 8. No change
in fluorescence was observed when compound 3, 10, or methanol
Figure 5. Chloride efflux promoted by 1À10 (2% molar carrier to lipid)
from unilamellar POPC vesicles loaded with 450 mM NaCl buffered to
pH 7.2 with 20 mM sodium phosphate salts. The vesicles were dispersed
in 162 mM Na2SO4 buffered to pH 7.2 with 20 mM sodium phosphate
salts. At t = 120 s, a solution of NaHCO3 was added to give a 40 mM
external concentration. At the end of the experiment, detergent was
added to lyse the vesicles and calibrate the ISE to 100% chloride efflux.
Each point represents the average of three trials. DMSO was used as
control. (a) Urea compounds 1À5. (b) Thiourea compounds 6À10.
anion with the highest lipophilicity (nitrate), rather than the anion
with the highest binding constant (sulfate).
However, it must be noted that there is one exception to the
general trend in this series, namely pentafluorophenyl thiourÀea 8.
Even though the transport rates still follow NO3À > HCO3
.
SO42À, significant chloride efflux can be observed when sulfate is
the external anion (Figure 5). The most likely explanation for this
observation is H+/ClÀ symport, because the X-ray structure
obtained for compound 8 in the presence of TEAHCO3 revealed
that the thiourea NH functionality of this compound can be
easily deprotonated. Therefore a potential mechanism could be a
neutral receptor transporting chloride across a membrane and
then loss of HCl with the resulting anionic form of the receptor
diffusing back across the membrane. This would explain why the
chloride efflux is most pronounced for the compound with the
most acidic (thio)urea functionality. To test this hypothesis we
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Evidence for Mobile Carrier Mechanism. There are three
main mechanisms by which ion transport can occur: mobile
carrier mechanism,4 relay mechanism,41 or pore formation (ion
channel).4 On the basis of the X-ray data, a mobile carrier
mechanism seems the most likely. Crystal structures of the free
ligands indicate a preference for a closed cavity-like conforma-
tion, while the anion complexes form well-defined 1:1 or 2:1
entities with the anion situated inside the cavity. It seems
therefore plausible that the ion transport occurs via diffusion
through the bilayer of the 1:1 or 2:1 complexes observed in
solution and in the solid state, rather than via the formation of
channels.
To test this hypothesis, the ISE vesicle studies were repeated
for vesicles consisting of POPC with 30% cholesterol. It is often
suggested that cholesterol decreases the fluidity of a lipid
bilayer42 and that this would have a negative effect on the
transport ability of a mobile carrier, which is largely dependent
on diffusion within the membrane.4 Comparing the anion
transport rates of compounds 1À10 in POPC vesicles and in
7:3 POPC:cholesterol vesicles shows an observable decrease in
transport activity in the presence of cholesterol for the same
carrier to lipid ratio (Supporting Information, Figures S99 and
S100), in agreement with a mobile carrier mechanism.
Further evidence for a carrier mechanism was obtained
through classic U-tube experiments on receptors 4, 6, and 9. In
these experiments the membrane is replaced by a bulk organic
phase separating two aqueous phases. Transport due to the
formation of channels is impossible in this case because of the
large dimensions of the organic phase.4,43 The setup of the
experiment involved a U-shaped tube filled with the organic
phase containing 1 mM carrier. For solubility reasons, nitroben-
zene was used as the organic phase. One arm of the tube was filled
with an aqueous NaNO3 solution, while the other arm was filled
with a NaCl solution and the chloride concentration of the
NaNO3 phase was monitored with an ISE to quantify the
transport through nitrobenzene. No change in chloride concen-
tration was observed in the absence of carrier, but an increase in
chloride concentration could be detected when carrier was
present, indicating that these compounds can act as mobile
carriers (Supporting Information, Figure S101). The extent of
U-tube transport was dependent on the type of carrier according
to 9, 6 > 4, the same trend as observed during the vesicle studies.
This suggests that the mobile carrier mechanism shown in a bulk
organic phase is also present in lipid bilayers. Even though Hill
analysis of the transport data revealed increasing n values for the
thioureas (Table 2) as the hydrophobicity of the compounds
increases, we believe the results of the POPC/cholesterol trans-
port studies (reduced transport rates) and U-tube experiments
remain evidence in favor of a mobile carrier mechanism, and we
propose in this case that we may be seeing a ‘mobile aggregate’
mediating transport rather than a channel possibly due to the
hydrophobic compounds aggregating at the polar waterÀbilayer
interface.
Figure 6. Evidence for transmembrane transport of sulfate. Unilamellar
POPC vesicles were loaded with 100 mM NaCl and 2 mM lucigenin
buffered to pH 7.2 with 20 mM sodium phosphate salts and dispersed in
a 100 mM NaCl solution (buffered to pH 7.2). At t = 30 s, a solution of
the appropriate anion was added (final concentration of 40 mM NaNO3
(red), 40 mM Na2SO4 (green) or 40 mM NaCl (blue)). At t = 60 s, a
methanol solution of the putative transporter was added (2% molar
carrier to lipid). At the end of the experiment (240 s), detergent was added
to lyse the vesicles. Each point represents the average of three trials. A blank
experiment was performed by addition of Na2SO4, followed by addition of
methanol; (a) compound 8. (b) compound 3.
(blank) was added (Supporting Information, Figures S97 and
S98), indicating that only 8 is able to transport sulfate.
However, a small amount of chloride/sulfate antiport was
observed for compound 5, which was the only other receptor
that showed a significant back transport in the HPTS assays—
indicating that the oÀbserved chloride/sulfate antiport might be
due to HCl/HSO4 exchange. To the authors’ knowledge,
this is the first time that a ClÀ/SO42À antiport mechanism has
been observed for a simple synthetic molecule. Although
the selectivity and activity for sulfate transport is poor, it is
still a remarkable result that a simple easy-to-make com-
pound can transport a highly hydrophilic, doubly negatively
charged anion across a lipid bilayer. We believe that the tripodal
(thio)urea based compounds 1À10 transport anions predomi-
nantly by an antiport mechanism and that this scaffold will
provide a good starting point for the challenging design of sulfate
transporters.
StructureÀActivity Relationship. To quantify the transport
abilities of 1À10, we performed a series of Hill analyses44 for the
chloride efflux in both the nitrate and bicarbonate antiport tests
(see Supporting Information for details). This enables us to
calculate EC50,
values, defined as the concentration of
270s
transporter required to achieve 50% chloride release 270 s after
the addition of carrier (or after the addition of bicarbonate). The
values are summarized in Table 2 together with the Hill
coefficients and the initial rate of chloride release promoted by
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Table 2. Overview of Transport Assays and Lipophilicity of Compounds 1À10
À
À
À
À
À
À
compound clog Pa kinib (ClÀ/NO3
)
EC50, 270sc (ClÀ/NO3
)
nd (ClÀ/NO3
)
kinie (ClÀ/HCO3
)
EC50, 270sc (ClÀ/HCO3
)
nd (ClÀ/HCO3
)
Urea-Based Compounds
f
1
2
3
4
5
2.06
2.53
4.43
4.82
7.59
0.081
0.571
1.84
5.6
1.2
1.4
1.4
1.1
1.6
0.024
0.081
0.250
0.46
>5 f
>5 f
À
f
0.43
À
g
g
0.24
À
0.24
À
1.35
0.052
0.0044
1.2
1.5
1.01
0.77
0.036
Thiourea-Based Compounds
6
5.50
5.97
0.97
3.3
0.31
1.9
2.9
2.4
4.8
5.0
0.186
0.47
0.38
0.47
0.76
2.3
1.0
1.2
7
0.042
0.032
0.077
0.042
0.35
g
g
8
7.87
3.2
À
À
4.8
9
8.26
1.18
0.11
10
11.03
0.90
0.14
3.8
a clog P calculated using Spartan ’08 for Macintosh (GhoseÀCrippen model). b Initial rate of chloride efflux for 2% molar carrier to lipid (% sÀ1).
c EC50, 270s defined as concentration (mol % carrier to lipid) needed to obtain 50% efflux after 270 s. d Hill coefficient. e Initial rate of chloride efflux
(after addition of NaHCO3) for 2% carrier to lipid (% sÀ1). f Accurate Hill analysis could not be performed due to low activity. g Meaningful Hill analysis
could not be performed due to significant background transport in the absence of NaHCO3 (HCl symport and/or ClÀ/SO42À antiport).
0.02 molar equivalent of transporter. These values clearly show a
profound fluorination effect, with EC50, 270s values that are at
least 1 order of magnitude smaller than for the unfluorinated
analogue (up to 3 orders of magnitude when comparing 5
with 1). Previous studies have shown that fluorination of
aromatic rings increases the lipophilicity of the molecule and
that this effect is even more pronounced for the addition of a
ÀCF3 group.26 We therefore calculated GhoseÀCrippen log P
values45 for the receptors using the Spartan ’08 computer
program46 (Table 2). When considering only the urea com-
pounds 1À5, a clear correlation between the lipophilicity and
the EC50, 270s values can be discerned for both chloride/nitrate
and chloride/bicarbonate exchange. The higher the calculated
log P value (clog P), the lower the EC50, 270s value—with an
EC50, 270s value for the most lipophilic compound (5) as low as
0.0044 mol % (1:22500 carrier to lipid ratio). The lipophilicityÀ
transport relationship is less pronounced for the thioureas 6À10.
Up to a clog P ≈ 8 the transport activity increases with increasing
lipophilicity, but higher clog P values do not result in lower
EC50, 270s values. In fact, the EC50, 270s values and the initial rate
of chloride release for chloride/nitrate antiport indicate a slight
decrease in activity for the most lipophilic compounds 9 and 10.
An explanation for this might be found in the mobile carrier
mechanism. For a mobile carrier to work, it has to diffuse into the
aqueous phase or to the more polar headgroups of the lipid
bilayer (at the membrane/water interphase) in order to extract
and release an anion in the aqueous phase and transport it
through the membrane. When the carrier is too lipophilic, this
process becomes the limiting step as the carrier remains inside
the lipid bilayer, hence resulting in slower transport of ions. The
same argument can explain the differences in transport ability
between the ureas and the thioureas. The substitution of oxygen
by a sulfur atom in thioureas is known to increase the lipophi-
licity. This explains why compounds 6À8 show a transport
activity more significantly enhanced than the analogous ureas,
and why thioureas 9 and 10 (clog P 8.26 and 11.03, respectively)
prove to be too lipophilic and show decreased transport activity
compared to those of ureas 4 and 5.
transport ability.13,35 However, such a correlation is not observed
in this series of tripodal anion transporters. Comparison of the
association constants of 1À10 with chloride (Table 1) with the
EC50, 270s values (Table 2) reveals a reverse trend where the best
transport ability is observed for the receptor with the lowest
binding constant. However, this correlation is not exact, and
discrepancies can be found for both the ureas and the thioureas
(e.g., compounds 3 and 8 have the lowest binding constants but
not the lowest EC50, 270s values). Overall it seems that the
observed enhanced transport rates of the fluorinated receptors
are mainly due to the impact of fluorination on lipophilicity,
rather than the influence on binding strength. Although there is
clearly an upper limit to the lipophilicity of potential mobile
carriers, this can provide a useful guideline in the design of future
anion transporters.
The exceptionally low EC50, 270s value for compound 5 led us
to investigate the lowest concentration able to mediate
observable chloride transport (Figure 7). Carrier to lipid ratios as
low as 1:100000 and even 1:250000 were still able to mediate
reasonable amounts of chloride efflux within the time scale of the
experiment—which is a remarkable result for such a structurally
simple molecule (and is in the range of the more complex
cholapods, still some of the best mobile carriers reported to
date).13 Despite the activity at low concentrations, Figure 7 also
shows that compound 5 never reaches 100% chloride efflux. At a
loading of 2 mol % (1:50 carrier to lipid) an initial fast release of
chloride is observed, but after one minute the chloride efflux
slows down dramatically, and the maximum chloride release that
could be achieved was 70%, in both chloride/nitrate and
chloride/bicarbonate assays. Changing the carrier concentration
did not improve chloride effluxes, and similar behavior was
observed for compounds 9 and 10 (see Figures 4 and 5). There
are various processes that could cause this limited maximum
efflux, but none could be determined with reasonable certainty.
It was thought that competition with binding events to phos-
phate buffer or to phospholipid headgroups—resulting in so-
called flippase activity24—could explain the sudden change in
chloride release rate; however, exchanging phosphate for
HEPES buffer resulted in the same behavior, and no significant
flippase activity could be detected within a reasonable time-scale
Previous reports on cholapod-based anion transporters indi-
cate that increased binding constants can lead to increased
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the pH changes in acidic cell compartments by acridine orange
staining and by nuclear Hoechst staining for the assessment of
apoptotic cell death induction (experimental details are listed in
the Supporting Information). Apoptosis is an important biolo-
gical consequence of exposure to extrinsic agents, and has been
claimed to be modulated or triggered when the intracellular pH
(pHi) drops below 7.0.47 The pHi in transformed or cancerous
cells generally remains neutral or slightly more alkaline than in
normal cells and is regulated by a variety of pHi homeostatic
mechanisms, including Na+/H+ cotransport, Na+-dependent
and -independent ClÀ/HCO3À exchangers, vacuolar type H+-
ATPase (V-ATPase), and other mechanisms.48 Matsuyama and
co-workers suggested in 2000 that alteration of pHi regulation is
an early event in apoptosis—preceding cytochrome C release
from mitochondria and facilitating caspase activation—and they
proposed that pHi regulatory mechanisms may offer new
approaches for tumor therapy.6 Natural molecules like prodigio-
sins and tambjamines and their derivatives, as well as bafilomycin
A1, are able to lower pHi through HCl transport across cell
membranes and thereby lead to apoptosis.49 It has been sug-
gested that this represents the main reason for the anticancer
activity of the prodigiosins.7
The in vitro cytotoxic activity of receptors 1À10 was tested on
a collection of different cancer cell lines of diverse origin, in casu
human small-cell lung carcinoma (SCLC) cell line GLC4, human
colon adenocarcinoma cell lines HT29 and DLD1, human colon
adenocarcinoma from lymph node metastasis cell line SW620,
squamous cell carcinoma from tongue (CAL27 cell line)
and from mouth floor (HN4 cell line). Initially, a single-point
(10 μM) screening assay was used to evaluate the cytotoxicity of
receptors 1À10 (Figure 8). In this test, the cells were counted, and
an MTT assay was performed in order to evaluate cell viability and
proliferation by measuring the level of mitochondrial dehydrogen-
ase activity.50 When only considering the single-point cell viability
data for cell line GLC4, it is clear that receptors 4, 5, and 7À10
display significant cytotoxic effects, while no effect is observed for
receptors 1À3 and 6. Comparing this result with the EC50, 270s
values of Table 2 reveals that only the receptors possessing
significant anion antiport activity can function as anticancer agents,
since the EC50, 270s values of the cytotoxic molecules are at least 1
order of magnitude better than those obtained for the noncytotoxic
compounds 1À3 and 6. It is also evident from Figure 8 that all
thioureas (and in particular receptors 8, 9, and 10) display a more
widespread cytotoxic effect across the panel of cell lines used in this
work, which might be due to the general increased toxicity of
thioureas compared to that of ureas.51 GLC4, HN4, and CAL27
were identified as the most sensitive cell lines for these types of
compounds, and on the basis of the promising single-point data on
these cell lines, we decided to quantify the cytotoxicity by collecting
doseÀresponse curves for receptors 4À10 in a 24 h viability assay
in GLC4, HN4, and CAL27 cell lines (Supporting Information,
Figures S123ÀS125). The doseÀresponse curves allowed us to
determine the IC50 values reported in Table 3. This data suggests
that receptors 8 and 9 are the most efficient molecules to reduce cell
viability followed by receptor 10.
Figure 7. Chloride efflux promoted by various concentrations of 5 from
unilamellar POPC vesicles loaded with 489 mM NaCl buffered to pH 7.2
with 5 mM sodium phosphate salts and dispersed in 489 mM NaNO3
buffered to pH 7.2 with 5 mM sodium phosphate salts. At the end of the
experiment, detergent was added to lyse the vesicles and calibrate the
ISE to 100% chloride efflux. Each point represents the average of three
trials. DMSO was used as control. Carrier to lipid ratios of 1:50, 1:10000,
1:33000, 1:100000, and 1:250000 were used.
(Supporting Information, Figure S118 and S120). Another
explanation may be that at 70% efflux, the chloride gradient is
too small to allow transport by carrier 5, 9, or 10. However, this
was contradicted by repeating the ISE experiment with a smaller
initial gradient (200 mM NaCl instead of 500 mM), which again
resulted in exactly the same behavior where the chloride efflux
stops when 70% efflux is reached (Supporting Information,
Figure S119). It is also possible that the chloride efflux does
not reach levels higher than 70% because the externally added
transporters cannot reach all of the vesicles. This suggestion
becomes more likely when considering the relatively high Hill
coefficients of the thiourea compounds (n = 3À5, Table 2),
indicating that these compounds can form aggregates (or show
other forms of cooperativity, although channel formation seems
unlikely as discussed above). Since preincorporation into the
vesicles was not possible due to poor solubility in organic
solvents, the theory was tested by repeating the ISE experiments
using more dilute vesicle solutions or by adding the transporters
in discrete steps to enhance distribution into all of the vesicles.
However, this only led to a small increase in chloride efflux,
indicating that delivery to the vesicles is not a major problem
(Supporting Information, Figures S121 and S122). Since this
behavior was observed for the three most lipophilic compounds,
it is possible that lipophilicity plays an important role (e.g.,
competition from precipitation in the aqueous phase), but this
could not be proven. However, a maximum chloride efflux might
be considered a positive characteristic in a biological setting
because a total efflux of all chloride would kill any healthy cell,
and the fact remains that the fluorinated compounds retain high
transport activity at low concentrations.
To further investigate the mechanism by which receptors
1À10 can display cytotoxic effects and to test the in vitro iono-
phoric activity of these compounds, the effect of the tripodal
receptors on GLC4 cell lines was studied using vital staining
with acridine orange (AO). This cell-permeable dye accumulates
in acidic compartments, such as lysosomes, exhibiting a char-
acteristic orange fluorescence emission in its protonated state,
In Vitro Cytotoxicity in Cancer Cell Lines. The anion
transport activity of ureas 1À5 and thioureas 6À10 prompted
us to investigate the in vitro bioactivity of these receptors on
tumor cell lines by the evaluation of cell viability, estimation of
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Figure 9. Acridine orange staining of GLC4 cell line after exposure of
1 h to different receptors: (a) untreated cells (control), (b) cells treated
with receptor 2, (c) cells treated with receptor 3, (d) cells treated with
receptor 7, (e) cells treated with receptor 8, (f) cells treated with
receptor 9. (aÀc) Cells with granular orange fluorescence in the
cytoplasm; (dÀf) cells with complete disappearance of orange fluores-
cence cytoplasm granules.
Figure 10. Hoechst 33342 staining of GLC4 cell line after exposure to
different receptors for 24 h: (a) untreated cells (control), (b) cells
treated with receptor 2, (c) cells treated with receptor 4, (d) cells treated
with receptor 7, (e) cells treated with receptor 8, (f) cells treated with
receptor 9. (aÀc) Cells with normal nuclear morphology; (dÀf) cells
with condensation of the nuclei and nuclei with “bean shape”.
Figure 8. Single-point screening of receptors 1À10 (10 μM) tested on
a collection of different cancer cell lines, from left to right, GLC4, HT29,
DLD1, HN4, CAL27, and SW620; (a) 24-h cell viability of cell exposure
to ureas 1À5; (b) 24-h cell viability of cell exposure to thioureas 6À10.
Table 3. IC50 Values of Receptors 4, 5, 8, 9, and 10 Obtained
from MTT Assays on GLC4, HN4 and CAL27 Cell Lines at 24
h Exposure Timea
intracellular pH. Partial disappearance of the orange staining of
the granules was obtained after exposure to receptors 5 and 10
(data not shown). On the other hand, cells treated with receptors
1, 2, 3, and 6 showed no changes in fluorescence. The cells were
incubated for 1 h with 10 μM (receptors 1, 2, 3, 6, and 7) or the
IC50 dose (receptors 4, 5, 8, 9, and 10) of the relevant receptors,
and the fluorescence due to AO was monitored. Representative
results are depicted in Figure 9 (additional results can be found in
the Supporting Information, Figures S126 and S127). The
obtained results correlate well with the activity observed in the
vesicle assays. Only active anionophores (EC50, 270s < 0.1 mol %
for chloride/nitrate antiportor EC50, 270s < 1.0 mol % for chloride/
bicarbonate antiport) induce an increase in the lysosomal pH,
whereas the inactive receptors 1, 2, 3, and 6 did not affect
intracellular pH. Combining the results obtained in the vesicle
assays with the in vitro AO staining results leads us to Àsuggest that
an anion antiport mechanism such as ClÀ/HCO3 exchange
could be responsible for the increase in internal pH, similar to
recently reported synthetic tambjamines,54 although other
receptors
GLC4
CAL27
HN4
4
7.18 ( 0.91
5.12 ( 0.98
2.43 ( 0.14
2.70 ( 0.04
3.05 ( 0.06
25.77 ( 8.31
10.93 ( 1.88
12.16 ( 1.61
10.98 ( 0.80
10.76 ( 0.82
27.63 ( 3.35
14.73 ( 0.81
11.04 ( 0.04
8.71 ( 0.79
9.05 ( 0.52
5
8
9
10
a Results represent a mean of three independent experiments with
standard deviation and are in μM.
whereas it emits green fluorescence at higher pH.53 When GLC4
cells were stained with AO, granular orange fluorescence was
observed in the cytoplasm (Figure 9a), suggesting that the
orange fluorescence is due to acidified lysosomes. Cells treated
with receptors 4, 7, 8, and 9 showed a complete disappearance of
orange emission, indicating that these receptors can alter the
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ARTICLE
mechanisms such as H+/ClÀ cotransport processes cannot be
ruled out. If the lipophilicity is the main determining factor for
the antiport activity, it is reasonable to suggest that the most
lipophilic compounds are also the best symporters. This is even
more likely for compounds 1À10, since the most lipophilic
compounds also contain the most acidic (thio)urea functional-
ities and are therefore likely to be good HCl cotransporters.
Because HPTS fluorescence assays indicated that 1À10 can
transport HCl, H+/ClÀ cotransport can also be another cause for
the increase in lysosomal pH.
plots, U-tube experiments, cell viability assays, acridine orange
staining assays, Hoechst 33342 staining assays, supporting
figures, and complete ref 38b. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
philip.gale@soton.ac.uk
’ ACKNOWLEDGMENT
Apoptotic stimuli induce changes in the nuclear morphology,
including nuclear condensation, fragmentation, holes in the
nuclei of dead cells and appearance of apoptotic bodies, which
can be identified by fluorescence microscopy. Hoechst 33342
staining was performed in order to confirm that apoptosis was
the cell death mechanism following exposure to tripodal com-
pounds 1À10. In GLC4 cell line treated with receptors 4, 7, 8,
and 9, we were able to identify condensation of the nuclei
(Figure 10c, d, e, respectively) and the presence of ‘bean-shaped’
nuclei (Figure 10f). Comparable results were obtained with cells
exposed to receptors 5 and 10 (data not shown). On the other
hand, cells treated with receptors 1, 2, 3, and 6 showed no nuclear
morphology changes (Figure 10b and Figures S128 and S129 in
Supporting Information). Again, these results correlate well with
the activity as anion exchangers studied in liposome models,
where only the most active transporters are able to induce
apoptosis. In summary, it seems that the cytotoxicity displayed
by receptors 1À10 is due to apoptosis, which is induced by
changes in the internal pH regulation due to the chloride/
bicarbonate antiport or HCl symport activity of the receptors.
P.A.G. thanks the EPSRC and the NSF for funding (M.W.)
and the EPSRC for access to the crystallographic facilties at the
University of Southampton. Additionally we thank the University
of Southampton for a postgraduate scholarship (N.B.). This
work has been partially supported by a grant from the Spanish
government (F.I.S.) and the European Union (PI10/00338).
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Supporting Information. Synthesis and characterization
b
of the receptors, stability constant determination, stack plots and
fitplots of the 1H NMR titrations with various anions in DMSO/
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