Acylthioureas as anion transporters: The effect of intramolecular hydrogen bonding

Article abstract of DOI:10.1039/c3ob41522h

Small molecule synthetic anion transporters may have potential application as therapeutic agents for the treatment of diseases including cystic fibrosis and cancer. Understanding the factors that can dictate the anion transport activity of such transporte

Full text of DOI:10.1039/c3ob41522h

Volume 12 Number 1 7 January 2014 Pages 1–202
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
Philip A. Gale et al.
Acylthioureas as anion transporters: the effect of intramolecular
hydrogen bonding

Organic &
Biomolecular Chemistry
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PAPER
Acylthioureas as anion transporters: the effect of
intramolecular hydrogen bonding†
Cite this: Org. Biomol. Chem., 2014,
12, 62
Cally J. E. Haynes,a Nathalie Busschaert,a Isabelle L. Kirby,a Julie Herniman,a
Mark E. Light,a Neil J. Wells,a Igor Marques,b Vítor Félixb and Philip A. Gale*a
Small molecule synthetic anion transporters may have potential application as therapeutic agents for the
treatment of diseases including cystic fibrosis and cancer. Understanding the factors that can dictate the
anion transport activity of such transporters is a crucial step towards their application in biological
systems. In this study a series of acylthiourea anion transporters were synthesised and their anion binding
and transport properties in POPC bilayers have been investigated. The transport activity of these receptors
is dominated by their lipophilicity, which is in turn dependent on both substituent effects and the for-
mation and strength of an intramolecular hydrogen bond as inferred from DFT calculations. This is in con-
trast to simpler thiourea systems, in which the lipophilicity depends predominantly on substituent effects
alone.
Received 24th July 2013,
Accepted 4th September 2013
DOI: 10.1039/c3ob41522h
www.rsc.org/obc
The transport of inorganic anions such as chloride across cell channels that exhibit significant biological activity. In these
membranes is an important biological process, and is regu- previous studies it was observed that the activity of anion car-
lated in the body by proteins embedded in the phospholipid riers is often strongly linked to the lipophilicity of the trans-
bilayer. When naturally occurring anion transport processes porter. We have seen that substituent effects on a thiourea
are defective, a number of diseases (channelopathies) are scaffold can dramatically modulate the transport activity. In a
known to result, including cystic fibrosis. There is a growing recent example, we reported a QSAR analysis of the anion
interest in the development of small molecular carriers for transport by a series of 1-hexyl-3-phenylthioureas containing
anions due to the hypothesis that they may serve as thera- various substituents in the 4-phenyl position, including recep-
peutic replacements for defective transport proteins for the tors 1–4, in which we correlated the transport activity to a
treatment of channelopathies or as tools for physiologists to range of molecular descriptors.5a We found that the variation
study these conditions.1 Additionally, there is increasing evi- in anion transport activity was mainly due to the varying lipo-
dence that anion carriers can induce apoptosis in various philicity of the receptors due to the substituent effects, with
cancer cell lines, indicating a further possible use for these smaller contributions from the molecular size and hydrogen
molecules as anticancer agents.1a,2
Research by our group and others has shown that small thiourea NH groups.
bond acidity (and hence anion binding capability) of the
molecule anion receptors based on motifs including isophtha-
Controlling the lipophilicity of a scaffold is a key concept
lamides,3 ureas,2b,4 thioureas,2a,c,5 squaramides,6 cyanoguani- in drug design, as the lipophilicity of a potential drug mole-
dines7 and tambjamines2d,8 are effective anion antiporters. cule will greatly affect its membrane permeability and hence
Schmitzer9 and Yang10 have reported small molecule anion its absorption and distribution. Increasing the lipophilicity is
often achieved through appending lipophilic functional
groups onto the scaffold. Another strategy to achieve this
aChemistry, University of Southampton, Southampton SO17 1BJ, UK.
when designing a compound is to introduce the possibility of
E-mail: philip.gale@soton.ac.uk; Tel: +44 (0)23 8059 3332
forming intramolecular hydrogen bonds.11 Intramolecular
bDepartamento de Química, CICECO and Secção Autónoma de Ciências da Saúde,
hydrogen bonds decrease the ability of the molecule to
interact with the environment (e.g. water), and hence the
hydrophilicity is reduced. This effect has been previously
Universidade de Aveiro, 3810-193 Aveiro, Portugal. E-mail: vitor.felix@ua.pt
†Electronic supplementary information (ESI) available: Synthesis and character-
isation of the receptors, details of X-ray crystallography,39 stack plots for the
1H NMR titrations of the receptors with TBACl in DMSO–water and CDCl3, various
vesicles assay methods, Hill plots and DFT calculations. CCDC 941594–941597.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c3ob41522h
reported as
a
strategy to improve brain penetration,12
bone tissue penetration,13 oral absorption14 and membrane
permeability.15 A recent report by Shalaeva et al. considers
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methods to assess the propensity of drug molecules to form
intramolecular hydrogen bonds16 and notes that the conse-
quences of such interactions are of great significance to medi-
cinal chemists but are often under-recognized and seldom
predicted.
In this study, we examine the role that intramolecular
hydrogen bonding has on the efficacy of a series of anion
transporters. Given that simple thioureas are effective anion
transporters, we decided to study related acylthiourea com-
pounds as putative anion transporters. Acylthioureas may
form six-membered intramolecular hydrogen-bonded rings
that mask the hydrophilicity of the receptor. We found that
acylthioureas are more lipophilic than analogous thioureas as
a result of the intramolecular hydrogen bond. When compar-
ing parent compounds of both series, the acylthiourea is a sig-
nificantly more effective chloride/nitrate antiport agent than
the thiourea. However, we found that if the transporters were
delivered to a pre-formed lipid bilayer then the more lipophilic
species resulted in less efficient transport, presumably due to
the barrier to their delivery through the aqueous phase,
whereas if these species were pre-incorporated into a lipid
bilayer then the more lipophilic compounds were the best
anion transporters.
Synthesis
Acylthiourea receptors 4–9 were synthesised by the reaction of
the corresponding benzoyl isothiocyanate with 1-hexylamine
or dipropylamine in ethyl acetate (Scheme 1).17b In the case of
6–8, the isothiocyanate was prepared in situ by the reaction of
an acid chloride with ammonium thiocyanate, using PEG-400
as a phase transfer catalyst. Receptor 10 was similarly syn-
thesised by the reaction of benzoyl isocyanate with 1-hexyl-
amine in ethyl acetate, and was isolated in 30% yield. A number
of these compounds have been previously reported.18
Previously, Wei and co-workers have reported that recep-
tors based on an acylthiourea motif form an intramolecular
hydrogen bond, as shown in Fig. 1, and this makes them
highly selective receptors for basic anions such as F− and
AcO−, as only these anions are strongly coordinating enough
to disrupt this interaction.17 Acylthiourea based receptors
have also been extensively studied for their affinity for soft
transition metal ions, with the S and O atoms able to form a
chelate ring.18 For example, Luckay et al., have reported the
use of acylthioureas (including 9) for the extraction and trans-
port of gold(III) ions.18d Acylthiourea derivatives have also
been reported to have herbicidal,19 fungicidal,19 bacteri-
cidal,19 insecticidal20 and plant growth regulatory21 activity,
and have been patented as anti-diabetic,22 anti-arthritic,23
anti-neoplastic24 and anti-coagulant25 agents for the treat-
ment of cognitive problems26 and prostate disorder.27 There-
fore their suitability for application to biological systems has
been previously explored. Our group, in collaboration with
J. T. Davis and Quesada, have previously used intramole-
cular hydrogen bonding to preorganise the anion-binding
site of an anion receptor and transporter based on an iso-
phthalamide structure.3
Receptors 11 and 12 were formed by the reaction of thio-
benzamide with 1-hexyl isocyanate or 1-hexyl isothiocyanate in
acetonitrile in the presence of potassium hydroxide pellets
(Scheme 2). However, receptor 12 was found to be unstable. It
appeared to decompose during purification by chromato-
graphic methods, and as such was isolated with 15–20% im-
purities (see ESI†). After a period of 6 months, a sample of this
receptor was found to have formed crystals of elemental
sulfur (S8), and emitted a strong smell of hydrogen sulfide.
Anion transport experiments were performed on a freshly
prepared sample of this receptor, although it should be
noted that the stability of this compound in buffered aqueous
solutions is not known.
Scheme 1 Synthesis of acylthiourea receptors 5–9. Reagents and con-
ditions: (i) NH4SCN, PEG-400, EtOAc; (ii) 1-hexylamine (5–8) or dipropyl-
amine (9).
Scheme 2 Synthesis of thioacyl receptors 11 and 12. Reagents and
conditions: (i) KOH, 1-hexyl isocyanate, yield = 30% (11) or 1-hexyl iso-
thiocyanate (12), MeCN, yield 70%.
Fig. 1 Competition between intramolecular hydrogen bond formation
and anion binding in acylthiourea receptors.
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therefore collected using a different batch of DMSO-d6 (with
presumably a different water content), therefore the precise
values are not directly comparable; however, the large magni-
tude of the difference in chemical shift of NHa between the
thiourea and the acyl thiourea suggests that this comparison is
still valid. The spectra for receptors 5–11 were all collected
from samples prepared at the same concentration using the
same batch of DMSO-d6. For all acyl thiourea receptors the
NHb resonance also appears significantly downfield by com-
parison to the analogous thiourea, as a result of the extra
acidity inferred by conjugation to both a CvO and CvS
group.
According to Baell,29 and Llinas and Klein,30 comparing the
chemical shift of this NH resonance in DMSO-d6 to the posi-
tion of the resonance in CDCl3 can give an insight into how
“solvent accessible” the NH group is. If the NH is strongly
intramolecularly hydrogen bonded, it will have minimal inter-
action with solvent and thus a downfield shift should not be
observed on moving to a more strongly coordinating system.
We compared the chemical shift of NHa and NHb in both
DMSO-d6 and CDCl3 for receptors 5–11. The NHa resonance
was identified either by its multiplicity (triplet), or if the multi-
plicity of the NH peaks was not clear due to broadening, by
COSY NMR (see ESI†). We found that, on moving from CDCl3
to DMSO-d6 as expected the NHb resonance of all of these
receptors appeared significantly downfield in DMSO-d6 (for
example from 8.95 ppm in CDCl3 to 11.27 ppm in DMSO-d6
for receptor 5). However, the resonance of NHa appeared at a
similar chemical shift in both DMSO-d6 and CDCl3, which is
evidence that leads us to suggest that this NH group is strongly
intramolecularly hydrogen bonded and thus shielded from
solvent effects.
Fig. 2 Asymmetric unit of the X-ray crystal structure of compound 6
showing the anti conformation with intramolecular hydrogen bonds
drawn as dashed lines. Non-acidic hydrogen atoms have been omitted
for clarity.
X-ray crystallography
Compounds 6, 7, 10 and 11 were isolated as crystalline solids,
and their crystal structures were elucidated by single crystal
X-ray diffraction.‡ Full details of these structures can be found
in the ESI.† The asymmetric unit of 6, composed by two inde-
pendent molecules, is shown in Fig. 2. As with all of the struc-
tures elucidated here (and as previously observed for
analogous systems17c,18a,28), this receptor forms a 6-membered
hydrogen-bonded ring with the thiourea binding unit adopting
an anti conformation. The donor–acceptor hydrogen bond dis-
tances (D⋯A) for this interaction is 2.619(2) and 2.620(2) Å,
and the corresponding DH⋯A angles are 134 and 135°. By
comparison, the analogous intramolecular hydrogen bonding
lengths in receptor 7 and 10 are 2.678(3) Å and 2.941(3) Å
respectively.
Evidence for intramolecular hydrogen
bonding in solution
The X-ray crystal structures obtained indicated the formation
of an intramolecular hydrogen bond in the solid state. Wei
et al., have reported that acylthiourea receptors form an intra-
molecular hydrogen bond in DMSO–water solutions, and that
this interaction competes with anion complexation.17b,c The
1H NMR spectra of 5–8 in DMSO-d6 show that the resonance of
the NHa proton adjacent to the alkyl group, which is involved
in the intramolecular hydrogen bonding, is shifted downfield
compared to the NHa resonance of an analogous thiourea
(Table 1). For example, the NHa resonance of thiourea 1
appears at 7.71 ppm, whilst the NHa resonance of analogous
acyl thiourea 5 appears at 10.88 ppm, a difference of 3.17 ppm.
The values for receptors 1–4 were previously reported and were
Table 1 Chemical shift of the NHa and NHb resonances of receptors
1–11 in DMSO-d6 and CDCl3
δ NHa
(DMSO-d6)
(ppm)
δ NHb
(DMSO-d6)
(ppm)
δ NHa
(CDCl3)
(ppm)
δ NHb
(CDCl3)
(ppm)
Receptor
1
2
3
4
7.71b
8.03b
7.48b
7.60b
10.88
10.78
10.95
10.92
8.66
9.43
9.77
9.20
—
—
—
—
10.73
10.62
10.79
10.77
8.20
8.64
9.93
—
—
—
—
8.95
8.99
8.89
8.94
—
8.51
9.18
9.31
5a
6a
7a
8a
9a
10a
11a
11.27
11.56
11.07
11.14
—
10.65
11.74
8.66
9.26
‡Structures were deposited with the Cambridge Crystallographic Database
Centre (CCDC) and given the numbers CCDC 941594–941597.
a Sample concentration 10 mM. b Previously reported.2c,7
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Again, the same observation was made in the experiments
with acylthioureas 6–8 (see ESI†). However, in the experiments
with (thio)acyl ureas 10 and 11 the NHa resonance was found
to retain its resistance to exchange in the more competitive
solvent mixture. This indicates a difference in the behaviour of
the acylthiourea receptors and their analogues.
Anion transport
The anion transport activity of receptors 5–12 was investigated
using vesicle based methods.31 A sample of unilamellar POPC
vesicles was prepared containing 489 mM NaCl buffered to pH
7.2 with 5 mM sodium phosphate salts. The vesicles were sus-
pended at a lipid concentration of 1 mM in 489 mM NaNO3
buffered to pH 7.2 with 5 mM sodium phosphate salts. The
experiment was initiated by the addition of a small amount of
a DMSO solution of the receptor (2 mol% with respect to
lipid), and the resulting chloride efflux was monitored using a
chloride ion selective electrode (ISE). At the end of the experi-
ment (300 s), the vesicles were lysed by adding a detergent,
and the final electrode reading was used to calibrate 100%
chloride release. Fig. 4 shows the chloride efflux mediated by
receptor 5 in comparison to receptor 1, indicating that in this
case the acylthiourea receptor is more active than its thiourea
analogue.
These studies showed that acylthioureas 5–8 are able to
facilitate chloride transport under these conditions, and that
each of these receptors could mediate 100% chloride efflux by
the end of the experiment. However, analogous receptors 9–12
did not mediate any detectable chloride transport under these
conditions.
In order to determine the mechanism of chloride release by
receptors 5–8, we performed a second experiment using POPC
vesicles containing 450 mM NaCl buffered to pH 7.2 with
Fig. 3 Changes to the downfield region of the 1H NMR spectrum of
receptor 5 (5 mL of 0.01 M solution) in (a) DMSO-d6 and (b) DMSO-d6
with 1% H2O (v/v) upon the addition of D2O.
A slow rate of deuterium exchange can also be used to indi-
cate internal hydrogen bonding. We attempted a series of D2O
shake experiments with receptors 5–8 and 10–11 in order to
see if NHa is less readily exchanged than NHb. We prepared a
0.01 M solution of the receptors in DMSO-d6 (as supplied i.e.
1
not dry), and monitored the H NMR spectrum on addition of
2 and 4 μL of D2O. A stack plot for the experiment with recep-
tor 5 is shown in Fig. 3(a). We found that for every receptor,
the resonance of NHa did not decrease in intensity as quickly
as the resonance of NHb, indicating that the formation of the
intramolecular hydrogen bond provides
a barrier to its
exchange. This is consistent with the comparison of the
chemical shifts of NHa in DMSO-d6 and CDCl3, which indi-
cated the presence of an intramolecular hydrogen bond in
both solvent systems.
However, it is reasonable to assume that the strength of an
intramolecular hydrogen bond should be weakened in the
presence of a more competitive solvent. Therefore, we repeated
the D2O shake experiments using DMSO-d6 with 1% H2O
added (v/v). A stack plot for this experiment with receptor 5 is
shown in Fig. 3(b). In this case, a larger volume of D2O was
required to cause the NH peaks to decrease in intensity, pre-
sumably because of competing deuterium exchange with the
added H2O. We found that the resonance associated with NHa
was less resistant to exchange in this case, which serves to
reiterate that the formation and stability of a hydrogen bond is
dynamic and highly dependent on the environment, and can
be weakened in the presence of competitive solvent mixtures.
Fig. 4 Chloride efflux mediated by unsubstituted receptors 1 and 5
(2 mol% with respect to lipid) from unilamellar POPC vesicles containing
489 mM NaCl buffered to pH 7.2 with 5 mM sodium phosphate salts.
The vesicles were suspended in 489 mM NaNO3 buffered to pH 7.2 with
5 mM sodium phosphate salts. At the end of the experiment the vesicle
were lysed with detergent in order to calibrate 100% chloride release.
Each point represents the average of 3 trials.
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20 mM sodium phosphate salts. This time, the vesicles were receptors into the bilayer as well as the viscosity of the mem-
suspended in 162 mM Na2SO4 buffered to pH 7.2 with 20 mM brane, therefore interpreting the results of such assays is not
sodium phosphate salts. On addition of a sample of receptors as straightforward as previously believed. However, receptors 6
5–8, there was negligible chloride release. This indicates that and 8 were found to have a limited but measurable ability to
these receptors function by an anion exchange mechanism. In transport chloride through a nitrobenzene organic phase in a
−
the first assay, the receptors were able to facilitate Cl−/NO3
U-tube assay (see ESI†). This supports a mobile carrier mecha-
exchange, but in this second experiment their activity was nism, as the assembly of a multicomponent channel across a
2−
diminished due to the high hydrophilicity of the SO4
anion,32 which in most cases prevents its transport by syn-
large organic phase is highly implausible.
We have previously reported that across the series of thio-
thetic transporters. After 120 s, we added a pulse of NaHCO3 ureas 1–4 there is large variation in the transport activity.5a In
solution, and found that the receptors were able to begin me- this case, the nature of the substituent is clearly dominating
diating chloride efflux, implying that these receptors are also the transport properties of these molecules. In contrast, we
able to facilitate a Cl−/HCO3− antiport mechanism. The lack of found that the EC50 values for Cl−/NO3 antiport by 5–8 were
−
transport in the absence of a readily transportable counter- similar. For this reason, each Hill plot was repeated 3 times in
anion was not perturbed by changing the encapsulated cation, order to assess the reproducibility of this data and to deter-
thus implying that a M+/Cl− co-transport mechanism is not mine whether these values were statistically distinct. These
operating (see ESI†).
results indicate that the order of transport efficiency is 7 ≈ 5 <
In order to quantify these anion transport processes, we 6 < 8. However, for reasons discussed in more detail below, the
performed a Hill analysis for Cl−/NO3 and Cl−/HCO3 anti- EC50 value for receptor 8 should be treated with caution as
port by receptors 5–8.33 These experiments allowed us to calcu- the activity was found to be highly dependent on the stock
late the EC50 for these processes, a measure of activity defined receptor concentration. We also performed Hill plots for
−
−
as the effective receptor concentration required to mediate Cl−/HCO3 antiport by these molecules. The same approxi-
−
50% chloride efflux after a specified time period. As is stan- mate trend in EC50 values for this process was obtained as for
dard in our studies, this period is 270 s for Cl−/NO3 experi- Cl−/NO3 exchange, although receptor 8 performed signifi-
−
−
−
ments and 390 s for Cl−/HCO3 experiments (corresponding cantly more poorly.
to 270 s after the HCO3 spike). We were also able to deter-
−
In some cases, the acylthiourea was significantly more
mine the Hill coefficient (n), which can be used to give an indi- active than the thiourea analogue. For example, receptor 7 is
cation of the number of monomers that form the active approximately 20 times more active than receptor 3 (i.e. the
transport system.34 These values, and the comparable values EC50 value of 7 is 20 times smaller than that of receptor 3).
for receptors 1–4 are shown in Table 1.
However, in the case of the –CF3 and –pentyl substituted recep-
For each receptor, the Hill coefficient is relatively small (n < tors, the thioureas were more active than the acylthiourea
3). This provides evidence that these receptors do not assem- (receptor 4 is 14 times more active than receptor 8).
ble into membrane spanning channels, as given the size of the
The small variation in activity across the series 5–8 is evi-
receptors, this would require the aggregation of a large dence that substituent effects are not dominating the anion
number of molecules. However, in each case for Cl−/NO3− anti- transport properties of these receptors. Adding lipophilic sub-
port n > 1 therefore the transport process does not necessarily stituents (such as –CF3 or –pentyl) did not enhance the trans-
occur via a 1 : 1 receptor : anion complex. Matile and co-workers port activity.
have previously reported anion transport by a capsule system, in
During the testing of compound 8 we observed some
which more than one receptor molecule surrounds the anion, unusual behaviour, which implied that the delivery of the
after which the complex is able to diffuse across the lipid receptor through the aqueous phase was problematic. Firstly,
bilayer,35 and we have observed high Hill coefficients for anion the chloride efflux profiles we obtained appeared to be sigmoi-
transport by tren based anion receptors.2a Transport by an aggre- dal, as the receptor seemed to take more time to begin trans-
gate species cannot be ruled out in this case, especially given the porting chloride after being introduced to the system than we
ambiguity as to how these receptors interact with anions.
have observed previously for other transporter systems (this
In order to gather further evidence for a mobile carrier effect can be seen in Fig. 5). Secondly, the activity of com-
mechanism, we tested the Cl−/NO3 antiport activity of 5–8 in pound 8 at a particular lipid : receptor ratio was highly depen-
−
vesicles composed of POPC–cholesterol (7 : 3). Cholesterol is dent on the concentration of the DMSO stock solution it was
known to order the bilayer, and it is thought that the resulting added from. Fig. 5 shows chloride efflux mediated by receptor
increased viscosity should slow the diffusion of a mobile 8 at a loading of 2 mol% with respect to lipid, which was
carrier. Compounds 5 and 7 did show a reduced rate of added to the experiment using different volumes of different
transport in the POPC–cholesterol system; however, com- concentration stock solutions. The more concentrated the
pounds 6 and 8 showed an increased rate of transport in the stock solution is, the lower the anion transport activity observed,
presence of cholesterol. This has been previously observed in despite the overall quantity of receptor added being the same.
the study of receptors bearing lipophilic substituents includ- This implies that the greater volume of DMSO present, when
ing –CF3 and alkyl groups. We propose that the addition of using the more dilute stock solution, was aiding the delivery of
cholesterol to the bilayer also affects the partitioning of the receptor to the lipid bilayer. It has been previously reported
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Fig. 5 Chloride efflux mediated by compound 8 (2 mol% w.r.t. lipid),
added from stock DMSO solutions of the receptor at varying concen-
tration. The receptor was added to POPC vesicles containing 489 mM
NaCl and suspended in 489 mM NaNO3, both buffered to pH 7.2 with
5 mM sodium phosphate salts. At the end of the experiment the vesicles
were lysed with detergent in order to calibrate 100% chloride efflux.
Each point represents the average of 3 trials.
Fig. 6 Chloride influx mediated by receptors 5–8 (preincorporated at
0.5 mol% with respect to lipid) into vesicles containing 225 mM NaNO3
buffered to pH 7.2 with sodium phosphate salts and 1 mM lucigenin. The
vesicles were suspended in 225 mM NaNO3 and the experiment was
initiated by adding 25 mM NaCl. Each experiment was repeated in
triplicate.
determining factor for all of the receptors, as when this barrier
to transport is removed the expected trend in activity is
observed.
that across a series of receptors, there will be an optimum lipo-
philicity for transport.4e,8 Less lipophilic compounds will par-
tition less effectively with the lipid bilayer, while more
lipophilic compounds will not be able to effectively pass the
aqueous phase. We propose that compound 8 might therefore
be too lipophilic. In contrast, compound 4 is the most efficient
of the thiourea series, indicating that across that series, the
optimum lipophilicity has not yet been exceeded. The Hill plot
for compound 8 was performed using only a 5 mM stock solu-
tion of the compound (whereas commonly a number of serial
dilutions of the stock solution are performed during a Hill
plot). Clearly, the results would have been very different if a
more concentrated stock solution was used.
In order to test if the poor activity of receptor 8 was due to
its problematic delivery to the bilayer, we adopted an anion
transport assay based on the experiment reported by Davis and
co-workers, in which the receptor is dispersed within the phos-
pholipid prior to vesicle formation.5d As such, there is no
aqueous barrier to delivering the receptor into the lipid
bilayer, meaning highly lipophilic transporters can be tested.
We prepared a sample of POPC vesicles (1 mM lipid) with
receptors 4–8 preincorporated at a concentration of 0.5 mol%
with respect to lipid. The vesicles were prepared with internal
and external NaNO3 (225 mM buffered to pH 7.2 with 5 mM
sodium phosphate salts). The vesicles also contained 1 mM
lucigenin, a fluorescent probe that is quenched in the presence
of halide anions. On addition of a pulse of NaCl (25 mM) any
resulting chloride influx was monitored via the corresponding
decrease in fluorescence intensity of the lucigenin. The results
are shown in Fig. 6. In this experiment, the trend in transport
activity is reversed. The order of activity is 5 ≈ 7 < 6 < 8. This
implies that although receptor 8 was the only receptor to show
obvious signs of being too lipophilic to pass readily through the
aqueous phase in the ISE experiments, perhaps this is a
Receptor lipophilicity
A common technique to quantitatively assess the lipophilicity
of a molecule is to determine a value of log P (the octanol–
water partition coefficient). The log P values of a series of com-
pounds have previously been shown to correlate to the reten-
tion time of the compounds on a reverse phase HPLC
column.36 We performed an HPLC experiment using a reverse
phase C18 column. The retention times of receptors 1–11 are
shown in Table 2. The retention times of 1–4 correlate well
with those previously reported (see ESI†).5a
In general, all of the acylthiourea receptors are more lipo-
philic than the analogous thioureas despite containing an
additional oxygen atom. However, tertiary acylthiourea 9,
which cannot form an intramolecular hydrogen bond, is by far
the least lipophilic compound studied. This indicates that the
increased lipophilicity of 5–8 is not simply due to the func-
tional groups present, implying that the formation of the intra-
molecular hydrogen bond is crucial. Further insights were
obtained from DFT calculations on compounds 5 and 10–12
presented below.
Across the series 5–8, the varying substituent R-groups
impose the same trend in lipophilicity as 1–4, i.e. –H < –OMe <
–CF3 < –pentyl. However, the range of values is smaller across
the acylthiourea series. The difference in retention time for
receptors 1 and 4 is 5.9 min, while the difference between 5
and 8 is 4 min. Excluding receptors 4 and 8 (containing the
most inherently lipophilic –pentyl substituent), the range is
3.4 min for the thioureas and 1.6 min for the acylthioureas.
This implies that the overall lipophilicity of the acylthioureas
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Table 2 Retention times (determined by reverse phase C18 HPLC experiment) and transport data (determined by Hill plot) for compounds 1–11.
EC50 is defined as the concentration (mol% with respect to lipid) of receptor required to mediate 50% chloride efflux after 270 s; n is the Hill
coefficient
Cl−/NO3
Cl−/HCO3
− a
− b
Receptor
Retention time (min)
EC50
Error
n
Error
EC50
n
1c
2c
3c
4c
5
6
7
8
9
4.9
8.3
5.1
10.8
8.9
10.5
9.4
12.9
3.9
7.3
2.7
0.44
5.5
0.6
0.04
0.9
0.90
1.58
1.26
0.86
2.07
2.76
2.03
2.3d
0.10
0.17
0.12
0.17
0.27
0.24
0.11
0.21d
3.1
0.89
5.8
0.10
0.27
8.1
0.6
0.1
0.74
1.5
0.08
0.33
0.69
0.28
1.1d
0.01
0.06
0.01
0.02
0.1d
0.78
0.77
0.91
0.28
6.5d
1.5d
e
e
e
e
e
e
—
—
—
—
—
—
—
—
—
e
e
e
e
e
e
e
10
11
—
—
—
—
e
e
e
e
e
9.4
—
—
—
—
—
a Transport data is the average of a minimum or 3 repeated Hill plot experiments. The error given is the standard deviation. b Transport data is
the result of a single Hill plot analysis (therefore no errors are given). c Some transport data has been previously reported.2c,5a d Activity
dependent on stock concentration of receptor solution – data gathered using only a 5 mM solution. e No anion transport activity detected.
is a balance between the lipophilicity of the substituents and performed the best if they were pre-incorporated into the lipid
the additional lipophilicity imparted by the intramolecular bilayer. This provides further proof that within this series, the
hydrogen bond. This balance is tipped in the favour of substi- delivery of the receptors to the lipid bilayer is problematic (as
tuent effects in the case of the –pentyl substituted receptor, as reflected by the most lipophilic compounds being the poorest
increasing the length of an alkyl substituent results in an transporters in the ISE assay). However, if the receptors are pre-
increase in lipophilicity without significant variation in elec- delivered to the lipid bilayer and this barrier is removed, the
tron donating character.
most lipophilic carriers are the most active, as might be
We examined the correlation between the activity of recep- expected. This rules out the possibility that the trends observed
tors 1–8 (represented by log(1/EC50)) and the retention time. in the ISE assays were due to the more lipophilic carriers becom-
The results are shown in Fig. 7. There is a negative correlation ing trapped within the hydrophobic interior of the bilayer and
between the activity of the acylthioureas and their lipophilicity. thus unable to extract the anions from the aqueous interface.
Although the results for receptor 8 should be treated with
caution due to the difficulties encountered in testing this com-
pound, the negative correlation would still be evident exclud-
Chloride binding in solution
ing this data point.
The trend in activity when the receptors were pre-incorpor-
ated is the opposite of the trend during the ISE assays. There-
Receptors 1–4 have been previously reported to interact weakly
with tetrabutylammonium chloride in DMSO-d6–H2O 0.5%,
fore, the compounds with the longest retention times
with binding constants in the range of 10.6–27.9 M−1
.
The strength of binding was related to the Hammett constant
of the R-group, with the strongest binding for compound 2
(electron withdrawing –CF3 group) and the weakest binding
for compound 3 (electron donating –OMe group).5a
In order to assess the solution phase chloride binding pro-
perties of receptors 5–11, we performed 1H NMR titrations
with tetrabutylammonium chloride in both a competitive
solvent mixture (DMSO-d6–H2O 0.5%) and a non competitive
solvent (CDCl3) at 298 K. Titrations were not performed with
receptor 12 due to its instability and the presence of residual
impurities.
Wei and co-workers have previously reported that acylthiourea
receptors do not interact detectably with chloride anions
under similar conditions as the intramolecular hydrogen bond
effectively “locks” the receptor into a conformation that does
not allow the anion to bind.17b No significant change in the 1H
NMR spectrum was observed on the addition of up to ∼6
equivalents of chloride to receptor 5 in DMSO-d6–H2O 0.5%.
Fig. 7 The relationship between retention time (determined by C18
HPLC) and transport activity of thioureas 1–4 and acylthioureas 5–8 as
determined by an ISE assay.
68 | Org. Biomol. Chem., 2014, 12, 62–72
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Table 3 VS,max Values (kcal mol−1) calculated for compounds 1–8 and
10–12
In CDCl3, the NHa resonance was observed to broaden slightly,
which may be indicative of a small change in the hydrogen
bonding environment of this group due to interaction with the
anion. Stack plots for all of the titrations performed can be
found in the ESI.†
We reasoned that if the receptor is unable to interact with
chloride due to the intramolecular hydrogen bond, it may be
possible to observe a binding effect by first breaking the hydro-
gen bond by heating the system. We repeated the titration of
receptor 5 with TBACl in DMSO-d6–H2O 0.5% at 323 K. In this
experiment we observed a small change in chemical shift of
the NH resonances on addition of the anion (see ESI†). This
implies that chloride may be weakly coordinated by this recep-
tor if the intramolecular hydrogen bonding interaction is first
broken or weakened.
Receptor
Substituent
syna
antia
1
2
3
4
5
6
7
8
H
59.8
66.6
59.1
58.7
64.7
70.6
64.0
62.9
64.0
63.2
62.6
—
—
—
—
35.3
42.0
33.2
34.4
39.3
36.7
31.6
CF3
OMe
n-Pentyl
H
CF3
OMe
n-Pentyl
H
10
11
12
H
H
a syn and anti refer to the two possible conformations adopted by the
thiourea or urea binding units.
Across the series 6–8, the results of the titrations in both
solvent systems were largely similar to those obtained for
receptor 5, with negligible interaction observed in DMSO-d6–
H2O 0.5% and peak broadening of the NHa resonance in
CDCl3 (see ESI†). However, for receptors 9–11 a clearer inter-
action was observed in CDCl3. The only NH resonance of com-
pound 9 and the NHa resonance in compound 11 were
observed to undergo a small downfield shift indicative of
anion binding, while the NHb resonance shifted slightly
upfield. In compound 11 the NHa resonance shifted downfield
and broadened, while NHb shifted upfield. All of these
binding effects were too small to quantify; however, it is inter-
esting that the compounds that were not able to mediate chlor-
ide transport again gave a different response to those that were
effective transporters.
It is important to consider that, although the chloride
binding by acylthioureas under the conditions of a 1H NMR
titration experiment seems to be minimal, these conditions
are not representative of a vesicle bilayer–aqueous phase inter-
face. Additionally, the acylthiourea receptors only transport
anions and not cations. Therefore it is reasonable to assume that
there must be some interaction between the receptor and the
anion under the conditions of the transport experiments, and
that the lipophilicity of the scaffold cannot be the only determin-
ing factor towards transport, as this would impart no selectivity.
The D2O shake experiments indicated that the strength of the
intramolecular hydrogen bond is perturbed in the presence of
increased aqueous content, which may imply binding is more
likely to be possible at the bilayer–water interface.
surface, the VS,max, the importance of which in the characteri-
zation of anion transporters has previously been demonstrated,5a
while the strength of the intramolecular NH⋯O/S bonds was
described with second order interaction energies (E2).37 With
the analysis of these parameters, along with anion efflux data,
we attempted to establish how these quantum descriptors
determine the transport ability of this series of molecules.
The VS,max is related to the hydrogen bonding capacity and
acidity38 and the values computed for compounds 1–8 and
10–12 are gathered in Table 3.
In the subsequent analysis the compounds must be clus-
tered in three sets: thioureas 1–4, acylthioureas 5–8 and com-
pounds containing acylthiourea 5, acylurea 10, thioacylurea 11
and thioacylthiourea 12 motifs.
As would be expected, the VS,max values of the syn confor-
mations of acylthioureas 5–8 are systematically higher when
compared to the analogous thioureas due to the acyl motif. It
is also evident that in both series the VS,max values increase,
according to the electronic nature of the substituent, following
the trend –pentyl < –OMe < –H < –CF3, which means that 2
and 6 with a para-electron withdrawing –CF3 group are the
most acidic ones and, consequently, with higher binding
affinity for the chloride anion. Furthermore, when the log(VS,max
are plotted against the corresponding EC50 values, a good
correlation is obtained with both series of compounds (R2
)
=
0.77 for thioureas and R2 = 1.00 for acylthioureas, see
Fig. SB3†), apart from the receptors with an n-pentyl substitu-
ent. In other words, in compounds 1–3 and 5–7 the transport
activity appears to be related to the binding affinity, which is,
in turn, mainly determined by the electronic nature of the
different substituents. By contrast, the transport activity of 4 and
8, with a long n-pentyl alkyl chain is governed by the individual
high lipophilicity (see Table 2), in agreement with the experi-
mental transport studies reported above. It is also important to
emphasise that the relationship between EC50 and VS,max para-
meters for thioureas and acylthioureas follow reverse trends,
which seems to indicate that the intramolecular hydrogen
bond in these molecules plays a role in the transport activity of
these receptors, as apparent from the subsequent analysis.
Quantum calculations
Receptors 5–8 and 10–12 were optimised by DFT methods
(B3LYP/6-311+G(d,p)) (see ESI† for computational details) to
characterise the receptors’ binding affinity for anions, as well
as the strength of the intramolecular hydrogen bonding inter-
actions in the stabilization of the anti conformation of
acylthioureas 6, 7, 10 and 11 found in the solid state (see
Fig. 2). Therefore, the binding affinity was assessed through
the most positive values mapped on the electrostatic potential
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Table 4 Second-order interaction energy (E2, in kcal mol−1
NHa⋯O and NHa⋯S hydrogen bonds in the receptors 5–8 and 10–12
)
for
The conformational change from syn to anti in acylthiour-
eas leads to a significant decrease in VS,max values, with the –
pentyl and –OMe substituents changing the order in the trend.
The lower VS,max values for the anti structures of 5–8, which
have an intramolecular hydrogen bond, indicate that these
conformations are less acidic than the syn ones. Hence, the
anti conformations are less able to bind the chloride anion,
promoting its transport, in agreement with the absence of
experimental transport activity found for 9, with a single NH
binding group. However, it is noteworthy that the correlation
found between EC50 and VS,max values of the anti structures is
maintained with syn structures, when compound 8 is excluded
from the data set, yielding a R2 = 0.98 (see Fig. SB3†). These
correlations suggest that both conformations are relevant for
the transmembrane transport of chloride. In the syn confor-
mation of 5–8 the VS,max is positioned at the binding pocket
defined by the two NH groups, while in the anti conformation
it was found to be close of the free NHb group. The electro-
static potential mapped on the electron density surface of 6
together with the location VS,max is illustrated in Fig. 8 for syn
(top) and anti (bottom) conformations. Equivalent graphical
depictions are shown in Fig. SB1† for compounds 5, 7, 8 and
10–12.
*
*
*
a
Receptor
LP1(X) → σNH
LP2(X) → σNH
LP1,2(X) → σNH
a
a
5
6
7
8
10
11
12
2.97
2.80
3.09
3.04
2.19
2.04
2.78
7.87
7.47
8.18
8.05
6.31
14.41
18.49
10.84
10.27
11.27
11.09
8.50
16.45
21.27
The interaction energy associated with the formation of an
intramolecular hydrogen bond in compounds 5–8 and 10–12
was estimated for the anti conformations via perturbation
theory using the natural bond orbitals (NBO) analysis. The
interaction energy values computed for NHa⋯O or NHa⋯S
intramolecular bonding interactions are collected in Table 4.
A graphical depiction of the interaction between the NBOs is
given in Fig. SB2† for 5, which shows the interaction between the
lone pair (LP) orbitals and the NHa antibonding (σ*) orbitals.
The active anion transporters 5–8 exhibit energies in a
narrow range from 10.27 to 11.27 kcal mol−1. Across this
series, as expected, receptor 7 has the strongest interaction as
a result of the electron donating –OMe group, while the
weakest interaction is found in receptor 6 with the electron
withdrawing –CF3 group. In contrast, the strength of this inter-
action in receptor 10 (8.50 kcal mol−1) and receptors 11 and 12
(16.45 and 21.27 kcal mol−1) are outside of this energy range.
These data suggest that chloride transport is dependent on an
ideal interval of interaction energies, defined by the
acylthiourea unit of 5–8. Thus, 10, with the lowest E2 energy,
does not promote anion transport, while 11 and 12, although
with higher E2 energy values, may be able to passively diffuse
across the bilayer, but are almost unable to bind the anion,
which is a necessary condition for the anion transmembrane
transport. In other words, a significant amount of the energy
should be required to break the NHa⋯S hydrogen bond in
11 or 12.
As observed for acylthioureas 5–8, the VS,max calculated for
compounds 10–12 in both conformations indicate that the syn
conformation is more acidic than the anti one. Furthermore,
among the series composed of 5 and 10–12, the anti confor-
mation in 10 with VS,max of 39.3 kcal mol−1 is slightly more
acidic than in the remaining compounds with VS,max values
ranging between 31.6 and 36.7 kcal mol−1
.
As previously found for the VS,max values, the E2 energies
also seem to correlate well with the EC50 values (R2 = 0.90, see
Fig. SB4†), thus suggesting the dependence between the
strength of the intramolecular hydrogen bond and the trans-
port ability. Also, the match between E2 and VS,max values for
anti conformations of the transporters yields a good corre-
lation (R2 = 0.97, Fig. SB5†), which indicates that a high VS,max
value corresponds to a weak intramolecular hydrogen bond
and vice-versa.
In summary, this quantum analysis shows that the inter-
action energies for this series of molecules are relatively low
with exception of 11 and 12. Therefore, the intramolecular
hydrogen bond is easily broken which leads to a conformation-
al change. In this context, a delicate balance between the
hydrogen bond strength, the anion binding affinity and the
lipophilicity dictates the transport ability of the transports 5–8.
Obviously, this analysis is limited by the number of studied
molecules.
Fig. 8 Computed electrostatic potential, mapped on the 0.001 elec-
trons per Bohr3 surface of 6 for syn (top) and anti (bottom) confor-
mations, with colours ranging between blue (+70.6 kcal mol−1) and red
(−64.2 kcal mol−1) for the more positive and negative values, respect-
ively. The location of VS,max for both conformations is emphasised as a
pink dot.
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S. J. Moore, N. J. Wells, J. Herniman, G. J. Langley,
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Conclusions
In conclusion, acylthioureas have been found to function as
efficient anion antiporters. They are more lipophilic than ana-
logous thioureas due to the formation of an intramolecular
hydrogen bond, which shields the binding site from inter-
actions with water. As such, the incorporation of lipophilic
substituents results in the receptors becoming too lipophilic if
passage through an aqueous phase is required.
It is clear that the lipophilicity of simple anion transporters
is crucial to their transport activity. Therefore, understanding
the factors that dictate the lipophilicity and anion binding
affinity of a structure is imperative. Intramolecular hydrogen
bonding could be used as a design tool to enable the use of
more hydrophilic scaffolds in transporter design. This would
be advantageous in the design of new transporters for medi-
cinal applications, in which absorption and distribution
throughout the body would require a balance between solubi-
lity in the blood and in lipid bilayers.
Acknowledgements
We thank the EPSRC for funding (CJEH – EP/J009687/1) and
for access to the crystallographic facilities at the University of
Southampton. We thank the University of Southampton and
A*STAR for a postgraduate scholarship (NB) and the University
of Southampton for a teaching assistantship (ILK). PAG thanks
the Royal Society and the Wolfson Foundation for a Royal
Society Wolfson Research Merit Award. IM thanks the FCT
(Fundação para a Ciência e a Tecnologia) for the PhD scholar-
ship SFRH/BD/87520/2012. VF acknowledges the funding from
QREN-FEDER, through the Operational Program Competitive-
ness Factors – COMPETE and National Funds through the FCT
under project PTDC/QUI-QUI/101022/2008.
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