Cationic calix[4]arenes as anion-selective ionophores

Article abstract of DOI:10.1039/b719482j

1,3-Alternate cationic calix[4]arene 1 proved highly selective for proton/halogens symport transport and showed antiproliferative activity against murine monocyte/macrophage J774.A1 cancer cells. The Royal Society of Chemistry.

Full text of DOI:10.1039/b719482j

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Chemical Communications
www.rsc.org/chemcomm
Number 26 | 14 July 2008 | Pages 2941–3072
ISSN 1359-7345
FEATURE ARTICLE
COMMUNICATION
Hiroko Yamada, Tetsuo Okujima
and Noboru Ono
Irene Izzo, Paolo Tecilla,
Francesco De Riccardis et al.
Cationic calix[4]arenes as anion-
selective ionophores
Organic semiconductors based on
small molecules with thermally or
photochemically removable groups
1359-7345(2008)26;1-0

COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Cationic calix[4]arenes as anion-selective ionophoresw
Irene Izzo,*a Sabina Licen,b Nakia Maulucci,a Giuseppina Autore,c Stefania Marzocco,c
Paolo Tecilla*b and Francesco De Riccardis*a
Received (in Cambridge, UK) 19th December 2007, Accepted 5th March 2008
First published as an Advance Article on the web 31st March 2008
DOI: 10.1039/b719482j
1,3-Alternate cationic calix[4]arene 1 proved highly selective for
proton/halogens symport transport and showed antiproliferative
activity against murine monocyte/macrophage J774.A1 cancer
cells.
The ion-coupled processes occurring in the plasma membrane
regulate most of the physiologically relevant cell processes,1
however, in contrast with the inspiring specificity of natural
ion channels2 and despite the advances in the ion-transport
Fig. 1 Chemical structures of ionophores 1 and 2.
field, only a restricted number of synthetic ionophores demon-
strated efficient anion transport and selectivity.3 The quest for
the design and synthesis of artificial anion conductors, is
justified by the surprising scarcity of anion-transporting sec-
ondary metabolites4 and by the concurrent unfortunate high
number of diseases due to insufficient chloride transport.5 In
this context, newly conceived transporters may represent
interesting leads for the treatment of these channelopathies.3
Calix[4]arene represents a versatile scaffold for the design of
synthetic ionophores.6,7 Recent work from the group of J. T.
Davis8 showed that a 1,3-alternate derivative of a tetrabutyl-
amide calix[4]arene, transports HCl across a liposome mem-
brane.8a We reasoned that cationic spermidine appendages,
serving as anion extracting devices and intrapore water stabi-
lizers, could regulate transmembrane anion traffic and elicit
biological activity. We thus designed the D2d symmetric 1,3-
alternate calix[4]arenes 1 and 2 (Fig. 1), confiding in a favour-
able balance between the polar set of spermidine chains and
the lipophilic calixarene scaffold, once decorated with four
non-polar benzyloxymethyl (1) or methyl (2) substituents.
Membrane spanning9 derivatives 1 and 2 were assembled,
both in 19% overall yield, from the known tetrachloromethyl
calix[4]arene 3,10 as shown in Scheme 1.
Carboxyethyl hydrolysis and subsequent reaction of the
tetra-acid 6 with the bis-Boc protected spermidine 7,11 yielded
the fully protected adduct 8. Finally, amino groups deprotec-
tion furnished target 1. The more polar conjugate 2 was
similarly obtained from 8, once effected a Pd-mediated benzyl
deoxygenation.
The ionophoric activities of compound 1 and 2 were in-
vestigated using the HPTS assay (Fig. 2).8a,12 In this test a pH
gradient is established across the liposomial membrane and
the increase of the HPTS fluorescence indicates H+ efflux or
OHÀ influx. The transmembrane charge translocations depend
on four possible overall processes: H+/Na+ antiport, OHÀ/
ClÀ antiport, H+/ClÀ symport and Na+/OHÀ symport
(Fig. 2(a)). The effect of 1 and 2 on ion transport, is reported
in Fig. 2(b) and (c), respectively.
Compound 1 shows a powerful ionophoric activity: the pH
gradient is completely discharged after about 15 min in the
presence of 1% ionophore (it still shows activity at 0.01%
concentration, Fig. 2(b)). The more polar 2 requires a 3%
The synthesis started with a sodium benzylate-induced
chlorines substitution, to give the ether 4. The lower rim
phenolic groups were subsequently alkylated, in the presence
of cesium carbonate, to afford the conformationally-immobi-
lized 1,3-alternate 5,11,17,23-tetrabenzyloxymethyl-25,26,-
27,28-tetrakis(ethoxycarbonylmethoxy)calix[4]arene (5).
a Department of Chemistry, University of Salerno, Via Ponte Don
Melillo, I-84084 Fisciano Salerno, Italy. E-mail: dericca@unisa.it;
iizzo@unisa.it; Fax: +39 089 969603; Tel: +39 089 969552
b Department of Chemical Sciences, University of Trieste, Via L.
Giorgieri, I-34127 Trieste, Italy. E-mail: ptecilla@units.it;
Fax: +39 040 5583903; Tel: +39 040 5583925
c Department of Pharmaceutical Sciences, University of Salerno, Via
Ponte Don Melillo, I-84084 Fisciano Salerno, Italy
w Electronic supplementary information (ESI) available: Complete
experimental synthetic procedures, ionophoric activity studies, and
biological tests. See DOI: 10.1039/b719482j
Scheme 1 Synthesis of 1 and 2. Reagents and conditions: (a) Na,
BnOH, THF; (b) Cs2CO3, BrCH2CO2Et, acetone, 56%, over two
steps; (c) KOH(aq), THF–MeOH (1 : 1), 49%; (d) (i) SOCl2, toluene,
80 1C; (ii) H2N(CH2)3N(Boc)(CH2)4NHBoc (7), NEt3, toluene, 68%;
(e) CF3COOH, CH2Cl2, quant.; (f) H2, Pd/C, MeOH, quant.
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2986 | Chem. Commun., 2008, 2986–2988

Fig. 4 Normalized fluorescence change in lucigenin emission (FI, lex
455 nm, lem 506 nm) in the presence of different concentrations of
ionophore 1 after the addition of NaCl (50 mL of 1.46 M solution, final
external concentration 24 mM) to 95 : 5 EYPC–EYPG LUVs loaded
with lucigenin (1 mM lucigenin, 0.4 mM total lipid concentration,
25 mM HEPES, 225 mM NaNO3, pH 7, total volume 3 mL). The
concentration of ionophore is given in percentage with respect to the
total concentration of lipid. In two runs, 5 min before the addition of
NaCl, an equimolar amount of sodium glutamate (Glu) and NaClO4
was added to the vesicular suspension (50 mL of 1.46 M solution, final
external concentration 24 mM NaX and 24 mM NaCl).
Fig. 2 (a) Schematic representation of the four possible mechanisms
for pH gradient collapse in the HPTS assay. (b) and (c) Normalized
fluorescence change in HPTS emission (FI, lex 460 nm, lem 510 nm) as
a function of time after the addition of base (50 mL of 0.5 M NaOH),
in the presence of different concentrations of ionophore 1 (b) and 2 (c),
to 95 : 5 EYPC–EYPG LUVs loaded with HPTS (0.1 mM HPTS, 0.17
mM total lipid concentration, 25 mM HEPES, 100 mM NaCl, pH 7.0,
total volume 3 mL). The ionophore concentration is given in percent
with respect to the total concentration of lipid. (HPTS: 8-hydroxy-
1,3,6-pyrenetrisulfonic acid, trisodium salt; HEPES: 4-(2-hydro-
xyethyl)-1-piperazine ethanesulfonic acid; EYPC: egg yolk phospha-
tidyl choline; EYPG: egg yolk phosphatidyl glycerol).
ionophore concentration): an almost instantaneous fluores-
cence ‘‘burst’’,14 and a subsequent slower first-order rate law
increase.15
Selectivity in ion transport was investigated using lower
concentrations of ionophores (1: 0.03%; 2: 2%) in order to
suppress the intrinsically unselective fluorescence ‘‘burst’’
(Fig. 3). Inspection of profiles (a) and (b) in Fig. 3, attests
independence of the ion fluxes from group 1 alkali metals,16
confirming the unfitness of polypositive ionophores to trans-
port alkaline cations.17 The observed results imply that the pH
gradient decay correlates with OHÀ/ClÀ antiport or to
H+/ClÀ symport, excluding the group 1 alkali metals in the
ion permeation processes.
concentration to fully collapse the pH gradient in equal times
(Fig. 2(c)).13 Both the kinetic profiles appear complex, being
composed by two different processes (both dependent on the
The results obtained with anions, evidenced in Fig. 3(c) and
(d), demonstrate efficient halide transport (with some selectiv-
ity toward iodine and bromide over chloride) and low tranÀs-
membrane conductance of oxygenated anions (ClO4
,
glutamate, NO3À, SO42À).18 These data correlate well with a
halide transport based on the lyotropic sequence (with the less
hydrated anion transported more efficiently), but exclude from
this trend the little hydrated nitrate ion and some other
oxygenated anions, suggesting, for the latter, a certain degree
of anion binding with the ionophores. It is interesting to note
that the fully protected neutral Boc-derivative 8 shows lower
activity with respect to 1 and, more importantly, it demon-
strates a low selectivity towards the anion transport.19 These
data attest the relevance of the cationic spermidine chains in
the overall transport processes.
The transport of chloride across the lipid bilayer was
independently evinced using lucigenin, a dye whose fluores-
cence emission is quenched by chloride ion.20 The results,
reported in Fig. 4, clearly show that 1 transports the chloride
anion across the bilayer.21 Interestingly, the chloride conduc-
tance is almost completely suppressed in the presence of
equimolar concentration of perchlorate and glutamate (with
respect to the added chloride), as shown in the two top curves
of Fig. 4 (indicated by the arrows) suggesting an interference
of these two not transported anions.22 This effect reminds,
Fig. 3 Determination of cation (a), (b) and anion (c), (d) selectivities
for ionophore 1 (0.03% concentration; (a) and (c)) and 2 (2.0%
concentration; (b) and (d)), using the HPTS assay (lex 460 nm, lem
510 nm) in 95 : 5 EYPC–EYPG LUVs (0.17 mM total lipid concen-
tration, total volume 3 mL). Conditions: (a) and (b) 25 mM HEPES,
100 mM MCl, pH 7.0, base pulse by addition of 50 mL of 0.5 M MOH;
(c) and (d) 25 mM HEPES, 100 mM NaX, pH 7.0, base pulse by
addition of 50 mL of 0.5 M NaOH. The rates of transport of anions
have been corrected for the membrane permeability of the different
anions in the absence of ionophore. (Glu: glutamate).
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Chem. Commun., 2008, 2986–2988 | 2987

respectively, the ClÀ current reduction induced by xenobiotic
anions on the cystic fibrosis chloride channel pore,23 and the
intrapore regulatory activity of the Glu148 in the EcClC
channels.24
Considering the strong correlation between H+/ClÀ sym-
port transport rates and in vitro cytotoxic activity demon-
strated for prodigiosin analogues,4a we evaluated the cytotoxic
potential of calix[4]arenes 1 and 8, against the J774.A1
(murine monocyte/macrophage) cancer cell line.
In the proliferation assays,25 compound 1 inhibited the cell
growth with an IC50 value of 47.2 Æ 0.5 mM, while compound
8 showed a much lower potency (IC50 = 99 Æ 0.5 mM).
In conclusion, in this contribution we demonstrated that
cationic calix[4]arenes mediate HX efflux, induce block of the
chloride transport in the presence of appropriate interfering
anions, and show a moderate antiproliferative activity against
murine monocyte/macrophage J774.A1 cancer cells. These last
two properties seem to be directly related to the presence of the
cationic spermidine chains. Efforts currently in progress are
aimed at understanding the complex interplay among iono-
phore structure, anion permeation, and biological activity.
Financial support from the University of Salerno, MIUR
(contract 2006039071) and Regione FVG is gratefully ac-
knowledged. We thank Ms C. Martone for experimental work.
8. (a) V. Sidorov, F. W. Kotch, G. Abdrakhmanova, R. Mizani,
J. C. Fettinger and J. T. Davis, J. Am. Chem. Soc., 2002, 124,
2267; (b) V. Sidorov, F. W. Kotch, J. L. Kuebler, Y.-F. Lam and
J. T. Davis, J. Am. Chem. Soc., 2003, 125, 2840; (c) J. L. Seganish,
P. V. Santacroce, K. J. Salimian, J. C. Fettinger, P. Zavalij and
J. T. Davis, Angew. Chem., Int. Ed., 2006, 45, 3334;
(d) O. A. Okunola, J. L. Seganish, K. J. Salimian, P. Y. Zavalij
and J. T. Davis, Tetrahedron, 2007, 63, 10743.
9. The length of 1 and 2 in their ‘‘extended’’ conformations (mole-
cular mechanic calculations using the MM3 software) is 3.2 Æ 0.2
nm, comparable to the phospholipid membrane thickness
(B3.5 nm).
10. F. W. Kotch, V. Sidorov, Y.-F. Lam, K. J. Kayser, H. Li,
M. S. Kaucher and J. T. Davis, J. Am. Chem. Soc., 2003, 125,
15140.
11. (a) R. A. Gardner, R. Kinkade, C. Wang and O. Phanstiel, J. Org.
Chem., 2004, 69, 3530; (b) W. Hu and M. Hesse, Helv. Chim. Acta,
1996, 79, 548; (c) R. Goodnow Jr, K. Konno, M. Niwa,
T. Kallimopoulos, R. Bukownik, D. Lenares and K. Nakanishi,
Tetrahedron, 1990, 46, 3267.
12. N. Sakai and S. Matile, J. Phys. Org. Chem., 2006, 19, 452.
13. The lower activity of 2 is ascribable to its higher hydrophilicity,
which limits its partition into the membrane (see ESIw).
14. The activity/concentration profiles and control experiments ex-
cluding that the ‘‘burst’’ is due to partial lysis or fusion of the
liposomes are reported in the ESIw.
15. This trend is compatible with a model recently proposed by Chiu
and co-workers: C. L. Kuyper, J. S. Kuo, S. A. Mutch and
D. T. Chiu, J. Am. Chem. Soc., 2006, 128, 3233. In this model
the ‘‘burst’’ is associated to ion flux across randomly formed
membrane pores while the slower process is governed by the
diffusion of the ions into the bilayer. Accordingly, the burst is
intrinsically unselective toward anion and cation transport (see
ESIw).
Notes and references
1. (a) B. Hille, in Ionic Channels of Excitable Membranes, Sinauer
Associates, Sunderland, MA, 3rd edn, 2001; (b) I. R. Booth,
M. D. Edwards and S. Miller, Biochemistry, 2003, 42, 10045.
2. (a) R. Dutzler, Curr. Opin. Struct. Biol., 2006, 16, 439;
(b) R. MacKinnon, Angew. Chem., Int. Ed., 2004, 43, 4265.
3. For a review see: A. P. Davis, D. N. Sheppard and B. D. Smith,
Chem. Soc. Rev., 2007, 36, 348. For more recent contributions,
see: (a) R. Ferdani, R. Li, R. Pajewski, J. Pajewska, R. K. Winter
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Chem., 2007, 5, 3000; (c) X. Li, B. Chen, X.-Q. Yao and D. Yang,
J. Am. Chem. Soc., 2007, 129, 7264; (d) N. Madhavan and
M. S. Gin, ChemBioChem, 2007, 8, 1834; (e) P. A. Gale,
J. Garric, M. E. Light, B. A. McNally and B. D. Smith, Chem.
Commun., 2007, 1736; (f) P. V. Santacroce, J. T. Davis,
16. These data are also confirmed by 23Na+-NMR experiments (see
ESIw) showing that Na+ is not transported across the lipid
bilayer.
17. (a) G. Deng, T. Dewa and S. L. Regen, J. Am. Chem. Soc., 1996,
118, 8975; (b) C. Jiang, E. R. Lee, M. B. Lane, Y.-F. Xiao,
D. J. Harris and S. H. Cheng, Am. J. Physiol. Lung Cell. Mol.
Physiol., 2001, 281, L1164.
18. With ionophore 2 we have also tested a slightly different protocol,
proposed by Matile (ref. 12) in which liposomes prepared in NaCl
solution are diluted in a buffer containing the ion under study.
This experiment confirms the independence of the transport
process from the cation (Li+, Na+, K+, Rb+, Cs+) and shows
a simÀilar trend for anions (IÀ 4 BrÀ E ClÀ c SO42À E NO3À c
ClO4 E FÀ 4 acetate), see ESIw.
19. The anions selectivity ratios for 1 and 8 are, respectively: ClÀ/Glu
M. E. Light, P. A. Gale, J. C. Iglesias-Sanchez, P. Prados and
´
R. Quesada, J. Am. Chem. Soc., 2007, 129, 1886.
= 9.4 and 1.3; ClÀ/ClO4 = 5.4 and 1.3; see ESIw.
À
20. B. A. McNally, A. V. Koulov, B. D. Smith, J.-B. Joos and
A. P. Davis, Chem. Commun., 2005, 1087.
4. Prodigiosins: (a) J. L. Sessler, L. R. Eller, W.-S. Cho, S. Nicolaou,
A. Aguilar, J. T. Lee, V. M. Lynch and D. J. Magda, Angew.
Chem., Int. Ed., 2005, 44, 5989; (b) Duramycins: F. Hayashi,
K. Nagashima, Y. Terui, Y. Kawamura, K. Matsumoto and
H. Itazaki, J. Antibiot., 1990, 63, 1421; (c) Pamamycin:
S. Lanners, H. Norouzi-Arasi, X. J. Salom-Roig and
G. Hanquet, Angew. Chem., Int. Ed., 2007, 46, 7086.
21. The chloride transport is associated with an increase in the
liposome internal pH as shown by an experiment performed in
similar conditions but using HPTS as internal pH probe (see
ESIw). This further supports that the anion translocation is
coupled with H+ symport or OHÀ antiport.
22. This effect is confirmed by a substantial anomalous mole fraction
effect (AMFE) observed with glutamate. Moreover, the glutamate
induced blockage is almost absent with compound 8 (see ESIw).
23. P. Linsdell, J. Physiol., 2001, 531, 51.
24. R. Dutzler, E. B. Campbell and R. Mackinnon, Science, 2003,
300, 108.
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L. Fanfoni, P. Tecilla and F. De Riccardis, Tetrahedron, 2006,
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A. Casapullo, E. Cressina, M. Fregonese, P. Tecilla and I. Izzo,
Chem. Commun., 2005, 1354.
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J. Opipari Jr, H. M. Hu, R. Yabkowitz and V. M. Dixit, J. Biol.
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2988 | Chem. Commun., 2008, 2986–2988