C2-symmetrical sterol-polyether conjugates as highly efficient synthetic ionophores

Article abstract of DOI:10.1016/S0040-4039(03)01454-0

A new class of artificial ionophores has been rationally designed and synthesized linking to a tetrafunctionalized L-treitol spacer two rigid hydrophobic 3β-hydroxy-5α-23,24-bisnorcholanic units and two flexible hydrophilic oligo(ethylene glycol) chains.

Full text of DOI:10.1016/S0040-4039(03)01454-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 6121–6124
C2-symmetrical sterol–polyether conjugates as highly efficient
synthetic ionophores
Elvira Avallone,a Irene Izzo,a Gerardo Vuolo,a Monica Costabile,a Davide Garrisi,b Lucia Pasquato,c
Paolo Scrimin,b Paolo Tecillac,* and Francesco De Riccardisa,*
aUniversity of Salerno, Department of Chemistry, Via S. Allende, I-84081, Baronissi, Salerno, Italy
bUniversity of Padova, Department of Organic Chemistry and ITM-CNR, Padova Section, via L. Marzolo 1, I-35131 Padova,
Italy
cUniversity of Trieste, Department of Chemical Sciences, via L. Giorgieri, I-34127, Trieste, Italy
Received 6 June 2003; accepted 7 June 2003
Abstract—A new class of artificial ionophores has been rationally designed and synthesized linking to a tetrafunctionalized
L
-treitol spacer two rigid hydrophobic 3b-hydroxy-5a-23,24-bisnorcholanic units and two flexible hydrophilic oligo(ethylene
glycol) chains. Compounds 1a and 1b were incorporated into phospholipid vesicles and shown to facilitate Na+-transport.
© 2003 Elsevier Ltd. All rights reserved.
The bio- and pharmacological implication of synthetic
channel-forming molecules is, currently, a focal point of
attention.1 Their activities give insight into the mem-
branes’ permeability mechanisms and may provide new
chemical tools useful to circumvent drug resistance,2
acting as ionic nano-devices3 or able to interfere with
the cellular transduction of the electrical signal.4
Tremendous efforts have gone into chemical studies of
peptide-based ion-channels5 but alternative systems
have also been explored.6
whose synthesis would be more convenient and whose
structural features could be easily controlled in order to
tune membrane interaction and activity.
With these ideas in mind we designed the two C2-sym-
metric sterol–oligo(ethylene glycol) conjugates proto-
types 1a and 1b.
Recently, we have succeeded in constructing an artifi-
cial non peptide ionophore7 inspired by membrane-
interfering natural polyhydroxysteroids derivatives.8
The active species were suggested to be supramolecular
aggregates in which the steroidal amphipatic frame-
work preorganizes into the membrane, promoting the
formation of hydrophilic pores through the lipid
bilayer.
Figure 1 shows their three main structural elements: a
L
-treitol-based central ‘core’, lying near the bilayer mid-
plane, two hydrophobic steroidal ‘wall’ units and two
partially hydrophilic oligo(ethylene glycol) moieties,10
radiating from the alditol.
However, because the synthetic pathway to the C2-sym-
metric polyhydroxylated steroid dimer derivative was
elaborate (16 steps)9 and not amenable to a modular
approach, we sought alternative sterol-based targets
,
The ‘wall’ units should provide, with their 40 A long
extended conformation,11 structural control to the
transmembrane aggregate and drag the polar polyether
chains into the bilayer. These last should point toward
the hydrophilic face of a nearest-neighbor identical
molecule, inducing a localized increase in membrane
hydrophilicity. The four distal hydroxy polar ‘head’
groups should enforce the correct orientation of the
molecule through the phospholipid bilayer.
Keywords: ionophore; steroid; amphiphiles.
* Corresponding authors. Tel.: +39 040 558 3925; fax: +39 040 558
3903 (P.T.); Tel.: +39 089 965 230; fax: +39 089 965 296 (F.D.R.);
e-mail: tecilla@dsch.univ.trieste.it; dericca@unisa.it
0040-4039/$ - see front matter © 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01454-0

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E. A6allone et al. / Tetrahedron Letters 44 (2003) 6121–6124
The starting material for the synthesis of the sterol–
polyether conjugates 1a and 1b was commercially avail-
able epi-androsterone (3, Scheme 1). This was
converted into the (Z)-17(20)-ethylidene steroid 412 in
83% yield by means of a highly stereoselective Wittig
coupling.13 C-3 PDC oxidation14 and a stereospecific
ene reaction of the ketone 5 with paraformaldehyde in
the presence of catalytic amount of boron trifluoride-
diethyl ether,15 afforded 5a-23,24-bisnorchol-16-en-22-
ol-3-one (6) in 84% yield. The omoallyl alcohol 6 was
stereoselectively hydrogenated at D16 to give a 9:1 mix-
ture of 3b- and 3a-alcohols, both showing a 17b side
chain. Jones oxidation16 of the crude reaction mixture
yielded the expected 3-oxo-5a-23,24-bisnorcholanic acid
8 in good overall yields (63%, five steps).
In this communication we report the synthesis and the
Na+ ionophoric activity of compounds 1a and 1b across
a lipid bilayer using a 23Na+ NMR based assay.7
The synthesis of the two polar oligo(ethylene glycol)
moieties started from penta- and hexa(ethylene glycols)
9a and 9b. They were easily transformed in the
monoprotected carboxyl derivatives 11a and 11b,
according the two-step synthetic sequence reported in
Scheme 2.
Figure 1. Design proposal for 1b inserted into a phospholipid
bilayer.
Assembling of bisnorcholanic acid derivative 8 and
monoprotected derivatives 11a–11b, with the commer-
cially available C-2,3 bis-protected (−)-2,3-O-benzyli-
dene-L-treitol, by two subsequent double con-
densations, made use of the 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide (EDC) hydrochloride.17
The first double esterification reaction between the pro-
tected treitol and the steroid 8, gave the non-symmetric
adduct 12 (Scheme 3).
C2-symmetry was established by removal of the benzyl-
idene protecting group, through Pd-mediated reductive
debenzylation. 13 was thus formed in quantitative yield.
A second EDC-mediated double condensation of the
free C-2,3 treitol hydroxy groups with the oligo-(ethyl-
ene glycol) acid derivatives 11a and 11b, gave the
bis-silyl protected adducts 14a and 14b. BH3 induced18
stereoselective reduction of the C-3 carbonyl, gave the
b-alcohols 15a,b in good yields. Primary ‘head-group’
desilylation with HF/pyridine19 furnished target com-
pounds 1a20 and 1b.21
Scheme 1. Reagents and conditions: (a)
3
equiv. of
EtP(Ph)3Br, 2.7 equiv. of t-BuOK, THF, reflux, 3 h, 83%; (b)
1.5 equiv. of PDC, m.s., CH2Cl2, rt, 2 h, 86%; (c) 5.5 equiv.
of paraformaldehyde, 0.1 equiv. of BF3·Et2O, CH2Cl2, rt, 0.1
h, 98%; (d) H2, Pt/C, EtOH, 1 h; (e) Jones reagent, acetone,
rt, 2 h, 90% (two steps).
The ionophoric properties of the target compounds
were investigated using a 23Na+ NMR based assay.7 In
brief, a solution of NaCl (75 mM) plus a membrane-
impermeable paramagnetic shift reagent (DyCl3–
tripolyphosphate complex, 4.0 mM) were added to a
95:5 egg phosphatidylcholine (PC) and egg phos-
phatidylglycerol (PG) dispersion (100 nm diameter,
large unilamellar vesicles) prepared in aqueous LiCl
(100 mM). Compounds 1a and 1b were incorporated in
the lipid mixture before the formation of vesicles which
were then prepared by extrusion through polycarbonate
filters with a 100 nm pore diameter. Because the shift
reagent is confined in the external bulk aqueous phase,
the Na+ entering the vesicular compartment appears as
a separate (unshifted) resonance and integration of
internal Na+ signal, as a function of time, yields the
kinetic profiles shown in Figure 2.
Scheme 2. Reagents and conditions: (a) 1.8 equiv. of DBU, 1.0
equiv. of TPS–Cl, CH2Cl2, 3 h, 40%; (b) Jones reagent,
acetone, 1 h, 62%.

E. A6allone et al. / Tetrahedron Letters 44 (2003) 6121–6124
6123
Figure 3. Plot of kobs for Na+ permeation as a function of
mol% of 1a.
case of compound 1a we have also investigated the
activity/concentration dependence. The kinetic profile is
shown in Figure 3.
Increasing the concentration of 1a the rate of Na+ entry
increases showing a saturation behavior which can
probably be attributed to a limited solubility of the
ionophore in the lipid bilayer.22 In any case, the
observed activities are comparable to those of a previ-
ously reported polyhydroxysteroid derivative7 and,
more importantly, also to that of the natural occurring
ionophore amphotericin B, which is often taken as the
reference compound.23
In conclusion the present communication reports on a
convenient approach towards the enantiospecific syn-
thesis of C2-symmetric sterol–oligo(ethylene glycol)
derivatives with high ionophoric activity. These results
may be considered as the starting point for a new class
of easily accessible sterol-polyether-conjugate dimers
with promising properties of altering the permeability
of biological membranes and potentially useful as
antibacterial drugs.
Scheme 3. Reagents and conditions: (a) 3.0 equiv. of DMAP,
1.0 equiv. of (−)-2,3-O-benzyliden- -threitol, 3.0 equiv. of
EDC, CH2Cl2, 48 h, 55%; (b) H2, Pd/C, AcOEt, rt overnight,
quant; (c) 10 equiv. of DMAP, 9 equiv. of EDC, 2 equiv. of
11a or 11b, CH2Cl2, 48 h, 72%; (d) 2 equiv. of BH3·SMe2,
THF, 1 h, 70%; (e) HF, Py, 2 h, 70%.
L
Acknowledgements
This work has been supported by the MIUR (‘PRIN:
Chimica dei Composti Organici di Interesse Biologico’)
and by the Universita` degli Studi di Salerno.
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Figure 2. Kinetic profiles for the entry of Na+ into 95:5 egg
PC/PG vesicles containing 1a (1.0%, ꢀ), 1b (1%, ꢁ), and
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derivative is given in percent with respect to the total concen-
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