On the importance of the pore inner cavity for the ionophoric activity of 1,3-alternate calix[4]arene/steroid conjugates

Article abstract of DOI:10.1016/j.tet.2006.04.028

1,3-Alternate calix[4]arenes, decorated with four nonpolar 'all-trans' tetracyclic nuclei and cation-stabilizing β-methoxyethoxy appendages, were synthesized from commercially available starting materials and through straightforward functional groups tran

Full text of DOI:10.1016/j.tet.2006.04.028

Tetrahedron 62 (2006) 5385–5391
On the importance of the pore inner cavity for the ionophoric
activity of 1,3-alternate calix[4]arene/steroid conjugates
a,*
a
Irene Izzo, Nakia Maulucci, Cristina Martone, Agostino Casapullo,
a
b
c
c
Lidia Fanfoni, Paolo Tecilla and Francesco De Riccardis
a
a
Department of Chemistry, University of Salerno, via S. Allende, Baronissi, I-84081 Salerno, Italy
Department of Pharmaceutical Science, University of Salerno, Via Ponte Don Melillo, Fisciano, I-84084 Salerno, Italy
b
c
Department of Chemical Sciences, University of Trieste, via L. Giorgieri, I-34127 Trieste, Italy
Received 16 February 2006; revised 15 March 2006; accepted 6 April 2006
Available online 2 May 2006
Abstract—1,3-Alternate calix[4]arenes, decorated with four nonpolar ‘all-trans’ tetracyclic nuclei and cation-stabilizing b-methoxyethoxy
appendages, were synthesized from commercially available starting materials and through straightforward functional groups transformations/
+
couplings. Their Na -transport activities, when compared with those exerted by the known conformationally-rigidified 1,3-alternate calix[4]-
arene AB/cis cholic acid conjugates, suggest that the cation conductance is related to the morphology of the pendant steroids.
Ó 2006 Published by Elsevier Ltd.
1
. Introduction
1
, X = NH,
R
X
R
X
R =
In living organisms, ion transport is mediated by integral
membrane proteins that span from the internal to the external
surface of the lipid bilayer. The intimate chemical details of
the cation transport in biological systems still remain elu-
sive, but details of the conductance mechanisms are emerg-
ing thanks to the structure/function relationship analysis of
natural and artificial transporters.
OAc
O
O
O
O
OH
2
, X = NH,
1
O
R =
O
HO
OAc
2
AcO
O
O
X
R
X
R
3
, X = O,
3
In this context, we have recently demonstrated that in
R =
HO
conformationally-rigidified 1,3-alternate (i.e., 1, Fig. 1)
and cone calix[4]arene/hydroxycholanic derivatives, the
observed unimolecular ion transport is dependent on the
length of the channel.
O
Figure 1. Calix[4]arenes conjugates 1–3.
In this paper we try to assess how the presence of a pre-orga-
nized pore cavity influences the cation transport process.
This is a crucial issue for the design of artificial ion channels.
Compound 2 was designed in order to have approximately
˚
the same length as 1 (35ꢀ2 A), an AB-trans ring junction
and two equatorial acetoxy groups, useful for transient cat-
ion stabilization, lying in the same plane of the tetracyclic
nucleus.
With the idea of clarifying the structural requisites useful
for the ionophoric properties, we embarked on the synthesis
of two conformationally immobilized 1,3-alternate calix[4]
arene/all-trans steroid conjugates 2 and 3 (Fig. 1), and
compared their ionophoric activity with that of the powerful
In conjugate 3, the steroid moiety was simplified to the flat
(20R)-3b-hydroxy-chol-5-enoate residue, showing a slightly
˚
4
longer side chain (estimated overall length of 3: 39ꢀ2 A)
+
3
Na -transporter 1, showing a ‘folded’ AB-cis-cholane
framework.
and no oxygenated substituents, apart from the hydroxy
group at C-3.
The absence of a pre-existing inner cavity in the N- and
O-linked calix[4]arene/sterol 2 and 3 is suggested by molec-
5
ular modeling studies. Figure 2 displays the extended low-
est energy conformations of conjugates 1–3. In the case of
the ‘folded’ AB-cis framework, as in 1, the presence of an
Keywords: Ionophores; Amphiphiles; Steroids; Calix[4]arenes.
*
Corresponding author. Tel.: +39 089965230; fax: +39 089965296; e-mail:
iizzo@unisa.it
0
040–4020/$ - see front matter Ó 2006 Published by Elsevier Ltd.
doi:10.1016/j.tet.2006.04.028

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I. Izzo et al. / Tetrahedron 62 (2006) 5385–5391
O
O
O
O
O
O
8
O
O
O
O
O
O
1
2,3
calix[4]arene-1,3-alt tetra-aldehyde 8
and, to our delight,
we isolated, after chromatographic purification, the tetra-
adduct 9 in a satisfying 83% yield (Scheme 2). HF/pyridine-
mediated tert-butyldiphenylsilyl deprotection, gave the
expected target 2.
R
R
NH
HN
O
O
O
O
9
, R =
a
TPSO
7
OAc
AcO
O
O
b
O
O
2
HN
NH
R
R
Figure 2. Energy-minimized structures obtained by molecular modeling of
compounds 1–3 in their extended conformations.
Scheme 2. Reagents and conditions: (a) 8, AcOH, NaBH(OAc)
3
,
ClCH CH Cl, 83% and (b) HF, Py, 48%.
2
2
intramolecular hollow is evident. In derivatives 2 and 3, Van
der Waals forces stabilize the interactions of the facing all-
trans steroid pairs, packing the nonpolar appendages in
a more ‘compact’ morphology.
2
.2. Attempted synthesis of conjugate 10
Having secured the first 1,3-alternate calix[4]arene/all-trans
steroid derivative, we turned our attention to the synthesis of
the D -conjugate 10.
5
2
. Results and discussion
R
2
.1. Synthesis of conjugate 2
R
NH
HN
O
O
The 1,3-alternate calix[4]arene/6a,7b-diacetoxy-5a-23,24-
bisnorcholan-3b-ol-22-amino derivative was synthesized
starting from the known (20S)-6a,7b-diacetoxy-3b-[(tert-
O
O
6
10, R =
HO
butyldiphenylsilyl)oxy]-5a-23,24-bisnorchol-16-en-22-ol (4,
Scheme 1). This was first tosylated at the primary hydroxy
O
7
O
8
group and then substituted in the presence of sodium azide
to yield 6. The C-22 azide was subsequently reduced, with
O
O
9
HN
NH
molecular hydrogen and Adam’s catalyst, to give, in good
yield and high stereoselectivity, the saturated (20S)-
R
R
1
0
Its supposedly easier convergent synthesis (Scheme 3),
started from a C-3 acetylation of the commercially available
cholenic acid (11) to give the (20R)-3b-acetoxy-chol-5-enic
acid (12). This was transformed into the carboxamide 13
(99% yield), using, as condensing agent, the diphenylphos-
phorylazide (DPPA) and then reduced to the required
(20R)-24-amino-chol-5-ene-3b-ol (14) with LiAlH . This
time we failed to synthesize the requested tetra-amino
6
5
a,7b-diacetoxy-3b-[(tert-butyldiphenylsilyl)oxy]-22-amino-
a-23,24-bisnorcholane (7).
OH
a
OTs
NH2
1
3
TPSO
b
OAc
5
4
AcO
4
c
N3
calix[4]arene 10 using the NaBH(OAc) -mediated reductive
3
7
6
amination conditions obtaining, instead, a complex mixture
containing the mono and the bis-adducts (ESI-MS analysis).
In order to improve the efficiency of the coupling reaction
we decided to change the reaction conditions. In particular,
considering the low solubility of the amine 14 in halogenated
solvents, we turned to the more polar solvent DMF and,
in a subsequent experiment, we increased the number of
Scheme 1. Reagents and conditions: (a) TsCl, Py, CH Cl
2
2
, 91%; (b) NaN
3
,
ꢁ
DMF, 60 C, 96% and (c) H
2
, PtO
2
, EtOH, 79%.
With the aminosteroid 7 in our hands we performed the well
described NaBH(OAc) -mediated reductive amination in
the presence of the known conformationally immobilized
1
1
3

I. Izzo et al. / Tetrahedron 62 (2006) 5385–5391
5387
equivalents of the steroid primary amine (see Section 4).
Unfortunately, none of the attempted reactions gave the
desired tetra-aminated calix[4]arene 10. We invariably
observed the formation of a complex mixture of lower
adducts (ESI-MS analysis, see Section 4) and no trace of
the required 10.
enic acid (16), easily synthesized in two steps from the com-
mercially available cholenic acid (11, Scheme 4).
The esterification reaction went smoothly and gave, after
chromatographic purification, the pure silylated tetra-adduct
17 (46% yield). Finally, HF-mediated desilylation, produced
the C symmetric target 3.
4
OH
+
b
NH2
2.4. Na -transporting activities of conjugates 2 and 3
RO
O
O
1
1, R = H
2, R = Ac
To investigate the ionophoric properties of conjugates 2 and
3 we studied their ability to promote the transport of Na
across a lipid bilayer using a Na NMR based methodol-
a
13
+
1
2
3
+
1
4
ogy. Figure 3 shows that conjugates 2 and 3 behave as
poor ionophores: in the presence of 1% of each of them (per-
cent of steroids with respect to the total concentration of
lipid, curve B for 2 and C for 3), and after more than
c
NH2
d
10
HO
1
4
+
Scheme 3. Reagents and conditions: (a) Ac
ꢁ
2
O, Py, CH
2
Cl ; (b) DPPA,
2
13 h, the amount of Na entering the internal vesicular com-
partment was only slightly higher than in the absence of any
NH
5
4
Cl, NEt
3
, DMF, 0 C, 99%, for two steps; (c) LiAlH
4% and (d) 8, AcOH, NaBH(OAc) , ClCH CH Cl or DMF (see Section 4).
4
, THF, reflux,
3
2
2
3
additive (curve A). On the contrary, as previously reported,
conjugate 1 efficiently induces the transport of Na across
+
2
.3. Synthesis of conjugate 3
the lipid bilayer and its transmembrane gradient is fully dis-
charged in almost 12 h (curve -).
1
5
To overcome the difficulties posed by the reductive amination,
we decided to link the two coupling partners (1,3-alternate
calix[4]arene and the all-trans sterol) via an ester bond.
Having the cholenic acid ready for use, we focused our atten-
tion to the reduction of the tetra-aldehyde 8, in order to obtain
thetetra-alcohol15. Theconformationallyrestricted1,3-alter-
nate calix[4]arene tetraol 15 was thus easily formed through
100
80
60
40
20
0
NaBH -induced carbonyl reduction of the tetra-aldehyde 8.
4
OH
HO
O
O
O
O
15
0
200
400
600
800
O
O
time, minutes
O
O
+
Figure 3. Kinetic profiles for the entry of Na into 95:5 egg PC/PG vesicles
containing 1 (1%, -), 2 (1%, B), and 3 (1%, C), and without additives
ꢁ
HO
OH
(
A) at 25 C. The total concentration of lipid was 10 mM.
The sterol partner used in the EDC-mediated condensation
reaction was the 3b-[(tert-butyldimethylsilyl)oxy]-chol-5-
3
. Conclusions
The results of this endeavor clarify the relevance of an
extended cavity throughout the pore for the transport of
Na across a lipid bilayer. The two newly prepared 1,3-alter-
+
OH
b
nate calix[4]arene/steroid conjugates characterized by the
presence of all-trans junctions behave as poor ionophores
regardless to the presence of the acetoxy moieties. As sug-
gested by the molecular modeling studies, in these conju-
gates the ‘flat’ steroid nucleus adopts a compact packing
hampering the cation transport. On the other hand, the
‘folded’ AB-cis geometry of the steroid appendages present
in 1 favors the formation of an inner cavity, which appears
essential for the transport process.
RO
O
11, R = H
a
16, R = TBS
R
O
R
O
O
O
O
17, R =
TBSO
O
O
c
O
O
3
O
O
4. Experimental
O
O
R
R
4.1. General methods
ꢁ
Scheme 4. Reagents and conditions: (a) TBSCl, imidazole, DMF, 0 C then
ꢁ
All reactions were carried out under a dry argon atmosphere
using freshly distilled and dried solvents, unless otherwise
K
2
CO
3
, MeOH, THF, H
2
O, 79%; (b) EDC, DMAP, CH
2
Cl
2
, 0 C, 46% and
(c) HF (48% in H
2
O), THF, quant.

5388
I. Izzo et al. / Tetrahedron 62 (2006) 5385–5391
1
noted. Tetrahydrofuran (THF) was distilled from LiAlH4.
Toluene, methylene chloride, and diethyl ether were dis-
tilled from calcium hydride. Glassware was flame-dried
6: [a] +9.2 (c 1.7, CHCl ). H NMR (CDCl , 400 MHz) d:
D 3 3
0.77 (3H, s, CH -18), 0.96 (3H, s, CH -19), 1.03 (12H, s,
3
(CH ) CSi– and CH -21), 1.87 (3H, s, COCH ), 1.97 (3H,
3
3
3
3
3
(
dried in vacuo over P O or by azeotropic removal of water
0.05 Torr) prior to use. When necessary, compounds were
s, COCH ), 2.32 (1H, m, H-20), 3.16 (1H, dd, J¼12.0,
3
0
7.7 Hz, H-22), 3.35 (1H, dd, J¼12.0, 5.9 Hz, H -22), 3.54
2
5
with toluene under reduced pressure. Starting materials and
reagents purchased from commercial suppliers were gener-
ally used without purification. Reaction temperatures were
measured externally. Reactions were monitored by TLC on
Merck silica gel plates (0.25 mm), visualized by UV light
or with H SO –Ce(SO ) or ninhydrin solutions. Flash chro-
(1H, m, H-3), 4.68 (1H, br t, J¼9.5 Hz, H-6 or H-7), 4.80
(1H, br t, J¼9.5 Hz, H-6 or H-7), 5.34 (1H, br s, H-16),
1
3
7.37 (6H, m, ArH), 7.64 (4H, m, ArH). C NMR (CDCl ,
3
100 MHz) d: 13.3, 15.9, 19.0, 19.6, 20.6, 21.0, 21.4, 26.9
(ꢂ3), 31.1, 32.0, 32.1, 32.3, 34.4, 36.0, 36.8, 37.9, 46.4,
48.0, 52.1, 54.8, 56.8, 72.0, 74.2, 77.6, 123.1, 127.4 (ꢂ4),
129.5 (ꢂ2), 134.6 (ꢂ2), 135.7 (ꢂ4), 155.8, 170.5, 170.7.
HRES-MS, m/z: 712.4119 (calcd 712.4146 for
2
4
4 2
matography was performed on Merck silica gel (60, particle
size: 0.040–0.063 mm). Yields refer to chromatographically
1
13
+
and spectroscopically ( H and C NMR) pure materials.
The NMR spectra were recorded at rt or, when indicated,
C H N O Si) [MH ].
42 58 3 5
ꢁ
C at 100 MHz) and a Bruker DRX 300 ( H at 300 MHz,
C at 75 MHz) spectrometers. Chemical shifts are reported
ꢁ
1
at 80 C or 100 C on a Bruker DRX 400 ( H at 400 MHz,
4.1.3. Compound 7. To a solution of the azide 6 (0.069 g,
0.097 mmol) in ethyl acetate (1.0 ml), PtO (0.007 g) was
1
1
3
3
1
2
added and the reaction mixture was stirred under hydrogen
atmosphere for 48 h. The catalyst was then filtered off on
a short pad of CeliteÔ washing with ethyl acetate. The solvent
was removed under reduced pressure and the crude product
was flash-chromatographed (10–20% methanol in chloro-
form) to give 7 (0.053 g, 79%) as a white amorphous solid.
relative to the residual solvent peak (CHCl : d¼7.26,
3
1
3
13
CDCl : d¼77.0; C H Cl : d¼5.80, C D Cl (TCDE):
3
2
2
4
2
2
4
d¼72.1). HRES-MS was performed on a Q-Star Applied
Biosystem mass spectrometer. Optical rotations were mea-
sured with a JASCO DIP-1000 polarimeter.
1
4
0
0
0
.1.1. Compound 5. To a solution of the alcohol 4 (0.100 g,
.145 mmol) in CH Cl (1.5 ml), pyridine (0.035 ml,
.435 mmol) and p-toluenesulfonylchloride (0.055 g,
.290 mmol) were added and the reaction mixture was
7: [a] +11.8 (c 1.6, CHCl ). H NMR (CDCl , 400 MHz) d:
3
D
3
0.65 (3H, s, CH -18), 0.92 (3H, s, CH -19), 0.97 (3H, d,
3 3
2
2
J¼6.9 Hz, CH -21), 1.03 (9H, s, (CH ) CSi–), 1.86 (3H, s,
3
3 3
COCH ), 1.93 (3H, s, COCH ), 2.38 (1H, dd, J¼11.9,
3 3
0
stirred overnight. Pyridine (0.023 ml, 0.29 mmol) and p-tol-
uenesulfonylchloride (0.042 g, 0.217 mmol) were further
added. After 24 h the reaction was treated with a saturated
solution of NH Cl (4 ml), extracted with CH Cl
7.2 Hz, H-22 or H -22), 2.71 (1H, br d, J¼11.9 Hz, H-22
0
or H -22), 3.51 (1H, m, H-3), 4.62 (1H, br t, J¼9.5 Hz, H-
6 or H-7), 4.75 (1H, br t, J¼9.5 Hz, H-6 or H-7), 7.37 (6H,
1
3
m, ArH), 7.63 (4H, m, ArH). C NMR (CDCl , 100 MHz)
3
4
2
2
(
3ꢂ5 ml), dried over Na SO , filtered, and concentrated in
d: 12.0, 13.3, 16.9, 19.1, 20.7, 21.2, 21.5, 24.8, 26.9 (ꢂ3),
28.0, 31.1, 32.1, 35.7, 37.0, 39.0 (ꢂ2), 39.3, 43.6, 46.1,
46.9, 51.7, 52.4, 54.6, 72.0, 74.5, 77.9, 127.5 (ꢂ4), 129.5
(ꢂ2), 134.5 (ꢂ2), 135.7 (ꢂ4), 170.6, 170.9. HRES-MS,
2
4
vacuo. The residue was flash-chromatographed (10–40%
ethyl acetate in petroleum ether) to give 5 (0.111 g, 91%)
as a white amorphous solid.
+
m/z: 688.4377 (calcd 688.4397 for C H NO Si) [MH ].
4
2
62
5
1
5
: [a] +17.3 (c 2.4, CHCl ). H NMR (CDCl , 400 MHz) d:
D
3
3
0
.67 (3H, s, CH -18), 0.94 (3H, s, CH -19), 0.97 (3H, d,
3
4.1.4. Compound 9. To a solution of aldehyde 8 (0.051 g,
0.066 mmol) and 7 (0.454 g, 0.660 mmol) in 1,2-dichloro-
ethane (2.5 ml) NaBH(OAc) (0.084 g, 0.396 mmol) and
3
J¼6.9 Hz, CH -21), 1.03 (9H, s, (CH ) CSi–), 1.87 (3H, s,
3
3 3
COCH ), 1.96 (3H, s, COCH ), 2.38 (1H, m, H-20), 2.44
3
3
3
(
3H, s, CH (Ts)), 3.53 (1H, m, H-3), 3.83 (1H, br t,
3
AcOH (15 ml, 0.264 mmol) were added. The reaction mix-
ture was stirred at rt overnight, quenched with 1 M NaOH so-
lution and extracted with CH Cl . The combined organic
0
J¼9.4 Hz, H-22), 3.98 (1H, dd, J¼9.4, 5.9 Hz, H -22),
4
.65 (1H, br t, J¼9.5 Hz, H-6 or H-7), 4.79 (1H, br t,
2
2
J¼9.5 Hz, H-6 or H-7), 5.20 (1H, br s, H-16), 7.36 (8H,
phases were washed with brine, dried (MgSO ) and concen-
4
m, ArH and ArH(Ts)), 7.63 (4H, m, ArH), 7.65 (2H, d,
trated in vacuo. The crude residue was flash-chromato-
graphed (5–20% of methanol in dichloromethane) to give
9 (0.190 g, 83%) as a white amorphous solid.
1
3
J¼7.2 Hz, ArH(Ts)). C NMR (CDCl , 100 MHz) d:
3
1
3
5
(
(
3.3, 15.9, 18.3, 19.0, 20.6, 20.9, 21.3, 21.5, 26.9 (ꢂ3),
1.0, 31.5, 32.0, 32.1, 34.1, 35.9, 36.8, 37.8, 46.4, 47.8,
2.0, 54.6, 71.9, 73.8, 74.2, 77.5, 123.6, 127.4 (ꢂ4), 127.8
1
ꢁ
400 MHz) d: 0.52 (12H, s, CH -18), 0.77 (24H, m, CH -19
9: [a]D +12.2 (c 1.0, CHCl ). H NMR (TCDE, 100 C,
3
ꢂ2), 129.5 (ꢂ2), 129.7 (ꢂ2), 133.2, 134.5, 134.6, 135.7
3
3
ꢂ4), 144.5, 154.2, 170.5, 170.6. HRES-MS, m/z:
and CH -21 overlapped), 0.91 (36H, s, (CH ) CSi–), 1.69
3 3 3
(12H, s, COCH ), 1.75 (12H, s, COCH ), 2.46 (8H, br m,
3 3
+
8
41.4135 (calcd 841.4169 for C H O SSi) [MH ].
65
4
9
8
CH NH), 3.16 (12H, br s, OCH ), 3.16–3.67 (36H, m,
2
3
4
0
0
.1.2. Compound 6. To a solution of the tosylate 5 (0.111 g,
.132 mmol) in dry DMF (1 ml), sodium azide (0.043 g,
.661 mmol) was added and the reaction mixture was stirred
ꢁ
ArOCH CH OCH , ArCH Ar, NHCH Ar and H-3 overlap-
2 2 3 2 2
ped), 4.48 (4H, m, H-6 or H-7), 4.61 (4H, m, H-6 or H-7),
7.07 (8H, br s, ArH), 7.17–7.24 (24H, m, ArH(TPS)), 7.50
13
ꢁ
at 60 C for 22 h. The reaction was quenched with brine
(2 ml) and then extracted with Et O (3ꢂ4 ml). The com-
bined organic phases were dried over K CO , filtered, and
concentrated in vacuo. The residue was flash-chromato-
graphed (10–30% ethyl acetate in petroleum ether) to give
6
(16H, m, ArH(TPS)). C NMR (TCDE, 80 C, 100 MHz)
d: 10.2 (ꢂ4), 11.5 (ꢂ4), 16.3 (ꢂ4), 17.2 (ꢂ4), 18.7 (ꢂ4),
19.5 (ꢂ4), 19.6 (ꢂ4), 22.9 (ꢂ4), 25.3 (ꢂ12), 25.8 (br,
ꢂ4), 27.8 (ꢂ4), 29.4 (ꢂ4), 30.5 (ꢂ4), 32.0 (br, ꢂ4), 33.9
(ꢂ4), 35.4 (ꢂ4), 37.4 (ꢂ8), 42.0 (ꢂ4), 44.6 (ꢂ4), 48.0 (br,
ꢂ4), 50.1 (ꢂ4), 51.0 (ꢂ8), 52.8 (ꢂ4), 57.0 (ꢂ4), 68.9 (br,
2
2 3
(0.090 g, 96%) as a white amorphous solid.

I. Izzo et al. / Tetrahedron 62 (2006) 5385–5391
5389
ꢂ4), 69.8 (ꢂ4), 70.4 (ꢂ4), 72.7 (ꢂ4), 76.2 (ꢂ4), 121.0 (br,
ꢂ4), 125.7 (ꢂ16), 127.6 (ꢂ8), 130.2 (br, ꢂ8), 132.4 (br,
ꢂ8), 133.0 (ꢂ4), 133.1 (ꢂ4), 133.9 (ꢂ16), 155.4 (br, ꢂ4),
4.1.7. Compound 14. To a solution of 13 (0.387 g,
0.93 mmol) in THF, LiAlH (1 M in THF, 2.3 mmol) was
added. The reaction mixture was refluxed at 65 C for 3 h,
then water (0.1 ml) and NaOH (15% solution of in water,
0.1 ml) were added. The resulting mixture was vigorously
stirred, filtered under reduced pressure washed with 30%
4
ꢁ
1
3
68.4 (ꢂ4), 168.6 (ꢂ4). ES-MS, m/z: 3455.0501 (calcd
+
455.0703 for C
H
12 293 4 28 4
N O Si ) [MH ].
2
4
0
7
.1.5. Compound 2. To a solution of 9 (0.064 g,
ꢁ
CH OH in ethyl acetate. The solvent was dried over
3
.018 mmol) in pyridine (0.5 ml) at 0 C, a solution of
0% hydrofluoric acid in pyridine (0.3 ml) was added. The
Na SO , filtered, and concentrated in vacuo. The residue
2
4
was flash-chromatographed (2–25% methanol in CH Cl /
2 2
reaction mixture was stirred overnight and concentrated
under a stream of N . The residue was purified by flash chro-
CH COOH 99:1) to give 13 (0.180 g, 54%) as a white amor-
3
phous solid.
2
matography (silica gel, 8–20% methanol in CHCl ) to afford
3
1
2
(0.022 g, 48%) as a white amorphous solid.
14: [a] ꢃ12.8 (c 1.0, MeOH). H NMR (CD OD, 300 MHz)
D
3
d: 0.76 (3H, s, CH -18), 1.01 (3H, d, J¼6.7 Hz, CH -21), 1.05
3
3
2
: R ¼0.4 (10% methanol in CH Cl ). [a] +25.1 (c 1.0,
(3H, s, CH -19), 2.90 (3H, s, CH NH ), 3.43 (1H, m, H-3),
3 2 2
f
2
2
D
1
ꢁ
13
MeOH). H NMR (TCDE, 80 C, 400 MHz) d: 0.56 (12H,
s, CH -18), 0.77 (24H, m, CH -19 and CH -21 overlapped),
5.36 (1H, br d, J¼4.8 Hz, H-6). C NMR (CD OD,
3
100 MHz) d: 12.3, 19.1, 19.9, 22.1, 25.3 (ꢂ2), 29.2, 32.3,
3
3
3
1
m, CH NH), 3.23 (12H, br s, OCH ), 3.35–3.98 (36H, m,
.79 (12H, s, COCH ), 1.82 (12H, s, COCH ), 2.67 (8H, br
3 3
33.0, 33.2, 33.8, 36.8, 37.6, 38.5, 41.2 (ꢂ2), 43.0, 43.5,
51.6, 57.2, 58.1, 72.4, 122.4, 142.2. HRES-MS, m/z:
360.3285 (calcd 360.3266 for C H NO) [MH ].
2
3
+
ArOCH CH OCH , ArCH Ar, NHCH Ar and H-3 overlap-
2
2
3
2
2
24 42
ped), 4.57 (4H, m, H-6 or H-7), 4.68 (4H, m, H-6 or H-7),
.07 (8H, br s, ArH), 7.17–7.24 (24H, m, ArH(TPS)), 7.50
7
4.1.8. First attempted synthesis of 10. To a solution
of amine 14 (0.105 g, 0.29 mmol) in 1,2-dichloroethane
(1 ml), aldehyde 8 (0.038 g, 0.049 mmol), NaBH(OAc)3
(0.062 g, 0.29 mmol), and AcOH (11 ml, 0.20 mmol), were
added. The reaction mixture was stirred at rt overnight,
quenched with 1 N NaOH solution and extracted with
CH Cl . The combined organic phases were washed with
1
3
ꢁ
(
16H, m, ArH(TPS)). C NMR (TCDE, 80 C, 100 MHz)
d: 10.2 (ꢂ4), 11.5 (ꢂ4), 15.2 (ꢂ4), 18.9 (ꢂ4), 19.6 (ꢂ8),
2
3
4
5
1
2.9 (ꢂ4), 25.7 (ꢂ4), 27.8 (ꢂ4), 29.3 (ꢂ4), 30.5 (ꢂ4),
1.9 (ꢂ4), 34.0 (ꢂ4), 35.3 (ꢂ4), 37.4 (ꢂ8), 42.2 (ꢂ4),
4.6 (ꢂ4), 48.0 (br, ꢂ4), 50.1 (ꢂ4), 50.8 (ꢂ8), 52.7 (ꢂ4),
6.8 (ꢂ4), 68.8 (ꢂ4), 70.0 (ꢂ8), 72.8 (ꢂ4), 76.1 (ꢂ4),
21.0 (br, ꢂ4), 129.8 (ꢂ8), 132.6 (ꢂ8), 155.4 (ꢂ4), 168.6
2
2
brine, dried (MgSO ) and concentrated in vacuo. The crude
4
(
ꢂ8). ES-MS, m/z: 2502.7001 (calcd 2502.5992 for
residue was flash-chromatographed (2–30% methanol in
dichloromethane) to give a complex mixture containing
the mono and the bis-adduct. ES-MS, m/z: 1111.6 (mono-
adduct); m/z: 1455.0 (bis-adduct). Mass peaks ratio: 4:1.
+
C
H
48 221 4 28
N O ) [MH ].
1
4
.1.6. Compound 13. To a solution of cholenic acid
0.288 g, 0.77 mmol) in dry CH Cl (20 ml), pyridine
(
(
2
2
1.00 ml, 12.3 mmol), DMAP (0.005 g, 0.04 mmol), and
4.1.9. Second attempted synthesis of 10. To a solution
of amine 14 (0.105 g, 0.29 mmol) in 1,2-DMF (1 ml), alde-
hyde 8 (0.038 g, 0.049 mmol), NaBH(OAc)3 (0.062 g,
0.29 mmol), and AcOH (11 ml, 0.20 mmol), were added.
The reaction mixture was stirred at rt overnight, quenched
with 1 M NaOH solution and extracted with CH Cl . The
Ac O (0.145 ml, 1.54 mmol) were added. The resulting mix-
2
ture was stirred for 3 h, diluted with CH Cl2 (10 ml),
2
quenched with 1 M HCl (10 ml) and extracted with
CH Cl (2ꢂ10 ml). The combined organic phases were
2
2
dried over Na SO , filtered, and concentrated in vacuo to
2
4
2
2
give a crude product, which was used in the next step without
further purification. To a solution of the crude compound in
combined organic phases were washed with brine, dried
(MgSO ), and concentrated in vacuo. The crude residue
was flash-chromatographed (2–30% methanol in dichloro-
methane) to give a complex mixture containing the mono
and the bis-adduct. ES-MS, m/z: 1111.6 (mono-adduct);
m/z: 1455.0 (bis-adduct). Mass peaks ratio: 3:1.
4
ꢁ
DMF (10 ml) at 0 C, diphenylphosphorylazide (DPPA,
0
.40, 1.85 mol), NH Cl (0.099 g, 1.85 mol), and Et N
4 3
(
0.054 ml, 3.85 mmol) were sequentially added. The mix-
ꢁ
acetate (10 ml), treated with a saturated solution of NH Cl
ture was stirred at ꢃ10 C overnight, diluted with ethyl
4
(
10 ml) and extracted with ethyl acetate (2ꢂ15 ml). The
4.1.10. Third attempted synthesis of 10. To a solution
of amine 14 (0.217 g, 0.60 mmol) in DMF (4 ml), alde-
hyde 8 (0.061 g, 0.079 mmol), NaBH(OAc)3 (0.127 g,
0.60 mmol), and AcOH (22 ml, 0.39 mmol) were added.
The reaction mixture was stirred at rt overnight, quenched
with 1 N NaOH solution and extracted with CH Cl . The
combined organic phases were washed with a solution of
NaHCO (10% in water), brine, dried over Na SO , filtered,
and concentrated in vacuo. The residue was flash-chromato-
3
2
4
graphed (2–10% ethyl acetate in petroleum ether) to give 13
(
0.317 g, 99%, for two steps) as a white amorphous solid.
2
2
combined organic phases were washed with brine, dried
(MgSO ), and concentrated in vacuo. The crude residue
was flash-chromatographed (2–30% of methanol in dichloro-
methane) to give a complex mixture containing the mono and
the bis-adduct. ES-MS, m/z: 1111.6 (mono-adduct); m/z:
1455.0 (bis-adduct). Mass peaks ratio: 3:1.
1
1
3: [a]D ꢃ27.9 (c 1.0, CHCl ). H NMR (CD OD,
3
3
4
300 MHz) d: 0.76 (3H, s, CH -18), 1.01 (3H, d, J¼6.7 Hz,
CH -21), 1.08 (3H, s, CH -19), 2.05 (3H, s, CH CO), 4.56
3
3
3
3
1
3
(
(
1H, m, H-3), 5.42 (1H, br d, J¼4.8 Hz, H-6). C NMR
CD OD, 100 MHz) d: 12.3, 18.9, 19.7, 21.2, 22.1, 25.3,
3
2
4
1
8.8, 29.2, 33.0, 33.2 (ꢂ2), 33.5, 36.9, 37.7, 38.2, 39.1,
1.0, 43.5, 51.5, 57.2, 58.0, 75.4, 123.6, 141.0, 172.4,
79.8. HRES-MS, m/z: 416.3209 (calcd 416.3165 for
4.1.11. Compound 15. To a solution of aldehyde 8 (0.143 g,
0.19 mmol) in EtOH (2 ml) at 0 C, NaBH (0.032 g,
ꢁ
0.84 mmol), was added. The resulting mixture was stirred
4
+
C H NO ) [MH ].
6
2
42
3

5390
I. Izzo et al. / Tetrahedron 62 (2006) 5385–5391
at rt for 2 h, quenched with HCl (10% solution in water,
ml) and concentrated under reduced pressure to remove
(36H, s, (CH ) Si–), 0.92 (12H, d, J¼6.7 Hz, CH -21),
3 3
3
3
3
5
0.99 (12H, s, CH -19), 3.44 (12H, s, CH O–), 3.45 (4H,
m, H-3), 3.54 (8H, s, Ar–CH –Ar), 3.63 (8H, m,
the excess of EtOH. The aqueous layer was extracted with
ethyl acetate (3ꢂ10 ml) and the combined organic phases
were dried over Na SO , filtered, and concentrated in vacuo
2
–OCH CH O–), 3.83 (8H, m, –OCH CH O–), 4.92 (8H,
2
2
2
2
br s, –CH OCO), 5.31 (1H, br s, H-6), 7.08 (8 H, s, Ar).
2
2
4
1
3
to give a residue, which was purified by flash chromato-
graphy (2–15% MeOH in EtOAc) to afford 15 (0.112 g,
C NMR (CDCl , 100 MHz) d: ꢃ4.6 (ꢂ8), 11.8 (ꢂ4),
3
18.2 (ꢂ4), 18.3 (ꢂ4), 19.4 (ꢂ4), 21.0 (ꢂ4), 24.2 (ꢂ4),
25.9 (ꢂ12), 28.1 (ꢂ4), 30.9 (ꢂ4), 31.3 (ꢂ4), 31.9 (ꢂ8),
7
6%) as a white amorphous solid.
3
2.0 (ꢂ4), 34.8 (ꢂ4), 35.3 (ꢂ4), 36.5 (ꢂ4), 37.3 (ꢂ4),
1
1
3
3
5: H NMR (CDCl , 400 MHz) d: 3.50 (12H, s, CH O–),
3
39.7 (ꢂ4), 42.3 (ꢂ4), 42.8 (ꢂ4), 50.1 (ꢂ4), 55.8 (ꢂ4),
56.7 (ꢂ4), 58.8 (ꢂ4), 66.2 (ꢂ4), 71.2 (ꢂ4), 71.9 (ꢂ4),
72.6 (ꢂ4), 121.1 (ꢂ4), 128.9 (ꢂ4), 130.5 (ꢂ8), 133.3
(ꢂ8), 141.5 (ꢂ4), 155.7 (ꢂ4), 174.0 (ꢂ4). ES-MS, m/z:
3
.67 (8H, s, Ar–CH –Ar), 3.71 (8H, m, –OCH CH O–),
2
.91 (8H, m, –OCH CH O–), 4.45 (8H, s, –CH OH), 7.05
2
2
2
2
2
1
3
(
8H, s, Ar). C NMR (CDCl , 100 MHz) d: 35.7 (ꢂ4),
3
5
1
7
8.7 (ꢂ4), 65.3 (ꢂ4), 71.5 (ꢂ4), 72.0 (ꢂ4), 130.3 (ꢂ8),
2680.6845 (2680.7990 calcd for
C
H
NaO Si )
1
64 256 20 4
+
33.4 (ꢂ8), 135.0 (ꢂ4), 155.3 (ꢂ4). HRES-MS, m/z:
[MNa ].
+
77.3811 (calcd 777.3850 for C H O ) [MH ].
44 57 12
4
.1.14. Compound 3. To a solution of 17 (0.098 g,
ꢁ
4
1
1
3
0
.1.12. Compound 16. To a solution of 11 (0.612 g,
ꢁ
0.0368 mmol) in THF (1.0 ml) at 0 C, a solution of 48%
hydrofluoric acid in water (40 ml, 2.21 mmol) was added.
The reaction mixture was stirred overnight then concen-
.63 mmol) in DMF (14 ml) at 0 C, imidazole (0.888 g,
3.04 mmol), 4-dimethylaminopyridine (DMAP, 0.398 g,
.26 mmol), and tert-butyldimethylsilyl chloride (TBSCl,
.737 g, 4.89 mmol), were added. The resulting mixture
trated under a stream of N . The residue was purified by flash
2
chromatography (silica gel, 1–10% methanol in CHCl ) to
3
was stirred at rt overnight, then quenched with HCl (1 M,
0 ml), extracted with ethyl acetate (3ꢂ30 ml), washed
with brine, dried on Na SO , and concentrated in vacuo to
afford 3 (0.081 g, quant.) as a white amorphous solid.
1
1
3: [a] ꢃ23.1 (c 0.3, CHCl ). H NMR (CDCl , 400 MHz)
2
4
D
3
3
give the crude, which was used in the next step without fur-
ther purification. To a solution of the crude material in
MeOH (14 ml) and THF (14 ml), a solution of K CO
d: 0.65 (12H, s, CH -18), 0.91 (12H, d, J¼6.7 Hz, CH -21),
3
3
0.99 (12H, s, CH -19), 3.45 (12H, s, CH O–), 3.50 (4H, m,
3 3
H-3), 3.53 (8H, s, Ar–CH –Ar), 3.61 (8H, m,
2
2
3
(
10% w/w, 7 ml) in water, was added. The resulting mixture
–OCH CH O–), 3.81 (8H, m, –OCH CH O–), 4.90 (4H, d,
2
2
2
2
was stirred at rt for 2 h then concentrated under reduced
pressure to remove the excess of THF and MeOH. A satu-
rated solution of NaCl was added and the aqueous layer
was acidified with HCl (5%) to pH 3. The mixture was ex-
tracted with ethyl acetate (3ꢂ40 ml), dried on Na SO and
J¼12.1 Hz, –CHHOCO), 4.93 (4H, d, J¼12.1 Hz,
–CHHOCO), 5.33 (1H, br d, J¼4.8 Hz, H-6), 7.07 (8H, s,
1
3
Ar). C NMR (CDCl , 100 MHz) d: 11.8 (ꢂ4), 18.3 (ꢂ4),
3
19.4 (ꢂ4), 21.0 (ꢂ4), 24.2 (ꢂ4), 28.1 (ꢂ4), 30.9 (ꢂ4),
31.6 (ꢂ4), 31.8 (ꢂ12), 34.9 (ꢂ4), 35.3 (ꢂ4), 36.4 (ꢂ4),
37.2 (ꢂ4), 39.7 (ꢂ4), 42.2, (ꢂ4), 42.3 (ꢂ4), 50.0 (ꢂ4),
55.8 (ꢂ4), 56.7 (ꢂ4), 58.8 (ꢂ4), 66.2 (ꢂ4), 71.2 (ꢂ4),
2
4
concentrated in vacuo to give pure 16 (0.630 g, 79%) as
a white amorphous solid.
7
1.6 (ꢂ4), 71.8 (ꢂ4), 121.5 (ꢂ4), 128.9 (ꢂ4), 130.5 (ꢂ8),
1
1
d: 0.06 (6H, s, (CH ) CSi–), 0.67 (3H, s, CH -18), 0.88 (9H,
6: [a] ꢃ29.3 (c 1.0, CHCl ). H NMR (CDCl , 400 MHz)
133.3 (ꢂ8), 140.8 (ꢂ4), 155.7 (ꢂ4), 174.0 (ꢂ4). ES-MS,
D
3
3
m/z: 2224.4679 (2224.4531 calcd for C
[MNa ].
H NaO )
140 200 20
3
2
3
+
s, (CH ) Si–), 0.93 (3H, d, J¼6.7 Hz, CH -21), 0.99 (3H, s,
3
3
3
CH -19), 3.48 (1H, m, H-3), 5.31 (1H, br s, H-6), 10.30 (1H,
3
1
3
br s, COOH). C NMR (CDCl , 100 MHz) d: ꢃ4 (ꢂ2),
3
Acknowledgements
1
3
5
1.9, 18.3 (ꢂ2), 19.4, 21.0, 24.2, 25.9 (ꢂ3), 28.1, 30.8,
1.1, 31.9, 32.0, 35.3 (ꢂ2), 36.5, 37.4, 39.7, 42.4, 42.8,
This work has been supported by the MIUR (‘Sintesi di sos-
`
tanze naturali e di loro analoghi sintetici con attivita antitu-
morale’) and by the Universit a` degli Studi di Salerno.
0.1, 55.7, 56.7, 72.6, 121.1, 141.5, 180.6. HRES-MS,
+
m/z: 489.3711 (calcd 489.3764 for C H O Si) [MH ].
30 53 3
4
0
0
.1.13. Compound 17. To a solution of 16 (0.41 g,
.84 mmol) in dry CH Cl (8 ml) at 0 C, DMAP (0.048 g,
ꢁ
.39 mmol), a solution of 15 (0.11 g, 0.14 mmol) in dry
2
2
References and notes
CH Cl (2 ml) and EDC (0.17 g, 0.90 mmol) were sequen-
2
2
tially added. The reaction mixture was stirred for 16 h,
quenched with water (5 ml) and extracted with ethyl acetate
1. (a) Aidley, D. J.; Stanfield, P. R. Ion Channels: Molecules in
Action; Cambridge University Press: Cambridge, 1996; (b)
Nicholls, D. G. Proteins, Transmitters, and Synapses;
Blackwell: Oxford, 1994; (c) Shai, Y. Biochim. Biophys. Acta
1999, 1462, 55–70.
2. (a) Sakai, N.; Mareda, J.; Matile, S. Acc. Chem. Res. 2005, 38,
79–87; (b) Matile, S.; Som, A.; Sord e´ , N. Tetrahedron 2004, 60,
6405–6435; (c) Yoshii, M.; Yamamura, M.; Satake, A.;
Kobuke, Y. Org. Biomol. Chem. 2004, 2, 2619–2623; (d)
Gokel, G. W.; Schlesinger, P. H.; Djedovi ꢀc , N. K.; Ferdani,
R.; Harder, E. C.; Hu, J.; Leevy, W. M.; Pajewska, J.;
Pajewski, R.; Weber, M. E. Bioorg. Med. Chem. 2004, 12,
(
solution of NaHCO and water, dried over Na SO , filtered,
20 mlꢂ3). The organic layer was washed with a saturated
3
2
4
and concentrated in vacuo. The crude was purified by flash
chromatography (1–25% of a solution of 1% AcOH in
EtOAc in petroleum ether), to furnish 17 (0.172 g, 46%) as
a white amorphous solid and 0.060 g of a mixture containing
1
6 and 17 in a 6:1 ratio.
1
1
d: 0.06 (24H, s, (CH ) CSi–), 0.67 (12H, s, CH -18), 0.89
7: [a] ꢃ20.2 (c 0.4, CHCl ). H NMR (CDCl , 400 MHz)
D
3
3
3
2
3

I. Izzo et al. / Tetrahedron 62 (2006) 5385–5391
291–1304; (e) Koulov, A. V.; Lambert, T. N.; Shukla, R.; Jain,
M.; Boon, J. M.; Smith, B. D.; Li, H.; Sheppard, D. N.; Joos,
5391
1
hydrogenation (‘unnatural’ 17a-side chain). See: Van Horn,
A. R.; Djerassi, C. J. Am. Chem. Soc. 1967, 89, 651–664; and
also: Izzo, I.; Di Filippo, M.; Napolitano, R.; De Riccardis, F.
Eur. J. Org. Chem. 1999, 3505–3510. In this case no. 17a-epi-
mer was isolated from the reaction mixture.
J. B.; Clare, J. P.; Davis, A. P. Angew. Chem., Int. Ed. 2003,
4
2, 4931–4933; (f) Bandyopadhyay, P.; Regen, S. L. J. Am.
Chem. Soc. 2002, 124, 11254–11255; (g) Gokel, G. W.;
Mukhopadhyay, A. Chem. Soc. Rev. 2001, 30, 274–286.
. Maulucci, N.; De Riccardis, F.; Botta, C. B.; Casapullo, A.;
Cressina, E.; Fregonese, M.; Tecilla, P.; Izzo, I. Chem.
Commun. 2005, 1354–1356.
. Other examples of ionophores showing hydrophobic steroid
scaffolds can be found in: (a) Avallone, E.; Cressina, E.;
Fregonese, M.; Tecilla, P.; Izzo, I.; De Riccardis, F.
Tetrahedron 2005, 61, 10689–10698; (b) Avallone, E.; Izzo,
I.; Vuolo, G.; Costabile, M.; Garrisi, D.; Pasquato, L.;
Scrimin, P.; Tecilla, P.; De Riccardis, F. Tetrahedron Lett.
11. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Marynoff,
C. A.; Shah, R. D. J. Org. Chem. 1996, 61, 3849–3862.
12. Arduini, A.; Fanni, S.; Manfredi, G.; Pochini, A.; Ungaro, R.;
Sicuri, A. R.; Ugozzoli, F. J. Org. Chem. 1995, 60, 1448–1453.
13. Uwai, K.; Ohashi, K.; Takaya, Y.; Ohta, T.; Tadano, T.; Kisara,
K.; Shibusawa, K.; Sakakibara, R.; Oshima, Y. J. Med. Chem.
2000, 43, 4508–4515.
14. As previously described (De Riccardis, F.; Di Filippo, M.;
Garrisi, D.; Izzo, I.; Mancin, F.; Pasquato, L.; Scrimin, P.;
Tecilla, P. Chem. Commun. 2002, 3066–3067), a solution of
NaCl (75.0 mM) plus a membrane-impermeable paramagnetic
3
4
2
003, 44, 6121–6124; (c) Otto, S.; Janout, V.; DiGiorgio,
A. F.; Young, M.; Regen, S. L. J. Am. Chem. Soc. 2000, 122,
200–1204; (d) Pechulis, A. D.; Thompson, R. J.; Fojtik,
3
shift reagent (DyCl –tripolyphosphate complex, 4.0 mM) were
1
added to a 95:5 egg phosphatidylcholine (PC) and egg phos-
phatidylglycerol (PG) dispersion (100 nm diameter, large uni-
lamellar vesicles) prepared in aqueous LiCl (100.0 mM).
Compounds 2 and 3 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.
J. P.; Schwartz, H. M.; Lisek, C. A.; Frye, L. L. Biorg. Med.
Chem. 1997, 5, 1893–1901. For general design principles, see
Ref. 4b.
. Lower energies of the extended conformations were calculated
using MM3 molecular mechanic package.
. Di Filippo, M.; Izzo, I.; Savignano, L.; Tecilla, P.; De Riccardis,
F. Tetrahedron 2003, 59, 1711–1717.
. Kabalka, G. W.; Varma, M.; Varma, R. S.; Srivastava, P. C.;
Knapp, F. F. J. Org. Chem. 1986, 51, 2386–2388.
. Taber, D. F.; You, K. K. J. Am. Chem. Soc. 1995, 117,
5
6
7
8
9
+
5757–5762.
. Mungall, W. S.; Greene, G. L.; Heavner, G. A.; Letsinger, R. L.
J. Org. Chem. 1975, 40, 1659–1662.
15. Also the protonophoric activity (measured using an assay based
on the response of the intravesicular pH-sensitive pyranine
fluorophore. See: Clement, N. R.; Gould, J. M. Biochemistry
1981, 20, 1534–1538), even at 5% molar concentration (per-
cent of steroids with respect to the total concentration of lipid,
data not shown), was only slightly higher than in the absence of
any additive.
1
6
1
0. It is known that the stereoselectivity of the D hydrogenation
is strictly dependent on the C-14 stereochemistry: a 14a-H con-
figuration leads to a a-face hydrogenation (‘natural’ 17b-side
chain), whereas a 14b-H configuration leads to a b-face