Synthesis, self-aggregation and bioactivity properties of a cationic aminoacyl surfactant, based on a new class of highly functionalized nucleolipids

Article abstract of DOI:10.1016/j.ejmech.2012.06.044

A highly functionalized aminoacyl nucleolipid based on uridine is here proposed as a novel cationic surfactant. To achieve this, a straightforward, high yielding and versatile protocol has been devised, in principle providing synthetic access to a variety

Full text of DOI:10.1016/j.ejmech.2012.06.044

European Journal of Medicinal Chemistry 57 (2012) 429e440
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, self-aggregation and bioactivity properties of a cationic aminoacyl
surfactant, based on a new class of highly functionalized nucleolipids
,
Luca Simeone a, Carlo Irace b, Antonio Di Pascale b, Donato Ciccarelli a, Gerardino D’Errico a
,
*
,
Daniela Montesarchio a
*
a Dipartimento di Scienze Chimiche, Università di Napoli “Federico II”, Complesso Universitario di Monte S. Angelo, via Cintia, 4, I-80126 Napoli, Italy
b Dipartimento di Farmacologia Sperimentale, Università di Napoli “Federico II”, via D. Montesano, 48, I-80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A highly functionalized aminoacyl nucleolipid based on uridine is here proposed as a novel cationic
surfactant. To achieve this, a straightforward, high yielding and versatile protocol has been devised, in
principle providing synthetic access to a variety of different, related analogs. Self-aggregation properties
Received 16 May 2012
Received in revised form
20 June 2012
Accepted 21 June 2012
Available online 3 July 2012
of this nucleolipid were determined by using
a combined approach, including surface tension,
conductivity and DLS measurements. Above the critical micellar concentration of 4 ꢀ 10ꢁ5 mol kgꢁ1
,
large supramolecular assemblies with a counterion condensation degree of 0.25 were observed. The
bioactivity profile of this new compound was investigated on cancer and non cancer cell lines.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Nucleolipid
Cationic surfactant
Chemical synthesis
Micellization
Biocompatibility
1. Introduction
cell membranes [3]. Nucleolipid-based aggregates may typically be
exploited as drug-carriers, being able to incorporate bioactive
The peculiar features of nucleolipids, associating the ability of
nucleosides to specifically recognize complementary bases via
Watson-Crick or Hoogsteen H-bonds with the strong tendency of
lipids to spontaneously self-assemble in aqueous solutions, have
attracted wide interest in several fields, with major impact on
biotechnological and pharmacological applications [1e4]. In the
last decades many research groups have investigated the micro-
structural properties of natural and artificial nucleolipids, forming
distinctive, well-defined aggregates, as fibers, vesicles, micro-
spheres [5e9]. Given the high structural variability of these
compounds, all the so called “weak interactions”, including H-
bonds, p-stacking, electrostatic, hydrophobic forces, as well as the
specific recognition between nucleobases, may contribute to the
stabilization of the self-assembled aggregates.
In most cases, known nucleolipids are nucleosides decorated
with a polar group e either charged or not e attached at the 50
position of the ribosidic moiety, and lipophilic chains at the 20 and/
or 30-OH groups, assembled in a molecular skeleton resembling
naturally occurring glycerophospholipids, the main constituents of
compounds in high concentration, protecting them against extra-
cellular enzymatic degradation and allowing efficient delivery
within cells by endocytosis.
To ensure an efficient and fast internalization in target cells,
cationic drug-carriers are, in principle, a good option. Indeed, cell
membranes usually present a negatively charged external surface,
due to the presence of anionic phospholipids and glycolipids in the
membrane composition. Oppositely charged compounds - in
monomeric forms or as supramolecular aggregates - should favor-
ably interact with the membrane, possibly translocating in the
cytosol through a variety of mechanisms (e.g., endocytosis, pora-
tion, etc.). However, typical cationic surfactants, such as quaternary
ammonium salts, are toxic, being able to completely destabilize
lipid membranes and thus induce cellular death [10]. Therefore
many efforts have been devoted to develop new, more biocom-
patible cationic surfactants. A promising approach is based on
modified cationic aminoacids, bearing a long acyl chain linked
through an amide bond [11].
Cationic nucleolipids have also attracted the researchers’
interest because of their intrinsic molecular recognition and cell
penetrating abilities. A recent work by Yang et al. showed very
interesting examples of cationic nucleolipids obtained from
uridine, linking one arginine residue at the 50-OH via an ester bond,
* Corresponding authors. Tel.: þ39 (0) 81 674126; fax: þ39 (0) 81 674393.
E-mail
addresses:
gerardino.derrico@unina.it
(G.
D’Errico),
daniela.montesarchio@unina.it (D. Montesarchio).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.044

430
L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
and two fatty acid chains at the 20 and 30-OH positions, attached
through carbamate bonds: both these linkages are chemically
stable but easily degradable in vivo. These derivatives, exhibiting
poor cytotoxicity, were able to self-aggregate into stable liposomes
and, in this form, efficiently vehiculate siRNA in vitro [12]. Another
intriguing cationic nucleolipid construct has been described by
Grinstaff, Camplo et al., who synthesized the tosylate salts of 10-
ester linkage
O
Hydrophilic group
H
O
N
O
R
HC
NH3
O
O
Lipophilic group
(20,30-dioleyl-50-trimethylammonium-
a,b-D-ribofuranosyl)-3-
O
nitropyrrole, found to be efficient nanovectors in siRNA knockdown
experiments [13].
amide linkage
ester linkage
Taking inspiration from these systems, we reasoned that a more
general and versatile design allowing a large diversification of the
target compounds would be desirable in order to expand the
repertoire of available functional nucleolipids. In this frame we here
describe a novel synthetic platform introducing - for these hybrid
compounds - a higher degree of functionalization than generally
allowed starting from natural ribo- or deoxyribonucleosides. Using
modified nucleoside 1 (Fig. 1) as the starting material, model
compound 2 (Fig. 2) has been prepared and characterized to test the
feasibility of the here proposed synthetic scheme. The self-
aggregation behavior of nucleolipid 2 has been studied by an
experimental approach combining surface tension, conductivity
and dynamic light scattering (DLS) measurements. Finally, its
bioactivity has been investigated on a panel of tumoral and non
tumoral cell lines.
R = aminoacid side chain
B
= purinic or pyrimidinic nucleobase
= in vivo hydrolyzable bond
Fig. 2. Schematic representation of the family of aminoacyl nucleolipids accessible
through the here described methodology.
In order to test the feasibility of the proposed synthetic plat-
form, a tailored model compound has been designed and synthe-
sized. Nucleoside analog 1 was thus exploited to build a novel
cationic aminoacyl nucleolipid, decorated with the following
groups: i) a lipophilic fatty acid chain at the 20 position, imparting
the intrinsic ability to generate large aggregates in aqueous solu-
tions; ii) an
easily available
L
-proline residue at the 30 position, as a natural and
-aminoacid ionized under physiological condi-
2. Results and discussion
a
tions, introducing the desired positive charge; iii) a polyether chain
at the 50 position, which increases the hydrophilicity of the
amphiphile [14] and makes its aggregates stealth to the human
immune system [15].
2.1. Synthesis of cationic nucleolipid 2
Nucleosides are polyfunctional scaffolds. In order to prepare
nucleolipids, all the modifications required to obtain hybrid
derivatives have to be introduced at the level of the sugar moiety,
being desirable to maintain unaltered the recognition abilities due
to the nucleobases.
The here described compound 2 is reported in Fig. 3.
Key intermediate in the synthesis of this compound is 30-azido-
30-deoxy-1-
b-D-xylofuranosyluracil 1. This modified nucleoside has
been previously described in the literature and synthesized
through a procedure involving several, troublesome steps in very
low yields [16]. In this work we have developed a new, optimized
protocol, requiring only straightforward and high yielding steps, as
described in Scheme 1.
Starting from uridine, the reaction with 4-monomethoxytrit
ylchloride (MMTrCl) and AgNO3, both in 2.8 equiv excess with
respect to the nucleoside, in pyridine/THF [17] led to a mixture
of two regioisomers in 92% overall yields, with desired compound
3 isolated in 72% yield. The obtained nucleoside 3, equipped with
one free hydroxyl group in the 30 position, could undergo the key
Our idea was to select, as the starting material, a nucleoside
equipped with at least three, easily differentiable, functional
groups. Each of them could then be exploited to attach a different
residue, conferring specific properties, using very simple, efficient
and selective procedures. As the starting scaffold we have chosen
the modified nucleoside 30-azido-30-deoxy-1-
b-D-xylofuranosylur-
acil (1, Fig. 1), carrying one primary and one secondary hydroxyl
group, respectively at the 50 and 20 positions, and one xylo-config-
ured azido function at the 30 position, as a masked precursor of
a primary amine. These functions well satisfy the cited require-
ments, being possible to alternately activate or deactivate them in
a highly regioselective derivatization scheme. A relevant advantage
is that, upon couplings with suitable carboxylic acids, chemical
bonds of different stability in physiological conditions, i.e. esters
and amides, can be introduced on the same scaffold, and a fine
tuning of the nucleolipid design is thus realizable.
OBn
O
In Fig. 2, a schematic representation of the class of nucleolipids
accessible starting from 1 is reported.
Cl-
O
N
O
O
+
NH2
O
NH
O
O
NH
O
N
O
NH
O
N3
O
O
O
HO
O
O
O
OH
O
1
2
Fig. 1. 30-azido-30-deoxy-1-
b-D-xylofuranosyluracil.
Fig. 3. Cationic aminoacyl nucleolipid 2.

L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
431
O
N
O
O
N
NH
NH
NH
O
N
O
O
b
a
HO
MMTrO
MMTrO
O
O
O
OH OH
OH OMMTr
OAc OMMTr
4
3
c
O
O
O
N
O
O
N
O
O
O
N
O
N
O
N
O
O
N
e
d
MMTrO
N
MMTrO
3O
MMTrO
O
O
OAc OMMTr
OH OMMTr
OMMTr
7
6
5
f
O
NH
N
O
N3
HO
O
OH
1
Scheme 1. Synthesis of nucleoside 1. Reagents and conditions: a) MMTrCl, AgNO3, Py, THF, 1 h, r.t., 72% yield for isolated regioisomer 3; b) Ac2O, Py, 1 h, r.t., 98% yield; c) Boc2O, TEA,
DMAP, CH2Cl2, 1 h, r.t., 80% yield; d) K2CO3, MeOH/Et3N (3:1), 24 h, r.t., 99% yield; e) PPh3, DIAD, DPPA, THF, 24 h, r.t., 92% yield; f) TFA, TIS/CH2Cl2 (1:9), 1 h, r.t., 97% yield.
conversion of the 30-OH group into azide by a Mitsunobu reaction
[18e20]. However, before undertaking this reaction, an efficient
protection of the N-3 nucleobase position was introduced to
prevent the formation of the corresponding 2,30-anhydro derivative
under the Mitsunobu reaction conditions. This goal was achieved
using Boc as the protecting group, inserted by a simple procedure
recently developed by some of us. [21].
successive reduction of the 30-azido group to 30-amine was carried out
by catalytic hydrogenation (P ¼ 1 atm) in the presence of Pd/C for 12 h.
Amino-nucleoside 10 was obtained in almost quantitative yields in
a pure form, simply filtering off and washing the catalyst.
As shown in Scheme 2, the successive step involved the
condensation of 10 with a residue of N-Boc L-Proline, realized by
DCC activation, giving aminoacyl nucleolipid 11 with 98% yield.
This amide was then deprotected at the 50 position by treatment
with Et3N 3xHF in THF to give nucleoside 12 with 93% yield. At this
stage a hydrophilic residue of BnO(CH2CH2O)6CH2COOH, synthe-
sized following a straightforward procedure, previously reported
by some of us, [22], was attached at the 50 position through an
ester linkage, thus giving nucleoside 13 with 87% yield. The final
step required a mild acidic treatment (10% TFA in CH2Cl2), ensuring
the complete and clean Boc removal, to give aminoacyl nucleolipid
14 as a trifluoroacetate salt. To obtain the target compound in
To this purpose nucleoside 3 was first acetylated in position 30,
obtaining 4 with 98% yield; this was then reacted with Boc2O, TEA
and DMAP in CH2Cl2 to give N-3 Boc-protected nucleoside 5 in 80%
isolated yield. The acetyl group was then removed by treatment
with catalytic K2CO3 in CH3OH/TEA, thus giving nucleoside 6 in
almost quantitative yields.
Remarkably, the acetylation and deacetylation procedures have
required prolonged reaction times, probably due to the high steric
hindrance of the 30 position. The Mitsunobu reaction was then
carried out treating nucleoside 6 with PPh3, DIAD and DPPA,
leading to the MMTr-protected 30-azido nucleoside 7 in 92% yield,
which was then fully deprotected by reaction with 10% TFA and TIS
in CH2Cl2 with 97% yield. Following this synthetic scheme, desired
nucleoside 1 was obtained in 6 simple steps with 50% overall yields
from uridine.
a
presumably more biocompatible form, the trifluoroacetate
counterion was then replaced with chloride. This was realized by
treatment of 14 with an equimolar amount of NHþ4 Clꢁ and subse-
quent repeated lyophilizations from water, removing the volatile
ammonium trifluoroacetate salt.
The complete absence of trifluoroacetate ions from salt 2 was
confirmed by 13C NMR analysis, which in concentrated samples
after extremely long acquisition times (>24 h) did not show the
signals diagnostic of trifluoroacetate carbons, even in traces.
Final compound 2, as well as all the synthesized intermediates,
have been fullycharacterized by 1H and 13C NMR and ESI-MS analysis.
Using this substrate, target aminoacyl nucleolipid 2 was then
prepared in 7 steps, as described in Scheme 2. First of all, the 50 position
of 1 was protected with the TBDMS group with 80% yield; resulting
compound 8 was then reacted with oleic acid, using DCC as the
condensing agent, thus obtaining 20-oleyl derivative 9 in 74% yield. The

432
L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
O
O
N
O
NH
NH
NH
N
O
O
N
O
b
a
N3
N3
N3
TBDMSO
HO
TBDMSO
O
O
O
O
OH
OH
1
8
9
O
c
O
N
O
N
NBoc
NH
NH
O
O
d
O
NH
TBDMSO
NH2
O
O
TBDMSO
O
O
O
11
10
O
OBn
e
O
O
N
NBoc
O
NH
O
O
O
NBoc
NH
O
O
O
NH
HO
f
N
O
O
NH
O
O
O
O
O
O
O
O
12
13
O
OBn
g
O
CF3COO-
O
O
O
+
NH2
NH
O
O
N
O
NH
O
O
O
O
O
O
O
14
Scheme 2. Synthesis of nucleolipid 14. Reagents and conditions: a) TBDMSCl, imidazole, DMF, 1 h, r.t., 80% yield; b) oleic acid, DCC, DMAP, CH2Cl2, 2 h, r.t.,74% yield; c) H2, (P ¼ 1 atm),
Pd/C 10% p.p., AcOEt, 12 h, r.t., 99% yield; d) N-Boc L-Proline, DCC, DMAP, CH2Cl2, 12 h, r.t., 98% yield; e) Et3N 3xHF, THF, 12 h, r.t., 93% yield; f) BnO(CH2CH2O)6CH2COOH, DCC, DMAP,
CH2Cl2, 2 h, r.t., 87% yield; g) 10% TFA/TIS, CH2Cl2, 1 h, r.t., 99% yield.
2.2. Physico-chemical characterization of the self-aggregation
behavior of compound 2 in water
In this work, the micellization behavior of compound 2 in
aqueous mixtures was investigated by means of a combined
experimental strategy: surface tension measurements were used to
detect the critical micellar concentration, cmc; conductivity
measurements were used to confirm the cmc value and to estimate
the ratio between the number of bound counterions and the
number of micellized amphiphiles, usually named counterion
condensation degree, b; dynamic light scattering (DLS) measure-
ments gave information on the mean dimension and distribution of
the micelles.
Because of its amphiphilic character, compound 2 is a suitable
candidate to form supramolecular assemblies in aqueous mixtures.
The major driving force for the self-aggregation process is furnished
by hydrophobic interactions among the oleyl chains. Further
possible assistance to self-aggregation is given by the cooperative
H-bonding among the hydrated polyether chains [23], as well as by
intermolecular interactions among nucleobases.

L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
433
Fig. 5. Binary mixtures waterecompound 2 (m [ 4 ꢀ 10ꢁ4 mol kgꢁ1). Relaxation time
Fig. 4. Binary mixtures waterecompound 2. Surface tension (full circles) and specific
conductivity (full squares) vs. nucleolipid molality at 25 ꢃC.
distribution obtained from DLS measurements at 90ꢃ and 25 ꢃC.
The surface tension data relative to the binary system
waterecompound 2, reported as a function of the amphiphile
molality, m, are shown in Fig. 4. On increasing m, the surface
ðd
ðd
k
=dmÞm>cmc
=dmÞm<cmc
b
¼ 1 ꢁ
k
The determined counterion binding degree was
This value is much lower than those observed for typical cationic
surfactants (e.g.,
¼ 0.6e0.8 for alkyltrimethylammonium halides
[29]) and aminoacid-based amphiphiles (e.g.,
¼ 0.7 for surfactants
b
¼ 0.25 ꢂ 0.05.
tension, g, decreases, as a consequence of the compound 2 tendency
to locate at the airesolution interface. Upon further increasing the
tends to a constant value, (34.0 ꢂ 0.2) mN mꢁ1
.
b
solute molality,
g
b
This evidence indicates that compound 2 concentration at the
interface doesn’t increase anymore, all the added molecules being
involved in the formation of micelles in the solution bulk. Thus, the
derived from arginine [30]). This result could be due to the inter-
position of the oligoethylene glycol chains between the charged
proline residues on the micelle surface. Indeed, a similar effect was
found in mixed micelles formed by cationic and non-ionic
ethoxylated surfactants [31].
molality at which a constant
g value is reached corresponds to
compound 2 cmc, estimated to be equal to y 4 ꢀ 10ꢁ5 mol kgꢁ1
.
Remarkably, this cmc value is at least one order of magnitude
lower than those observed for other oleyl-derivatized ionic
amphiphiles (e.g., cmc ¼ 3 ꢀ 10ꢁ4 mol kgꢁ1 for oleyl lysophos-
phatidic acid [24], cmc ¼ 5 ꢀ 10ꢁ3 mol kgꢁ1 for sodium oleyl sar-
cosinate [25]) and of the same order of magnitude of the cmc
detected for nonionic analogs (e.g., cmc ¼ 1.7 ꢀ 10ꢁ5 mol kgꢁ1 for
Brij 97 [26]). This evidence can be explained assuming that inter-
molecular interactions due to the nucleobases and the oligo-
ethylene chains provide additional contributions for the self-
aggregation process.
Information on the size and shape of the micellar aggregates
formed by compound 2 in water was obtained from DLS measure-
ments performed on mixtures with concentration ranging between
2ꢀ10ꢁ4 mol kgꢁ1 and 4 ꢀ10ꢁ4 mol kgꢁ1, i.e., from five to ten times the
cmc. More dilute mixtures could not be considered because of the too
low intensity of the scattered light. In all the measurements, the
relaxation time distribution is monomodal, indicating the presence of
only one kind of aggregates in solution (see Fig. 5 for an example).
For all the samples, measurements were performed at various
scattering angles, , and the measured G was reported as a function
q
Further information can be obtained from a quantitative anal-
of q. By fitting a quadratic polynomial to the data, we estimated the
average diffusion coefficient of the nucleolipid aggregates. An
example of this procedure is shown in Fig. 2 of the Supplementary
Material. For diluted mixtures, it is possible to correlate D to the
apparent hydrodynamic radius of the diffusing particles through
the equation:
ysis of the g data. Premicellar g data, reported as a function of ln(m),
present an almost linear trend (see Fig. 1 in the Supplementary
Material). The area at the airesolution interface per nucleolipid
molecule at the cmc, A, was estimated through the relation:
ꢀ
ꢁ
ꢂ
ꢃꢁ1
1
1
v
g
A ¼ ꢁ
NA zRT vlnm
T;p
kT
ph0
D
RH
¼
6
where NA is the Avogadro number, R is the gas constant, and T is the
temperature. The factor z depends on the surfactant type; its value
is 1 for most nonionic and zwitterionic surfactants, while it is 2 for
most 1:1 ionic surfactants [27]. The surface tension derivative at the
cmc was evaluated by differentiating a polynomial equation fitted
to the premicellar data. The determined interfacial area was
A ¼ (190 ꢂ 16) Å2 per molecule.
Where k is the Boltzmann constant, T is the absolute temperature
and h0 is the solvent viscosity. For not spherical particles, RH
represents the radius of equivalent spherical aggregates. The RH
and D values obtained for the micelles of compound 2 are collected
in Table 1.
Conductivity
data
relative
to
the
binary
system
waterecompound 2 are also shown in Fig. 4.
k
increases with the
Table 1
molality, as expected on increasing ions concentration. A slight but
significant slope change is observed at w4 ꢀ 10ꢁ5 mol kgꢁ1, thus
confirming the cmc value estimated by surface tension measure-
Diffusion coefficients and hydrodynamic radii of compound 2 micelles, as derived by
DLS measurements at 25 ꢃC.
Nucleolipid
D ꢀ 108 /m2 sꢁ1
RH /nm
ments. The lower rate of the k increment above the cmc is due to the
concentration ꢀ 104 /mol kgꢁ1
partial condensation of counterions on the micelles surface, which
reduces both the ions concentration and the micelle charge [28]. The
fraction of counterions condensed on the surface of the aggregates
was obtained from the slope variation according to the relation:
2.0
3.0
4.0
10.4 ꢂ 0.9
6.8 ꢂ 1.1
21 ꢂ 2
32 ꢂ 5
68 ꢂ 4
3.21 ꢂ 0.16

434
L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
Table 2
The obtainedRH values are higher than those expected for spherical
IC50 values (mM) for nucleolipid 2 in the indicated cell
lines after 48 h from incubation. IC50 values are re-
ported as mean ꢂ SEM.
micelles and increase with the nucleolipid concentration. This
behavior could be due to strong interactions between micelles and/or
micelle growth [32]. Being the here investigated concentrations quite
low, the second factor is probably more relevant. Furthermore, since
the scattered intensity measured by the DLS technique is weighed by
the particles volumes, this experimental approach could overestimate
the average dimension of polydispersed micelles [33]. Consequently,
the RH reported above has to be considered only as a qualitative
indication that quite large, non spherical micelles form in the consid-
ered mixture. Mostprobably, theypresent acylindrical, rod-likeshape,
as previously observed for other cationic surfactants at concentrations
largely exceeding the cmc. [34].
Cell line
Nucleolipid 2
MCF-7
WiDr
C6
51 ꢂ 7
19 ꢂ 5
86 ꢂ 11
98 ꢂ 9
3T3-L1
incubated
under
the
same
conditions
with
cetyl-
trimethylammonium bromide (CTAB), a typical cationic surfactant
endowed with antiseptic activity against bacteria and fungi, that
showed relevant cell toxicity [37]. Findings concerning the nucle-
olipid 2 bioactivity were further verified by examination of cell
cultures under phase-contrast light microscopy. Indeed, as reported
in the representative microphotographs of Fig. 3 in the Supple-
mentary Material, subsequent to incubation with 100 mM nucleo-
lipid 2 and CTAB, in vitro signs of injuries clearly appeared, coupled
to modifications of the cell morphology.
2.3. In vitro bioactivity studies
Nucleolipid 2 was designed as a potential nanocarrier. It can also
be regarded as a prodrug of aminoacyl nucleosides [35,36], a class
of compounds intrinsically endowed with a variety of biological
properties and still not well explored in their cytotoxic potential.
Therefore, we considered of interest to investigate the bioactivity of
2 on a panel of human, rat and murine cell lines and the corre-
sponding data are here reported. As shown in Fig. 6, in a 48 h in vitro
Interestingly, from the concentration/response data of 2 re-
ported in Fig. 6, a negligible cytotoxic effect on C6 and 3T3-L1 cells
is observed at 10
mM, with ca. 80% cell survival index at concen-
tration values close to its cmc (ca. 40
mM). Therefore, these
bioscreening, nucleolipid
dependent cytotoxicity against normal and cancer cells with no
apparent selectivity, albeit at low concentrations (10e25 M) an
2 exhibited a moderate and dose-
preliminary results indicate that nucleolipid 2 exhibits an accept-
able biocompatibility on some cell lines at low concentrations. We
expect that a relevant enhancement of the biological properties of
cationic surfactants related to 2 can be presumably achieved by
simply varying the functionalizing arms (cationic aminoacid, oli-
goethylene glycol and lipid chains) using different natural and/or
artificial analogs, within the same general design.
m
interesting cytotoxic effect was observed exclusively on human
MCF-7 and WiDr cancer cells. The calculated IC50 values - in the
tens-to-one hundred micromolar concentration range (Table 2) e
confirmed that nucleolipid 2 shows some ability to interfere with
the cell growth or survival. In parallel, as positive control, cells were
Fig. 6. Concentration/response curves and cell survival index, evaluated by MTT assay and total cell count, in MCF-7 (panel A), WiDr (panel B), C6 (panel C) and 3T3-L1 (panel D) cell
lines incubated for 48 h with different concentrations of 2 and CTAB, as indicated in the legend. Data are expressed as percentage of untreated control cells and are reported as mean
of three independent experiments ꢂ SEM.

L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
435
3. Conclusions
4.2. Synthesis of compound 3
The chemical synthesis of amphiphilic uridine analog 2,
combining in its skeleton an oleic acid chain, a positively charged L-
Uridine (1.0 g, 4.09 mmol), suspended in anhydrous THF
(120 mL), was treated with pyridine (3.3 mL, 40.9 mmol), AgNO3
(1.95 g, 11.5 mmol) and MMTrCl (3.55 g, 11.5 mmol). The mixture
was stirred at room temperature for 12 h, then the suspension was
filtered and concentrated under reduced pressure. The crude was
then diluted with CH2Cl2, transferred into a separatory funnel and
washed twice with an aq. solution of NaHCO3 5% p.p. The collected
organic phases were dried over anhydrous Na2SO4, concentrated
under reduced pressure and purified on a silica gel column, eluted
with n-hexane/AcOEt (1:1, v/v), added with 1% TEA. Nucleoside 3
was recovered in 72% yield (2.33 g, 2.95 mmol), while the 50,30-bis-
monomethoxytritylated regioisomer was obtained in 19% yield
(614 mg, 0.778 mmol).
proline residue and a polar oligoethylene glycol chain, is here
described. This highly functionalized cationic surfactant was
thought as a simple prototype of a novel class of nucleolipid-based
nanocarriers. 2 was prepared through straightforward and high
yielding steps starting from the modified nucleoside 30-azido-30-
deoxy-1-b-D-xylofuranosyluracil (1). This useful precursor, already
known in the literature, was in turn obtained by exploiting a novel,
convenient synthetic procedure. Self-aggregation properties in
aqueous solution of amphiphilic nucleolipid 2 were determined by
using a combined approach, including surface tension, conductivity
and DLS measurements. Above the critical micellar concentration
of 4 ꢀ 10ꢁ5 mol kgꢁ1, large supramolecular assemblies with
a counterion condensation degree of 0.25 were observed. Thus,
nucleolipid 2 behaves as a weakly charged cationic surfactant, the
properties of which are also influenced by the presence of the
polyether chain. Its bioactivity profile was investigated on a panel
of human and non human cells. Preliminary data show that the
here presented compound is endowed with a not negligible
bioactivity, more marked against human cancer cells MCF-7 and
WiDr, although is by far less cytotoxic than the common cationic
surfactant CTAB, here used as a reference compound.
Finally, the here presented results demonstrate that a tailored
design of nucleolipid-based systems can be profitably exploited to
obtain cationic surfactants with acceptable biocompatibility. In
principle, the high versatility of the here presented synthetic
strategy allows to prepare a large variety of diverse nucleolipid
analogs, having distinctive features which can be finely modulated
by proper choice of the ribose decorations.
As a future development, aminoacyl nucleolipid 2 and related,
optimized analogs will be studied in formulation with anionic
derivatives, so to obtain catanionic vesicles [38]. Particularly,
studies are in progress to investigate catanionic vesicles formed
by the here described cationic nucleolipid 2 in mixture with
amphiphilic nucleolipid-based Ru(III) complexes [39,40], intrinsi-
cally having an anionic nature. The final goal is to generate more
stable and effective formulations for ruthenium-containing anti-
cancer drugs, aiming at enhanced cell delivery and prolonged
in vivo half-life.
Nucleoside 3: white, amorphous solid; Rf ¼ 0.8 [n-hexane/AcOEt
(2:3, v/v)]. 1H NMR (C6D6, 200 MHz):
d 10.86 (1H, bs, NeH); 7.70
(1H, d, J ¼ 8.2 Hz, H-6); 7.64e6.64 (28H, overlapped signals,
aromatic protons of MMTr); 6.61 (1H, d, J ¼ 7.2 Hz, H-10); 5.22 (1H,
d, J ¼ 8.4 Hz, H-5); 4.73e4.67 (1H, dd, J ¼ 7.2 and 4.6 Hz, H-20); 4.03
(1H, bs, H-30); 3.33 and 3.29 (3H each, s’s, 2x OCH3); 3.13e2.92 (3H,
overlapped signals, H-50a, H-50b and H-40). 13C NMR (C6D6, 50 MHz):
d
163.3 (C-4); 159.7, 159.4, 149.5, 144.9, 144.3, 143.9, 135.9, 135.2,
131.0, 130.6, 128.7, 128.5, 128.2, 128.0, 127.5, 123.6, 114.1, 113.6
(aromatic carbons of MMTr); 151.8 (C-2); 140.8 (C-6); 103.2 (C-5);
88.0 and 87.8 (quaternary carbons of MMTr); 87.1 (C-10); 84.7 (C-40);
78.2 (C-30); 71.3 (C-20); 64.7 (C-50); 54.9 (OCH3). ESI-MS (positive
ions): for C49H44N2O8, calcd. 788.3098; found m/z: 811.97
[M
þ
Naþ]; 827.84 [M
þ
Kþ]. HRMS (MALDI-TOF): calcd.
forC49H44N2O8Na ¼ 811.2995; found m/z: 811.3021 [M þ Naþ].
5’,3’-di(monomethoxytrityl)uridine: white, amorphous solid;
Rf ¼ 0.6 [n-hexane/AcOEt (2:3, v/v)]. 1H-NMR (C6D6, 200 MHz):
d 7.78e6.69 (29H, overlapped signals, H-6 and aromatic protons of
MMTr); 6.35 (1H, d, J ¼ 4.6 Hz, H-10); 5.27 (1H, d, J ¼ 8.6 Hz, H-5);
5.02 (1H, d, J ¼ 7.2 Hz, OH-20, exchangeable proton); 4.60 (1H, dd,
J ¼ 7.2 and 4.6 Hz, H-20); 4.14 (2H, bs, overlapped H-30 and H-40);
3.52e3.46 (1H, m, H-50a); 3.35 and 3.33 (3H each, s’s, 2x OCH3);
3.18e3.12 (1H, m, H-50b). 13C-NMR (C6D6, 50 MHz):
d 163.3 (C-4);
159.5, 145.2, 144.5, 135.8, 135.4, 131.4, 130.9, 129.3, 129.0, 128.2,
128.0, 127.0, 113.6 (aromatic carbons of MMTr); 151.4 (C-2); 140.2
(C-6); 102.7 (C-5); 90.2 (C-10); 88.2 and 87.7 (quaternary carbons of
MMTr); 83.3 (C-40); 75.2 (C-30); 73.8 (C-20); 63.2 (C-50); 54.8 (OCH3).
ESI-MS (positive ions): for C49H44N2O8, calcd. 788.3098; found m/z:
811.94 [M þ Naþ]; 827.79 [M þ Kþ].
4. Experimental section
4.1. General methods
4.3. Synthesis of compound 4
All the reagents were of the highest commercially available
quality and were used as received. TLC analyses were carried out on
silica gel plates from Merck (60, F254). Reaction products on TLC
plates were visualized by UV light and then by treatment with a 10%
Ce(SO4)2/H2SO4 aq. solution. For column chromatography, silica gel
from Merck (Kieselgel 40, 0.063e0.200 mm) was used. NMR
spectra were recorded on Varian XR 200 and Varian Inova 500
spectrometers, as specified. All the chemical shifts are expressed in
ppm with respect to the residual solvent signal. Peak assignments
have been carried out on the basis of standard 1H-1H COSY and
HSQC experiments. For the ESI MS analyses, a Waters Micromass ZQ
instrument e equipped with an Electrospray source e was used in
the positive and/or negative mode. MALDI TOF mass spectrometric
analyses were performed on a PerSeptive Biosystems Voyager-De
Pro MALDI mass spectrometer in the Linear mode using 3,4-
dihydroxybenzoic acid as the matrix. UV measurements were
carried out on a Jasco V-530 UV spectrophotometer equipped with
a Jasco ETC-505T temperature controller unit.
Nucleoside 3 (870 mg, 1.10 mmol) was dissolved in pyridine
(3.0 mL) and reacted with acetic anhydride (1.5 mL). The solution
was stirred at room temperature for 12 h, then the reaction was
quenched by addition of CH3OH (2 mL) and taken to dryness under
reduced pressure. The crude was then diluted with CH2Cl2, trans-
ferred into a separatory funnel and washed twice with an aq.
solution of NaHCO3 5% p.p.; the collected organic phases were dried
over anhydrous Na2SO4 and concentrated under reduced pressure
to give compound 4 in 98% isolated yield (898 mg, 1.08 mmol).
4: oil, Rf ¼ 0.3 [n-hexane/AcOEt (1:1, v/v)]. 1H-NMR (CDCl3,
500 MHz):
d
7.60 (1H, d, J ¼ 8.5 Hz, H-6); 7.48e6.71 (28H, overlapped
signals, aromatic protons of MMTr); 6.55 (1H, d, J ¼ 7.5 Hz, H-10); 5.12
(1H, d, J ¼ 8.5 Hz, H-5); 4.48 (1H, dd, J ¼ 8.0 and 7.5 Hz, H-20); 3.83 (2H,
bs, H-30 and H-40); 3.79 and 3.78 (3H each, s’s, 2x OCH3); 3.30e3.06
(2H, ABX system, J ¼ 11.0 and 1.5 Hz, H-50a and H-50b); 1.99 (3H, s,
CH3C]O). 13C-NMR (CDCl3, 50 MHz):
d 169.7 (C]O); 163.6 (C-4);
159.7, 159.4, 149.5, 144.9, 144.5, 144.4, 144.3, 143.9, 135.9, 135.1, 131.1,
130.6, 129.3, 128.8, 128.5, 128.2, 128.0, 127.5, 125.7, 123.8, 113.9, 113.7

436
L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
(aromatic carbons of MMTr); 151.6 (C-2); 140.3 (C-6); 103.2 (C-5); 87.9
and 87.7 (quaternary carbons of MMTr); 86.8 (C-10); 84.4 (C-40); 75.9
(C-30); 72.8 (C-20); 64.2 (C-50); 54.9 (OCH3); 20.5 (CH3C]O). ESI-MS
(positive ions): for C51H46N2O9, calcd. 830.3203; found m/z: 854.10
[M þ Naþ]; 870.09 [M þ Kþ]. HRMS (MALDI-TOF): calcd. for
C51H46N2O9Na ¼ 853.3101; found m/z: 853.3127 [M þ Naþ].
were sequentially added to the solution. The reaction mixture was
stirred at room temperature for 5 min, then DPPA (336 L,1.56 mmol)
m
was added and the resulting mixture left overnight at room temper-
ature. The solvent was then removed under vacuum and the crude
was purified on a silica gel column, eluted with n-hexane/AcOEt
(75:25, v/v), containing a few drops of TEA. Desired product 7 was
obtained in 92% isolated yield (440 mg, 0.481 mmol).
4.4. Synthesis of compound 5
7: oil, Rf ¼ 0.5 [n-hexane/AcOEt (3:2, v/v)]. FT-IR (liquid film):
significant band centered at 2112.9 cmꢁ1 1H-NMR (CDCl3,
Nucleoside 4 (889 mg, 1.07 mmol), dissolved in anhydrous
500 MHz):
d
7.48 (1H, d, J ¼ 8.0 Hz, H-6); 7.34e6.81 (28H, over-
CH2Cl2 (6 mL), was reacted with TEA (448
mL, 3.21 mmol), Boc2O
lapped signals, aromatic protons of MMTr); 6.49 (1H, bs, H-10); 5.61
(1H, d, J ¼ 8.0 Hz, H-5); 4.12e4.08 (1H, m, H-20); 3.94 (1H, bs, H-40);
3.79 (6H, overlapped s’s, 2x OCH3); 3.53e3.51 (1H, m, H-50a);
3.08e3.06 (1H, m, H-50b), 2.63 (1H, d, J ¼ 3.0 Hz, H-30); 1.64 (9H, s,
(467 mg, 2.14 mmol) and DMAP (65 mg, 0.535 mmol). The solution
was stirred at room temperature for 1 h, then the solvent was
removed under reduced pressure and the crude was purified on
a silica gel column, eluted with n-hexane/AcOEt (6:4, v/v) added
with 1% TEA. The desired product 5 was obtained in 80% isolated
yield (793 mg, 0.856 mmol).
3x CH3 Boc). 13C-NMR (CDCl3, 50 MHz):
d 159.8 (C-4); 159.2, 158.6,
143.9,143.6,143.2, 134.7,134.5,130.4,130.1,127.9,127.7,127.4,126.9,
113.5, 112.9 (aromatic carbons of MMTr); 148.3 (C-2); 147.4 (C]O
Boc); 140.0 (C-6); 102.6 (C-5); 90.5 (C-10); 88.7 (C-40); 86.8 and
86.6 (quaternary carbons of MMTr); 83.4 (C(CH3)3 Boc); 78.9 (C-
20); 66.6 (C-30); 60.9 (C-50); 54.9 (OCH3); 27.3 (C(CH3)3 Boc). ESI-
MS (positive ions): for C54H51N5O9, calcd. 913.3687; found m/z:
5: oil, Rf ¼ 0.6 [n-hexane/AcOEt (1:1, v/v)]. 1H-NMR (CDCl3,
500 MHz):
d
7.57 (1H, d, J ¼ 8.0 Hz, H-6); 7.50e6.72 (28H, overlapped
signals, aromatic protonsof MMTr); 6.52(1H, d, J ¼ 8.5 Hz, H-10);5.06
(1H, d, J ¼ 8.0 Hz, H-5); 4.42 (1H, dd, J ¼ 6.0 and 8.5 Hz, H-20); 3.80
(2H, bs, H-30 and H-40); 3.79 and 3.77 (3H each, s’s, 2x OCH3);
3.30e3.06 (2H, ABX system, J ¼ 9.5 and 2.0 Hz, H-50a and H-50b); 2.00
(3H, s, CH3C]O); 1.60 (9H, s, 3x CH3 Boc).13C-NMR (CDCl3, 50 MHz):
814.90
[M-BocþHþ].
HRMS
(MALDI-TOF):
calcd.
for
C54H51N5O9Na ¼ 936.3584; found m/z: 936.3606 [M þ Naþ].
4.7. Synthesis of compound 1
d
169.9 (C]O); 160.2 (C-4); 159.1, 158.7, 143.9, 143.6, 143.5, 143.0,
134.5, 134.4, 130.4, 130.1, 128.2, 128.1, 128.0, 127.9, 127.8, 127.3, 113.4,
113.2 (aromatic carbons of MMTr); 148.9 (C-2); 147.6 (C]O Boc);
139.5 (C-6); 102.1 (C-5); 87.6 (C-10); 87.0 (quaternary carbon of
MMTr); 86.6 (C(CH3)3Boc); 84.3 (C-40); 75.6 (C-20); 72.5 (C-30); 63.7
(C-50); 55.2 and 55.1 (2x OCH3); 27.3 (C(CH3)3 Boc); 20.5 (CH3C]O).
ESI-MS (positive ions): for C56H54N2O11, calcd. 930.3729; found m/z:
954.11 [M þ Naþ]; 970.30 [M þ Kþ]. HRMS (MALDI-TOF): calcd. for
C56H54N2O11Na ¼ 953.3625; found m/z: 953.3651 [M þ Naþ].
Nucleoside 7 (440 mg, 0.481 mmol) was dissolved in anhydrous
CH2Cl2 (2.7 mL) and then TFA (0.15 mL) and TIS (0.15 mL) were
added. The solution was stirred at room temperature for 1 h, then
the solvent was removed under reduced pressure and the crude
was purified on a silica gel column, eluted with n-hexane/
AcOEt(1:4, v/v). The desired product 1 was obtained in 97% isolated
yield (126 mg, 0.468 mmol).
1: oil, Rf ¼ 0.2 [n-hexane/AcOEt (1:4, v/v)]. 1H-NMR (CD3OD,
4.5. Synthesis of compound 6
500 MHz):
d
7.86 (1H, d, J ¼ 8.0 Hz, H-6); 5.79 (1H, d, J ¼ 3.0 Hz, H-
10); 5.71 (1H, d, J ¼ 8.0 Hz, H-5); 4.38e4-36 (1H, m, H-40); 4.34 (1H,
dd, J ¼ 3.5 and 3.0 Hz, H-20); 4.18 (1H, dd, J ¼ 4.0 and 3.5, H-30); 3.85
(2H, d, J ¼ 4.5 Hz, H-50a and H-50b). 13C-NMR (CD3OD, 125 MHz):
Nucleoside 5 (965 mg, 1.04 mmol), dissolved in CH3OH (3.0 mL),
was treated with TEA (1.0 mL, 7.17 mmol) and K2CO3 (29 mg,
0.208 mmol) and the resulting mixture was stirred at room
temperature for 12 h. Then the solution was taken to dryness, the
crude was diluted with CH2Cl2, transferred into a separatory funnel
and washed twice with water; the collected organic phases were
dried over anhydrous Na2SO4 and concentrated under reduced
pressure to give compound 6 in almost quantitative yields (921 mg,
1.04 mmol), pure by TLC and NMR control.
d
166.7 (C-4); 152.9 (C-2); 142.8 (C-6); 102.9 (C-5); 92.2 (C-10); 82.9
(C-40); 80.3 (C-20); 68.3 (C-30); 61.7 (C-50). ESI-MS (positive ions):
for C9H11N5O5, calcd. 269.0760; found m/z: 270.98 [M þ Hþ];
292.97 [M
þ
Naþ]. HRMS (MALDI-TOF): calcd. for
C9H11N5O5Na ¼ 292.0658; found m/z: 292.0662 [M þ Naþ].
4.8. Synthesis of compound 8
6: oil, Rf ¼ 0.4 [n-hexane/AcOEt (3:2, v/v)]. 1H-NMR (CDCl3,
200 MHz):
d
7.79 (1H, d, J ¼ 8.2 Hz, H-6); 7.60e6.75 (28H, overlapped
Nucleoside 1 (197 mg, 0.732 mmol) was dissolved in anhydrous
DMF (1.0 mL), then imidazole (119 mg, 1.76 mmol) and TBDMSCl
(132 mg, 0.878 mmol) were added. The solution was stirred at r.t.
for 1 h, then the solvent was removed under reduced pressure and
the crude was purified on a silica gel column, eluted with n-hexane/
AcOEt (1:1, v/v). Target nucleoside 8 was obtained in 80% isolated
yield (224 mg, 0.586 mmol).
signals, aromatic protons of MMTr); 6.59 (1H, d, J ¼ 5.4 Hz, H-10); 5.11
(1H, d, J ¼ 8.2 Hz, H-5); 4.52e4.48 (1H, m, H-20); 4.05 (1H, bs, H-40);
3.81 (7H, overlapped s’s, 2x OCH3 and H-30); 3.18 (2H, m, H-50a and H-
50b); 2.88 (1H, bd, OH, exchangeable proton); 1.65 (9H, s, 3x CH3 Boc).
13C-NMR (CDCl3, 50 MHz):
d 160.3 (C-4); 159.2, 158.7, 143.9, 143.4,
143.3,143.0,134.3,134.1,130.3,130.1,128.2,128.0,127.8,127.4,127.3,
113.7, 113.2 (aromatic carbons of MMTr); 149.0 (C-2); 147.6 (C]O
Boc); 140.0 (C-6); 102.0 (C-5); 87.6 (C-10); 87.0 and 86.6 (quaternary
carbons of MMTr and C(CH3)3Boc); 84.6 (C-40); 75.6 (C-20); 70.7
(C-30); 64.2 (C-50); 55.1 (OCH3); 27.3 (C(CH3)3 Boc). ESI-MS (positive
8: oil, Rf ¼ 0.5 [n-hexane/AcOEt (1:1, v/v)]. 1H-NMR (CDCl3,
200 MHz):
d
10.53 (1H, bs, NeH); 7.73 (1H, d, J ¼ 8.4 Hz, H-6); 5.79
(1H, d, J ¼ 1.4 Hz, H-10); 5.71 (1H, d, J ¼ 8.0 Hz, H-5); 4.45 (overlapped
signals, H-20 and H-40); 4.21e4.19 (1H, m, H-30); 3.93 (2H, d,
J ¼ 5.2 Hz, H-50a and H-50b), 0.92 (9H, s, SiC(CH3)3), 0.12 (6H, s,
ions): for C54H52N2O10
, calcd. 888.3622; found m/z: 911.22
[M þ Naþ]; 927.32 [M þ Kþ]. HRMS (MALDI-TOF): calcd. for
Si(CH3)2).13C-NMR (CDCl3, 50 MHz):
d 164.0 (C-4); 151.2 (C-2); 140.3
C54H52N2O10Na ¼ 911.3520; found m/z: 911.3539 [M þ Naþ].
(C-6); 101.8 (C-5); 91.8 (C-10); 81.7 (C-40); 79.4 (C-20); 65.9 (C-30);
61.0 (C-50); 25.8 (SiC(CH3)3); 18.2 (SiC(CH3)3); -5.5 and ꢁ5.6
(Si(CH3)2). ESI-MS (positive ions): for C15H25N5O5Si, calcd. 383.1625;
found m/z: 384.14 [M þ Hþ]; 406.06 [M þ Naþ]; 422.00 [M þ Kþ].
HRMS (MALDI-TOF): calcd. for C15H25N5O5SiNa ¼ 406.1523; found
m/z: 406.1544 [M þ Naþ].
4.6. Synthesis of compound 7
Nucleoside 6 (464 mg, 0.520 mmol) was dissolved in anhydrous
THF (5.0 mL); PPh3 (410 mg,1.56 mmol) and DIAD (307 mL,1.56 mmol)

L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
437
4.9. Synthesis of compound 9
on a silica gel column, eluted with n-hexane/AcOEt (7:3, v/v). The
desired product 11 was obtained in 98% isolated yield (36 mg,
0.044 mmol).
Nucleoside 8 (100 mg, 0.261 mmol) was dissolved in anhydrous
CH2Cl2 (2.0 mL), then DMAP (16 mg, 0.131 mmol), oleic acid (100
m
L,
11: oil, Rf ¼ 0.5 [n-hexane/AcOEt (1:1, v/v)]. 1H NMR (DMSO-d6,
89 mg, 0.315 mmol) and DCC (81 mg, 0.392 mmol) were added. The
solution was stirred at room temperature for 2 h, then the solvent
was removed under reduced pressure and the crude purified on
a silica gel column, eluted with n-hexane/AcOEt (7:3, v/v). The
desired product 9 was obtained in 74% isolated yield (125 mg,
0.193 mmol).
500 MHz):
d
11.41 (1H, bs, NH-3); 7.96 (1H, d, J ¼ 7.0 Hz, NH-30); 7.75
(1H, d, J ¼ 8.0 Hz, H-6); 5.83 (1H, d, J ¼ 6.0 Hz, H-10); 5.74 (1H, bs,
H-5); 5.56 (1H, d, J ¼ 8.0 Hz, Ha Proline); 5.32e5.30 (2H, m, H-9 and
H-10 oleic acid); 5.24 (1H, bs, H-20); 4.63e4.61 (1H, m, H-30); 4.27
(1H, bs, H-40); 3.58e3.55 (1H, m, H2-50); 3.30 (2H, overlapped
signals, 2x Hb Proline); 2.27 (2H, bs, CH2C]O oleic acid); 1.97 (4H,
d, J ¼ 6.0 Hz, 2x H-8 and 2x H-11 oleic acid); 1.77e1.75 (2H, m, Hg
Proline); 1.48e1.24 (33H, overlapped signals, aliphatic protons of
oleic acid, Boc and 2x Hd Proline); 0.85 (12H, overlapped signals,
9: oil, Rf ¼ 0.7 [n-hexane/AcOEt(1:1, v/v)]. 1H-NMR (CDCl3,
200 MHz):
d
7.70 (1H, d, J ¼ 8.2 Hz, H-6); 6.05 (1H, d, J ¼ 3.8 Hz,
H-10); 5.76 (1H, dd, J ¼ 1.4 and 8.2 Hz, H-5); 5.34e5.32 (2H, m, H-9
and H-10 oleic acid); 5.23 (1H, dd, J ¼ 3.2 and 3.8 Hz, H-20);
4.23e4.21 (2H, m, H-30 and H-40); 3.91 (2H, d, J ¼ 4.8 Hz, H-50a and
H-50b), 2.39 (2H, dd, J ¼ 6.8 and 7.8 Hz, 2x H-2 oleic acid); 2.03e2.01
(4H, m, 2x H-8 and 2x H-11 oleic acid); 1.64e1.62 (2H, m, H-3 oleic
acid), 1.28 (20H, overlapped signals, aliphatic protons of oleic acid);
0.92 (9H, s, SiC(CH3)3), 0.87 (3H, t, J ¼ 6.6 and 6.6 Hz, CH3 oleic acid);
CH3 oleic acid and SiC(CH3)3), 0.07 and 0.05 (6H, s’s, Si(CH3)2). 13
NMR (DMSO-d6, 125 MHz): 172.3 (C]O); 162.9 (C-4); 156.5 (C]O
C
d
Proline); 153.1 (C]O Boc); 150.4 (C-2); 140.6 (C-6); 129.5 (C-9 and
C-10 oleic acid); 102.1 (C-5); 85.8 (C-10); 78.6 (C-20 and C-40); 77.3
(Ca Proline); 62.3 (C-50); 60.0 (C-30), 53.1 (Cd Proline); 47.5 (Cg
Proline); 33.3 (Cb Proline); 31.2, 29.1, 28.8, 28.6, 28.2, 28.0, 26.6,
25.3, 24.4, 24.2, 22.1 (aliphatic carbons of oleic acid and Boc
eC(CH3)3); 25.8 (SiC(CH3)3); 17.8 (SiC(CH3)3); 14.0 (-CH3 oleic acid);
-5.5 (Si(CH3)2). ESI-MS (positive ions): for C43H74N4O9Si, calcd.
818.5225; found m/z: 820.41 [M þ Hþ]; 842.13 [M þ Naþ]; 858.25
0.12 (6H, s, Si(CH3)2).13C-NMR (CDCl3, 50 MHz):
d 172.4 (C]O);
163.1 (C-4); 150.3 (C-2); 139.8 (C-6); 130.0 and 129.6 (C-9 and C-10
oleic acid); 102.9 (C-5); 87.2 (C-10); 80.2 (C-40); 79.5 (C-20); 64.6 (C-
30); 61.0 (C-50); 33.8, 31.8, 29.7, 29.6, 29.4, 29.2, 29.0, 28.9, 27.1, 24.6,
22.6 (aliphatic carbons of oleic acid); 25.8 (SiC(CH3)3); 18.2
(SiC(CH3)3); 14.0 (-CH3 oleic acid); -5.5 and ꢁ5.7 (Si(CH3)2). ESI-MS
(positive ions): for C33H57N5O6Si, calcd. 647.4078; found m/z:
648.12 [M þ Hþ]; 670.01 [M þ Naþ]; 686.07 [M þ Kþ]. HRMS
(MALDI-TOF): calcd. for C33H57N5O6SiNa ¼ 670.3976; found m/z:
670.3989 [M þ Naþ].
[M
þ
Kþ].
HRMS
(MALDI-TOF):
calcd.
for
C43H74N4O9SiNa ¼ 841.5123; found m/z: 841.5150 [M þ Naþ].
4.12. Synthesis of compound 12
Nucleolipid 11 (60 mg, 0.073 mmol), dissolved in anhydrous THF
(1.0 mL), was treated with Et3N,3xHF (35 L, 0.22 mmol). The
m
4.10. Synthesis of compound 10
solution was stirred at r.t. for 12 h, then the solvent was removed
under reduced pressure and the crude was purified on a silica gel
column, eluted with n-hexane/AcOEt (1:4, v/v). The desired
product 12 was obtained in 93% isolated yield (48 mg, 0.068 mmol).
12: oil, Rf ¼ 0.2 [n-hexane/AcOEt (3:7, v/v)]. 1H NMR (DMSO-d6,
Nucleolipid 9 (31 mg, 0.048 mmol) was dissolved in anhydrous
AcOEt (0.7 mL) and then Pd/C 10% p.p. (10 mg, 0.01 mmol) was
added. The solution was stirred at r.t. for 12 h under H2 pressure
(1 atm), then the solvent was removed under vacuum and the crude
was filtered on a short column of silica gel, eluted with AcOEt. The
desired product 10 was obtained in almost quantitative yields
(30 mg, 0.048 mmol).
500 MHz):
d
11.34 (1H, bs, NH-3); 8.06 (1H, bs, NH-30); 7.99 (1H, d,
J ¼ 8.0 Hz, H-6); 5.89 (1H, bs, H-10); 5.73 (1H, d, J ¼ 7.5 Hz, H-5);
5.56 (1H, d, J ¼ 8.0 Hz, Ha Proline); 5.33e5.31 (3H, m, H-20, H-9 and
H-10 oleic acid); 5.17 (1H, m, OH-50); 4.76e4.73 (1H, m, H-30); 4.24
(1H, bs, H-40); 3.59e3.56 (2H, m, H-50a and H-50b); 3.29 (2H, over-
lapped signals, 2x Hb Proline); 2.27 (2H, bs, CH2C]O oleic acid);
1.97 (4H, d, J ¼ 5.5 Hz, 2x H-8 and 2x H-11 oleic acid); 1.74e1.72 (2H,
m, Hg Proline); 1.52e1.24 (33H, overlapped signals, aliphatic
protons of oleic acid, Boc and 2x Hd Proline); 0.85 (3H, dd, J ¼ 6.5
10: oil, Rf ¼ 0.2 [n-hexane/AcOEt (3:2, v/v)]. 1H-NMR (CDCl3,
200 MHz):
d
8.04 (1H, d, J ¼ 8.4 Hz, H-6); 6.03 (1H, d, J ¼ 4.8 Hz,
H-10); 5.72 (1H, d, J ¼ 8.4 Hz, H-5); 5.37e5.33 (2H, m, H-9 and H-10
oleic acid); 5.03 (1H, dd, J ¼ 4.8 and 5.2 Hz, H-20); 4.22e4.18 (2H, m,
H-40); 3.93 (2H, m, H-50a and H-50b), 3.68 (1H, dd, J ¼ 6.4 and 5.8 Hz,
H-30); 2.36 (2H, t, J ¼ 7.4 Hz, 2x H-2 oleic acid); 2.03e2.01 (4H, m, 2x
H-8 and 2x H-11 oleic acid); 1.60e1.57 (2H, m, H-3 oleic acid),
1.28e1.25 (20H, overlapped signals, aliphatic protons of oleic acid);
0.94e0.91 (12H, overlapped signals, CH3 oleic acid and SiC(CH3)3),
and 7.0 Hz, CH3 oleic acid). 13C NMR (DMSO-d6, 125 MHz):
d 172.7
(C]O); 163.1 (C-4); 156.8 (C]O Proline); 153.4 (C]O Boc); 150.6
(C-2); 140.9 (C-6); 129.8 (C-9 and C-10 oleic acid); 102.4 (C-5); 85.2
(C-10); 78.9 (C-40); 78.5 (C-20); 77.2 (Ca Proline); 60.5 (C-50); 60.0
(C-30); 52.9 (Cd Proline); 47.7 (Cg Proline); 33.5 (Cb Proline); 31.5,
29.2, 29.0, 28.9, 28.7, 28.6, 28.4, 28.2, 26.8, 25.5, 24.6, 24.4, 22.3
(aliphatic carbons of oleic acid and Boc eC(CH3)3); 14.2 (-CH3 oleic
acid). ESI-MS (positive ions): for C37H60N4O9, calcd. 704.4360;
found m/z: 705.98 [M þ Hþ]; 728.03 [M þ Naþ]; 743.97 [M þ Kþ].
HRMS (MALDI-TOF): calcd. for C37H60N4O9Na ¼ 727.4258; found m/
z: 727.4273[M þ Naþ].
0.14 (6H, s, Si(CH3)2).13C-NMR (CDCl3, 50 MHz):
d 173.2 (C]O);
162.7 (C-4); 150.2 (C-2); 140.8 (C-6); 130.0 and 129.6 (C-9 and C-10
oleic acid); 102.4 (C-5); 86.2 (C-10); 81.5 (C-40); 80.2 (C-20); 61.9
(C-50); 56.6 (C-30); 33.9, 31.8, 29.6, 29.4, 29.2, 29.0, 27.1, 24.6, 22.5
(aliphatic carbons of oleic acid); 25.8 (SiC(CH3)3); 18.0 (SiC(CH3)3);
14.0 (-CH3 oleic acid); -5.7 (Si(CH3)2). ESI-MS (positive ions): for
C33H59N3O6Si, calcd. 621.4173; found m/z: 622.15 [M þ Hþ]; 644.07
[M þ Naþ]; 660.13 [M þ Kþ]. HRMS (MALDI-TOF): calcd. for
C33H59N3O6SiNa ¼ 644.4071; found m/z: 644.4092 [M þ Naþ].
4.13. Synthesis of compound 13
4.11. Synthesis of compound 11
Nucleolipid 12 (45 mg, 0.064 mmol), dissolved in anhydrous
CH2Cl2 (1.0 mL), was treated with DMAP (4.0 mg, 0.032 mmol),
BnOCH2CH2(OCH2CH2)5OCH2COOH (36 mg, 0.083 mmol), synthe-
sized according to reported procedures [22], and DCC (20 mg,
0.096 mmol), added in the order. The solution was stirred at r.t. for
2 h, then the solvent was removed under reduced pressure and the
crude was purified on a silica gel column, eluted with n-hexane/
Nucleolipid 10 (28 mg, 0.045 mmol), dissolved in anhydrous
CH2Cl2 (0.50 mL), was reacted with DMAP (3.0 mg, 0.025 mmol),
Boc-L-Proline (13 mg, 0.060 mmol) and DCC (14 mg, 0.068 mmol).
The resulting solution was stirred at r.t. for 12 h, then the solvent
was removed under reduced pressure and the crude was purified

438
L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
acetone (1:1, v/v). The desired product 13 was obtained in 87%
isolated yield (62 mg, 0.056 mmol).
4.15. Surface tension measurements
The surface tension, , of binary mixtures waterecompound 2
was measured by the DeNouy ring method using a KSV Sigma 70
digital tensiometer equipped with an automatic device to select the
rising speed of the platinum ring and to set the time between two
consecutive measurements. Weighed amounts of a compound 2
solution at a concentration well above its cmc were added to
a weighed amount of water into the tensiometer circular vessel,
thermostatted at 25.0 ꢂ 0.1 ꢃC.
13: oil, Rf ¼ 0.4 [n-hexane/acetone (1:1, v/v)]. 1H NMR (DMSO-
g
d6, 500 MHz):
d
11.40 (1H, bs, NH-3); 8.23 (1H, bs, NH-30); 7.70 (1H,
d, J ¼ 7.5 Hz, H-6); 7.34e7.27 (5H, m, aromatic protons of Bn); 5.87
(1H, d, J ¼ 5.5, H-10); 5.78 (1H, bs, H-5); 5.32 (3H, d, J ¼ 8.0 Hz, Ha
Proline, H-9 and H-10 oleic acid); 5.42 (1H, dd, J ¼ 6.5 and 6.0 Hz, H-
20); 5.34e5.32 (2H, m, CH2Bn); 4.71e4.69 (1H, m, H-30); 4.48 (3H,
bs, H-40 and CH2C]O); 4.11e4.09 (2H, m, H-50a and H-50b); 3.50
(24H, overlapped signals, -OCH2CH2O-); 3.30 (2H, overlapped
signals, 2x Hb Proline); 2.27 (2H, m, CH2C]O oleic acid); 1.97 (4H, d,
J ¼ 5.5 Hz, 2x H-8 and 2x H-11 oleic acid); 1.73e1.71 (2H, m, Hg
Proline); 1.52e1.00 (33H, overlapped signals, aliphatic CH2 protons
of oleic acid, Boc and 2x Hd Proline); 0.85 (3H, dd, J ¼ 6.5 and 7.0 Hz,
4.16. Conductivity measurements
The conductivity experiments were carried out using a conduc-
tivity meter YSI model 3200, with an accuracy of ꢂ0.1% full scale, at
1010 Hz frequency. A YSI model 3252 conductivity cell was used for
all measurements. The compound 2 aqueous solutions were con-
tained in a double-wall glass vessel maintained at 25.0 ꢂ 0.1 ꢃC.
Mixture concentration was varied by adding weighed amounts of
a compound 2 solution at a concentration well above its cmc to
a weighed amount of water initially put into the thermostatted
vessel.
CH3 oleic acid). 13C NMR (CD3OD, 50 MHz):
d 172.1 and 170.7 (2x C]
O); 166.1 (C-4); 156.8 (C]O Proline); 152.7 (C]O Boc); 146.6 (C-2);
140.2 (C-6); 139.8 (quaternary carbon of Bn group); 131.4, 131.3,
129.9,129.4,129.2 (C-9 and C-10 oleic acid and aromatic CH carbons
of Bn); 103.9 (C-5); 82.5 (C-10); 81.8 (C-40), 79.0 (C-20); 74.6
(OCH2C]O); 72.4 (Ca Proline); 72.0, 71.6, 71.1 (overlapped signals,
OCH2CH2O); 69.8 (-OCH2Bn); 63.8 (C-50); 63.2 (C-30), 56.3 (Cd
Proline); 33.5 (Cb Proline); 31.5, 29.2, 29.0, 28.9, 28.7, 28.6, 28.4,
28.2, 26.8, 25.5, 24.2 (aliphatic CH2 carbons of oleic acid and Boc-
C(CH3)3); 15.0 (-CH3 oleic acid). Cg Proline is buried under the
solvent signal. ESI-MS (positive ions): for C58H92N4O17, calcd.
1116.6457; found m/z: 1118.88 [M þ Hþ]; 1140.66 [M þ Naþ];
4.17. DLS measurements
1156.65 [M
þ
Kþ]. HRMS (MALDI-TOF): calcd. for
DLS measurements were performed with a home-made instru-
ment composed by a Photocor compact goniometer, an SMD 6000
Laser Quantum 50 mW light source operating at 5325 Å, a photo-
multiplier (PMT-120-OP/B) and a correlator (Flex02-01D) from Cor-
relator.com. Allthe measurements were performed at (25.00 ꢂ 0.05)ꢃC
with the temperature controlled through the use of a thermostat bath.
In elaborating the DLS data, the distribution of relaxation times,
C58H92N4O17Na ¼ 1139.6355; found m/z: 1139.6380 [M þ Naþ].
4.14. Synthesis of compound 2
Nucleolipid 13 (54 mg, 0.048 mmol), dissolved in anhydrous
CH2Cl2 (0.85 mL), was reacted with TFA (0.15 mL). The reaction was
stirred at r.t. for 1 h, then the solvent was removed under reduced
pressure and the crude was coevaporated with 2-propanol (1.0 mL
for 3 times). The desired final compound was thus obtained in
almost quantitative yields in the form of trifluoroacetate salt (14,
55 mg, 0.048 mmol).
Successive treatment with one equivalent of ammonium chlo-
ride (2.6 mg, 0.048 mmol), followed by repeated lyophilizations
from water, gave target compound 2 in the form of chloride salt.
2:white powder, Rf ¼ 0.2 [n-hexane/acetone (1:4, v/v)]. 1H NMR
s
, is obtained by regularized inverse Laplace transformation of the
measured correlation functions. Furthermore, the relaxation rates,
, are used to calculate the translational diffusion coeffi-
G
¼ 1=
s
cient, D, according to the relation:
G
D ¼ lim
q/0 q2
where q ¼ 4
n0 is the refractive index of the solution,
length and represents the scattering angle. Thus D is obtained
from the limit slope of is measured at
as a function of q2, where
p
n0=
l
sinð
q
=2Þ is the modulus of the scattering vector,
l
is the incident wave-
(DMSO-d6, 500 MHz):
d
11.40 (1H, bs, NH-3); 8.81 (1H, d, J ¼ 8.0 Hz,
q
NH-30); 7.70 (1H, d, J ¼ 8.0 Hz, H-6); 7.34e7.26 (5H, complex signals,
aromatic protons of Bn); 5.89 (1H, d, J ¼ 6.0, H-10); 5.78 (1H, d,
J ¼ 7.5 Hz, H-5); 5.32e5.29 (3H, m, H-20, H-9 and H-10 oleic acid);
4.73e4.71 (1H, m, H-30); 4.84 (3H, bs, H-40 and OCH2C]O);
4.16e4.14 (3H, overlapped signals, Ha proline, H-50a and H-50b); 3.51
(24H, overlapped signals, OCH2CH2O); 3.35e3.33 (2H, m, CH2C]O
oleic acid); 2.31e2.29(2H, m, 2 x Hb Proline); 1.98e1.95 (4H, m, 2x H-
8 and 2x H-11 oleic acid); 1.51e1.49 (2H, overlapped signals, Hg
Proline and CH2CH2C]O); 1.26e1.00 (24H, overlapped signals,
aliphatic CH2 protons of oleic acid and 2x Hd Proline); 0.83 (3H, m,
G
G
different scattering angles. In this work, relaxation times were
acquired at six different angles ranging between 30ꢃ and 110ꢃ, and
three or more measurements per angle were performed in order to
improve statistical accuracy.
4.18. Cell cultures
Human WiDr epithelial colorectal adenocarcinoma cells and
MCF-7 breast adenocarcinoma cells, rat C6 glioma cell line and
murine 3T3-L1 fibroblasts were purchased from ATCCÒ (American
Type Culture Collection, Manassas, Virginia, USA). C6 and 3T3-L1
cells were grown in Dulbecco’s modified Eagle’s medium (DMEM,
Invitrogen, Paisley, UK) containing high glucose (4.5 g/l); MCF-7
and WiDr were grown in RPMI 1640 medium (Invitrogen,
Paisley, UK). Media were supplemented with 10% fetal bovine
CH3 oleic acid).13C NMR (CD3OD, 50 MHz):
d 172.5 and 170.5 (2xC]
O); 166.2 (C-4); 160.3 (C]O Proline); 152.6 (C-2); 144.3 (C-6); 139.9
(quaternarycarbon of Bn); 131.4,131.3,130.0,129.5,129.3 (C-9 and C-
10 oleic acid and aromatic CH carbons of Bn); 104.1 (C-5); 91.2 (C-10);
80.3 (C-40); 78.0 (C-20); 74.5 (Ca Proline); 72.2, 71.7, 70.9 (-OCH2
-
CH2O-); 69.8 (-OCH2Bn); 69.5 (OCH2CO); 64.3 (C-30); 61.7 (C-50), 56.8
(Cd Proline); 48.0 (Cg Proline); 35.2 (Cb Proline); 33.5, 32.3, 31.3, 31.1,
30.8, 28.6, 27.5, 27.2, 27.0, 26.5, 26.2, 25.5, 24.2 (aliphatic CH2 carbons
of oleic acid); 15.0 (-CH3 oleic acid). ESI-MS (positive ions): for
C53H84N4O15, calcd. 1016.5933; found m/z: 1018.57 [M þ Hþ];
1040.56 [M þ Naþ]; 1056.44 [M þ Kþ]. HRMS (MALDI-TOF): calcd.
forC53H84N4O15Na ¼ 1039.5831; found m/z: 1039.5856 [M þ Naþ].
serum (FBS, Cambrex, Verviers, Belgium),
Sigma, Milan, Italy), penicillin (100 units/ml, Sigma) and strepto-
mycin (100 g/ml, Sigma), according to ATCC recommendations.
L-glutamine (2 mM,
m
All the cells were cultured in a humidified 5% carbon dioxide
atmosphere at 37 ꢃC.

L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
439
4.19. Microculture bioassay
Appendix A. Supplementary material
The cytotoxic activity of nucleolipid 2 and of cetrimonium
bromide (cetyltrimethylammonium bromide, CTAB), used as
positive control, were investigated by the estimation of a “cell
survival index”, arising from the combination of cell viability
evaluation with cell counting. WiDr, MCF-7, C6 and 3T3-L1 cells
were washed with PBS buffer solution (Sigma), collected by
trypsine (Sigma) and then inoculated in a 96-microwell culture
plates at density of 104 cells/well. Cells were allowed to grow for
24 h, then the medium was replaced with fresh medium and cells
were treated for further 48 h with a range of concentrations
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.044.
Abbreviations
Ac
acetyl
AcOEt
Bn
ethyl acetate
benzyl
Boc
DCC
DIAD
tert-butoxycarbonyl
dicyclohexylcarbodiimide
diisopropylazodicarboxylate
(10e250
mM) of nucleolipid 2 and of CTAB. Cell viability was
evaluated with an MTT assay procedure, which measures the
level of mitochondrial dehydrogenase activity using the yellow
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide
(MTT, Sigma) as substrate. The assay was based on the redox
ability of living mitochondria to convert dissolved MTT into
insoluble purple formazan. Briefly, after the treatments the
DMAP 4-(N,N-dimethylamino)pyridine
DMF
N,N-dimethylformamide
DPPA
MMTr
diphenylphosphorylazide
4-monomethoxytriphenylmethyl
TBDMS tert-butyldimethylsilyl
medium was removed and the cells were incubated with 20 ml/
TEA
TFA
THF
TIS
triethylamine
well of an MTT solution (5 mg/ml) for 1 h in a humidified 5% CO2
trifluoroacetic acid
tetrahydrofuran
triisopropylsilane
incubator at 37 ꢃC. The incubation was stopped by removing the
MTT solution and by adding 100 ml/well of DMSO to solubilize the
purple formazan. Finally, the absorbance was monitored at
530 nm by using a multiwell plate reader in a PerkineElmer LS 55
Luminescence Spectrometer (PerkineElmer Ltd, Beaconsfield,
UK) [41].
Cell number and proliferation was determined by TC10 auto-
mated cell counter (Bio-Rad, Milan, Italy), providing an accurate
and reproducible total count of mammalian cells and a live/dead
ratio in one step.
The calculation of the concentration required to inhibit the net
increase in the cell number and viability by 50% (IC50) is based on
plots of data carried out in triplicates and repeated three times. IC50
values were obtained using a dose response curve by nonlinear
regression using a curve fitting program, GraphPad Prism 5.0, and
are expressed as mean ꢂ SEM.
References
[1] V. Allain, C. Bourgaux, P. Couvreur, Self-assembled nucleolipids: from supra-
molecular structure to soft nucleic acid and drug delivery devices, Nucl. Acids
Res. 40 (2012) 1891e1903.
[2] D. Berti, C. Montis, P. Baglioni, Self-assembly of designer biosurfactants, Soft
Matter 7 (2011) 7150e7158.
[3] A. Gissot, M. Camplo, M.W. Grinstaff, P. Barthélémy, Nucleoside, nucleotide
and oligonucleotide based amphiphiles: a successful marriage of nucleic acids
with lipids, Org. Biomol. Chem. 6 (2008) 1324e1333.
[4] H. Rosemeyer, Nucleolipids: natural occurrence, synthesis, molecular recog-
nition, and supramolecular assemblies as potential precursors of life and
bioorganic materials, Chem. Biodiversity 2 (2005) 977e1062.
[5] X. Mulet, T. Kaasgaard, C.E. Conn, L.J. Waddington, D.F. Kennedy,
A. Weerawardena, C.J. Drummond, Nanostructured nonionic thymidine
nucleolipid self-assembly materials, Langmuir 26 (2010) 18415e18423.
[6] F. Cuomo, F. Lopez, R. Angelico, G. Colafemmina, A. Ceglie, Nucleotides and
nucleolipids derivatives interaction effects during multi-lamellar vesicles
formation, Colloid Surf. B 64 (2008) 184e193.
4.20. Light microscopy
[7] J. Huang, C. Li, Y. Liang, FT-SERS studies on molecular recognition capabilities
of monolayers of novel nucleolipid amphiphiles, Langmuir 16 (2000)
3937e3940.
[8] D. Berti, P. Baglioni, S. Bonaccio, G. Barsacchi-Bo, P.L. Luisi, Base complemen-
tarity and nucleoside recognition in phosphatidylnucleoside vesicles, J. Phys.
Chem. B 102 (1998) 303e308.
[9] D. Berti, L. Franchi, P. Baglioni, P.L. Luisi, Molecular recognition in monolayers.
Complementary base pairing in dioleoylphosphatidyl derivatives of adeno-
sine, uridine, and cytidine, Langmuir 13 (1997) 3438e3444.
[10] H.P. Drobeck, Current topics on the toxicity of cationic surfactants, in: J. Cross,
E.J. Singer (Eds.), Cationic Surfactants, Marcel Dekker, NY, USA, 1994, pp.
61e94.
In microphotography experiments, MCF-7, WiDr, C6 and 3T3-L1
cell lines were grown on 100 mm standard culture dishes (Tissue
Culture Dish, Falcon) by plating 3 ꢀ 106 cells in 10 ml of culture
medium (see above). After reaching the confluence, cells were
incubated for 48 h with 100
Finally, cells were placed on a contrast-phase light microscope
(Labovert microscope, Leizt). Microphotographs at an 100
mM of nucleolipid 2 and of CTAB.
x
magnification were taken with a standard VCR camera (Nikon).
[11] M.R. Infante, L. Perez, M.C. Moran, R. Pons, M. Mitjans, M.P. Vinardell,
M.T. Garcia, A. Pinazo, Biocompatible surfactants from renewable hydrophiles,
Eur. J. Lipid Sci. Technol. 112 (2010) 110e121.
[12] H.W. Yang, J.W. Yi, E.-K. Bang, E.M. Jeon, B.H. Kim, Cationic nucleolipids as
efficient siRNA carriers, Org. Biomol. Chem. 9 (2011) 291e296.
[13] C. Ceballos, C.A.H. Prata, S. Giorgio, F. Garzino, D. Payet, P. Barthélémy,
4.21. Statistical analysis
All data were presented as mean ꢂ SEM (standard error of the
media). The statistical analysis was performed using Graph-Pad
Prism (Graph-Pad software Inc., San Diego, CA) and ANOVA test
for multiple comparisons was performed followed by Bonferroni’s
test.
M.W. Grinstaff, M. Camplo, Cationic nucleoside lipids based on
a 3-
nitropyrrole universal base for siRNA delivery, Bioconjug. Chem. 20 (2009)
193e196.
[14] L. Costantino, G. D’Errico, P. Rescigno, V. Vitagliano, Effect of urea and alky-
lureas on micelle formation by a nonionic surfactant with short hydrophobic
tail at 25 ꢃC, J. Phys. Chem. B 104 (2000) 7326e7333.
[15] M.L. Immordino, F. Dosio, L. Cattel, Stealth liposomes: review of the basic
science, rationale, and clinical applications, existing and potential, Int. J.
Nanomedicine 1 (2006) 297e315.
Acknowledgments
[16] J.G. Buchanan, D.R. Clark, Studies of the interconversion of 2,3’-anhydro-1-b-
D-xylofuranosyluracil and 2,2’-anhydro-1-
bohydr. Res. 68 (1979) 331e341.
[17] G.H. Hakimelahi, Z.A. Proba, K.K. Olgivie, New catalysts and procedures for the
dimethoxytritylation and selective silylation of ribonucleosides, Can. J. Chem.
60 (1982) 1106e1113.
b-D-arabinofuranosyluracil, Car-
We thank MIUR (PRIN 2008-prot. 20087K9A2J) for grants in
support of this investigation and Centro di Metodologie Chimico-
Fisiche (CIMCF), Università di Napoli “Federico II”, for the MS and
NMR facilities.

440
L. Simeone et al. / European Journal of Medicinal Chemistry 57 (2012) 429e440
[18] O. Mitsunobu, The use of diethyl azodicarboxylate and triphenylphosphine
in synthesis and transformation of natural products, Synthesis 1 (1981)
1e28.
[30] A. Pinazo, X. Wen, L. Pérez, M.R. Infante, E.I. Frances, Aggregation behavior in
water of monomeric and gemini cationic surfactants derived from arginine,
Langmuir 15 (1999) 3134e3142.
[19] K.C. Kumara Swamy, N.N. Bhuvan Kumar, E. Balaraman, K.V.P. Pavan Kumar,
Mitsunobu and related reactions: advances and applications, Chem. Rev. 109
(2009) 2551e2651.
[31] G. D’Errico, O. Ortona, L. Paduano, A.M. Tedeschi, V. Vitagliano, Mixed micellar
aggregates of cationic and nonionic surfactants with short hydrophobic tails.
An intradiffusion study, Phys. Chem. Chem. Phys. 4 (2002) 5317e5324.
[32] L. Pérez, A. Pinazo, M.T. Garcia, M. Lozano, A. Manresa, M. Angelet,
M.P. Vinardell, M. Mitjans, R. Pons, M.R. Infante, Cationic surfactants from
lysine: synthesis, micellization and biological evaluation, Eur. J. Med. Chem.
44 (2009) 1884e1892.
[33] L. Pérez, A. Pinazo, M.R. Infante, R. Pons, Investigation of the micellization
process of single and gemini surfactants from arginine by SAXS, NMR self-
diffusion, and light scattering, J. Phys. Chem. B 111 (2007) 11379e11387.
[34] F. Li, J.Z. Li, H.Q. Wang, Q.J. Xue, Studies on cetyltrimethylammonium bromide
(CTAB) micellar solution and CTAB reversed microemulsion by ESR and 2H
NMR, Colloids Surf. A 127 (1997) 89e96.
[35] L. De Napoli, G. Di Fabio, J. D’Onofrio, D. Montesarchio, New nucleoside-based
polymeric supports for the solid phase synthesis of ribose-modified nucleo-
side analogs, Synlett 11 (2004) 1975e1979.
[36] A.P. Spork, D. Wiegmann, M. Granitzka, D. Stalke, C. Ducho, Stereoselective
synthesis of uridine-derived nucleosyl amino acids, J. Org. Chem. 76 (2011)
10083e10098.
[20] For a recent application of the Mitsunobu reactions to obtain azides from
alcohols, see J.P. Scott, M. Alam, N. Bremeyer, A. Goodyear, T. Lam, R.D. Wilson,
G. Zhou, Mitsunobu inversion of a secondary alcohol with diphenylphos-
phoryl azide. Application to the enantioselective multikilogram synthesis of
a HCV polymerase inhibitor, Org. Process Res. Dev. 15 (2011) 1116e1123.
[21] L. Simeone, L. De Napoli, D. Montesarchio, N(3)-protection of thymidine with
Boc for an easy synthetic access to sugar-alkylated nucleoside analogs, Chem.
Biodiversity 3 (2012) 589e597.
[22] L. Simeone, G. Mangiapia, C. Irace, A. Di Pascale, A. Colonna, O. Ortona, L. De
Napoli, D. Montesarchio, L. Paduano, Nucleolipid nanovectors as molecular
carriers for potential applications in drug delivery, Mol. BioSyst. 7 (2011)
3075e3086.
[23] G. D’Errico, On the segregative tendency of ethoxylated surfactants in
nonionic mixed micelles, Langmuir 27 (2011) 3317e3323.
[24] Z. Li, E. Mintzer, R. Bittman, The critical micelle concentrations of lysophos-
phatidic acid and sphingosylphosphorylcholine, Chem. Phys. Lipids 130
(2004) 197e201.
[37] E. Ito, K.W. Yip, D. Katz, S.B. Fonseca, D.W. Hedley, S. Chow, G.W. Xu,
T.E. Wood, C. Bastianutto, A.D. Schimmer, S.O. Kelley, F.-F. Liu, Potential use of
cetrimonium bromide as an apoptosis-promoting anticancer agent for head
and neck cancer, Mol. Pharmacol. 76 (2009) 969e983.
[25] E.A.M. Gad, M.M.A. El-Sukkary, D.A. Ismail, Surface and thermodynamic
parameters of sodium N-acyl sarcosine surfactant solutions, J. Am. Oil Chem.
Soc. 74 (1997) 43e47.
[26] L. Zakharova, F. Valeeva, A. Zakharov, A. Ibragimova, L. Kudryavtseva,
H. Harlampidi, Micellization and catalytic activity of the cetyl-
trimethylammonium bromide-Brij 97-water mixed micellar system, J. Colloid
Interface Sci. 263 (2003) 597e605.
[27] J. Eastoe, S. Nave, A. Downer, A. Paul, A. Rankin, K. Tribe, J. Penfold,
Adsorption of ionic surfactants at the air-solution interface, Langmuir 16
(2000) 4511e4518.
[38] E. Soussan, S. Cassel, M. Blanzat, I. Rico-Lattes, Drug delivery by soft matter:
matrix and vesicular carriers, Angew. Chem. Int. Ed. 48 (2009) 274e288.
[39] G. Mangiapia, G. D’Errico, L. Simeone, C. Irace, A. Radulescu, A. Di Pascale,
A. Colonna, D. Montesarchio, L. Paduano, Ruthenium-based complex nano-
carriers for cancer therapy, Biomaterials 33 (2012) 3770e3782.
[40] L. Simeone, G. Mangiapia, G. Vitiello, C. Irace, A. Colonna, O. Ortona,
D. Montesarchio, L. Paduano,
A cholesterol-based nucleolipid-Ruthenium
[28] B. Orioni, M. Roversi, C. La Mesa, F. Asaro, G. Pellizer, G. D’ Errico, Poly-
morphic behavior in protein-surfactant mixtures: the water-bovine serum
albumin-sodium taurodeoxycholate system, J. Phys. Chem. B 110 (2006)
12129e12140.
[29] N.M. Van Os, J.R. Haak, L.A.M. Rupert, Physico-chemical Properties of Selected
Anionic, Cationic and Nonionic Surfactants, Elsevier, Amsterdam, NL, 1993.
complex stabilized by lipid aggregates for antineoplastic therapy, Bioconjug.
Chem. 23 (2012) 758e770.
[41] C. Irace, A. Scorziello, C. Maffettone, G. Pignataro, C. Matrone, A. Adornetto,
R. Santamaria, L. Annunziato, A. Colonna, Divergent modulation of iron
regulatory proteins and ferritin biosynthesis by hypoxia/reoxygenation in
neurones and glial cells, J. Neurochem. 95 (2005) 1321e1331.