Synthesis and NMR characterization of a novel crown-ether ring-fused uridine analogue

Article abstract of DOI:10.1016/j.tet.2010.06.073

The chemical synthesis and ¹H NMR analysis of a novel bicyclic uridine derivative, with a 18-crown-6 ether moiety fused at the ribose 2- and 3-positions, as first example of a hitherto unknown class of ribose-modified nucleosides, are here desc

Full text of DOI:10.1016/j.tet.2010.06.073

Tetrahedron 66 (2010) 6769e6774
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Synthesis and NMR characterization of a novel crown-ether ring-fused uridine
analogue
Cinzia Coppola a, Luca Simeone a, Roberta Trotta b, Lorenzo De Napoli a, Antonio Randazzo b,
,
Daniela Montesarchio a
*
a Dipartimento di Chimica Organica e Biochimica, Complesso Universitario di Monte S. Angelo, via Cintia, 4, Università degli Studi di Napoli ‘Federico II’, I-80126 Napoli, Italy
b Dipartimento di Chimica delle Sostanze Naturali, via D. Montesano 48, Università degli Studi di Napoli ‘Federico II’, I-80131 Napoli, Italy
a r t i c l e i n f o
a b s t r a c t
The chemical synthesis and 1H NMR analysis of a novel bicyclic uridine derivative, with a 18-crown-6
ether moiety fused at the ribose 2- and 3-positions, as first example of a hitherto unknown class of ri-
bose-modified nucleosides, are here described. NMR-based conformational analysis studies showed for
the modified nucleoside a marked preference for an N-type sugar puckering and the nucleobase in the
anti conformation, with the uracil favouring the coordination of a sodium ion hosted in the crown ether.
Ó 2010 Elsevier Ltd. All rights reserved.
Article history:
Received 11 February 2010
Received in revised form
13 May 2010
Accepted 24 June 2010
Available online 3 July 2010
Keywords:
Nucleosides
Crown ether derivatives
Cyclization
Protecting groups
NMR analysis
1. Introduction
DDU (20,30-dideoxyuridine), proposed as the first prototypes of
a new class of nucleotide prodrugs.5 The design and synthesis of
Since Cram and Pedersen first proposed the synthesis of mac-
rocyclic polyether compounds, commonly defined as ‘crown ethers’,
a plethora of different crown ether derivatives have been described
in the literature over the past three decades and their synthetic and
analytical potential investigated.1 The incorporation of crown ether
moieties into chiral scaffolds is interesting, providing effective chiral
receptors, potentially useful as chiral reagents and catalysts for
enantioselective reactions, or chiral selectors in chromatographic
techniques.2 In this context, many carbohydrate-based crown
ethers, with one or more crown ethers attached to pyranosidic or
furanosidic skeletons, have been described, proving to be suitable
chiral phase transfer catalysts and/or models for the study of chiral
recognition in enzymatic processes,3 or useful amphiphilic macro-
cycles with tunable complexation and self-aggregation properties.4
To the best of our knowledge, very few examples of hybrid
molecules combining nucleoside scaffolds with these macrocycles
have appeared in the literature. The first report concerned the
synthesis and biological evaluation of crown ether-linked aryl-50-
phosphate diesters derivatives of AZT (30-azido-thymidine) and
another class of AZT prodrugs, 50-conjugated to cyclam- and bicy-
clam residues through a flexible five carbon atom linker, have been
described.6 Remarkably, the conjugation of DNA fragments with
amphiphilic polyaza crown ethers, attached at opposite ends of the
oligonucleotide backbone, has been shown to significantly stabilize
DNA duplexes.7 More recently, the synthesis and physico-chemical
characterization of an adenosine derivative bearing a benzo-15-
crown-5 ether moiety at the N6 nitrogen atom have also been
reported.8,9
In the search for effective, selective and non-toxic antiviral and/or
antitumoral therapeutics, a variety of strategies have been devised to
design novel nucleoside analogues, mostly modified at the level of
the sugar moiety. Among the plethora of known ribose-modified
nucleosides, considerable attention has been focused on four, five
and six atom ring-fused bicyclic nucleosides as conformationally
locked substrates, preferentially recognized by cellular kinases10 or
privileged building blocks for biologically active modified oligonu-
cleotides.11 Nucleoside ribose-fused crown ethers have not been
described in the literature and the effect of the insertion of these
macrocycles on the nucleoside sugar conformation cannot be easily
a priori predicted. Further interest in combining nucleosides with
crown ethers may be found in the excellent binding abilities of these
macrocycles towards biologically relevant cations as Naþ and Kþ,
* Corresponding author. E-mail address: daniela.montesarchio@unina.it
(D. Montesarchio).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.073

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C. Coppola et al. / Tetrahedron 66 (2010) 6769e6774
expected to confer partial positive charging to the modified
nucleosides: these can show interesting biological properties per se
or, if attached to an oligonucleotide chain, enhance their cell-
membrane permeability. Potential ionophoric activity may also be
envisaged in a design where a central nucleosidic core is decorated
with crown ether moieties and amphiphilic appendages.
We have recently anticipated a general, straightforward syn-
thetic scheme for the preparation of new bicyclic ribonucleoside
derivatives, carrying a 18-crown-6 ether moiety fused at the
cis-oriented 2- and 3-hydroxyls of the ribose.12 To prove the fea-
sibility of this strategy, we here describe novel uridine derivative 7
(Scheme 1), chosen as a model synthetic target, and analyze its
peculiar conformational behaviour in solution as inferred from
NMR data.
steps: first, a mild oxidation promotes the conversion of the thio-
ether to sulfone, followed by its effective removal through an aq
basic treatment. It can be easily inserted into the uracil moiety
through a high yielding Mitsunobu condensation of the sugar-
protected ribonucleoside with 2-(phenylthio)ethanol and is very
stable to a variety of conditions, including strongly basic solutions.
Starting from commercially available 50-O-DMT-uridine 1, ad-
dition of an excess of acetic anhydride in pyridine gave sugar-
protected derivative 2. This was then reacted with 2-(phenylthio)
ethanol in the presence of tri-n-butylphosphine and ADDP in
benzene, giving fully protected nucleoside 3 in 89% yields. Suc-
cessive treatment of this substrate with a 2 M NaOH solution in
dioxane/H2O 1:1 (v/v) allowed the clean deprotection of the 20,30-
hydroxyls, thus affording 4 in almost quantitative yields. Desired
18-crown-6-ether-fused derivative 5 was obtained by reacting 4
with a small excess of penta(ethylene glycol) ditosylate in the
presence of NaH in THF. Appropriate dilution conditions were
adopted to avoid undesired polymerization events; indeed, TLC
monitoring of the reaction mixture showed the formation of the
target compound in very satisfactory yields. Not unexpectedly, as-
suming, as the first event, the alkylation on one OH group, the close
proximity of the vicinal OH associated with the templating effect of
the metal cation (Naþ in this case) strongly favoured the cyclization
process over undesired dimerization, as deduced from the com-
plete absence, in the reaction mixture, of side products. As the
starting nucleoside disappeared, the resulting thioether was di-
rectly oxidizeddby addition of m-chloroperbenzoic acid (m-CPBA),
in a one pot proceduredto sulfone 5, isolated after column chro-
matography in 70% for the two steps. Complete removal of the 2-
(phenylsulfonyl)ethyl group was then achieved by treatment with
2 M NaOH solution in dioxane/H2O 1:1 (v/v). Final deprotection
involved the reaction of 50-O-DMT-protected nucleoside 6 with a 1%
TFA solution in CH2Cl2 for 1 h at rt. After repeated washings with
organic solvents, the reaction mixture was simply purified by gel
filtration chromatography on a G25 Sephadex column, eluted with
H2O/EtOH 4:1 (v/v), giving the pure target compound 7 in 80%
yields. Following the described procedures, 7 was prepared in
seven steps and 46% overall yield from commercially available 50-O-
DMT-uridine 1.
O
O
NH
NH
N
O
N
O
DMTO
DMTO
a
O
O
OH OH
O
O
O
O
1
2
O
S
b
N
N
O
O
DMTO
S
O
N
c
OH OH
N
O
DMTO
4
O
d, e
O
O
O
O
3
O
O
S
O
NH
N
O
N
O
N
O
DMTO
DMTO
O
c
O
O
O
O
O
All the synthesized compounds were purified by column chro-
matography, in all cases allowing the isolation of homogeneous
compounds, as checked by HPLC analysis, and fully characterized by
1H and 13C NMR spectroscopy and MALDI and/or ESI-MS methods.
O
O
O
O
O
O
O
O
O
NH
6
5
f
N
O
HO
3. NMR analysis and structural studies
O
O
O
In order to elucidate the conformational features of synthesized
compound 7, a detailed NMR analysis was performed. The 1D 1H
NMR proton spectrum of 7 (700 MHz, D2O, T¼25 ꢀC) showed the
presence of signals at dH 7.90 (H6), 6.09 (H10), 5.89 (H5), 4.32 (H20),
4.23 (H40), 4.12 (H30), 3.97 and 3.83 ppm (H50a and H50b, re-
spectively). Furthermore, a severe overlapping of signals was ob-
served at dH 3.70 ppm, attributed to the methylene groups of the
crown ether moiety. Proton (700 MHz, T¼25 ꢀC) and carbon
(175 MHz, T¼25 ꢀC) assignments were obtained through an in-
depth analysis of two-dimensional COSY, HSQC and HMBC NMR
experiments. 2D NOESY experiments were also acquired and
studied in order to confirm the assignment and obtain structural
information. The NOE pattern was consistent with the assignment
we have done. Among others, we observed strong and diagnostic
NOE effects between H6 and H10, H20, H30, and between H50a/H50b
and H20 that could be indicative of the conformation adopted by 7.
Thus, these NOE contacts have been used as restraints in
structural calculations. On this basis, an initial structure of 7 was
generated and minimized to eliminate any possible conformational
bias. The dynamics started at 1000 K using the consistent valence
O
O
7
O
O
Scheme 1. a) Acetic anhydride, pyridine, rt, 12 h; (b) 2-(phenylthio)ethanol, ADDP, tri-
n-butylphosphine, benzene, 10 min at 0 ꢀC, then 18 h at rt; (c) 2 M NaOH in dioxane/
H2O, 1:1 (v/v), 50 ꢀC, 8 h; (d) NaH, penta(ethylene glycol) di-p-toluenesulfonate, THF,
reflux, 12 h; (e) m-CPBA, rt, 1 h; (f) 1% TFA in CH2Cl2 (v/v), rt, 1 h.
2. Synthesis
The overall synthetic procedure to obtain crown-ether ring-
fused derivative 7 is described in Scheme 1. A crucial prerequisite
for the selective alkylation of the 20 and 30-OH of a ribonucleoside is
the choice of suitable protecting groups for the nucleobase, which
have to be highly resistant to very basic media required for the
crown ether formation step. Previously, some of us had showed the
2-(phenylthio)ethyl group as a very efficient protection to tran-
siently mask the N3 position of thymidine, thus allowing easy
manipulation of the ribose moieties.13 2-(Phenylthio)ethyl is
a ‘safety catch’ protecting group, which can be removed in two

C. Coppola et al. / Tetrahedron 66 (2010) 6769e6774
6771
force field, as implemented in the Discover software (Accelrys, San
Diego, USA). Thereafter, the temperature was decreased stepwise to
250 K. The final step was again to energy-minimize in order to
refine the structures thus obtained, successively using the steepest
descent and the quasi-NewtoneRaphson (VA09A) algorithms.
Out of 200 structures generated, 50 structures with the lowest
energies were selected. As expected, the superimposition of
these 50 structures (Fig. 1) (RMSD¼0.03ꢁ0.03) revealed that the
nucleosidic core of the molecule is well structured, while a confor-
mational heterogeneity is observed for the crown ether moiety. The
lack of violations and the low RMSD value suggest that the mini-
mized conformation of 7 is consistent with the experimentally
determined restraints. Interestingly, the nucleobase strongly pre-
fers an anti glycosidic conformation. Furthermore, whereas the
addition of sodium or potassium ions can only be explained as-
suming that this nucleoside is already coordinated with one sodium
ion, presumably incorporated during the NaH-promoted cyclization
step to give 5 and never lost in the successive synthetic steps.18 On
this basis, new sets of calculation were successively performed on 7
imposing the presence of one sodium ion. Particularly, the calcula-
tions were carried out by using the same constraints and procedures
used for those performed in the absence of the cation. Furthermore,
we have used additional distance restraints in the range of 2.4e5.0 Å
between the oxygens of the crown ether and the sodium ion. The
width of this range, if necessary, guarantees the Naþ ion to freely
move towards other functional groups of the molecule and, at the
same time, to remain in the proximity of the crown ether. Also in this
case, out of 200 structures generated, 50 structures with the lowest
energies were selected. The superimposition of these 50 structures
(Fig. 2) turned out to be excellent, having a root mean square de-
viation (RMSD) value of 0.03ꢁ0.02. The lack of violations and the low
RMSD value suggest that the minimized conformation of 7 in the
presence of sodium is also consistent with the experimentally de-
termined restraints. This superimposition clearly shows that the
sugar and the uracil base possess the same relative orientation in all
the structures (Fig. 2, panel A), which is also perfectly
Figure 1. Superimposition of the best 50 structures of 7 in the absence of coordinated
cations. The molecule is depicted in coloured ‘stick’ (carbons, green; oxygens, red;
hydrogens, white).
unmodified uridine is characterized by a balanced distribution of
the populations of ribose conformers between the North and South
conformation,14 it seems that the presence of the crown ether
strongly selects the N-type conformation (C20-exo) of the sugar. The
net prevalence of the N-type conformation has been also confirmed
by the coupling constants between H10 and H20, H20 and H30 and
H30 and H40, being 3.0, 5.3, 6.0 Hz, respectively. Using the
equation:15
%N ¼ J3040 =J1020 þ J2030
the population of the N-type conformation (%N) for 7 is found to be
72%, compared to 56% for uridine.16 VT-1H NMR experiments also
clearly indicated a high rigidity for the ribose moiety in 7, with no
detectable variation in the J values in the temperature range
288e328 K.
Taking into consideration the high binding abilities of 18-crown-6
ethers towards Naþ and Kþ, a series of 1H NMR-based titrations have
been performed. In all cases, when alkali metal cationsdi.e., sodium
and potassium, both given in the form of chlorides or per-
chloratesdwere progressively added to the host molecule solution
up to a large excess, no detectable changes, either at the level of the
chemical shifts or of J values, were observed in the 1H NMR spectra.
In analogy with the case of the carbohydrate-fused bis-crown ether
we have recently studied,17 the absence of effects in our system upon
Figure 2. Two different orientations of the superimposition of the best 50 structures of
7 in the presence of Naþ. The orientation showed in panel A highlights the perfect
superimposition of the base and sugar moieties. The structural heterogeneity of the
crown ether is reported in panel B. The four main conformations computed for 7 are
reported in red, yellow, blue and green.

6772
C. Coppola et al. / Tetrahedron 66 (2010) 6769e6774
superimposable to the structure obtained in the absence of Naþ
(Fig. 3). Nevertheless, in the first case, only four main conformations
have been found for the crown ether moiety (Fig. 2, panel B), very
similar to each other and with the sodium ion always fixed in the
same position.
5. Experimental section
5.1. Materials and methods
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 aqueous
solution. For column chromatography, silica gel from Merck (Kie-
selgel 40, 0.063e0.200 mm) was used. HPLC analyses were per-
formed on a Beckman System Gold instrument equipped with a UV
detector module 166 and a Shimadzu Chromatopac C-R6A in-
tegrator. By HPLC analysis on a Nucleosil 100-5 C18 Supelco ana-
lytical column (250ꢂ4.6 mm, 5
from 0 to 100% in 30 min of CH3CN in H2O, flow¼0.8 mL/min, de-
tection at
¼264 nm, all the synthesised compounds resulted to be
mm), eluted with a linear gradient
l
more than 98% pure. For the ESI-MS analyses a Waters Micromass
ZQ instrumentdequipped with an Electrospray sourcedwas used
in the positive and/or negative mode. MALDI TOF mass spectro-
metric analyses were performed on a PerSeptive Biosystems Voy-
ager-De Pro MALDI mass spectrometer in the Linear mode using
2,5-dihydroxybenzoic acid as the matrix. NMR spectra were
recorded on Bruker WM-400 or Varian INOVA 500 spectrometers.
All the chemical shifts are expressed in parts per million with re-
spect to the residual solvent signal; J values are in hertz. The fol-
lowing abbreviations were used to explain the multiplicities:
s¼singlet; d¼doublet; t¼triplet; q¼quartet; m¼multiplet;
b¼broad; dd¼double doublet. Peak assignments have been carried
out on the basis of standard 1He1H COSY and HSQC experiments.
Figure 3. Superimposition of the structures obtained for 7 without (yellow) and with
(magenta) the sodium ion.
Interestingly, the nucleobase is oriented in such a way that O2
points right towards the ion, providing an additional coordination
site for sodium binding (Fig. 3), thus resulting into a highly func-
tionalized lariat ether. The present results show that the 18-
membered macrocycle 18-crown-6 ether, when cis-fused to the 20
and 30 positions of a nucleoside moiety, is indeed able to partially
freeze the ribofuranose pseudorotational cycle, analogously to
three, four or five-membered ring-fused bicyclic nucleosides19 es-
sentially locking the sugar in a C30-endo (N-type) conformation.
5.2. Nuclear magnetic resonance
NMR spectra for compound 7 were determined in D2O (Aldrich,
Milwaukee, USA). The NMR samples were prepared at a concen-
tration range of 1.0e10.0 mM. Upon varying the concentration, no
significant change was observed in the shape or distribution of the
1H NMR resonances, thus allowing to exclude aggregation phe-
nomena under these conditions. NMR spectra were recorded with
a VarianUnity INOVA 700 MHz spectrometer. Phase sensitive NOESY
spectra20 were recorded with mixing times in the range
250e500 ms (T¼25 ꢀC). TOCSY spectrum21 with mixing time of
100 ms was recorded. ROESY and TOCSY were recorded using
TPPI22 procedure for quadrature detection. {1He13C}-HSQC23 and
4. Conclusions
The synthesis of a novel crown-ether ring-fused uridine de-
rivative is described here, realized through a simple, high yielding
procedure from 50-O-DMT-uridine. Key step allowing the straight-
forward manipulation of the ribose moieties was the selective
protection of the nucleobase with the two-stage protecting group
2-phenylthioethyl, inserted at N3 via a Mitsunobu reaction.
Interestingly, NMR-based conformational analysis studies car-
ried out on 7 show that the crown ether-fused ribofuranose pre-
dominantly adopts an N-type conformation, with the nucleobase in
an anti conformation about the N-glycosidic bond. Furthermore,
the well known cation binding properties of crown ethers could be
strongly enhanced in 7 because of the presence of uracil with the
O2 protruding towards the crown ether. This, in a sort of lariat
ether-structure, can provide an additional binding site for the co-
ordination of one sodium or potassium ion, thereby contributing to
a further stabilization of the cationecrown ether complex.
Preliminary biological evaluation on this modified nucleoside,
studied at a maximum concentration of 10 mM, showed no relevant
antiviral activity when tested against HIV-1 and a variety of other
viruses.
Future work will address the complete biological evaluation of
the synthesized compound, as well as its further elaboration into
a variety of amphiphilic conjugatesdthrough 50-derivatization
with different lipophilic moietiesdfor the development of poten-
tial artificial ionophores.
{1He13C}-HMBC24 were optimized for 1JCeH¼135 Hz and
2,3
J
¼10 Hz, respectively. In all the 2D experiments, time domain
CeH
data consisted of 2048 complex points in t2 and 400e512 fids in t1
dimension. The relaxation delay was kept at 1.2 s for all the ex-
periments. All the experiments were acquired at T¼25 ꢀC, and the
spectra were calibrated relative to HDO (4.75 ppm) as the internal
standard. The NMR data were processed on a SGI Octane work-
station using FELIX 98 software (Accelrys, San Diego, USA) and on
a iMAC using the software iNMR (www.inmr.net).
5.3. Structure calculations
Cross peak volume integrations were performed with the pro-
gram FELIX 98, using the NOESY experiment collected at mixing
time of 250 ms and with a relaxation delay of 3 s. The NOE volumes
were then converted to distance restraints after they were cali-
brated using known fixed distances of H50a and H50b of the ribose
and H5 and H6 of the base. Then an NOE restraint file was gener-
ated with three distance classifications as follows: strong NOEs
(1.5 Åꢃrijꢃ3.5 Å), medium NOEs (3.0 Åꢃrijꢃ4.5 Å) and weak NOEs
(4.0 Åꢃrijꢃ5.5 Å). A total of five NOEs derived distance restraints
were used. Three-dimensional structures, which satisfy NOE were
constructed by simulated annealing calculations. All the calcula-
tions used a distance dependant macroscopic dielectric constant of

C. Coppola et al. / Tetrahedron 66 (2010) 6769e6774
6773
80ꢂr and an infinite cut-off for nonbonded interactions to partially
compensate for the lack of the solvent.25 The initial structure of 7
was built using a completely random array of atoms. Using the
steepest descent followed by quasi-NewtoneRaphson method
(VA09A) the conformational energy was minimized. Restrained
simulations were carried out in vacuo for 42 ns at 1000 K using the
CVFF force field as implemented in Discover software (Accelrys, San
Diego, USA). Then, the temperature was decreased stepwise until
250 K. The final step was again to energy-minimize so to refine the
structures obtained, using successively the steepest descent and
the quasi-NewtoneRaphson (VA09A) algorithms. Both dynamic
and mechanic calculations were carried out by using a 10 (kcal/
mol)/Å2 flatwell restraints. A total of 200 structures were generated
for both calculations (with and without Naþ).
Na2SO4, was filtered, concentrated under reduced pressure and
purified by column chromatography eluted with CH2Cl2 containing
growing amounts of CH3OH (from 0 to 5%), in the presence of a few
drops of triethylamine. Desired compound 4 (550 mg, 0.80 mmol)
was isolated in a pure form in 97% yields.
Compound 4: oil, Rf¼0.5 [CH2Cl2/CH3OH, 97:3 (v/v)]. 1H NMR
(CDCl3, 500 MHz):
d
7.80 (1H, d, J¼8.0 Hz, H-6); 7.65e6.81 (18H,
overlapped signals, aromatic protons); 5.86 (1H, d, J¼3.5 Hz, H-10);
5.65 (2H, br s, exchangeable signal, 2ꢂ(OH)); 5.38 (1H, d, J¼8.0 Hz, H-
5); 4.37 (1H, t, J¼5.0 and 5.0 Hz, H-30); 4.24 (1H, apparent t, J¼3.5 and
4.0 Hz,
H-20);
4.19e4.14
(3H,
overlapped
signals,
(PheSeCH2eCH2eNeUridine) and H-40); 3.47e3.43 (2H, m, H2-50);
3.16 (2H, apparent t, J¼8.0 and 7.5 Hz, PheSeCH2eCH2eNeUridine).
13C NMR (CDCl3, 50 MHz):
d 162.3 (C-4); 151.0 (C-2); 144.2 (C-6);
Illustrations of structures were generated using the INSIGHT II
program, version ’98 (Accelrys, San Diego, USA). All the calculations
were performed on a PC running Linux RedHat Enterprise WS 4.0.
158.5, 138.1, 135.2, 129.9, 128.8, 128.4, 128.0, 127.8, 126.9, 125.8 and
113.1 (aromatic carbons); 101.3 (C-5); 91.0 (C-10); 86.8 (quaternary
carbon of DMTgroup); 83.9 (C-40); 75.2 (C-20); 69.7 (C-30); 62.1 (C-50);
55.0 (2ꢂ(OCH3 of DMT group)); 40.4 (PheSeCH2eCH2eNeUridine);
29.7 (PheSeCH2eCH2eNeUridine). ESI-MS (positive ions): calcd for
C38H38N2O8S, 682.2349; m/z, found 704.33 ([MþNa]þ, 100); 720.23
([MþK]þ, 40). HRMS (ESI-MS, positive ions): calcd for
C38H38N2O8SNa, 705.2247; m/z, found 705.2222 [MþNa]þ.
5.3.1. Synthesis of 3. 50-O-DMT-uridine 1 (500 mg, 0.92 mmol) was
treated with anhydrous pyridine (3 mL) and acetic anhydride
(1.5 mL). The reaction mixture was left under stirring at room tem-
perature. After 12 h, the reaction mixture was concentrated under
reduced pressure, diluted with CH2Cl2, transferred into a separatory
funnel and washed twice with water. The organic phase, dried over
anhydrous Na2SO4, was filtered, concentrated under reduced pres-
sure and purified by column chromatography. Elution of the column
with CH2Cl2 containing growing amounts of CH3OH (from 0 to 5%), in
the presence of a few drops of triethylamine, allowed desired com-
pound 2 (576 mg, 0.92 mmol) in almost quantitative yields.
5.3.3. Synthesis of 5. Compound 4 (550 mg, 0.80 mmol), dissolved
in anhydrous THF (17 mL) was treated with NaH (60% dispersion in
mineral oil, 96 mg, 2.40 mmol); after 10 min, pentaethylene glycol
di-p-toluenesulfonate (655 mg, 1.2 mmol) was added and the re-
action mixture was left under stirring at room temperature. After
20 min, anhydrous THF (12 mL) was added and the reaction mix-
ture was left at reflux for 12 h. Upon consumption of the starting
nucleoside, m-CPBA (414 mg, 2.4 mmol) was added to the mixture
and the reaction left at rt, under stirring for 1 h. The reaction
mixture was then concentrated under reduced pressure, redis-
solved in CH2Cl2 and washed twice with a satd. NaHCO3 solution in
water. The organic phase, dried over anhydrous Na2SO4, was fil-
tered, concentrated under reduced pressure and purified by col-
umn chromatography. Eluting the column with CH2Cl2 containing
growing amounts of CH3OH (from 0 to 5%), in the presence of a few
drops of triethylamine, gave pure compound 5 (512 mg, 0.56 mmol)
in 70% yield for the two steps.
Compound 2 (576 mg, 0.92 mmol) was dissolved in anhydrous
benzene (3 mL) at 0 ꢀC and treated with 2-(phenylthio)ethanol
(100 mL, 0.92 mmol) and n-tributylphosphine (270 mL, 1.1 mmol).
After 10 min, the reaction mixture was taken to rt, treated with
ADDP (280 mg, 1.1 mmol) and left, under stirring, at rt for 18 h. The
solution was then taken to dryness, redissolved in ethyl acetate and
washed twice with water. The organic layer was concentrated un-
der reduced pressure and purified by silica gel chromatography.
Eluting the column with n-hexane/ethyl acetate, 3:2 (v/v), in the
presence of a few drops of triethylamine, furnished desired com-
pound 3 in a pure form (639 mg, 0.82 mmol) in 89% yield.
Compound 3: oil, Rf¼0.3 [n-hexane/ethyl acetate, 7:3 (v/v)]. 1H
Compound 5: oil, Rf¼0.3 [CH2Cl2/CH3OH, 97:3 (v/v)]. 1H NMR
NMR (CDCl3, 500 MHz):
d
7.67 (1H, d, J¼7.5 Hz, H-6); 7.44e6.83
(CDCl3, 500 MHz):
d
7.97 (1H, d, J¼7.0 Hz, H-6); 7.68e6.82 (18H,
(18H, overlapped signals, aromatic protons); 6.22 (1H, d, J¼5.5 Hz,
H-10); 5.59e5.54 (2H, overlapped signals, H-20 and H-30); 5.37 (1H,
d, J¼7.5 Hz, H-5); 4.23 (1H, m, H-40); 4.17e4.13 (2H, m,
PheSeCH2eCH2eNeUridine); 3.78 (6H, s, 2ꢂ(OCH3 of DMT
group)); 3.52e3.45 (2H, m, H2-50); 3.15 (2H, t, J¼7.5 and 7.0 Hz,
PheSeCH2eCH2eNeUridine); 2.10 (6H, s, 2ꢂ(CH3C]O)). 13C NMR
overlapped signals, aromatic protons); 5.87 (1H, br s, H-10); 5.73
(1H, d, J¼8.0 Hz, H-5); 4.53 (1H, apparent t, J¼4.5 and 4.0 Hz, H-20);
4.37 (1H, t, J¼5.0 and 5.0 Hz, H-30); 4.26 (2H, t, J¼7.0 and 7.0 Hz,
PheSO2eCH2eCH2eNeUridine); 4.21 (1H, m, H-40); 3.98e3.57
(22H, overlapped signals, 5ꢂ(OeCH2eCH2eO) and H2-50); 3.49 (2H,
t, J¼7.0 and 7.0 Hz, PheSO2eCH2eCH2eNeUridine). 13C NMR
(CDCl3, 125 MHz):
d
169.5 and 169.4 (2ꢂ(CH3C]O)); 162.0 (C-4);
(CDCl3, 125 MHz): d 161.9 (C-4); 150.1 (C-2); 144.3 (C-6); 158.5,
150.8 (C-2); 143.7 (C-6); 158.6, 137.6, 135.4, 134.9, 134.8, 134.6,
130.0, 129.8, 128.8, 128.4, 127.9, 127.1, 126.3, 125.7 and 113.2 (aro-
matic carbons); 102.2 (C-5); 87.3 (quaternary carbon of DMT
group); 86.4 (C-10); 81.6 (C-40); 73.0 (C-20); 70.7 (C-30); 62.2 (C-50);
55.1 (2ꢂ(OCH3 of DMT group)); 40.6 (PheSeCH2eCH2eNeUridine);
29.5 (PheSeCH2eCH2eNeUridine); 20.4 and 20.3 (2ꢂ(CH3C]O)).
MALDI-MS (positive ions): calcd for C42H42N2O10S, 766.2560; m/z,
found 789.13 ([MþNa]þ, 100); 805.08 ([MþK]þ, 45); 486.26
([(MꢄDMT)þNa]þ, 10); 502.45 ([(MꢄDMT)þK]þ, 5). HRMS (ESI-MS,
positive ions): calcd for C42H42N2O10SNa, 789.2458; m/z, found
789.2439 [MþNa]þ.
138.9, 138.4, 135.3, 135.1, 133.7, 129.9, 129.6, 129.1, 128.0, 127.8, 126.9
and 113.1 (aromatic carbons); 100.9 (C-5); 89.0 (C-10); 86.7 (C-40
and quaternary carbon of DMT group); 80.9 (C-20); 75.3 (C-30); 70.6,
70.4, 70.3, 69.6 and 69.3 (5ꢂ(OeCH2eCH2eO)); 60.7 (C-50); 55.0
(OCH3 of DMT group); 52.4 (PheSO2eCH2eCH2eNeUridine); 34.6
(PheSO2eCH2eCH2eNeUridine). MALDI-MS (positive ions): calcd
for C48H56N2O14S, 916.3452; m/z, found 938.94 ([MþNa]þ, 100);
954.93 ([MþK]þ, 30); 636.68 ([(MꢄDMT)þNa]þ, 10); 652.77
([(MꢄDMT)þK]þ, 5). HRMS (ESI-MS, positive ions): calcd for
C38H38N2O8SNa, 939.3350; m/z, found 939.3319 [MþNa]þ.
5.3.4. Synthesis of 6. Compound
5 (512 mg, 0.56 mmol) was
5.3.2. Synthesis of 4. Compound
3
(639 mg, 0.82 mmol) was
treated with 5 mL of a 2 M solution of NaOH in dioxane/H2O,1:1 (v/v)
and left for 8 h at 50 ꢀC. The reaction mixture was then con-
centrated under reduced pressure, redissolved in CH2Cl2 and
washed twice with water. The organic phase, dried over anhydrous
Na2SO4, was filtered, concentrated under reduced pressure and
treated with 5 mL of a 2 M solution of NaOH in dioxane/H2O,1:1 (v/v)
and left for 8 h at 50 ꢀC. Then the reaction mixture was con-
centrated under reduced pressure, redissolved in CH2Cl2 and
washed twice with water. The organic phase, dried over anhydrous

6774
C. Coppola et al. / Tetrahedron 66 (2010) 6769e6774
purified by column chromatography. Eluting the column with
CH2Cl2 containing growing amounts of CH3OH (from 0 to 5%), in
the presence of a few drops of triethylamine, gave pure desired
compound 6 (400 mg, 0.53 mmol) in 95% yields.
References and notes
1. Molotov, Y. A. Macrocyclic Compounds in Analytical Chemistry; John Wiley &
Sons: New York, NY, 1997 and literature cited therein.
2. For example, see: Yongzhu, J.; Hirose, K.; Nakamura, T.; Nishioka, R.; Ueshige, T.;
Tobe, Y. J. Chromatogr., A 2006, 1129, 201e207.
3. For a review, see: (a) Miethchen, R.; Fehring, V. Liebigs Ann. 1997, 553e561; For
more recent examples, see: (b) Yamanoi, T.; Oda, Y.; Muraishi, H.; Matsuda, S.
Molecules 2008, 13, 1840e1845; (c) Bakó, P.; Makó, A.; Keglevich, G.; Menyhárt,
D. K.; Sefcsik, T.; Fekete, J. J. Inclusion Phenom. Macrocyclic Chem. 2006, 55,
295e302.
4. Larpent, C.; Laplace, A.; Zemb, T. Angew. Chem., Int. Ed. 2004, 43, 3163e3167.
5. Morin, G. T.; Smith, B. D. Tetrahedron Lett. 1996, 37, 3101e3104.
6. Dessolin, J.; Galea, P.; Vlieghe, P.; Chermann, J.-C.; Kraus, J.-L. J. Med. Chem. 1999,
42, 229e241.
Compound 6: oil, Rf¼0.3 [CH2Cl2/CH3OH, 95:5 (v/v)]. 1H NMR
(CDCl3, 500 MHz):
d
8.09 (1H, d, J¼8.0 Hz, H-6); 7.92e6.83 (13H,
overlapped signals, aromatic protons); 5.94 (1H, br s, H-10); 5.29
(1H, d, J¼8.0 Hz, H-5); 4.35 (1H, m, H-30); 4.23e4.21 (2H, br s, H-20
and
H-40);
3.96e3.59
(20H, overlapped signals,
5ꢂ
(OeCH2eCH2eO)); 3.79 (6H, s, 2ꢂ(OCH3)); 3.56e3.45 (2H, m, H2-
50). 13C NMR (CDCl3, 125 MHz):
d
162.7 (C-4); 149.6 (C-2); 144.3 (C-
6); 158.5, 140.2, 135.2, 135.0, 130.0, 129.4, 128.0, 127.9, 127.0 and
113.1 (aromatic carbons); 101.7 (C-5); 88.3 (C-10); 86.8 (quaternary
carbon of DMT group); 81.0 (C-40); 80.8 (C-20); 75.4 (C-30); 70.6,
70.4, 70.3, 69.7 and 69.4 (5ꢂ(OeCH2eCH2eO)); 60.7 (C-50); 55.1
(OCH3 of DMT group). MALDI-MS (positive ions): calcd for
C40H48N2O12, 748.3207; m/z, found 468.49 ([(MꢄDMT)þNa]þ, 100);
484.45 ([(MꢄDMT)þK]þ, 30). HRMS (ESI-MS, positive ions): calcd
for C40H48N2O12Na, 771.3105; m/z, found 771.3119 [MþNa]þ.
7. Rohr, K.; Vogel, S. ChemBioChem 2006, 7, 463e470.
8. Milecki, J.; Schroeder, G. Pol. J. Chem. 2005, 79, 1781e1785.
9. Malek, K.; Podstawka, E.; Milecki, J.; Schroeder, G.; Proniewicz, L. M. Biophys.
Chem. 2009, 142, 17e26.
10. For recent reviews, see: (a) Modified Nucleosides; Herdewijn, P., Ed.; Wiley-VCH:
Weinheim, Germany, 2008; (b) Mathè, C.; Perigaud, C. Eur. J. Org. Chem. 2008, 9,
1489e1505; (c) Len, C.; Mondon, M.; Lebreton, J. Tetrahedron 2008, 64,
7453e7475; (d) Ichikawa, E.; Kato, K. Curr. Med. Chem. 2001, 8, 385e423; For
recent articles on conformationally constrained uridine or thymidine ana-
logues, see for example: (e) Enderlin, G.; Taillefumier, C.; Didierjean, C.;
Chapleur, Y. J. Org. Chem. 2009, 74, 8388e8391; (f) Mieczkowski, A.; Blu, J.; Roy,
V.; Agrofoglio, L. A. Tetrahedron 2009, 65, 9791e9796; (g) Bonache, M.-C.;
Cordeiro, A.; Carrero, P.; Quesada, E.; Camarasa, M.-J.; Jimeno, M.-L.; San-Felix,
A. J. Org. Chem. 2009, 74, 9071e9081.
11. (a) Herdewijn, P. Biochim. Biophys. Acta 1999, 1489, 167e179; (b) Cobb, A. J. A.
Org. Biomol. Chem. 2007, 5, 3260e3275; (c) Kaur, H.; Babu, B. R.; Maiti, S. Chem.
Rev. 2007, 107, 4672e4697.
12. Coppola, C.; D’Onofrio, J.; De Napoli, L.; Di Fabio, G.; Montesarchio, D. Nucleic
Acids Symp. Ser. 2008, 52, 667e668.
13. D’Onofrio, J.; De Napoli, L.; Di Fabio, G.; Montesarchio, D. Synlett 2006,
845e848.
14. (a) Blackburn, B. J.; Grey, A. A.; Smith, I. C. P.; Hruska, F. E. Can. J. Chem. 1970, 48,
2866e2870; (b) Neumann, J. M.; Bernassau, J. M.; Gukron, M.; Tran-Dinh, S. Eur.
J. Biochem. 1980, 108, 457e463.
15. Altona, C. Recl. Trav. Chim. Pays-Bas 1982, 101, 413e433.
16. Sejo, K.; Tawarada, R.; Sasami, T.; Serizawa, M.; Ise, M.; Ohkubo, A.; Sekine, M.
Bioorg. Med. Chem. 2009, 17, 7275e7280.
17. Coppola, C.; Virno, A.; De Napoli, L.; Randazzo, A.; Montesarchio, D. Tetrahedron
2009, 65, 9694e9701 and literature cited therein.
18. Competitive binding assays carried out by ESI-MS gave indirect, but clear
support to this hypothesis. When 7 was analyzed in bidistilled water, [MþNa]þ
was the prevailing m/z signal (100%), with [MþK]þ as the unique accompanying
ion (60%). When dissolved in a 0.1 M KCl solution, exclusively [MþK]þ was
observed, thus showing complete displacement of sodium by potassium ion,
with known higher affinity for 18-crown-6 moiety. On the contrary, when
dissolved in 0.1 M triethylammonium bicarbonate, [MþEt3NH]þ could not be
detected, even in traces, and [MþNa]þ and [MþK]þ were found almost in the
same ratio as in pure water. Since the big triethylammonium ion cannot dis-
place sodium or potassium, this result points to a ca. 100% complexation of the
crown ether and almost complete absence of unbound molecules, and can be
taken as an indirect evidence of the presence of sodium (and, in minor per-
centage, of potassium) fully complexing the crown ether ring in 7.
19. For a review on bicyclic nucleoside analogues, see: Meldgaard, M.; Wengel, J.
J. Chem. Soc., Perkin Trans. 1 2000, 21, 3539e3554.
5.3.5. Synthesis of 7. Compound
6 (50 mg, 0.067 mmol) was
treated with a 1% TFA solution in CH2Cl2 (v/v, 1 mL total volume)
under stirring at room temperature for 1 h. The reaction mixture
was then concentrated under reduced pressure, redissolved in
diethyl ether and washed twice with distilled water. The aqueous
phase was concentrated under reduced pressure and purified on
a Sephadex G25 column eluted with H2O/EtOH, 4:1 (v/v). From UV
spectrophotometric measurements, fractions absorbing at
l
¼264 nm were collected and taken to dryness, yielding pure 7
(24 mg, 0.053 mmol) with 80% yields.
Compound 7: colourless oil, Rf¼0.3 [CH2Cl2/CH3OH, 7:3 (v/v)]. 1H
NMR (D2O, 700 MHz, 25 ꢀC):
d
7.90 (1H, d, J¼8.0 Hz, H-6); 6.09 (1H,
d, J¼3.0 Hz, H-10); 5.89 (1H, d, J¼8.0 Hz, H-5); 4.32 (1H, dd, J¼5.3
and 3.0 Hz, H-20); 4.23 (1H, m, H-40); 4.12 (1H, dd, J¼5.3 and 6.0 Hz,
H-30); 3.97 (1H, AB part of an ABX system, J¼3.0 and 12.0 Hz, H-5a );
0
0
3.83 (1H, m, H-5b ); 3.80e3.69 (20H, overlapped signals, 5ꢂ
(OeCH2eCH2eO)). 13C NMR (D2O, 125 MHz):
d 161.7 (C-4); 152.0
(C-2); 143.4 (C-6); 104.8 (C-5); 90.5 (C-10); 84.3 (C-40); 82.4 (C-20);
78.6 (C-30); 72.4, 72.3, 72.1, 72.0, 71.8 and 71.7 (5ꢂ
(OeCH2eCH2eO)); 63.0 (C-50). MALDI-MS (positive ions): calcd for
C19H30N2O10, 446.1900; m/z, found 468.90 ([MþNa]þ, 100); 484.85
([MþK]þ, 25). HRMS (ESI-MS, positive ions): calcd for
C19H30N2O10Na, 469.1798; m/z, found 469.1821 [MþNa]þ.
Acknowledgements
20. Jeener, J.; Meier, B.; Bachmann, H. P.; Ernst, R. R. J. Chem. Phys. 1979, 71,
4546e4553.
We thank MIUR (PRIN) 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. We also thank Dr.
Jan Balzarini, from Rega Institute for Medical Research, Katholieke
Universiteit Leuven, Leuven, Belgium, for the antiviral experiments.
21. Braunschweiler, L.; Ernst, R. R. J. Magn. Reson. 1983, 53, 521e528.
22. Marion, D.; Wuthrich, K. Biochem. Biophys. Res. Commun. 1983, 113, 967e974.
23. Kay, L. E.; Keifer, P.; Saarinen, T. J. Am. Chem. Soc. 1992, 114, 10663e10665.
24. Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093e2094.
25. Weiner, S. J.; Kollman, P. A.; Case, D. A.; Singh, U. C.; Ghio, C.; Alagona, G.;
Profeta, S.; Weiner, P. J. J. Am. Chem. Soc. 1984, 106, 765e784.