Synthesis and biological properties of 5-(1H-1,2,3-triazol-4-yl)isoxazolidines: A new class of C-nucleosides

Article abstract of DOI:10.3390/molecules20045260

A novel series of C-nucleosides, featuring the presence of a 1,2,3-triazole ring linked to an isoxazolidine system, has been designed as mimetics of the pyrimidine nucleobases. An antiproliferative effect was observed for compounds 17a and 17b: the growth

Full text of DOI:10.3390/molecules20045260

Molecules 2015, 20, 5260-5275; doi:10.3390/molecules20045260
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Properties of 5-(1H-1,2,3-Triazol-4-
yl)isoxazolidines: A New Class of C-Nucleosides
Salvatore V. Giofrè 1,*, Roberto Romeo 1,*, Caterina Carnovale 1,†, Raffaella Mancuso 2,†
Santa Cirmi 1,†, Michele Navarra 1,†, Adriana Garozzo 3,† and Maria A. Chiacchio 4,†
,
1
Dipartimento Scienze del Farmaco e Prodotti per la Salute, Università di Messina,
Via S.S. Annunziata, Messina 98168, Italy; E-Mails: ccarnovale@unime.it (C.C.);
scirmi@unime.it (S.C.); mnavarra@unime.it (M.N.)
2
Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Via P. Bucci, 12/C,
Arcavacata di Rende (CS) 87036, Italy; E-Mail: raffaella.mancuso@unical.it
Dipartimento di Scienze Bio-mediche, Università di Catania, Via Androne 81, Catania 95124, Italy;
E-Mail: agar@unict.it
3
4
Dipartimento Scienze del Farmaco, Università di Catania, Viale A. Doria 6, Catania 95125, Italy;
E-Mail: ma.chiacchio@unict.it
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: sgiofre@unime.it (S.V.G.);
robromeo@unime.it (R.R.); Tel.: +39-090-356230 (S.V.G. & R.R.);
Fax: +39-090-676-6562 (S.V.G. & R.R).
Academic Editors: Mahesh K. Lakshman and Fumi Nagatsugi
Received: 4 February 2015 / Accepted: 13 March 2015 / Published: 24 March 2015
Abstract: A novel series of C-nucleosides, featuring the presence of a 1,2,3-triazole ring
linked to an isoxazolidine system, has been designed as mimetics of the pyrimidine
nucleobases. An antiproliferative effect was observed for compounds 17a and 17b: the
growth inhibitory effect reaches the 50% in HepG2 and HT-29 cells and increases up to
56% in the SH-SY5Y cell line after 72 h of incubation at a 100 µM concentration.
Keywords: vinyl triazoles; C-Nucleosides; 1,3-dipolar cycloaddition; antiproliferative
activity; microwave

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1. Introduction
Structural modification of natural N-nucleosides on either the sugar unit or the nucleobase has
led to the discovery of a variety of new therapeutic agents, which includes antiviral and anticancer
agents [1–9]. In this context, the family of C-nucleosides constitutes a group of molecules
characterized by the link of the sugar unit to a carbon atom of the pyrimidine/purine nucleobase: some
of these compounds have been reported to exhibit significant antibacterial, antiviral and antitumoral
activities [10].
While natural and synthetic N-nucleosides are vulnerable to enzymatic and acid-catalyzed
hydrolysis of nucleosidic bond, the C-analogues are much more stable. Many natural and synthetic
C-nucleosides, also containing modified heterocyclic bases, biologically active, have been described in
literature. Natural antibiotics formycins [11] (in particular FA, FB, and oxoformycin B OFB) have
been known since the early 1960s to possess antibiotic and cytotoxic properties; their antiparasitic
activity (antimalarial, antischisostoma, etc.) was unraveled later. Pseudouridine is the most abundant
natural C-nucleoside present in most tRNA and rRNA structures [12], where it has been shown to
stabilize RNA duplex [13,14]. Showdomycin [15] possesses well known antibiotic and cytotoxic
properties, involving inhibition of nucleoside transport into the cell [16]. Pyrazofurin [17] and
tiazofurin [18] have been shown to possess a wide range of medicinal properties, including antibiotic,
antiviral, and antitumor activity (Figure 1).
Figure 1. Examples of C-Nucleosides.
As part of ongoing efforts to identify new antiviral/antitumor agents, we were interested in the
investigation of N,O-nucleosides, where the ribose moiety has been replaced by an isoxazolidine
system, as mimetic of the sugar unit [19–23]. Phosphonated carbocyclic 2'-oxa-3'-azanucleosides 1
have shown to be potent inhibitors of RT of different retroviruses, following incubation with human
PBMCs crude extract [24–26]; truncated phosphonated azanucleosides 2 are able to inhibit HIV

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and HTLV-1 viruses at concentration in the nanomolar range [27]; truncated phosphonated
N,O-psiconucleosides 3 inhibit HIV in vitro infection with low or absent cytotoxicity (Figure 2) [28].
Figure 2. Phosphonated N,O-nucleosides.
New base-modified N,O-nucleosides have been also synthesized. N,O-pseudouridine 4 [29]
and N,O-tiazofurin 5 [30] are characterized by a C-C bond between the isoxazolidine and the
heterocyclic systems.
Recently, 1,2,3-triazolyl N,O-nucleosides have been designed: 3-hydroxymethyl-5-(1H-1,2,3-
triazol)isoxazolidine 6 are able to inhibit proliferation of follicular and anaplastic human thyroid
cancer cell lines, with IC50 values ranging from 3.87 to 8.76 mM (Figure 3) [31]. In the same context,
novel 1,2,3-triazole-appended N,O-nucleoside analogs 7 were developed [32]: some of these compounds
show a good anticancer activity against the follicular (FTC-133), the anaplastic (8305C) human thyroid
cancer cell lines, and especially on the U87MG human primary glioblastoma cell line (Figure 3).
Figure 3. Examples of N,O-Nucleosides.
According to these promising results, we have developed a novel series of C-nucleosides featured
by the presence of a 1,2,3-triazole ring, linked to an isoxazolidine system, as mimetic of the pyrimidine
nucleobases. We report in this paper the synthesis of 5-(1H-1,2,3-triazol-4-yl)isoxazolidines 8 and
their biological activities as antiviral and/or antitumoral agents.

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2. Results and Discussion
C-Vinyl triazoles 12a–g have been prepared according to Scheme 1 [33]. Thus, 3-butyn-1-ol 10 was
reacted with a variety of azides 9a–g, by click chemistry reactions, performed in H2O/tert-BuOH (1:1)
in the presence of sodium ascorbate, copper(II) sulfate and TEA at room temperature. The obtained
triazole derivatives have been tosylated and then converted into vinyl triazoles 12a–g by reaction with
potassium tert-butoxide in tert-butanol (78%–90% yields).
Reagents and conditions: (a) CuSO4·5H2O, sodium ascorbate, TEA, 4 h, rt; (b) tosyl chloride, TEA,
CH2Cl2, rt, 12 h; (c) t-BuOK, t-BuOH, 40 °C, 12 h.
Scheme 1. Synthesis of C-Vinyl triazoles 12a–g.
The 1,3-dipolar cycloaddition of 1-substituted-4-vinyl-1,2,3-triazoles 12a–g with C-[(tert-
butyldiphenylsilyl)oxy]-N-methylnitrone 13 [34,35], at 150 W, 80 °C for 2 h in CHCl3, proceeded with
a good yield and a complete regioselectivity to give a mixture of trans/cis isoxazolidines 14a–g and
15a–g in a 1:1.3 relative ratio (global yield 80%–85%). Removal of the TBDPS protecting group was
accomplished under standard conditions, by treating the diastereomeric mixture with TBAF in THF, to
afford the triazolyl nucleosides 16a–f and 17a–f, which were separated by silica gel chromatography
(Scheme 2, Table 1).
1
The diastereomeric ratio of the products was determined by H NMR spectroscopy of the crude
reaction mixture, whereas the relative configuration was assigned by NOEDS spectra. In particular, in
the cis derivative 17a, chosen as model compound, a positive NOE effect observed for H-4' and H-5'b
(the downfield resonance of protons at C-5', 2.91 ppm) upon irradiation of H-1'(δ = 5.25 ppm), is
clearly indicative of their cis relationship. Analogously, irradiation of H-4'(δ = 3.25 ppm) in the same
compound gives rise to an enhancement in the signals corresponding to H-1' and H-5'b (δ = 5.25 and
2.91 ppm, respectively). On the contrary, no NOE effect was detected between H-4' and H-1' in
compound 16a.
The absence of cis/trans diastereoselectivity can be rationalized by assuming that the E-endo attack
of the dipolarophile on the nitrone, which leads to cis adducts, competes efficiently with the E-exo
attack, the preferred reaction pathway (steric control) leading to trans adducts, because of secondary
orbital interactions exerted by the triazole ring. This behavior is also in agreement with literature
data [36].

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Reagents and conditions: (a) CHCl3, 150 W, 80 °C, 2 h, 85%; (b) TBAF, THF, r.t., 4–5 h, global
yield 93%–96% ratio trans/cis 1:1.3 or 1:1.
Scheme 2. Synthesis of isoxazolidinyl-triazoles 16a–g and 17a–g by 1,3-dipolar cycloaddition.
Table 1. Vinyl triazoles 12a–g and isoxazolidinyl-triazoles 16a–g and 17a–g produced via
Schemes 1 and 2.
R
Vinyl-Triazole Yield % Product Ratio trans:cis Yield % a
16a
12a
88
1:1.3
92
17a
16b
17b
16c
17c
12b
12c
85
78
1:1.3
1:1.3
94
93
16d
17d
12d
12e
82
85
1:1
93
93
16e
17e
1:1.3
16f
17f
12f
81
90
1:1.3
1:1
95
96
16g
17g
12g
a Combined yield.

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3. Experimental Section
3.1. General Information
Solvents and reagents were used as received from commercial sources. Melting points were
determined with a Kofler apparatus (Fisher Scientific, Loughborough, UK). Elemental analyses were
performed with a Perkin–Elmer elemental analyzer (PerkinElmer, Waltham, MA, USA). NMR spectra
(1H-NMR recorded at 500 MHz, 13C-NMR recorded at 125 MHz) were obtained with a Varian
instrument (Agilent Technologies, Palo Alto, CA, USA), and data are reported in ppm relative to
tetramethylsilane. Thin-layer chromatographic separations were carried out on Merck silica gel
60-F254 precoated aluminum plates (Merk, Darmstadt, Germany). Flash chromatography was carried
out using Merck silica gel (200–400 mesh). Preparative separations were carried out using an Büchi
C-601 MPLC instrument (BUCHI Italia S.r.l., Milano, Italy) using Merck silica gel 0.040–0.063 mm,
and the eluting solvents were delivered by a pump at the flow rate of 3.5–7.0 mL/min.
C-[(tert-Butyldiphenylsilyl)oxy]-N-methyl nitrone was prepared according to described procedures [34,35].
Benzyl/alkyl and aromatic azides were synthesized according to literature procedures [32].
3.2. General 1,3-Dipolar Cycloaddition Procedure
A solution of 12a (0.50 g, 2.92 mmol) and nitrone 13 (1.10 g, 3.50 mmol) in CHCl3 (5 mL) was put
in a sealed tube and irradiated under microwave conditions at 150 W, 80 °C, for 2 h. The removal of
the solvent in vacuo afforded a crude material which, after flash chromatography purification by using
as eluent a mixture of cyclohexane/ethyl acetate 7:3, gave the unseparable mixture (trans/cis) of
compound 14a and 15a, as yellow oil, that was used for the next reaction, yield 1.24 g (85%). The
1H-NMR spectrum of the crude reaction mixture shows the presence of trans and cis isomers in 1:1.3
ratio, respectively. Compounds 14b–g and 15b–g were prepared by the 1,3-dipolar cycloaddition
procedure in 80%–85% yield as yellow oil and then used for the next reaction.
3.3. General Desilylation of the Hydroxymethyl Group Procedure: Synthesis of 16 and 17
A solution of compounds 14a and 15a (1.24 g, 2.49 mmol) and TBAF (0.90 mL, 3.73 mmol) in
freshly distilled THF (30 mL) was stirred until desilylation was completed (TLC, 4–5 h). Volatiles
were flash evaporated, and the residue was purified by MPLC (CH2Cl2/MeOH, 98:2) to afford 17a–g
first eluted isomer (trans) and 16a–g second eluted isomer (cis) in 92% global yield.
((3RS,5RS)-2-Methyl-5-(1-phenyl-1H-1,2,3-triazol-4-yl)isoxazolidin-3-yl) methanol (16a): White solid,
1
52% yield, mp = 57–59 °C. H-NMR (CDCl3): δ = 8.00 (s, 1H), 7.70 (d, J = 7.7 Hz, 2H), 7.50 (t,
J = 7.8 Hz, 2H), 7.42 (t, J = 7.4 Hz, 1H), 5.52 (dd, J = 8.5, 6.6 Hz, 1H), 3.66–3.52 (m, 2H), 3.31–3.19
13
(m, 1H), 2.94 (dt, J = 12.8, 8.5 Hz, 2H), 2.81 (s, 3H), 2.36 (ddd, J = 12.8, 6.6, 4.8, 1H). C-NMR
(CDCl3): δ = 148.50, 137.07, 129.86, 128.95, 120.66, 119.84, 71.06, 69.53, 63.42, 44.94, 36.62; Anal.
Calcd for C13H16N4O2: C, 59.99; H, 6.20; N, 21.52; found C, 60.04; H, 6.27; N, 21.60.
((3RS,5SR)-2-Methyl-5-(1-phenyl-1H-1,2,3-triazol-4-yl)isoxazolidin-3-yl) methanol (17a): White solid,
1
40% yield, mp = 100–102 °C. H-NMR (CDCl3): δ = 7.97 (s, 1H), 7.71 (d, J = 7.8 Hz, 2H), 7.51 (t,

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J = 7.8 Hz, 2H), 7.43 (t, J = 7.4 Hz, 1H), 5.25 (t, J = 8.0 Hz, 1H), 3.68 (ddd, J = 17.2, 11.4, 4.8 Hz,
2H), 3.25–3.18 (m, 1H), 2.98–2.85 (m, 1H), 2.83 (s, 3H), 2.62 (ddd, J = 12.7, 8.0, 5.0 Hz, 1H).
13C-NMR (CDCl3): δ = 148.15, 137.06, 129.89, 128.99, 120.72, 120.49, 72.39, 69.36, 62.35, 45.70,
36.78; Anal. Calcd for C13H16N4O2: C, 59.99; H, 6.20; N, 21.52; found C, 60.08; H, 6.26; N, 21.48.
((3RS,5RS)-5-(1-Benzyl-1H-1,2,3-triazol-4-yl)-2-methylisoxazolidin-3-yl) methanol (16b): Compound 16b
1
was prepared by the general desilylation procedure in 53.1% yield as yellow oil. H-NMR (CDCl3):
δ = 7.46 (s, 1H), 7.38–7.30 (m, 3H), 7.26–7.23 (m, 2H), 5.51–5.43 (m, 2H), 5.40 (dd, J = 8.2, 7.0 Hz,
1H), 3.55–3.46 (m, 2H), 3.23–3.14 (m, 1H), 2.84 (dt, J = 12.8, 8.4 Hz, 1H), 2.74 (s, 3H), 2.26 (ddd,
J = 12.8, 6.7, 4.7, 1H), 2.15 (bs, 1H). 13C-NMR (CDCl3): δ = 147.87, 134.48, 129.21, 128.88, 128.24,
121.55, 71.00, 69.48, 63.37, 54.32, 44.90, 36.44; Anal. Calcd for C14H18N4O2: C, 61.30; H, 6.61; N,
20.42; found C, 61.36; H, 6.67; N, 20.45.
((3RS,5SR)-5-(1-Benzyl-1H-1,2,3-triazol-4-yl)-2-methylisoxazolidin-3-yl) methanol (17b): Compound 17b
was prepared by the general desilylation procedure in 40.9% yield as white solid, mp = 117–119 °C.
1H-NMR (CDCl3): δ = 7.43 (s, 1H), 7.39–7.33 (m, 3H), 7.28–7.24 (m, 2H), 5.54–5.44 (m, 2H), 5.12 (t,
J = 7.9 Hz, 1H), 3.63 (ddd, J = 17.2, 11.4, 4.8, 2H), 3.10–3.18 (m, 1H), 2.82 (dt, J = 12.7, 8.3, 1H),
2.76 (s, 3H), 2.52 (ddd, J = 12.7, 7.9, 5.0, 1H). 13C-NMR (CDCl3): δ = 147.72, 134.52, 129.24, 128.92,
128.26, 122.10, 72.35, 69.28, 62.31, 54.31, 45.55, 36.77; Anal. Calcd for C14H18N4O2: C, 61.30; H,
6.61; N, 20.42; found C, 61.38; H, 6.68; N, 20.47.
((3RS,5RS)-2-Methyl-5-(1-(pyridin-2-ylmethyl)-1H-1,2,3-triazol-4-yl) isoxazolidin-3-yl)methanol (16c):
Compound 16c was prepared by the general desilylation procedure in 52.6% yield as yellow oil.
1H-NMR (CDCl3): δ = 7.71 (s, 1H), 7.65 (td, J = 7.8, 1.7 Hz, 1H), 7.23 (dd, J = 7.0, 5.1 Hz, 1H), 7.16
(d, J = 7.8 Hz, 1H), 5.65–5.54 (m, 2H), 5.41 (dd, J = 8.5, 6.8, 1H), 3.58–3.48 (m, 2H), 3.23–3.15 (m,
1H), 2.84 (dt, J = 12.8, 8.5, 1H), 2.74 (s, 3H), 2.39 (bs, 1H), 2.28 (ddd, J = 12.8, 6.5, 4.8 Hz, 1H).
13C-NMR (CDCl3): δ = 154.31, 149.82, 147.92, 137.49, 123.55, 122.58, 122.37, 70.93, 69.50, 63.32,
55.68, 44.86, 36.42; Anal. Calcd for C13H17N5O2: C, 56.71; H, 6.22; N, 25.44; found C, 56.76; H, 6.27;
N, 25.48.
((3RS,5SR)-2-Methyl-5-(1-(pyridin-2-ylmethyl)-1H-1,2,3-triazol-4-yl) isoxazolidin-3-yl)methanol (17c):
Compound 17c was prepared by the general desilylation procedure in 40.4% yield as yellow solid,
mp = 76–78 °C. 1H-NMR (CDCl3): δ = 8.66–8.56 (m, 1H), 7.72 (s, 1H), 7.70 (dt, J = 7.7, 1.7 Hz, 1H),
7.33–7.26 (m, 1H), 7.23 (d, J = 7.8 Hz, 1H), 5.73–5.59 (m, 2H), 5.18 (t, J = 7.8 Hz, 1H), 3.66 (ddd,
J = 17.2, 11.4, 4.8 Hz, 2H), 3.17 (s, 1H), 2.93–2.81 (m, 1H), 2.80 (s, 3H), 2.62–2.52 (m, 1H).
13C-NMR (CDCl3): δ = 154.31, 149.82, 137.49, 123.55, 122.58, 122.37, 70.93, 69.50, 63.32, 55.68, 44.86,
36.42; Anal. Calcd for C13H17N5O2: C, 56.71; H, 6.22; N, 25.44; found C, 56.78; H, 6.30; N, 25.50.
((3RS,5RS)-5-(1-(4-Chloro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-2-methyl isoxazolidin-3-
yl)methanol (16d): Compound 16d was prepared by the general desilylation procedure in 46.5% yield
1
as yellow solid, mp = 94–96 °C. H-NMR (CDCl3): δ = 8.07 (s, 1H), 8.06 (d, J = 2.6 Hz, 1H), 7.86
(dd, J = 8.7, 2.6 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 5.49 (dd, J=8.7, 6.3 Hz, 1H), 3.58 (d,
J = 5.7 Hz, 2H), 3.26–3.18 (m, 1H), 2.94 (dt, J = 12.8, 8.7, 1H), 2.79 (s, 3H), 2.35 (ddd, J = 12.8, 6.3,

Molecules 2015, 20
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4.9, 1H). 13C-NMR (CDCl3): δ = 149.50, 135.51, 133.11, 132.57, 130.06 (q, J = 32.4 Hz), 124.38,
123.24, 121.07, 119.73, 119.64 (q, J = 5.5 Hz), 70.94, 69.55, 63.25, 44.92, 36.78, 29.78; Anal. Calcd
for C14H14ClF3N4O2: C, 46.36; H, 3.89; N, 15.45; found C, 46.41; H, 3.93; N, 15.48.
((3RS,5SR)-5-(1-(4-Chloro-3-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-2-methyl isoxazolidin-3-
yl)methanol (17d): Compound 17d was prepared by the general desilylation procedure in 46.5% yield
1
as yellow thick oil. H-NMR (CDCl3): δ = 8.08 (d, J = 2.5 Hz, 1H), 8.00 (s, 1H), 7.89 (dd,
J = 8.6, 2.5 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 5.26 (t, J = 7.9, 1H), 3.69 (ddd, J=17.1, 11.4, 4.7, 2H),
3.25–3.18 (s, 1H), 2.90 (dt, J = 12.8, 8.0, 1H), 2.84 (s, 3H), 2.66 (ddd, J = 12.8, 8.0, 5.0, 1H).
13C-NMR (CDCl3): δ = 149.02, 135.55, 134.94, 133.22, 132.77, 130.11, 127.85, 124.51, 119.77 (q,
J = 5.3), 72.29, 69.31, 62.28, 45.62, 36.84; Anal. Calcd for C14H14ClF3N4O2: C, 46.36; H, 3.89; N,
15.45; found C, 46.40; H, 3.95; N, 15.50.
((3RS,5RS)-5-(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)-2-methylisoxazolidin -3-yl)methanol (16e):
Compound 16e was prepared by the general desilylation procedure in 52.6% yield as white solid,
1
mp = 57–59 °C. H-NMR (CDCl3): δ = 7.91 (s, 1H), 7.56 (dd, J =8.9, 1.3 Hz, 2H), 6.96 (dd, J = 8.9,
1.3 Hz, 2H), 5.48 (dd, J = 8.3, 6.8 Hz, 1H), 3.82 (s, H), 3.62–3.53 (m, 2H), 3.25–3.18 (m, 1H),
13
2.94–2.85 (m, 1H), 2.78 (s, 3H), 2.37–2.29 (m, 1H). C-NMR (CDCl3): δ = 159.89, 148.21, 130.45,
122.21, 120.01, 114.81, 70.99, 69.53, 63.38, 55.67, 44.91, 36.64; Anal. Calcd for C14H18N4O3: C,
57.92; H, 6.25; N, 19.30; found C, 57.96; H, 6.28; N, 19.27.
((3RS,5SR)-5-(1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)-2-methyl isoxazolidin-3-yl)methanol (17e):
Compound 17e was prepared by the general desilylation procedure in 40.4% yield as white solid,
1
mp = 103–105 °C. H-NMR (CDCl3): δ = 7.88 (s, 1H), 7.60 (d, J = 8.9 Hz, 2H), 7.00 (d, J = 8.9 Hz,
2H), 5.24 (t, J = 7.8 Hz, 1H), 3.85 (s, 3H), 3.67 (ddd, J=17.1, 11.4, 4.8 Hz, 2H), 3.22–3.15 (m, 1H),
2.97–2.86 (m, 1H), 2.82 (s, 3H), 2.76 (bs, 1H), 2.60 (ddd, J = 12.7, 7.9, 5.0 Hz, 1H). 13C-NMR
(CDCl3): δ = 160.00, 147.89, 130.51, 122.36, 120.66, 114.89, 72.39, 69.35, 55.74, 45.68, 36.79; Anal.
Calcd for C14H18N4O3: C, 57.92; H, 6.25; N, 19.30; found C, 57.99; H, 6.30; N, 19.33.
((3RS,5RS)-5-(1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)-2-methylisoxazolidin-3-yl)methanol
(16f):
Compound 16f was prepared by the general desilylation procedure in 53.7% yield as white solid,
mp = 68–70 °C. 1H-NMR (CDCl3): δ = 7.96 (s, 1H), 7.71–7.64 (m, 2H), 7.20 (t, J = 8.5 Hz, 2H), 5.51
(dd, J = 8.5, 6.6 Hz, 1H), 3.58 (d, J = 5.7 Hz, 2H), 3.31–3.18 (m, 1H), 2.94 (dt, J = 12.8, 8.5 Hz, 1H),
2.80 (s, 3H), 2.36 (ddd, J = 12.8, 6.2, 4.9, 1H). 13C-NMR (CDCl3): δ = 163.56, 161.57, 148.73, 133.37,
122.65 (d, J = 8.7 Hz), 120.00, 116.84 (d, J = 23.2 Hz), 71.04, 69.51, 63.38, 44.93, 36.65; Anal. Calcd
for C13H15FN4O2: C, 56.11; H, 5.43; N, 20.13; found C, 56.14; H, 5.49; N, 20.17.
((3RS,5SR)-5-(1-(4-Fluorophenyl)-1H-1,2,3-triazol-4-yl)-2-methylisoxazolidin-3-yl)methanol
(17f):
Compound 17f was prepared by the general desilylation procedure in 41.3% yield as white solid,
mp = 102–104 °C. 1H-NMR (CDCl3): δ = 7.93 (s, J = 1.0, 1H), 7.73–7.63 (m, 2H), 7.24–7.14 (m, 2H),
5.24 (t, J = 7.8 Hz, 1H), 3.68 (ddd, J = 17.2, 11.5, 4.8 Hz, 2H), 3.24–3.17 (m, 1H), 2.97–2.86 (m, 1H),
13
2.82 (s, 3H), 2.66–2.56 (m, 1H). C-NMR (CDCl3): δ = 163.58, 161.59, 148.26, 133.34, 122.70 (d,

Molecules 2015, 20
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J = 8.5 Hz), 120.67, 116.86 (d, J = 23.2 Hz), 72.30, 69.38, 62.32, 45.68, 36.79; Anal. Calcd for
C13H15FN4O2: C, 56.11; H, 5.43; N, 20.13; found C, 56.15; H, 5.47; N, 20.19.
((3RS,5RS)-2-Methyl-5-(1-(naphthalen-2-ylmethyl)-1H-1,2,3-triazol-4-yl) isoxazolidin-3-yl)methanol
(16g): Compound 16g was prepared by the general desilylation procedure in 48.0% yield as white
solid, mp = 95–97 °C. 1H-NMR (CDCl3): δ = 7.83–7.80 (m, 3H), 7.74 (s, 1H), 7.51 (dd, J = 6.2, 3.3 Hz,
2H), 7.48 (s, 1H), 7.33 (dd, J = 8.4, 1.4 Hz, 1H), 5.70–5.56 (m, 2H), 5.41 (dd, J = 8.3, 7.0 Hz, 1H),
3.51 (d, J = 6.2 Hz, 2H), 3.21–3.17 (m, 1H), 2.84 (dt, J = 12.8, 8.3 Hz, 1H), 2.74 (s, 3H), 2.31–2.22
(m, 1H), 1.91 (bs, 1H). 13C-NMR (CDCl3): δ = 148.00, 133.32, 131.83, 129.32, 128.07, 127.90,
127.67, 126.87, 125.51, 71.06, 69.44, 63.40, 54.57, 44.89, 36.40; Anal. Calcd for C18H20N4O2: C,
66.65; H, 6.21; N, 17.27; found C, 66.68; H, 6.25; N, 17.30.
((3RS,5RS)-2-Methyl-5-(1-(naphthalen-2-ylmethyl)-1H-1,2,3-triazol-4-yl) isoxazolidin-3-yl)methanol
(17g): Compound 17g was prepared by the general desilylation procedure in 48.0% yield as white
solid, mp = 115–117 °C. 1H-NMR (CDCl3): δ = 7.88–7.79 (m, 3H), 7.74 (s, J = 6.8 Hz, 1H), 7.55–7.48
(m, 2H), 7.46 (s, 1H), 7.34 (d, J = 8.3 Hz, 1H), 5.72–5.60 (m, 2H), 5.12 (t, J = 7.9 Hz, 1H), 3.62 (ddd,
J = 17.2, 11.5, 4.8, 2H), 3.08–3.16 (m, 1H), 2.90–2.76 (m, 1H), 2.75 (s, 3H), 2.56–2.47 (m, 1H).
13C-NMR (CDCl3): δ = 147.79, 133.29, 131.85, 129.29, 128.03, 127.89, 127.62, 126.87, 126.84,
125.49, 122.15, 72.35, 69.27, 62.28, 54.50, 45.54, 36.80; Anal. Calcd for C18H20N4O2: C, 66.65; H,
6.21; N, 17.27; found C, 66.70; H, 6.27; N, 17.32.
3.4. Biological Tests
3.4.1. Antiviral Activity
Compounds 16–17 were evaluated for their ability to inhibit the replication of a variety of DNA and
RNA viruses, using the following cell-based assays: (a) Vero cell cultures: poliovirus 1, human
echovirus 9, coxsackievirus B4, adenovirus type 2, herpes simplex type 1 (HSV-1), herpes simplex
type 2 (HSV-2); (b) human embryonic lung fibroblast cells (MRC-5): cytomegalovirus (CMV: VR-538);
(c) African green monkey kidney cells (BS-C-1): varicella-zoster virus (VZV). Acyclovir was used as
the reference compounds. Unfortunately, no inhibitory activity against any virus was detected for the
evaluated compounds (data not shown).
3.4.2. Antiproliferative Activity
The antiproliferative activity of all the synthesized C-Nucleosides was evaluated. An
antiproliferative effect was observed for compounds 17a and 17b, while other derivatives show a IC50
in the range 150–200 μM. In particular, as shown in Figures 4A and 5A, cell culture incubation with
increasing concentration of 17a and 17b, ranging from 1 µM to 100 µM for 24, 48 and 72 h, reduced
the proliferation in all the cancer cell lines with a similar trend.

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HepG2
HT-29
SH-SY5Y
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100
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60
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20
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*
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48 h
72 h
24 h
48 h
72 h
24 h
48 h
72 h
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17a (M)
17a (M)
Figure 4. Compound 17a reduces cell proliferation and induce LDH release. HepG2,
HT-29 and SH-SY5Y cells were exposed to increased concentration of 17a compound for
24, 48 and 72 h. (A) Proliferation rate assessed by BrdU assay. (B) Cytotoxic effect
assessed in terms of LDH release after 24 h of exposure. Each value is the mean ± S.E.M.
of three experiments performed eight times (BrdU) or in triplicate (LDH) and repeated
three different times. * p < 0.05 vs. ctrl, ** p < 0.01 vs. ctrl, *** p < 0.001 vs. ctrl.
In particular, data of the BrdU assay shows that the growth inhibitory effect induced by 17a reaches
the 50% in both HepG2 and HT-29 cells and increases up to 56% in the SH-SY5Y cell line (p < 0.001
vs. control) after 72 h of incubation at a 100 µM concentration. Reduction of cell proliferation was also
observed when the cells were exposed for 48 (p < 0.001 vs. control) and 24 hours (p < 0.01 vs. control
in HepG2 and HT-29 cells and p < 0.001 in SH-SY5Y cell line; Figure 4A) to 100 µM concentration
of the compound 17a.
Lesser antiproliferative effect was observed treating the cells with compound 17a at concentrations
of 50 and 10 µM (expecially for longer time of exposure). Similar results were obtained by the
compound 17b (56% of cell growth inhibition for the HepG2, 62% for the HT-29 and the SH-SY5Y
cell lines at the 100 µM concentration for 72 h; Figure 5A). Results of the BrdU assay show very close
alignment with those of MTT test performed after 24, 48 and 72 h of incubation with both 17a and 17b
(see Supplementary Materials Figures S1A and S2A).

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HepG2
HT-29
SH-SY5Y
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120
100
80
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48 h
72 h
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48 h
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100
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50
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1
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17b (M)
17b (M)
17b (M)
Figure 5. Compound 17b inhibit cell growth and determine LDH release. (A) Proliferation
rate assessed by BrdU assay after 24–72 h of treatment. (B) Cytotoxic activities evaluated
by the test of LDH after 24 h of incubation. Each value is the mean ± S.E.M. of three
experiments performed in eight times (BrdU) or in triplicate (LDH) and repeated three
different times. * p < 0.05 vs. ctrl, ** p < 0.01 vs. ctrl, *** p < 0.001 vs. ctrl.
3.4.3. Cytoxicity Evaluation
The cytotoxic effect induced by 17a and 17b was evaluated through either LDH and trypan blue
dye exclusion assays. Figures 4B and 5B show that both 17a and 17b caused increase of the LDH
release at 50 and 100 µM concentration in all the investigated cell lines. Furthermore, LDH release
was accompanied by a significant increase of cell death, as detected by the trypan blue test (see
Supplementary Materials Figures S1B and S2B), underlying the cytotoxic activity of the compounds
17a and 17b.
3.4.4. Cell Culture and Treatments
HepG2 (hepatocellular carcinoma), HT-29 (colorectal adenocarcinoma) and SH-SY5Y
(neuroblastoma) cells were obtained originally from ATCC (Rockville, MD, USA). These cell lines,
maintained in RPMI supplemented with 10% heat-inactivated fetal bovine serum, 1 mM sodium
pyruvate, 2 mM glutamine, penicillin/streptomycin (100 units/mL and 100 µg/mL, respectively), were
grown at 37 °C and 5% CO2 conditions. All the reagents for cell cultures were from Gibco (Life
Technologies, Monza, Italy). For biological investigations, 100 mM stock solutions were prepared
dissolving the tested compounds in dimethyl sulfoxide (DMSO). Small aliquots were stored at −20 °C
and were diluted in culture media to the final concentration, ranging from 1 to 100 µM, just prior the

Molecules 2015, 20
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use. The highest DMSO concentration used in this study (0.1%) did not have any appreciable effect on
cell proliferation or cytotoxicity.
3.4.5. Antiproliferative Activity
The antiproliferative activity of all the synthesized compounds was evaluated by either BrdU and
MTT assays. For the first assay, the cells were seeded in 96-well plates at a density of 10 × 104 cell/well
(HT-29 cells and SH-SY5Y) and 12 × 104 (HepG2) and allowed to stand overnight. Then, the culture
medium was replaced with clean media containing the tested compounds at a concentrations ranging
from 1 to 100 µM or with media with equivalent dilutions of DMSO (control cultures). After 24, 48 or
72 h incubation, BrdU assay was performed as described [37]. BrdU is a uridine derivative, structural
analog of thymidine, which can be used as a marker for proliferation, because it is incorporated into
DNA during the synthesis-phase of the cell cycle as a substitute for thymidine. Cells marked by BrdU
incorporation may be detected after addition of goat anti-mouse IgG-peroxidase conjugated secondary
antibody. Results are expressed as percentages of the absorbance measured in control cells.
Cell growth was also detected by the MTT assay as reported with modification [38]. The cells
were seeded onto 96-well plates at a density of 5 × 103 cells/well (HT-29 cells and SH-SY5Y) or
6 × 103 cells/well (HepG2). The following day, cells were treated with the test compounds as described
above. At the end of the exposure time, the plates were centrifuged at 1200 rpm for 10 min, the
supernatants were replaced with clean medium without phenol red containing 0.5 mg/mL of
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich, Milan, Italy),
and the plates were returned in the incubator for 4 h. Then, the solutions were removed from the wells
and crystals of formazan (MTT metabolic product) were solubilised by 100 µL HCl/isopropanol 0.1 N
lysis buffer. The latter were spectrophotometrically quantified at a wavelength of 595 nm (iMark™
microplate reader, Bio-Rad Laboratories, Milan, Italy). Results of the cell proliferation assays are
expressed as percentages in untreated cultures. The tests were performed in triplicate (BrdU) or eight
times (MTT) and repeated three different times.
3.4.6. Detection of Cell Viability
Lactate dehydrogenase is a stable cytosolic enzyme which is released into the surrounding culture
medium when the plasma membrane is damaged, and can be considered a marker of cytotoxicity. The
released LDH can be quantified by a coupled enzymatic reaction. First, LDH catalyzes the conversion
of lactate to pyruvate via reduction of NAD+ to NADH. Second, diaphorase uses NADH to reduce a
tetrazolium salt to a red formazan product. Therefore, the level of formazan formation is directly
proportional to the amount of released LDH in the medium. HepG2, HT-29 and SH-SY5Y cells
were seeded in 96-well plates in a number of 15 × 103 cells/well. The following day, cells were
incubated with the synthesized C-Nucleosides for 24 h. Then the LDH concentration was measured as
reported [39] by using commercial LDH kit (CytoTox 96® Non-Radioactive Cytotoxicity Assay,
Promega, Milan, Italy), according to the manufacturer’s protocol. LDH levels are extrapolated as the
values detected in control cells, which are arbitrarily expressed as 1.

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Cell viability in presence of the tested compounds was assessed also by the trypan blue dye
(0.4% w/v) exclusion test [40] and cell death was reported as the percentage of stained (non-viable) vs.
total cells counted. All the experiments were carried out in triplicate and repeated three times.
3.4.7. Statistical Analysis
Data were expressed as mean ± S.E.M. and statistically evaluated for differences using one-way
analysis of variance (ANOVA), followed by Turkey-Kramer multiple comparisons test (GraphPAD
Software for Science, GraphPad Software, Inc., La Jolla, CA, USA).
4. Conclusions
We report in this paper the synthesis of 5-(1H-1,2,3-triazol-4-yl)isoxazolidines, a novel series of
C-nucleosides, featured by the presence of a 1,2,3-triazole ring linked to an isoxazolidine system, as
mimetic of the pyrimidine nucleobases. The synthesized compounds have been evaluated for their
ability to inhibit the replication of a variety of DNA and RNA viruses: unfortunately, no inhibitory
activity against any virus was detected for the evaluated compounds. The antiproliferative activity of
all the synthesized C-Nucleosides was also tested. An antiproliferative effect was observed for
compounds 17a and 17b: the induced growth inhibitory effect reaches the 50% in HepG2 and HT-29
cells and increases up to 56% in the SH-SY5Y cell line (p < 0.001 vs. control) after 72 h of incubation
at a 100 µM concentration.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/04/5260/s1.
Acknowledgments
We gratefully acknowledge the Italian Ministry of Education, Universities, and Research (MIUR),
the Universities of Messina and Catania, and the Interuniversity Consortium for Innovative
Methodologies and Processes for Synthesis (CINMPIS) for partial financial support.
Author Contributions
SVG and RR, designed research; MN, SC, AG performed biological data; SVG, RR, CC, RM and
MAC performed research and analyzed the data; SVG and RR wrote the paper. All authors read and
approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. De Clercq, E. The history of antiretrovirals: Key discoveries over the past 25 years. Rev. Med. Virol.
2009, 19, 287–299.

Molecules 2015, 20
5273
2. De Clercq, E. Antiviral Drug Strategies; Wiley-VCH: Weinheim, Germany, 2011.
3. Parker, W.B. Enzymology of Purine and Pyrimidine Antimetabolites Used in the Treatment of
Cancer. Chem. Rev. 2009, 109, 2880–2893.
4. Vanek, V.; Budesinsky, M.; Rinnova, M.; Rosenberg, I. Prolinol-based nucleoside phosphonic acids:
New isosteric conformationally flexible nucleotide analogues. Tetrahedron 2009, 65, 862–876.
5. Ishitsuka, H.; Shimma, N. Capecitabine Preclinical Studies: From Discovery to Translational
Research. In Modified Nucleosides in Biochemistry, Biotechnology and Medicine; Herdewijn, P.,
Ed.; Wiley-VCH: Weinheim, Germany, 2008; pp. 587–600.
6. Kumamoto, H.; Topalis, D.; Broggi, J.; Pradere, U.; Roi, V.; Berteina-Raboin, S.; Nolan, S.P.;
Deville-Bonne, D.; Andrei, G.; Snoeck, R.; et al. Preparation of acyclo nucleoside phosphonate
analogues based on cross-metathesis. Tetrahedron 2008, 64, 3517–3526.
7. Jung, K.-H.; Marx, A. Synthesis of 4'-C-modified 2'-Deoxyribonucleoside Analogues and
Oligonucleotides. Curr. Org. Chem. 2008, 12, 343–354.
8. Littler, E.; Zhou, X.-X. Deoxyribonucleic Acid Viruses: Antivirals for Herpesviruses and
Hepatitis B Virus. In Comprehensive Medicinal Chemistry II; Taylor, J.B., Triggle, D.J., Eds.;
Elsevier, Oxford, UK, 2006; Volume 7, pp. 295–327.
9. Thottassery, J.V.; Westbrook, L.; Someya, H.; Parker, W.B. c-Abl-independent p73 stabilization
during gemcitabine- or 4'-thio-beta-D-arabinofuranosylcytosine-induced apoptosis in wild-type
and p53-null colorectal cancer cells. Mol. Cancer Ther. 2006, 5, 400–410.
10. Stambasky, J.; Hocek, M.; Kocovsky, P. C-Nucleosides: Synthetic Strategies and Biological
Applications. Chem. Rev. 2009, 109, 6729–6764.
11. Bzowska, A. Formycins and their Analogues: Purine Nucleoside Phosphorylase Inhibitors and Their
Potential Application in Immunosuppression and Cancer. In Modified Nucleosides: in Biochemistry,
Biotechnology and Medicine; Herdewijn, P., Ed.; Wiley-VCH; Weinheim, Germany, 2008;
pp. 473–510.
12. Desaulniers, J.P.; Chang, Y.C.; Aduri, R.; Abeysirigunawardena, S.C.; Santa Lucia, J.; Chow, C.S.
Pseudouridines in rRNA helix 69 play a role in loop stacking interactions. Org. Biomol. Chem.
2008, 6, 3892–3895.
13. Matsuura, S.; Shiratori, O.; Katagiri, K. Antitumor activity of showdomycin. J. Antibiot. 1964, 17,
234–237.
14. Nakagawa, Y.; Kano, H.; Tsukuda, Y.; Koyama, H. Structure of a new class of C-nucleoside
antibiotic, showdomycin. Tetrahedron Lett. 1967, 42, 4105–4109.
15. Uehara, Y.I.; Rabinowicz, J. Showdomycin and its reactive moiety, maleimide. A comparison in
selective toxicity and mechanism of action in vitro. Biochem. Pharmacol. 1980, 29, 2199–2204.
16. Shaban, M.A. E.; Nasr, A.Z. The Chemistry of C-Nucleosides and Their Analogs I:
C-Nucleosides of Hetero Monocyclic Bases. Adv. Heterocycl. Chem. 1997, 68, 223–432.
17. Sallam, M.A.E.; Luis, F.F.; Cassady, J.M. Studies on Epimeric-D-arabino-and-D-ribo-tetritol-1-yl-
2-phenyl-2 H-1,2,3-triazoles. Synthesis and Anomeric Configuration of 4-(α- and β-D-
Erythrofuranosyl)-2-phenyl-2 H-1,2,3-Triazole C-Nucleoside Analogs. Nucleos. Nucleot. 1998,
17, 769–783.
18. Romeo, G.; Chiacchio, U.; Corsaro, A.; Merino, P. Chemical Synthesis of Heterocyclic−Sugar
Nucleoside Analogues. Chem. Rev. 2010, 110, 3337–3370.

Molecules 2015, 20
5274
19. Romeo, G.; Giofré, S.V.; Piperno, A.; Romeo, R.; Chiacchio, M.A. Synthesis of
N,O-homonucleosides with high conformational freedom. ARKIVOC 2009, viii, 168–176.
20. Romeo, R.; Giofrè, S.V.; Iaria, D.; Sciortino, M.T.; Ronsisvalle, S.; Chiacchio, M.A.; Scala, A.
Synthesis of 5-Alkynyl Isoxazolidinyl Nucleosides, Eur. J. Org. Chem. 2011, 28, 5690–5695.
21. Romeo, R.; Giofrè, S.V.; Macchi, B.; Balestrieri, E.; Mastino, A.; Merino, P.; Carnovale, C.;
Romeo, G.; Chiacchio, U. Truncated Reverse Isoxazolidinyl Nucleosides: A New Class of
Allosteric HIV-RT Inhibitors. ChemMedChem 2012, 7, 565–569.
22. Romeo, R.; Giofrè, S.V.; Garozzo, A.; Bisignano, B.; Corsaro, A.; Chiacchio, M.A. Synthesis and
biological evaluation of furopyrimidine N,O-nucleosides. Bioorg. Med. Chem. 2013, 21, 5688–
5693.
23. Chiacchio, U.; Rescifina, A.; Iannazzo, D.; Piperno, A.; Romeo, R.; Borrello, L.; Sciortino, M.T.;
Balestrieri, E.; Macchi, B.; Mastino, A.; et al. Phosphonated Carbocyclic 2'-Oxa-3'-azanucleosides
as New Antiretroviral Agents. J. Med. Chem. 2007, 50, 3747–3750.
24. Chiacchio, U.; Balestrieri, E.; Macchi, B.; Iannazzo, D.; Piperno, A.; Rescifina, A.; Romeo, R.;
Saglimbeni, M.; Sciortino, M.T.; Valveri, V.; et al. Synthesis of Phosphonated Carbocyclic
2'-Oxa-3'-aza-nucleosides:ꢀ Novel Inhibitors of Reverse Transcriptase. J. Med. Chem. 2005, 48,
1389–1394.
25. Chiacchio, U.; Iannazzo, D.; Piperno, A.; Romeo, R.; Romeo, G.; Rescifina, A.; Saglimbeni, M.
Synthesis and biological evaluation of phosphonated carbocyclic 2'-oxa-3'-aza-nucleosides.
Bioorg. Med. Chem. 2006, 14, 955–959.
26. Romeo, R.; Carnovale, C.; Giofrè, S.V.; Monciino, G.; Chiacchio, M.A.; Sanfilippo, C.; Macchi, B.
Enantiomerically Pure Phosphonated Carbocyclic 2'-Oxa-3'-Azanucleosides: Synthesis and
Biological Evaluation. Molecules 2014, 19, 14406–14416.
27. Piperno, A.; Giofrè, S.V.; Iannazzo, D.; Romeo, R.; Romeo, G.; Chiacchio, U.; Rescifina, A.;
Piotrowska, D.G. Synthesis of C-4' Truncated Phosphonated Carbocyclic 2'-Oxa-3'-azanucleosides
as Antiviral Agents. J. Org. Chem. 2010, 75, 2798–2805.
28. Romeo, R.; Carnovale, C.; Giofrè, S.V.; Romeo, G.; Macchi, B.; Frezza, C.; Merino-Merlo, F.;
Pistarà, V.; Chiacchio, U. Truncated phosphonated C-1'-branched N,O-nucleosides: A new class
of antiviral agents. Bioorg. Med. Chem. 2012, 20, 3652–3657.
29. Chiacchio, U.; Corsaro, A.; Mates, J.; Merino, P.; Piperno, A.; Rescifina, A.; Romeo, G.; Romeo, R.;
Tejero, T. Isoxazolidine analogues of pseudouridine: A new class of modified nucleosides.
Tetrahedron 2003, 59, 4733–4738.
30. Chiacchio, U.; Rescifina, A.; Saita, M.G.; Iannazzo, D.; Romeo, G.; Mates, J.A.; Tejero, T.;
Merino, P. Zinc(II) triflate-controlled 1,3-dipolar cycloadditions of C-(2-thiazolyl)nitrones:
application to the synthesis of a novel isoxazolidinyl analogue of tiazofurin. J. Org. Chem. 2005,
70, 8991–9001.
31. Romeo, R.; Giofrè, S.V.; Carnovale, C.; Campisi, A.; Parenti, R.; Bandini, L.; Chiacchio, M.A.
Synthesis and Biological Evaluation of 3-Hydroxymethyl-5-(1H-1,2,3-Triazol) Isoxazolidines.
Bioorg. Med. Chem. 2013, 21, 7929–7937.
32. Romeo, R.; Giofrè, S.V.; Carnovale, C.; Chiacchio, M.A.; Campisi, A.; Mancuso, R.; Cirmi, S.;
Navarra, M. Synthesis and Biological Activity of Triazole-Appended N,O-Nucleosides. Eur. J.
Org. Chem. 2014, 25, 5442–5447.

Molecules 2015, 20
5275
33. Nulwala, H.; Takizawa, K.; Odukale, A.; Anzar Khan, A.; Thibault, R.J.; Taft, B.R.;
Lipshutz, B.H.; Hawker, C.J. Synthesis and Characterization of Isomeric Vinyl-1,2,3-triazole
Materials by Azide−Alkyne Click Chemistry. Macromolecules 2009, 42, 6068–6074.
34. Iannazzo, D.; Piperno, A.; Pistarà, V.; Rescifina, A.; Romeo, R. Modified nucleosides. A general
and diastereoselective approach to N,O-psiconucleosides. Tetrahedron 2002, 58, 581–587.
35. Romeo, R.; Carnovale, C.; Giofrè, S.V.; Chiacchio, M.A.; Garozzo, A.; Amata, E.; Romeo, G.;
Chiacchio, U. C-5'-Triazolyl-2'-oxa-3'-aza-4'a-carbanucleosides: Synthesis and biological evaluation.
Beilstein J. Org. Chem. 2015, 11, 328–334.
36. Merino, P.; Revuelta, J.; Tejero, T.; Chiacchio, U.; Rescifina, A.; Romeo, G. A DFT study on the
1,3-dipolar cycloaddition reactions of C-(methoxycarbonyl)-N-methyl nitrone with methyl
acrylate and vinyl acetate. Tetrahedron 2003, 59, 3581–3592.
37. Visalli, G.; Ferlazzo, N.; Cirmi, S.; Campiglia, P.; Gangemi, S.; Pietro, A.D.; Calapai, G.;
Navarra, M. Bergamot juice extract inhibits proliferation by inducing apoptosis in human colon
cancer cells. Anticancer Agents Med. Chem. 2014, 14, 1402–1413.
38. Navarra, M.; Celano, M.; Maiuolo, J.; Schenone, S.; Botta, M.; Angelucci, A.; Bramanti, P.;
Russo, D. Antiproliferative and pro-apoptotic effects afforded by novel Src-kinase inhibitors in
human neuroblastoma cells. BMC Cancer 2010, 10, 602.
39. Romeo, R.; Navarra, M.; Giofrè, S.V.; Carnovale, C.; Cirmi, S.; Lanza, G.; Chiacchio, M.A.
Synthesis and biological activity of new arenediyne-linked isoxazolidines. Bioorg. Med. Chem.
2014, 22, 3379–3385.
40. Delle Monache, S.; Sanità, P.; Trapasso, E.; Ursino, M.R.; Dugo, P.; Russo, M.; Ferlazzo, N.;
Calapai, G.; Angelucci, A.; Navarra, M. Mechanisms Underlying the Anti-Tumoral Effects of
Citrus bergamia Juice. PLoS One 2013, 8, e61484.
Sample Availability: Samples of the compounds are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).