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Truncated phosphonated C-1′-branched N,O-nucleosides: A new class of antiviral agents

01 Jun 2012-Bioorganic & Medicinal Chemistry (Pergamon)-Vol. 20, Iss: 11, pp 3652-3657

TL;DR: Preliminary biological assays show that β-anomers are able to inhibit HIV in vitro infection at concentrations in the micromolar range and higher SI values with respect to AZT indicated that the compounds were endowed with low cytotoxicity.
Abstract: Truncated phosphonated C-1′-branched N,O-nucleosides have been synthesized in good yields by 1,3-dipolar cycloaddition methodology, starting from N-methyl-C-(diethoxyphosphoryl)nitrone 7. Preliminary biological assays show that β-anomers are able to inhibit HIV in vitro infection at concentrations in the micromolar range. Higher SI values with respect to AZT indicated that the compounds were endowed with low cytotoxicity.

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Truncated phosphonated C-1
0
-branched N,O-nucleosides: A new class
of antiviral agents
Roberto Romeo
a,
, Caterina Carnovale
a
, Salvatore V. Giofrè
a
, Giovanni Romeo
a
, Beatrice Macchi
b
,
Caterina Frezza
b
, Francesca Marino-Merlo
c
, Venerando Pistarà
d
, Ugo Chiacchio
d
a
Dipartimento Farmaco-Chimico, Università of Messina, 98168 Messina, Italy
b
Dipartimento di Neuroscienze, Università di Roma ‘Tor Vergata’, 00133 Roma, Italy
c
Dipartimento di Scienze della vita, Università di Messina, 98100 Messina, Italy
d
Dipartimento di Scienze del Farmaco, Università di Catania, 95125 Catania, Italy
article info
Article history:
Received 23 December 2011
Revised 21 March 2012
Accepted 22 March 2012
Available online 29 March 2012
Keywords:
C-1
0
-branched N,O-nucleosides
1,3-Dipolar cycloaddition
Antiviral agents
HIV in vitro infections
MTS assay
abstract
Truncated phosphonated C-1
0
-branched N,O-nucleosides have been synthesized in good yields by 1,3-
dipolar cycloaddition methodology, starting from N-methyl-C-(diethoxyphosphoryl)nitrone 7. Prelimin-
ary biological assays show that b-anomers are able to inhibit HIV in vitro infection at concentrations in
the micromolar range. Higher SI values with respect to AZT indicated that the compounds were endowed
with low cytotoxicity.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Natural psicofuranosyl nucleosides, bearing a hydroxymethyl
group at the anomeric carbon atom, have been reported to possess
different and relevant biological activities.
1
Typical examples are
represented by angustmycin A 1 and C 2, which show interesting
antimicrobial and antiviral properties,
2
and by hydantocidin 3,a
spironucleoside, which exhibits herbicidal activity, able to regulate
plant growth
3
(Fig. 1). Besides their potential biological activity as
antiviral agents, the C1
0
-branched nucleosides show further great
interest, linked to the availability of model nucleosides which
may allow the study of the formation and evolution of radical spe-
cies generated during DNA/RNA damage.
4
N,O-psiconucleosides 4 constitute a particular class of modified
psiconucleosides where an isoxazolidine system mimics the ribose
ring of natural nucleosides and a hydroxymethyl group is linked at
the anomeric carbon atom.
5
These derivatives show synthetic
interest for their potential antiviral or anticancer activity which
have been also discovered in other N,O-nucleosides.
6–11
Our research group has reported a versatile route towards the
synthesis of N,O-psiconucleosides both in racemic and in enantio-
pure form.
5,12–15
More recently, the use of a chiral auxiliary has promoted the
enantioselective synthesis of a series of psiconucleosides.
16
How-
ever, none of the reported compounds has shown a remarkable
biological activity, probably due to the lack of efficient phosphory-
lation towards the triphosphate derivatives, the active form of
nucleoside RT inhibitors.
In the field of nucleoside analogs, N,O-modified nucleosides
have been proved to efficiently block the in vitro and in vivo virus
infections caused by HIV, HBV, and HTLV-1.
17–22
Following intra-
cellular phosphorylation to their 5
0
-triphosphate forms, they are
able to serve as chain terminators, thus acting as inhibitors in
the viral reverse transcription reaction.
18,19
Several strategies to
overcome the initial selective phosphorylation step have been de-
signed;
23
in particular, phosphonate analogues,
20
by miming the
nucleoside monophosphates, overcome the instability of nucleo-
tides towards phosphodiesterase and enhance the cellular uptake
by bypassing the initial phosphorylation step.
In this context, we have recently reported the synthesis of phos-
phonated carbocyclic 2
0
-oxa-3
0
-azanucleosides (PCOANS) 5, which
have shown to be potent inhibitors of RT of different retroviruses
21
(Fig. 2). Also truncated phosphonated azanucleosides (TPCOANS) 6,
where the phosphonate group is directly linked to the C-4
0
position
of the pseudosugar moiety, are able to inhibit the HIV and HTLV-1
viruses at concentration in the nanomolar range, with a potency
comparable with that of Tenofovir.
22
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.03.047
Corresponding author.
E-mail address: robromeo@unime.it (R. Romeo).
Bioorganic & Medicinal Chemistry 20 (2012) 3652–3657
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc

In connection with our work addressed to the search of new and
potent antiviral agents, we have extended our interest to the syn-
thesis of the new generation of truncated phosphonated C-1
0
-
branched N,O-nucleosides. The rational of our choice is based on
the consideration that the presence of the phosphonic unit could
bypass the limiting monophosphorylation step, thus promoting
the cellular uptake and leading to biologically active compounds.
We report in this paper the synthetic approach towards these
derivatives and their preliminary biological evaluation. To the best
of our knowledge, no example of this kind of compounds has been
reported in the literature until now.
2. Results and discussion
2.1. Chemistry
According to the retrosynthetic analysis shown in Scheme 1, the
key step of the synthetic route involves the 1,3-dipolar cycloaddi-
tion of the N-methyl-C-(diethoxyphosphoryl)nitrone 7 with the
acrylate 8.
The nitrone 7 can be prepared from the commercially available
diethyl hydroxymethyl phosphonate 13 (route a), as previously de-
scribed.
24
We have designed an alternative methodology towards 7
(route b), which is based on the conversion of the phosphonated
alcohol 13 into the corresponding mesylate 15; the subsequent
reaction with N-methyl hydroxylamine afforded the derivative 16
which, by oxidation with MnO
2
, gave the target nitrone 7 in a
60% yield (Scheme 2).
In comparison to the route A, this second approach is performed
in milder conditions, at room temperature, thus allowing to avoid
the severe experimental conditions required by the Swern oxida-
tion (78 °C) and the possible decomposition of intermediate 14
to carbon monoxide and dialkyl phosphate, with the risk of explo-
sion, during the work up.
Dipolarophile 8 has been prepared starting from ethyl pyruvate,
by heating with acetic anhydride.
5,25
The cycloaddition of 7 with
ethyl 2-acetyloxyacrylate 8, in THF at reflux for 24 h, afforded a
mixture of cis/trans isoxazolidines 9 and 10 in an isomeric ratio
of 1:4.5 and a combined yield of 80% (Scheme 3).
The crude reaction mixture was purified by flash chromatogra-
phy (CH
2
Cl
2
/Me
2
CHOH 98:2 as eluent) and two cycloadducts 9 and
10 were obtained in pure form. The cis/trans stereochemistry of
both adducts was deduced on the basis of
1
H NMR spectroscopy
and by means of NOE experiments. Thus, in the major trans com-
pound 10, the resonance of H
4a
appears as ddd at 2.85 ppm, while
H
4b
resonates at 2.96 ppm (ddd). Moreover, H
3
resonates as ddd at
3.27 ppm. For the cis compound 9, the resonance of H
4a
and H
4b
ap-
pears as ddd at 2.81 and 2.93 ppm respectively; H
3
resonates as
ddd at 3.23 ppm. NOE difference experiments were conclusive in
the stereochemical assignment: (a) on irradiation at 2.96 ppm
(H
4b
of trans compound 10), a diagnostic positive NOE was ob-
served for H
3
(d = 3.27 ppm) and the methyl of the OAc group, thus
confirming their cis relationship, and (b) on irradiation of H
4b
of cis
derivative 9,atd = 2.93 ppm, a positive NOE was observed for H
3
(d = 3.23 ppm) but not for the methyl protons of OAc group.
O
OHOH
N
N
N
N
NH
2
OH
O
OHOH
N
N
N
N
NH
2
OH
HO
O
OHOH
HO NH
HN
O
O
Angustmycin A
Angustmycin C
Hydantocidin
1
2
3
N
O
Me
HO
B
OH
N,O-Psiconucleosides
4
Figure 1. Modified nucleosides, B = nucleobase.
N
O Me
P
O
OE
t
OEt
OAc
CO
2
Et
N
O
(EtO)
2
OP
OAc
CO
2
Et
Me
N
O
(EtO)
2
OP
OAc
CO
2
Et
Me
9
1
0
7
8
N
O
(EtO)
2
OP
B
CO
2
Et
Me
N
O
(EtO)
2
OP
B
CO
2
Et
Me
1
1
1
2
Scheme 1. Retrosynthetic analysis of truncated phosphonated N,O-
psiconucleosides.
route a
P
OH
O
EtO
OEt
P
O
O
EtO
OEt
H
7
13
14
route b
15
a
P
OSO
2
Me
O
EtO
OEt
b
P
N
O
EtO
OEt
OH
Me
c
16
N
O Me
P
O
OEt
OEt
a
b
Scheme 2. Synthesis of phosphonated nitrone 7. Reagents and conditions route a:
(a) Swern oxidation; (b) MeNHOHHCl, Et
3
N, 78 °C; route b: (a) MeSO
2
Cl, CH
2
Cl
2
,
Et
3
N, rt; (b) Et
3
N, MeNHOH, reflux, 6 h; (c) MnO
2
,CH
2
Cl
2
.
N
O Me
P
O
OEt
OEt
OAc
CO
2
Et
N
O
(EtO)
2
OP
OAc
CO
2
Et
Me
N
O
(EtO)
2
OP
OAc
CO
2
Et
Me
+
9
107
8
1:4.5
H
H
H
3
4a'
4b'
1
5
a
Scheme 3. Cycloaddition reaction. Reagents and conditions: nitrone 7, ethyl 2-
acetyloxyacrylate 8, THF, reflux, 24 h, overall yield 80%.
N
N
N
N
NH
2
N
O
Me
B
PCOANS
5
O
Me
P
O
HO
OH
Tenofovir
O
HO
B
R
Truncated Nucleosides
R = H, OH
B = purine or pyrimidine nucleobases
N
O
Me
PEtO
B
TPCOANS
6
B = Ty, 5-Fu, Cy B = Ty, 5-Fu
O
EtO
P
O
EtO
OEt
Figure 2. Tenofovir, truncated nucleosides and phosphonated N,O-nucleosides.
R. Romeo et al. / Bioorg. Med. Chem. 20 (2012) 3652–3657
3653

The crude mixture of isoxazolidines 9 and 10 was then trans-
formed into the pyrimidine nucleoside analogues 11 and 12, as de-
picted in Scheme 4.
The condensation with silylated thymine, uracil or acetylcyto-
sine, using the glycosylation methodology developed by Vorbrüg-
gen, resulted in nucleoside products consisting of b-11a,c,d and
a
-12a,c,d anomers in a 3:2 ratio and in 61–80% combined yield.
The nucleosidation reaction performed with 5-fluoruracil showed
a better diastereoselectivity in favor of the b-anomer 11b (b/
a
ratio
4:1; combined yield: 85%).
The ratio between
a
and b nucleosides did not change if the
nucleosidation was performed starting from the separated cis/trans
isoxazolidines 9 and 10. As previously reported for similar com-
pounds,
13
these results indicate that the coupling reaction occurs
without selectivity with respect to the anomeric center. The ano-
mers have been separated by flash chromatography, the cis isomer
(b) showing the lower rf. The anomeric configuration of obtained
modified nucleosides was assigned on the basis of
1
H NMR and
NOE experiments.
The
1
H NMR spectrum of the major isomer 11a, chosen as refer-
ence, showed a different set of signals in comparison with 12a.In
particular, the H4
0
proton resonated at 3.25 ppm (ddd), H5
0
b at
2.93 ppm (ddd) and H5
0
a
at 3.28 ppm (ddd). Positive NOE effects
were observed within H5
0
b–H6 and H4
0
–H5
0
a
pairs of protons
and fully supported the cis configuration of the substituents at
C1
0
(thymine) and C4
0
(diethoxyphosphoryl) in the b isomer 11a.
Analogous NOE effects have been detected for the fluorouracil, ura-
cil and cytosine derivatives 11bd.
Since values of all vicinal coupling constants were successfully
extracted from the
1
H and
13
C NMR spectra of 11a, detailed
conformational analysis was accomplished. On the basis of vicinal
couplings [J (H-C4
0
–C5
0
Hb) = 10.5 Hz, J (H-C4
0
–C5
0
H
a
) = 8.5 Hz,
J (P-C4
0
–C5
0
b) = 16.5 Hz, J (P-C4
0
–C5
0
a
) = 4.0 Hz, J (C1
0
-C–C-P) =
11.5 Hz] it was concluded that the isoxazolidine ring exists in an
E
1
0
conformation in which the thymine residue occupies the
equatorial position, while the ethoxyphosphoryl group is located
pseudoequatorially (Fig. 3).
2.2. Biological results
For testing the potential activity of the new truncated phospho-
nated C-1
0
-branched N,O-nucleosides against human retroviruses,
we assessed their ability to inhibit both HIV and HTLV-1 in vitro
infections. Zidovudine (AZT) was used as an internal positive con-
trol in the assay, since it is the prototype of nucleoside inhibitors of
HIV reverse transcriptase, acting as chain terminator, equally ac-
tive in comparison with other nucleoside and nucleotide ana-
logues, towards HIV in vitro infection
22
The results reported in
Table 1 showed that compounds 11a and 11d were not active,
while 11b and 11c inhibited HIV infection at an inhibitory concen-
tration 50 (IC
50
) of 220 and 132
l
M respectively. Conversely AZT
showed an IC
50
28 times lower in comparison with that of the
tested compounds. On the other hand, the compounds were unable
to inhibit HTLV-1 infection (data not shown). Moreover, cytotoxic-
ity indicated that 11b and 11c (CC
50
>1000
l
M) were at least
eighty three times less cytotoxic than AZT (CC
50
12.1
l
M), as dem-
onstrated by the values of cytotoxic concentration 50 (CC
50
) shown
in Table 1. Actually this is summarized by the values of the selec-
tivity index (SI) calculated on the basis of the ratio between the
CC
50
and IC
50
values. Table 1 shows the comparison between the
anti HIV activity of 11b and 11c versus AZT, by reporting the SI
for each compound. The SI of 11c, (7.6) was higher than that of
11b (4.5) and than that of AZT (1.57). A high SI value indicated that
the compounds were endowed with low cytotoxicity.
Thus, the low cytotoxic effect of the compounds could balance
their higher IC
50
with respect to the positive control
.
In addition,
the activity of 11b and 11c seems to be rather specific toward
HIV infection, since they were unable to inhibit HTLV-1 infection
(data not shown). Moreover, it is interesting to put in evidence that
compounds 12a–d did not show any activity against HIV and
HTLV-1 viruses, in agreement with the
a
-nature of these
derivatives.
3. Conclusion
Truncated phosphonated C-1
0
-branched N,O-nucleosides have
been synthesized in good yields by the 1,3-dipolar cycloaddition
methodology, starting from the nitrone 7. Preliminary biological
N
O
(EtO)
2
OP
OAc
CO
2
Et
Me
N
O
(EtO)
2
OP
OAc
CO
2
Et
Me
+
9
1
0
N
O
(EtO)
2
OP
B
CO
2
Et
Me
N
O
(EtO)
2
OP
B
CO
2
Et
Me
+
11a-d
12a-d
4'
5a'
H
H
5b '
1'
H
2'
a
HN
N
O
O
Me
HN
N
O
O
F
ab
HN
N
O
O
N
N
O
NHAc
cd
B
Scheme 4. Nucleosidation reaction. Reagents and conditions: isoxazolidines 9 and 10, MeCN, TMSOTf, silylated Thy (overall yield 72%), 5-Fu (overall yield 80%), U (overall
yield 71%), or Ac-Cy (overall yield 61%).
O
N
Me
H
(EtO)
2
OP
N
EtO
2
C
1'
5'
Hα
Hβ
NH
O
H
6
Me
O
4'
Figure 3. Preferred conformation of 11a.
Table 1
Inhibitory activity of the compounds 11ad on HIV infection
Compound HIV infection IC
50
a
(
l
M) SI
b
CC
50
Molt-3
c
(
l
M)
11a >1000 n.d >1000
11b 220 4.5 >1000
11c 132 7.57 >1000
11d >1000 n.d. >1000
11e 180 7.02 >1000
AZT 7.7 1.57 12.1
a
IC
50
: inhibitory concentration required to inhibit 50% HIV infection.
b
SI: selectivity index based on the ratio CC
50
/IC
50
.
c
CC
50
is the cytotoxic concentration 50% required to inhibit 50% metabolic
activity evaluated in cell line MOLT-3 by MTS.
3654 R. Romeo et al. / Bioorg. Med. Chem. 20 (2012) 3652–3657

assays show that the b-anomers are able to inhibit infection of HIV
at concentrations in the micromolar range. Although twenty eight
times less active than AZT, they are certainly less cytotoxic than
AZT, as deduced from the calculated SI values. In addition, they
seem to be rather specific in inhibiting HIV infection, while they
were unable to exert the same effect on HTLV-1 infection.
Truncated phosphonated represent a new template of cyclic
nucleoside analogs which deserve further investigations as lead
compounds for extending the current spectrum of antiviral
activity of modified nucleosides, avoiding some unwanted side
effects.
4. Experimental section
Melting points were recorded on a capillary melting point appa-
ratus and are uncorrected. Elemental analyses were recorded on a
Perkin-Elmer elemental analyzer. The elemental analyses of all fi-
nal compounds were within ±0.4% of the expected values. NMR
spectra were performed on a Varian instrument at 500 MHz (1H)
and at 125 MHz (13C) using deuterochloroform; chemical shifts
are given in ppm from TMS. The NOE difference spectra were
obtained by subtracting alternatively right off-resonance free
induction decays (FIDS) from right-on-resonance-induced FIDS.
Thin-layer chromatographic separations were performed on Merck
silica gel 60-F254 precoated aluminum plates. Preparative separa-
tions were carried out by flash chromatography using Merck silica
gel 0.035–0.070 mm. All reagents were purchased from Aldrich
Chemicals Ltd.
4.1. Synthesis of N-methyl-C-(diethoxyphosphoryl) nitrone 7
To a solution of diethyl hydroxymethyl phosphonate (500 mg,
2.97 mmol) in dry dichloromethane (20 mL), triethylamine
(2.4 mL, 17 mmol) and mesyl chloride (1.45 g, 12 mmol) were
added at 10 °C. The mixture was stirred at room temperature
for 2 h and, then, washed with a saturated solution of NaHCO
3
and concentrated in vacuo. The residue was purified by flash chro-
matography (dichloromethane/methanol 99:1 as eluent) to afford
the mesylate 15
26
in 90% yield. A solution of 15 in 25 mL of trieth-
ylamine was then reacted with methyl hydroxylamine (668 mg,
7.9 mmol) and left under reflux for 5 h. The reaction mixture was
evaporated and the residue was extracted with ether to afford
compound 16 as an yellow oil (50% yield).
1
H NMR (CDCl
3
):
d = 5.80 (bs s, OH), 4.30 (m, 4H, CH
2
OP), 3.32 (d, J = 14.9 Hz,
CH
2
P, 2H), 2.95 (s, NCH
3
), 1.45 (t, J = 7.0 Hz, 6H).
13
C NMR (CDCl
3
):
d = 62.02 (d,
2
J
POC
= 6.2 Hz), 57.99 (d,
1
J
PC
= 162.0 Hz), 49.87 (d,
3
J
PCNC
= 17.2 Hz), 16.22 (d,
2
J
POCC
= 6.2 Hz). Anal. Calcd for
C
6
H
16
NO
4
P: C, 36.55; H, 8.18; N, 7.10. Found: C, 36.69; H, 8.17;
N, 7..04.
Phosphonated hydroxylamine 16 (330 mg, 1.6 mmol) in 15 mL
of dichloromethane was oxidized with activated MnO
2
(145 mg,
1.7 mmol) at room temperature overnight to afford, after filtration
on celite, a residue which was purified by medium pressure chro-
matography to give nitrone 7. Spectral data correspond perfectly
with earlier reported data.
23
4.2. Synthesis of isoxazolidines 9 and 10
A solution of C-diethoxyethylphosphoryl-N-methyl nitrone (7;
3.0 g, 22.9 mmol) and ethyl 2-acetyloxyacrylate (8; 3.7 g, 23 mmol)
in THF (100 mL) was stirred at reflux for 24 h. The solvent was
evaporated and the residue was purified by flash chromatography
(dichloromethane/isopropanol 98:2). The product eluted first was
the ethyl (3SR,5RS)-5-(acetyloxy)-3-(diethoxyphosphoryl)-2-
methyl-isoxazolidine-5-carboxylate 9; 18% yield; yellow oil.
1
H
NMR (CDCl
3
): d = 4.28–4.12 (m, 6H, 2 CH
2
–O–P + CH
2
–O–C),
3.23 (ddd, J = 9.1, 6.9 and 6.5 Hz, H-C3), 2.93 (ddd, J = 12.5, 12.6
and 9.1 Hz, HbC4), 2.84 (s, 3 H, CH
3
–N), 2.81 (ddd, J = 18.8, 12.5
and 6.9 Hz, H
a
-C4), 2.07 (s, 3H, CH
3
–CO), 1.31 (t, 6H, J = 6.5 Hz),
1.24 (t, 3H, J = 7.0 Hz).
13
C NMR (CDCl
3
): d = 170.16 (s, C@O),
165.50 (s, C@O), 102.19 (d,
3
J
PCCC
= 9.5 Hz, C5), 64.57 (d,
1
J
PC
= 163.1, C3), 63.61 (d,
2
J
POC
= 6.0 Hz), 62.71 (d,
2
J
POC
= 7.8 Hz),
62.61 (s, CH
2
–O), 46.08 (s, CH
3
–N), 44.50 (d,
2
J
PCC
= 1.5 Hz, C4),
20.84 (s, CH
3
–C@O), 16.38 (d,
3
J
POCC
= 6.0 Hz), 13.86 (s, CH
3
);
31
P
NMR (121.5 MHz, CDCl
3
): d 22.77. Anal. Calcd for C
13
H
24
NO
8
P: C,
44.19; H, 6.85; N, 3.96. Found: C, 44.26; H, 6.88; N,3.94.
The fraction eluted second was the ethyl (3SR,5SR)-5-(acetyl-
oxy)-3-(diethoxyphosphoryl)-2-methylisoxazolidine-5-carboxyl-
ate 10; 62% yield; yellow oil.
1
H NMR (CDCl
3
): d = 4.27-4.09 (m, 6H,
2 CH
2
–O–P + CH
2
–O–C), 3.27 (ddd, J = 12.0, 6.1 and 1.5 Hz, H-C3),
2.96 (ddd, J = 16.5, 13.0 and 12.0 Hz, Hb-C4), 2.94 (d, J = 0.9 Hz, 3H,
CH
3
–N), 2.85 (ddd, J = 13.0, 6.1, 4.3 Hz, H
a
-C4), 2.05 (s, 3H, CH
3
CO), 1.32 (t, 6H, J = 6.5 Hz), 1.23 (t, 3H, J = 7.0 Hz).
13
C NMR (CDCl
3
)
d = 169.48 (s, C@O), 164.84 (s, C@O), 102.24 (d,
3
J
PCCC
= 13.5 Hz,
C5), 64.00 (d, J = 6.0 Hz, C–O–P), 62.78 (d,
1
J
PC
= 171.7, C3), 62.70
(d, J = 8.2 Hz, C–O–P), 62.63 (s, CH
2
–O), 49.24 (d, J = 4.5 Hz, C–N–
C–P), 43.01 (d,
2
J
PCC
= 1.5 Hz, C4), 21.07 (s, CH
3
–C@O), 16,38 (d,
J = 6.0 Hz), 13.86 (s, CH
3
);
31
P NMR (121.5 MHz, CDCl
3
): d 21.66.
Anal. Calcd for C
13
H
24
NO
8
P: C, 44.19; H, 6.85; N, 3.96. Found: C,
44.22; H,6.87; N, 3.99.
4.3. General procedure for the preparation of truncated
phosphonated N,O-psiconucleosides 11 and 12
A suspension of nucleobases (2 mmol) in dry acetonitrile
(30 mL) was treated with bis(trimethylsilyl)acetamide (1.5 mL,
6 mmol) and left under stirring until the solution was clear. A solu-
tion of a mixture of isozaxolidines 9 and 10 (282 mg, 1 mmol) in
dry acetonitrile (10 mL) and trimethylsilyl triflate (72
l
L,
0.4 mmol) was then added, and the reaction mixture was heated
at 70 °C for 5 h. After being cooled at 0 °C, the solution was care-
fully neutralized by addition of aqueous 5% sodium bicarbonate
and then concentrated in vacuo. After addition of dichloromethane
(20 mL), the organic phase was separated, washed with water
(2 10 mL), dried over sodium sulfate, filtered, and evaporated
to dryness. The
1
H NMR spectrum of the crude reaction mixture
shows the presence of b-anomers as major adducts, while the
a
-
anomers are present only in low amount. The residue was purified
by MPLC on a silica gel column using as eluent a mixture of CH
2
Cl
2
/
Me
2
CHOH 98:2 to afford b-nucleosides 11a–d and
a
-nucleosides
12a–d.
4.3.1. Ethyl (3SR,5RS)-3-(diethoxyphosphoryl)-2-methyl-5-(5-
methyl-2,4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)isoxazolidi-
ne-5-carboxylate 11a
Yield: 43.2%; white solid, mp 193–195 °C.
1
H NMR (CDCl
3
):
d = 8.71 (s, NH, 1H), 7.52 (br q, J = 1.0 Hz, CH@, 1H), 4.26 (q,
J = 7.0 Hz, 2H), 4.30–4.08 (m, 4H), 3.84 (ddd, J = 14.5, 8.5 and
4.0 Hz, H
a
–C5
0
, 1H), 3.25 (ddd, J = 10.5, 8.5 and 3.0 Hz, H–C4
0
,
1H), 3.06 (s, 3H), 2.88 (ddd, J = 16.5, 14.5 and 10.5 Hz, Hb–C5
0
,
1H), 1.97 (d, J = 1.0 Hz, 3H), 1.34 (t, J = 7.0 Hz, 3H), 1.28 (t,
J = 7.0 Hz, 3H), 1.25 (t, J = 7.0 Hz, 3H).
13
C NMR (CDCl
3
): d = 164.7,
164.2 (C4), 150.3 (C2), 134.7 (C6), 109.3 (C5), 92.5 (d,
3
J
PCCC
= 11.5 Hz, C1
0
), 65.2 (d,
1
J
PC
= 155.2 Hz, C4
0
), 63.5 (d,
2
J
POC
= 6.0 Hz), 63.3(CH
2
–O), 62.5 (d,
2
J
POC
= 6.8 Hz), 46.2 (CH
3
N),
45.4 (D,
2
J
PCC
= 4 Hz, C5
0
), 16.4 (d,
3
J
POCC
= 2.2 Hz), 16.3 (d,
3
J
POCC
= 2.2 Hz), 13.8 (CH
3
–CH@), 12.7.
31
P NMR (121.5 MHz,
CDCl
3
): d 21.52. Anal. Calcd for C
16
H
26
N
3
O
8
P: C, 45.82; H, 6.25; N,
10.02. Found: C, 45.86; H, 6.23; N, 10.01.
R. Romeo et al. / Bioorg. Med. Chem. 20 (2012) 3652–3657
3655

4.3.2. Ethyl (3SR,5RS)-3-(diethoxyphosphoryl)-5-(5-fluoro-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-
5-carboxylate 11b
Yield: 68%; white solid, mp 200–201 °C.
1
H NMR (CDCl
3
):
d = 8.91 (s, 1H), 7.77 (d, J = 6.5 Hz, 1H), 4.30–4.10 (m, 6H), 3.83
(ddd, J = 14.0, 8.5 and 4.0 Hz, 1H), 3.25 (ddd, J = 10.0, 8.5 and
3.0 Hz, 1H), 2.95 (s, 3H), 2.93 (ddd, J = 16.5, 14.0 and 10.0 Hz,
1H), 1.35 (t, J = 7.5 Hz, 3H), 1.28 (m, 6H).
13
C NMR (125 MHz,
CDCl
3
): d = 164.12, 156.93 (d, J = 26.7 Hz), 148.74, 139.90 (d,
J = 235.1 Hz), 123.73 (d, J = 35.2 Hz), 92.63 (d, J = 10.6 Hz), 65.13
(d, J = 163.7 Hz), 63.43 (d, J = 7.3 Hz), 63.35, 62.61 (d, J = 6.8 Hz),
46.20, 45.17, 16.41, 16.21, 13.84. Anal. Calcd for C
15
H
23
FN
3
O
8
P: C,
42.56; H, 5.48; N, 9.93. Found: C, 42.59; H, 5.50; N, 9.91.
4.3.3. Ethyl (3SR,5RS)-3-(diethoxyphosphoryl)-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-5-
carboxylate 11c
Yield: 47.61%; white solid, mp 171–174 °C.
1
H NMR (CDCl
3
):
d = 8.61 (bs s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 5.77 (d, J = 8.2 Hz, 1H),
4.37–4.02 (m, 6H), 3.92 (ddd, J = 14.2, 8.5 and 4.1 Hz, H
a
–C5
0
,
1H), 3.31 (ddd, J = 10.1, 8.5 and 2.7 Hz, H–C4
0
, 1H), 3.00 (ddd,
J = 16.3, 14.2 and 10.1 Hz, Hb–C5
0
, 1H),. 2.98 (s, 3H),), 1.34 (t,
J = 7.1 Hz, 3H), 1.32 (m, 6H).
13
C NMR (125 MHz, CDCl
3
):
d = 164.42, 163.31, 150.28, 139.01, 100.96, 92.84 (d, J = 12.0 Hz),
65.04 (d, J = 164.9 Hz), 63.69 (d, J = 6.0 Hz), 63.44, 62.49 (d,
J = 7.2 Hz), 46.11, 45.28, 16.50, 16.41, 16.31, 13.84. Anal. Calcd for
C
15
H
24
N
3
O
8
P: C, 44.45; H, 5,97; N, 10.37. Found: C, 44.41; H,
5.96 N, 10.40.
4.3.4. Ethyl (3SR,5RS)-5-[4-(acetylamino)-2-oxopyrimidin-
1(2H)-yl]-3-(diethoxyphosphoryl)-2-methylisoxazolidine-5-
carboxylate 11d
Yield: 41.27%; yellow sticky oil.
1
H NMR (CDCl
3
): d = 8.91 (bs s,
1H), 8.10 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 4.22–4.08 (m,
6H), 3.95 (ddd, J = 13.5, 8.4 and 3.4 Hz, H
a
–C5
0
, 1H), 3.28 (ddd,
J = 11.2, 8.4 and 2.9 Hz, H–C4
0
, 1H), 3.07 (s, 3H), 2.92 (ddd,
J = 16.2, 13.5 and 10.3 Hz, Hb–C5
0
, 1H), 2.26 (s, 3H), 1.35 (t,
J = 7.1 Hz, 3H), 1.33 (m, 6H).
13
C NMR (125 MHz, CDCl
3
):
d = 170.96, 164.32, 163.51, 155.18, 143.69, 95.92, 92.84 (d,
J = 10.6 Hz), 65.23 (d, J = 162.6 Hz), 63.61 (d, J = 69 Hz), 63.15,
62.41 (d, J = 6.6 Hz), 46.12, 44.86, 24.76, 16.41 (d, J = 4.4 Hz),
16.30 (d, J = 5.4 Hz), 13.77. Anal. Calcd for C
17
H
27
N
4
O
8
P: C, 45.74;
H, 6.10; N, 12.55. Found: C, 45.78; H, 6,16 N, 12.53.
4.3.5. Ethyl (3SR,5RS)-5-[4-amino-2-oxopyrimidin-1(2H)-yl]-3-
(diethoxyphosphoryl)-2-methylisoxazolidine-5-carboxylate 11e
Yield: 41.42%; yellow sticky oil.
1
H NMR (CDCl
3
): d = 8.15 (d,
J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 6.70 (bs s, 1H), 4.20–4.08
(m, 6H), 4.01 (ddd, J = 13.4, 8.2 and 3.4 Hz, H
a
–C5
0
, 1H), 3.30
(ddd, J = 11.2, 8.2 and 3.0 Hz, H–C4
0
, 1H), 3.02(s, 3H), 2.80 (ddd,
J = 16.2, 13.4 and 10.3 Hz, Hb–C5
0
, 1H), 1.35 (t, J = 7.1 Hz, 3H),
1.29 (m, 6H).
13
C NMR (125 MHz, CDCl
3
): d =165.06, 163.50,
154.89, 142.12, 96.28, 94.544 (d, J = 10.4 Hz), 66.02 (d,
J = 162.4 Hz), 64.05 (d, J = 67 Hz), 63.14, 62.38 (d, J = 6.4 Hz),
48.89, 46.85, 15.89 (d, J = 4.4 Hz), 16.22 (d, J = 5.5 Hz), 12.70. Ana-
l.Calcd for C
15
H
25
N
4
O
7
P: C, 44.56; H, 6.20, N, 13.88. Found: C,
44.59; H, 6.29 N, 13.84.
4.3.6. Ethyl (3SR,5SR)-3-(diethoxyphosphoryl)-2-methyl-5-(5-
methyl-2,4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)isoxazolidi-
ne-5-carboxylate 12a
Yield: 28.8%; white solid, mp 198–200 °C.
1
H NMR (CDCl
3
):
d = 8.23 (br s, NH, 1H), 7.61 (br q, J = 0.9 Hz, CH@, 1H), 4.30–4.11
(m, 6H), 3.84 (ddd, J = 15.0, 10.2 and 2.8 Hz, H
a
–C5
0
, 1H), 3.15–
3.08 (m, H–C4
0
and Hb–C5
0
, 2H), 3.13 (s, 3H), 1.97 (d, J = 0.9 Hz,
3H), 1.34 (t, J = 7.0 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.24 (t,
J = 7.0 Hz, 3H).
13
C NMR (CDCl
3
): d = 165.19, 163.46 (C4), 150.13
(C2), 134.61 (C6), 109.92 (C5), 92.69 (d, J = 14.2 Hz, C1
0
), 63.33
(CH
2
–O), 63.24 (d, J = 6.9 Hz), 62.15 (d, J = 163.2 Hz, C4
0
), 62.70
(d, J = 6.9 Hz), 45.43 (CH
3
N), 43.95 (C5
0
), 16.43 (d, J = 4.5 Hz),
13.95 (d, J = 7.5 Hz), 13.80 (CH
3
–CH@), 12.86.
31
PNMR
(121.5 MHz, CDCl
3
): d 20.79. Anal. Calcd for C
16
H
26
N
3
O
8
P: C,
45.82; H, 6.25; N, 10.02. Found: C, 45.87; H, 6.27; N, 9.98.
4.3.7. Ethyl (3SR,5SR)-3-(diethoxyphosphoryl)-5-(5-fluoro-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-
5-carboxylate 12b
Yield: 12%; white solid, mp 193–195 °C.
1
H NMR (CDCl
3
):
d = 8.65 (bs s, 1H), 7.86 (d, J = 6.6 Hz), 4.29–4.14 (m, 6H), 3.81
(ddd, J = 15.0, 14.6 and 1.3 Hz, H-C5
0
, 1H), 3.18–3.02 (bm, H-C4
0
and H-C5
0
, 2H), 3.13 (s, N–CH
3
, 3H), 1.38 (t, J = 8.5 Hz, 3H), 1.35
(t, J = 8.2 Hz, 3H), 1.25 (t, J = 8.2 Hz, 3H).
13
C NMR (CDCl
3
):
d = 164.66, 159.82 (d, J = 30.4 Hz), 156.20, 139.95 (d, J = 237.0 Hz),
123.60 (d, J = 35.8 Hz), 92.55 (d, J = 12.5 Hz), 64.07 (d, J = 6.5 Hz),
63.88 (d, J = 175.0 Hz), 63.80, 62.90 (d, J = 6.5 Hz), 53.43, 42.62,
16.48, 16.37, 13.87. Anal. Calcd for C
15
H
23
FN
3
O
8
P: C, 42.56; H,
5.48; N, 9.93. Found: C, 42.53; H, 5.51; N, 9.94.
4.3.8. Ethyl (3SR,5SR)-3-(diethoxyphosphoryl)-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-5-
carboxylate 12c
Yield: 23.8%; white solid, mp 178–180 °C.
1
H NMR (CDCl
3
):
d = 8.49 (bs s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 5.79 (d, J = 8.3 Hz, 1H),
4.37–4.10 (m, 6H), 4.02 (ddd, J = 14.3, 8.2 and 1.3 Hz, H
a
–C5
0
,
1H), 3.16–3.05 (m, H–C4
0
, and H–C5
0
, 2H), 3.13 (s, 3H), 1.35 (t,
J = 7.2 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.24 (t, J = 7.0 Hz, 3H).
13
C
NMR (125 MHz, CDCl
3
): d = 169.33, 166.44, 150.16, 138.88,
101.53, 90.77 (d, J = 12.3 Hz), 64.07 (d, J = 6.0 Hz), 63.14 (d,
J = 176.8 Hz), 63.31, 62.81 (d, J = 7.1 Hz), 47.27, 42.67, 16.41 (d,
J = 4.5 Hz), 14.13, 13.86. Anal. Calcd for C
15
H
24
N
3
O
8
P: C, 44.45; H,
5,97; N, 10.37. Found: C, 44.47; H, 5.94 N, 10.41.
4.3.9. Ethyl (3SR,5SR)-5-[4-(acetylamino)-2-oxopyrimidin-
1(2H)-yl]-3-(diethoxyphosphoryl)-2-methylisoxazolidine-5-
carboxylate 12d
Yield: 19.84%; yellow sticky oil.
1
H NMR (CDCl
3
): d = 8.89 (bs s,
1H), 7.75 (d, J = 7.3 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 4.27–4.03 (m,
6H), 4.02 (ddd, J = 13.9, 7.8 and 1.7 Hz, H
a
–C5
0
, 1H), 3.33–3.22
(m, H–C4
0
, and H–C5
0
, 2H), 3.08 (s, 3H), 2.24 (s, 3H), 2.24 (s, 3H),
1.29 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz,
3H).
13
C NMR (125 MHz, CDCl
3
): d = 170.16, 164.30, 162. 22,
155.10, 143.87, 113.88, 93..82 (d, J = 11.9 Hz), 65.85 (d,
J = 105.0 Hz), 63.53 (d, J = 6.0 Hz), 63.21, 62.42 (d, J = 6.5 Hz),
46.17, 44.87, 24.95, 16.39 (d, J = 6.3 Hz), 16.41, 13.96 (d,
J = 16.0 Hz). Anal. Calcd for C
17
H
27
N
4
O
8
P: C, 45.74; H, 6.10; N,
12.55. Found: C, 45.70; H, 6.14; N, 12.58.
4.4. Biological assay
The compounds were tested for their inhibitory activity on
HTLV-1 and HIV infection. HTLV-1 infection was carried out as pre-
viously shown.
26
Peripheral blood mononuclear cells were
co-cultivated with a HTLV-1 chronically infected cell line and
infection was evaluated as production of the viral core protein
p19. HIV infection was carried on by using a stable T cell line
(CEM) containing a plasmid encoding a green fluorescence protein
(GFP) driven by the HIV-1 long terminal repeat.
27
Infection was
carried on as previouslyb shown with some modification.
28
Briefly,
5 10
5
CEM-GFP were infected with a volume of supernatant from
HIV chronically infected H9 cells equivalent to 20 ng/mL of HIV
p24, for 2 h in 100
l
l CM in presence of 1000, 100, 10 and 1
l
M
concentration of compounds. Then medium was added and the
3656 R. Romeo et al. / Bioorg. Med. Chem. 20 (2012) 3652–3657

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