Synthesis of C-4′Truncated Phosphonated Carbocyclic 2′-Oxa-3′-azanucleosides as Antiviral Agents

Article abstract of DOI:10.1021/jo902485m

A new template of C-4′-truncated phosphonated nucleosides (TPCOANs) has been obtained in good yields according to two different routes which exploit the reactivity of a phosphonated nitrone. The one-step procedure based on the 1,3-dipolar cycloaddition of a phosphonated nitrone with vinyl nucleobases leads to the unnatural α-nucleosides as the main adducts. On the other hand, the target β-anomers have been obtained in high yield by a two-step procedure based on the 1,3-dipolar cycloaddition of a phosphonated nitrone with vinyl acetate followed by nucleosidation reaction. The reactivity of the phosphonated nitrone has been investigated trough quantum mechanical DFT calculations at the B3LYP/D95+(d,p) theory level. Preliminary biological assays show that β-anomers of TPCOANs are able to inhibit the reverse trancriptase of different retroviruses at concentrations in the nanomolar range, with a potency comparable with that of tenofovir.

Full text of DOI:10.1021/jo902485m

pubs.acs.org/joc
Synthesis of C-40Truncated Phosphonated Carbocyclic
20-Oxa-30-azanucleosides as Antiviral Agents
,†
Anna Piperno,* Salvatore V. Giofre, Daniela Iannazzo, Roberto Romeo,
†
†
ꢀ †
Giovanni Romeo,† Ugo Chiacchio,‡ Antonio Rescifina,‡ and Dorota G. Piotrowska§
†Dipartimento Farmaco-Chimico, University of Messina, Via SS. Annunziata, Messina 98168, Italy,
‡Dipartimento di Scienze Chimiche, University of Catania, Viale A. Doria 6, Catania 95125, Italy, and
§
ꢁ ꢁ
ꢁ ꢁ
Bioorganic Chemistry Laboratory, Faculty of Pharmacy, Medical University of yodz, 90-151 yodz,
Muszynꢁskiego 1, Poland
apiperno@pharma.unime.it
Received December 10, 2009
A new template of C-40-truncated phosphonated nucleosides (TPCOANs) has been obtained in good yields
according to two different routes which exploit the reactivity of a phosphonated nitrone. The one-step pro-
cedure based on the 1,3-dipolar cycloaddition of a phosphonated nitrone with vinyl nucleobases leads to the
unnatural R-nucleosides as the main adducts. On the other hand, the target β-anomers have been obtained in
high yield by a two-step procedure based on the 1,3-dipolar cycloaddition of a phosphonated nitrone with
vinyl acetate followed by nucleosidation reaction. The reactivity of the phosphonated nitrone has been
investigated trough quantum mechanical DFT calculations at the B3LYP/D95þ(d,p) theory level. Preliminary
biological assays show that β-anomers of TPCOANs are able to inhibit the reverse trancriptase of different
retroviruses at concentrations in the nanomolar range, with a potency comparable with that of tenofovir.
Introduction
therapeutic position: (i) cidofovir3 in the treatment of papilloma,
herpes, adeno, and pox virus, (ii) adefovir4 in the treatment
of hepatitis B virus (HBV) infections, and (iii) tenofovir5 in
the treatment of retrovirus infections. New templates of both
acyclic and cyclic phosphonated nucleosides have been
intensively investigated in order to discover new lead com-
pounds for extending the current spectrum of antiviral
activity, avoiding some unwanted side effects.6
The introduction in chemotherapy of the phosphonated
nucleosides cidofovir, tenofovir, and adefovir represents a turn-
ing point in the fight against viral infections.1 Before their
discovery, the collection of nucleoside analogues was limited
to anti-HIV and anti-herpes agents.2 The acyclic phospho-
nated nucleosides have been shown to be novel selective
broad-spectrum anti-DNA virus agents, acquiring a prominent
ꢁ
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2798 J. Org. Chem. 2010, 75, 2798–2805
Published on Web 04/15/2010
DOI: 10.1021/jo902485m
r
2010 American Chemical Society

Piperno et al.
JOCArticle
FIGURE 2. Known C-phosphonated nitrones.
triphosphate form, the substrate of the polymerase in the
nucleic acid synthesis. The insertion of a phosphonic moiety
allows the construction of compounds in which the first
monophosphate group, mimed by the phosphonate moiety,
is already present in the structure: thus, the biological antiviral
effects could depend on the length of the chain linking the
nucleic acid base to the phosphorus atom and, imperatively,
from the presence in the chain of an heteroatom able to
coordinate metal ions.12 These considerations suggest that
truncated phosphonated carbocyclic 20-oxa-30-aza nucleosides
(TPCOANs) can be regarded as new potential antiviral agents,
since the nitrogen atom in the isoxazolidine ring could be
involved in metal ion coordination, with a role similar to that
of ethereal oxygen present in the adefovir.
FIGURE 1. Phosphonated nucleosides and general structure of
truncated nucleosides.
A phosphonated nucleoside is essentially a monophosphate
nucleoside analogue where the monophosphate group is re-
placed by a phosphonic or alkylphosphonic acid residue,
metabolically and chemically more stable.7 The presence of a
50-phosphonate group allows the reaction to overcome the first,
inefficient, and often rate-limiting step of monophosphoryla-
tion in the conversion to 50-triphosphate nucleotides.8
Recently, we have reported the synthesis of a new class
of phosphonated nucleosides, the phosphonated carbocyclic
20-oxa-30-azanucleosides (PCOANs), which have been shown
to be potent inhibitors of the reverse transcriptase of different
retroviruses and have been proposed for ensuring a long last
control of HTLV-1, an oncogen retrovirus associated with
adult T-cell leukemia/lymphoma.9 In our ongoing program,
aimed to the discovery of new polymerase inhibitors, our
interest was focused on the class of the truncated phospho-
nated carbocyclic 20-oxa-30-aza nucleosides (TPCOANs),
where the phosphonate group is directly linked to the C-40
position of the sugar moiety (Figure1).
Our target compounds, TPCOANs, were prepared by
exploiting the 1,3-dipolar cycloaddition of the phosphonated
nitrone 1, following two different approaches:13 the one-step
procedure, which employs vinyl nucleobases as dipolaro-
philes, and the two-step procedure, where the cycloaddition
€
with vinyl acetate is followed by Vorbruggen nucleosidation.
From these studies an outstanding reactivity of dipole 1 with
respect to the other C-phosphonated nitrones 2 and 3
emerged (Figure 2).7,9a,9b
In this paper, we report the synthesis of the second
generation of PCOANs, the TPCOANs, their preliminary
biological evaluation, and the mechanistic considerations for
the rationalization of the different reactivity of dipoles 1-3
in the 1,3-cycloaddition process.
Generally, truncated nucleosides, lacking of the C-40
hydroxymethyl group, have been designed as anticancer
agents10 or as ligands of adenosine receptors,11 while the
antiviral activity is unexpected since they cannot turn into their
Results and Discussion
(7) Piperno, A.; Chiacchio, M. A.; Iannazzo, D.; Romeo, R. Curr. Med.
Chem. 2006, 13, 3675–3695.
(8) Shirokova, E. A.; Maxim, V. J.; Khandiazhinskaya, A. L.; Ivanov, A.
V.; Yanvarev, D. V.; Skoblov, Y. S.; Mitkervich, V. A.; Bocharov, E. V.;
Pronyaeva, T. R.; Fedyuk, N. V.; Kuklanova, M. K.; Pokrovski, A. J. J.
Med. Chem. 2004, 47, 3606–3614.
(9) (a) Chiacchio, U.; Rescifina, A.; Iannazzo, D.; Piperno, A.; Romeo,
R.; Borrello, L.; Sciortino, M. T.; Balestrieri, E.; Macchi, B.; Mastino, A. J.
Med. Chem. 2007, 50, 3747–3750. (b) Chiacchio, U.; Balestrieri, E.; Macchi,
B.; Iannazzo, D.; Piperno, A.; Rescifina, A.; Romeo, R.; Saglimbeni, M.;
Sciortino, M. T.; Valveri, V.; Mastino, A.; Romeo, G. J. Med. Chem. 2005,
48, 1389–1394. (c) Balestrieri, E.; Matteucci, C.; Ascolani, A.; Piperno, A.;
Romeo, R.; Romeo, G.; Chiacchio, U.; Mastino, A.; Macchi, B. Antimicrob.
Agents and Chemoter. 2008, 52, 54–64.
1. One-Step Synthesis of TPCOANs. Truncated phospho-
nated carbocyclic 20-oxa-30-aza nucleosides 8a,b and 9a,b
were synthesized by 1,3-dipolar cycloaddition of nitrone 1,
prepared from the commercially available diethyl hydroxy-
methyl phosphonate, as previously described.14
A study of the optimal experimental conditions in the one-
step procedure, using equimolar amounts of nitrone 1 and
the respective dipolarophile 7, was carried out by employing
dipolarophiles 7a and 7b under conventional heating or
microwave irradiation (Scheme 1, Table 1, entries 1-4).
ꢀ
(10) (a) Chiacchio, U.; Corsaro, A.; Iannazzo, D.; Piperno, A.; Pistara,
V.; Rescifina, A.; Romeo, R.; Valveri, V.; Mastino, A.; Romeo, G. J. Med.
Chem. 2003, 46, 3696–3702. (b) Nishimura, G.; Yanomab, S.; Mizunoa, H.;
Satakea, K.; Taguchia, T.; Ikedac, Y.; Tsukuda, M. Cancer Lett. 2000, 159,
1–7. (c) Engel, D.; Nudelman, A.; Tarasenko, N.; Levovich, I.; Makarovsky,
I.; Sochotnikov, S.; Tarasenko, S.; Rephaeli, A. J. Med. Chem. 2008, 51, 314–
323.
(11) Jeong, L. S.; Pal, S.; Choe, S. A.; Choi, W. J.; Jacobson, K. A.; Gao,
Z. G.; Klutz, A. M.; Hou, X.; Kim, H. O.; Lee, H. W.; Lee, S. K.; Tosh, D. K.;
Moon, H. R. J. Med. Chem. 2008, 51, 6609–6613.
(12) (a) Sigel, H. Chem. Soc .Rev. 2004, 33, 191–200. (b) Sigel, H.;
Griesser, R. Chem. Soc . Rev. 2005, 34, 875–900.
(13) (a) Ichikawa, E.; Kato, K. Curr. Med. Chem. 2001, 8, 385–423. (b)
Merino, P. Curr. Med. Chem. 2006, 13, 539–545. (c) Chiacchio, U.; Genovese,
F.; Iannazzo, D.; Librando, V.; Merino, P.; Rescifina, A.; Romeo, R.; Procopio,
A.; Romeo, G. Tetrahedron 2004, 60, 441–448.
(14) Piotrowska, D. G. Tetrahedron Lett. 2006, 47, 5363–5366.
J. Org. Chem. Vol. 75, No. 9, 2010 2799

Piperno et al.
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SCHEME 1. One-Step Procedure
TABLE 1. Different Reaction Conditions for the One-Step Procedure
entrya
dipolarophile
conditions
9/8 ratio
yieldb (%)
1
2
3
4
7a
7a
7b
7b
EtOH, reflux, 12 h
CH3CN, MW, 90 °C, 5 h, 100W
EtOH, reflux, 12 h
CH3CN, MW, 90 °C, 5 h, 100W
5:1
5:1
5:1
5:1
30
70
35
73
aMolar ratio 7a,b/1 = 1:1. bTotal yields, calculated by the conversion of vinyl nucleobases.
The reaction of nitrone 1 in refluxing ethanol proceeded
slowly in moderate yield (entry 1). After switching to micro-
wave irradiation, at 100 W for 5 h at 90 °C, an acceleration of
the reaction time together with an increased yield was
observed (entry 2). In both experiments, a 5:1 mixture of
R- and β-anomers was obtained and the unreacted vinyl base
was easily recovered. Compounds 8a and 9a were separated
by flash chromatography, and the relative configurations
FIGURE 3. Preferred conformations of 8a and 9a together with 2D
NOESY correlations.
were assigned on the basis of 2D NOE measurements.
1
The H NMR spectrum of the major product 9a shows
the diagnostic resonances of H10 at 6.07 ppm (dd), H40 at
3.25 ppm (bm), H50R at 2.60 ppm (dddd), H50β at 3.00 ppm
(bm), and H6 at 7.25 ppm (q). In the 2D NOE NMR
spectrum of the isoxazolidine 9a, positive signals were
noticed between protons in the following pairs: H40-H50R and
H10-H50β. These observations prove the cis relationship
for H10 and H50β as well as H40 and H50R and thus allow
for assigning the trans configuration between the diethoxy-
ring. In this conformation the diethoxyphosphoryl group
occupies the equatorial position, whereas thymine substituent
is pseudoaxialy oriented (Figure 3). Such disposition of the
thymine group in the major isomer 9a is a result of the
additional stabilization by the anomeric effect.
Furthermore, a significant downfield shift of the H6 signal
noticed in 8a (δ 1H = 7.75 ppm) as compared to the signal of
the same proton in 9a (δ 1H = 7.25 ppm) may be attributed
to the deshielding effect of the PdO group. This effect, higher
in TPCOANs (0.5 ppm), is lower in the superior homologous
where the difference in chemical shift is, respectively, 0.43
and 0.26 ppm for the phosphonated nucleosides containing a
methylene or two extra methylene moieties, respectively.9b,15
This observation additionally supports the cis-relationship
of the substituents at C10 (thymine) and C40 (diethoxyphos-
phoryl) positions of the isoxazolidine ring in the minor
isomer 8a.
1
phosphoryl and thymine substituents in 9a. The H NMR
spectrum of the minor isomer 8a shows a different set of signals
in comparison to 9a. In particular, the H10 proton resonates at
6.23 ppm (dd), H40at 3.01 ppm (ddd), H50βat 2.70 ppm (dddd),
H50R at 3.20 ppm (dddd), and the H6 appears as a quartet at
7.75 ppm. The positive NOE signals were observed within
H10-H50R and H40-H10 pairs of protons and fully support the
cis configuration of the substituents at C10 (thymine) and C40
(diethoxyphosphoryl) in the β-isomer 8a.
Since values of all vicinal coupling constants were success-
fully extracted from the 1H and 13C NMR spectra of 8a and 9a,
detailed conformational analyses of the isoxazolidines 8a and
9a were accomplished. On the basis of the vicinal couplings
found for 8a [J(C10-C-C-P) = 10.6 Hz, J(H-C40-C50-
Hβ) = 9.6 Hz, J(H-C40-C50-HR) = 9.0 Hz, J(P-C40-
C50-Hβ) = 17.1 Hz, J(P-C40-C50-HR) = 4.8 Hz, J(H-
C10-C50-HR) = 7.8 Hz, J(H-C10-C50-Hβ) = 3.6 Hz], it
The synthetic methodology was also applied to the synth-
esis of the 5-fluorouracil derivatives 8b and 9b, by using the
vinyl base 7b as dipolarophile (Scheme 1 and Table 1, entries
3 and 4). Analogously to the cycloaddition with 7a, the best
results were obtained by microwave irradiation conditions,
affording a 5:1 mixture of R and β anomers in 73% yield.
2. Mechanistic Consideration. C-Phosphonated nitrones
have been poorly investigated as dipoles in the 1,3-dipolar
cycloadditions, and to the best of our knowledge, only seven
examples of N-substituted C-phosphonated nitrones (Figure 2)
have been reported. Nitrone 4 is the first reported phospho-
nated nitrone;16 recently, nitrones 2 and 3 have been exploited
as substrates for the synthesis of antiviral agents9a,b,15 while
0
was concluded that the isoxazolidine ring exists in an E1
conformation in which the thymine residue occupies the equa-
torial position while the diethoxyphosphoryl group is located
pseudoequatorially (Figure 3). On the other hand, from vicinal
couplings found for 9a [J(C10-C-C-P) = 10.2 Hz, J(H-
C40-C50-Hβ) = 9.6 Hz, J(H-C40-C50-HR) = 8.0 Hz,
J(P-C40-C50-Hβ) = 16.8 Hz, J(P-C40-C50-HR) = 6.8
Hz, J(H-C10-C50-HR) = 3.9 Hz, J(H-C10-C50-Hβ) =
(15) Chiacchio, U.; Iannazzo, D.; Piperno, A.; Romeo, R.; Romeo, G.;
Rescifina, A.; Saglimbeni, M. Bioorg. Med. Chem. 2006, 14, 955–959.
(16) Vasella, A.; Voeffray, R. Helv. Chim. Acta 1982, 65, 1953–1964.
0
7.7 Hz], an E4 conformation is assigned for the isoxazolidine
2800 J. Org. Chem. Vol. 75, No. 9, 2010

Piperno et al.
JOCArticle
FIGURE 4. Acetonitrile B3LYP/D95þ(d,p)-optimized structures of the reactants.
TABLE 2. B3LYP/D95þ(d,p) Free Energies G (Hartrees) and Relative
Free Energies ΔG (kcal/mol) for Nitrones 1 and 2 and Vinyl Thymine 7a
nitrones 5 and 6 appeared to be useful intermediates in the
syntheses of phosphonate analogues of various amino acids.17
During our study on the 1,3-dipolar cycloaddition we
observed a different reactivity of C-phosphonated nitrones
1-3. In particular, nitrone 1 easily reacts with various
electron-rich or electron-poor dipolarophiles,18 including
vinyl nucleobases. The cycloaddition reactions of nitrones
2 and 3 with vinyl acetate proceed in moderate to excellent
yields (90% for nitrone 2 and 50-65% for nitrone 3),9b,16
but no reaction was observed when vinyl nucleobases were
used under conventional heating or microwave irradia-
tion. In all cases, the electron-rich carbon of the olefin
tends to attack the nitrone carbon atom, leading to C5
regioisomers.
gas phase
acetonitrile
G
ΔG
G
ΔG
(E)-1
(Z)-1
(E)-2
(Z)-2
s-trans-7a
s-cis-7a
-934.022697
-934.021654
-973.321370
-973.329793
-531.529636
-531.526152
0.00
0.65
5.28
0.00
0.00
2.19
-934.028700
-934.028590
-973.331032
-973.335858
-531.535100
-531.531921
0.00
0.07
3.03
0.00
0.00
1.99
Relative energies shown in Table 2 indicate that the
energy difference among E and Z isomers of nitrone 1 is
small in the gas phase and practically zero in acetonitrile,
suggesting the instauration of a rapid equilibrium in the
polar solvent; on the contrary, for nitrone 2 the energy
difference is quite significant in both the gas phase and
acetonitrile in favor of the Z-isomer that should be the
only one present in solution. The same considerations are
valid for vinyl thymine: the s-trans isomer is clearly pre-
dominant in solution.
The values of μ, η, andωfor the starting compounds 1, 2, and
7a are listed in Table 3, besides the ΔNmax values, that are the
maximum amount of electronic charge that the electrophile
system may accept.20a The electronic chemical potential of
vinyl thymine 7a is lower than that of nitrone 1 so indicating
that a net charge transfer will take place from 7a to 1; i.e., a
HOMOdipolarophile-LUMOdipole interaction occurs. Conver-
sely, in the case of nitrone 2, the μ value is lower than that of
vinyl thymine 7a and the charge transfer will take place from 2
to 7a, indicating a HOMOdipole-LUMOdipolarophile interaction.
The same trend is observed considering the electrophilicity
power values, which measure the stabilization in energy when
the system acquires an additional electronic charge ΔN from
the environment; this is because the electrophilicity index
encompasses both the propensity of the electrophile to acquire
an additional electronic charge driven by μ2 and the resistance
of the system to exchange electronic charge with the environ-
ment described by η simultaneously. Thus, nitrone 1 possesses
For a rationalization of the experimental results, quantum
mechanical calculations have been carried out at DFT level
of theory19 in both the gas phase and solvent.
We have also analyzed the cycloaddition reactions using
the global indexes, as defined in the context of DFT,20
which are useful tools to understand the reactivity of
molecules in their ground states (see the Supporting
Information). So, the chemical potential μ, the chemical
hardness η, and the global electrophilicity power ω were
calculated according to the previous reported formulas.21
Nitrone 3, being electronically similar to nitrone 2, was not
considered in the calculations.
The calculated energies for nitrones 1 and 2, in both E
and Z configurations, and for the vinyl thymine 7, in both
s-trans and s-cis configurations (Figure 4), are reported in
Table 2.
(17) (a) Piotrowska, D. G.; Gzowacka, I. E. Tetrahedron: Asymmetry
2007, 18, 1351–1363. (b) Piotrowska, D. G.; Gzowacka, I. E. Tetrahedron:
Asymmetry 2007, 18, 2787–2790. (c) Piotrowska, D. G. Tetrahedron: Asym-
metry 2008, 19, 2323–2329.
(18) Piotrowska, D. G. Tetrahedron 2006, 62, 12306–12317.
(19) Hohenberg, P.; Kohn, W. Phys. Rev. 1964, 136, B864–B871.
(20) (a) Pearson, R. G. J. Chem. Educ. 1987, 64, 561–567. (b) Parr, R. G.;
Chattaraj, P. K. J. Am. Chem. Soc. 1991, 113, 1854–1855.
(21) (a) Merino, P.; Tejero, T.; Chiacchio, U.; Romeo, G.; Rescifina, A.
Tetrahedron 2007, 63, 1448–1458. (b) Merino, P.; Revuelta, J.; Tejero, T.;
Chiacchio, U.; Rescifina, A.; Romeo, G. Tetrahedron 2003, 59, 3581–3592.
J. Org. Chem. Vol. 75, No. 9, 2010 2801

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TABLE 3. HOMO, LUMO, and Global Propertiesa for Nitrones 1 and
2 and Vinyl Thymine 7a Calculated at the B3LYP/D95þ(d,p) Level in
Acetonitrile
TABLE 4. B3LYP/D95þ** Relative Electronic Energies ΔE (kcal/
mol), Relative Free Energies ΔG (kcal/mol), and Charge Transfer (au) in
Terms of the Residual Charge of the Nitrone Fragment in the Transition
State for the Reaction of Nitrones 1 and 2 with Vinyl Thymine 7a
HOMO
LUMO
μ
η
ω
ΔNmax
gas phase
acetonitrile
(E)-1
(Z)-1
(E)-2
(Z)-2
-0.26172 -0.08215 -0.17194 0.17957 2.24
-0.26256 -0.07955 -0.17106 0.18301 2.18
-0.23626 -0.05458 -0.14542 0.18168 1.58
-0.24161 -0.04900 -0.14531 0.19261 1.49
0.96
0.93
0.80
0.75
0.91
0.84
ΔEa
ΔGa
ΔEa
ΔGa
NPA qCT (e)
E-exo-TS
E-endo-TS
Z-exo-TS
Z-endo-TS
E-exo-TS-2
15.09
18.15
14.26
16.68
28.40
32.00
28.44
31.16
16.76
20.33
16.51
19.66
14.36
30.29
34.59
30.95
33.92
32.30
-0.07
-0.05
-0.07
-0.06
-0.03
s-trans-7a -0.24513 -0.07110 -0.15812 0.17403 1.95
s-cis-7a -0.24319 -0.06228 -0.15274 0.18091 1.75
aHOMO, LUMO, electronic chemical potential μ, and chemical
hardness η values are in au; electrophilicity power ω values are in eV.
aReferenced to (E)-1 or (Z)-2 þ s-trans-7a (see Table 2 for G values).
a high electrophilicity value, and according to the absolute scale
of electrophilicity based on the ω index,22 it can be classified as a
strong electrophile (ω = 2.24 eV); on the other hand, nitrone 2
is only a moderate electrophile (ω ∼ 1.54 eV). The s-trans vinyl
thymine 7a results as a strong electrophile (ω = 1.95 eV) with
the electrophilic value between those of nitrones 1 and 2;
compound 7a then behaves as nucleophile with respect to
nitrone 1 and as electrophile with respect to nitrone 2. More-
over, the electrophilicity differences between 7a and nitrones 1
and 2 (Δω = 0.29 and 0.37 eV, respectively) indicate a lower
polar character for these cycloadditions, and their values are
characteristic of nonpolar pericyclic reactions.23
Knowing that the C5-regioisomers are the only product
for this type of reactions,9b,16 we also studied diastereoselec-
tivities for the reaction of nitrone 1 with vinyl thymine 7a.
For each transition state, the most stable conformation of
vinyl thymine has been chosen. Both E and Z configurations
of nitrone 1 have been evaluated. The stability of the
reactants was evaluated, and the most stable conformations
were chosen for performing the study and employed for
locating the corresponding transition states.
The relative free and electronic energies, in both the gas phase
and acetonitrile, with respect to reactants for the transition
structures located for the reaction between nitrones 1 and vinyl
thymine 7a are collected in Table 4. The same table reports the
values for charge transfer, i.e., the charge transferred between
the two reactants at the TSs geometry, in terms of the residual
charge on the nitrone; these charges have been calculated by
natural population analysis (NPA).24 For nitrone 2, which does
not react with dipolarophile 7a, we have considered only the
E-exo-TS, the more stable of all TSs, to perform a comparison.
For nitrone 1, the analysis of relative electronic (ΔE)
shows that, both in gas-phase and acetonitrile, the exo
approach is the preferred one with the Z-exo-TS, which
leads to compound R-9a, more stable than that E-exo-TS,
which leads to compound β-8a. Considering the free energies
(ΔG), although the exo approach remains the lowest in
energy, the introduction of entropy inverts the stability of
Z- and E-TS and the energetic gap between these two TSs
(Figure 5) further increases in acetonitrile solvent; this gap in
free activation energies, in acetonitrile, equal to 0.66 kcal/
mol, inserted in the Boltzman’s equation gives a calculated
9a:8a ratio of 3:1 that is in good agreement with the experi-
mental one.
It is noteworthy that the activation’s free energies for the
cycloaddition of nitrone 1 with vinyl thymine 7a are in the range
of 30.29-30.95 kcal/mol, that is, almost 2-6 kcal/mol highest
than that of analogous cycloadditions of electron-poor nitrones,
e.g., C-methoxycarbonyl-N-methyl nitrone, with methyl acry-
late or vinyl acetate,21b and this implies the necessity of more
drastic conditions of reaction. The most stable TS for cyclo-
addition of nitrone 2 has a more high ΔGq, and this accounts for
the lack of cycloaddition reaction due to the instability of the
substrate 7a at more elevate temperatures.
Taking into account NPA charges, the negative values are
indicative of an electron flow from the HOMO of the vinyl
thymine to the LUMO of both nitrones 1 and 2. Whereas this
value is in agreement with the lower absolute value of the
electronic chemical potential of vinyl thymine (μ = -0.15812)
with respect to nitrone 1 (μ = -0.17194), in the case of reaction
with nitrone 2 the NPA charge transfer at TS shows an
inversion of electron demand respect to that evidenced by
global parameters. Because the NPA charge transfer at TSs
level is more reliable than any value calculated on the ground
state of the reagents, in this latter case the reaction takes place
as HOMOdipolarophile-LUMOdipole interplay; despite this, the
interaction is unfavored by 15.9 kcal/mol respect to the
HOMOdipole-LUMOdipolarophile. It probably occurs owing to
a better orbital coefficient superposition. Again, this result is in
agreement with more drastic experimental conditions and with
the failure of the reaction.
3. Two-Step Synthesis of TPCOANs. In order to obtain as
major products the β-anomers 8a,b, the biologically inter-
esting compounds,25 a two-step procedure was investigated.
The 1:9 mixture of cycloadducts 10 and 11, obtained from the
nitrone 1 and vinyl acetate as previously described,18 was
subjected to nucleosidation reaction with silylated nucleo-
bases (Scheme 2).
Nucleosidation of 10 and 11 with silylated thymine or
5-fluorouracil, in the presence of 0.4 equiv of TMSOTf as
catalyst in dry acetonitrile at room temperature, proceeded
with a low stereoselectivity to give the R- and β-anomers in a
2:3 ratio. When the reaction was carried out at higher
temperature, the β-anomers were obtained as almost exclu-
sive compounds in 70% yield for 8a and 75% for 8b. The β/R
ratio of β- and R-nucleosides does not change when the
(22) Perez, P.; Domingo, L. R.; Aurell, M. J.; Contreras, R. Tetrahedron
2003, 59, 3117–3125.
(23) (a) Chandra, A. K.; Nguyen, M. T. J. Phys. Chem. A 1998, 102, 6181–
6185. (b) Chandra, A. K.; Nguyen, M. T. J. Comput. Chem. 1998, 19, 195–
202. (c) Nguyen, T. L.; De Proft, F.; Chandra, A. K.; Uchimaru, T.; Nguyen,
M. T.; Geerlings, P. J. Org. Chem. 2001, 66, 6096–6103. (d) Sengupta, D.;
Chandra, A. K.; Nguyen, M. T. J. Org. Chem. 1997, 62, 6404–6406. (e)
Damoun, S.; Van de Woude, G.; Mendez, F.; Geerlings, P. J. Phys. Chem. A
1997, 101, 886–893.
(24) (a) Reed, A. E.; Weinstock, R. B.; Weinhold, F. J. Chem. Phys. 1985,
83, 735–746. (b) Carpenter, J. E.; Weinhold, F. J. Mol. Struct.: Theochem
1988, 169, 41–62.
(25) Chiacchio, U.; Borrello, L.; Crispino, L.; Rescifina, A.; Merino, P.;
Macchi, B.; Balestrieri, E.; Mastino, A.; Piperno, A.; Romeo, G. J. Med.
Chem. 2009, 52, 4054–4057.
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FIGURE 5. Optimized geometries at the B3LYP/D95þ(d,p) level, in acetonitrile, for the most stable transition structures leading to
compounds 9a and 8a. Distances of forming bonds are given in angstroms.
SCHEME 2. Two-Step Procedure
nucleosidation reaction was performed starting from the pure
trans- or cis-isoxazolidine. These results show that the coupling
reaction of isoxazolidines with silylated bases occurs with low
selectivity with respect to the anomeric center. As previously
reported,26 this is due to the formation of an intermediate
oxonium ion and an equilibration of the products is possible
under the reaction conditions. The nucleosidation reaction can
proceed under kinetic or thermodynamic control, but in our
case, 8a appears to be the thermodynamically controlled
compound. To confirm this hypothesis, compound 9a, which
was obtained as a major cycloadduct in the one step procedure
(vide supra), was heated with the silylated thymine in the
presence of TMSOTf in acetonitrile at 70 °C for12h to produce
the β-isomer 8a as a single product.
4. Biological Results. For testing the potential activity of
TPCOANs against human retroviruses, we determined their
ability to inhibit the reverse transcriptase activity of different
retroviruses by means of a cell-free assay, recently described
by us,9a-c after incubation with a crude extract from 1 ꢀ 106
PMMCs (human peripheral blood mononuclear cells) which
serve as enzyme supplier for phosphorylation processes. Zido-
vudine (AZT), a well-known nucleoside, and tenofovir, a phos-
phonated nucleoside actually used in antiviral chemotherapy,
were utilized in the assay as internal positive controls. The
obtained preliminary results show that compounds 8a and 8b
completely inhibit the reverse transcriptase activity of Avian
Moloney Virus (AMV) and Human Immunodeficiency Virus
(HIV), at 1 ( 0.1 nM, at a level comparable with that of
tenofovir (1 nM) and 10-fold lower than AZT (10 nM). The
R-anomers 9a and 9b are completely inactive at the higher
concentration tested (1000 nM). Moreover, the citotoxicity,
evaluated by MTS assay,25 indicates for 8a,b and 9a,b a very
low toxicity (CC50 > 500 μM) in comparison with AZT (CC50
12.14 μM).
The obtained results, in agreement with our previous data
on similar derivatives,9a-c indicate that diethyl esters of N,O-
phosphonated nucleosides seem to be a good delivery sys-
tem. Probably, by action of nonspecific cellular esterases,
they release the two ethanol unities, not toxic for the cell,
leading to the free phosphonic acid which can be subse-
quently phosphorylated by cellular kinases. In the cell free
assay, the cellular lysate is a source of enzymes which cleave
the estereal unit and promote the phosphorylation step.
The exact mechanism by which this class of compounds exert
RT inhibition has not been addressed, but it seems plausible
that they act as chain terminators as indicated by several
evidence. In fact, the R-anomers are completely inactive and
only the β-anomers, analogues of natural nucleosides are
active. Moreover, compounds 8a,b are active on RT assay only
after incubation with a crude extract from PBMCs and are
completely inactive without this pretreatment. These data
suggest that our compounds are not allosteric inhibitors and
that they act at the active site of the enzyme.
Biological assays in order to understand if the TPCOANs
possess the necessary requirements to efficiently inhibit the
transmission of human retroviruses, by specific cellular
assays, are actually in progress.
5. Conclusion. TPCOANs have been synthesized in good
yields by 1,3-dipolar cycloaddition methodology, according
to two different routes which exploit the reactivity of phos-
phonated nitrone 1. The obtained results show that the two-
step procedure gives in high yield the desired β-anomers,
while the unnatural R-nucleosides represent the main adduct
in the one-step methodology.
(26) Chiacchio, U.; Corsaro, A.; Iannazzo, D.; Piperno, A.; Rescifina, A.;
Romeo, R.; Romeo, G. Tetrahedron Lett. 2001, 42, 1777–1780.
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The different reactivity of phosphonated nitrones 1-3 in
the 1,3-dipolar cycloaddition has been rationalized through
quantum mechanical DFT calculations.
8.0, 6.8, 3.9 Hz, HR-C50, 1H), 1.96 (d, J = 0.95 Hz, 3H), 1.39 (t,
J = 7.0 Hz, 3H), 1.37 (t, J = 7.0 Hz, 3H); 1H NMR (700 MHz,
C6D6) δ 10.17 (s, 1H, NH), 6.67 (s, 1H, CHd), 5.97 (br s, 1H,
H-C10), 4.10-4.00 (m, 2H), 4.00-3.80 (m, 2H), 3.17 (very br t,
1H, H-C40), 2.87 (dddd, J = 16.8, 13.6, 9.6, 7.7 Hz, 1H, Hβ-C50),
2.96 (s, 3H), 2.39 (dddd, J = 13.6, 8.0, 6.8, 3.9 Hz, 1H, HR-C50),
Preliminary biological assays show that the β-anomers of
TPCOANs are able to inhibit the reverse transcriptase of
different retroviruses at concentrations in the nanomolar range,
with a potency comparable with Tenofovir. TPCOANs repre-
sent a new template of cyclic phosphonated nucleosides which
deserve further investigations as lead compounds for extending
the current spectrum of antiviral activity of the acyclic phos-
phonated nucleosides, avoiding some unwanted side effects.
1.70 (s, 3H), 1.06 (t, J = 7.0 Hz, 3H), 1.03 (t, J = 7.0 Hz, 3H); 13
C
NMR (176 MHz, CDCl3) δ 163.5 (C4), 150.1 (C2), 135.3 (C6),
111.3 (C5), 83.2 (d, J = 10.2 Hz, C10), 63.1 (d, J = 164.7 Hz, C40),
62.6 (d, J = 7.3 Hz), 62.6 (d, J = 7.3 Hz), 38.7 (CH3N), 29.7
(C50), 16.52 (d, J = 5.6 Hz), 16.50 (d, J = 6.3 Hz), 12.6 (CH3-
CHd); 31P NMR (121.5 MHz, CDCl3) δ 20.73; HRMS-EI (m/z)
[M]þ calcd for C13H22N3O6P 347.1246, found 347.1244.
Diethyl [(10SR,40SR)-10-[5-Fluoro-2,4-dioxo-3,4-dihydropyri-
mid-1(2H)-yl]-30-methyl-20-oxa-30-azacyclopent-40-yl]phosphonate
8b: sticky foam (263 mg, 75% yield by the two-step procedure;
43 g, 13% yield by the one step procedure); 1H NMR (500 MHz,
CDCl3) δ 10.01 (s, 1H), 8.01 (d, J = 6.5 Hz, 1H), 6.21 (d, J = 5.5
Hz, 1H), 4.30-4.15 (m, 4H), 3.22 (ddd, J = 13.5, 9.5, and 5.5 Hz,
1H), 3.03 (td, J =9.5 and 2.5Hz, 1H), 2.95 (s, 3H), 2.69 (ddd, J =
17.0, 9.5, and 2.5 Hz, 1H), 1.35 (m, 6H); 13C NMR (125 MHz,
CDCl3) δ 157.0 (d, J = 26.4 Hz), 149.2, 140.2 (d, J = 234.8 Hz),
124.7 (d, J = 34.4 Hz), 82.4 (d, J = 8.8 Hz), 63.42 (d, J = 6.2 Hz),
63.40 (d, J = 165.3 Hz), 62.7 (d, J = 6.9 Hz), 45.6, 41.0, 16.35,
16.34; HRMS-EI (m/z) [M]þ calcd for C12H19FN3O6P 351.0996,
found 351.0999.
Diethyl [(10SR,40RS)-10-[5-Fluoro-2,4-dioxo-3,4-dihydropyri-
mid-1(2H)-yl]-30-methyl-20-oxa-30-azacyclopent-40-yl]phosphonate
9b: stickyfoam (213 mg, 60%yieldbythe one-stepprocedure);1H
NMR (500 MHz, CDCl3, δ): 10.0 (bs, 1H), 7.29 (d, J = 9.5 Hz,
1H), 6.60 (dd, J = 7.0 Hz, 1H), 4.32- 4.15 (m, 4H), 3.94 (m, 1H),
3.0 (s, 3H), 2.95 (m, 1H), 2.40 (m, 1H), 1.35 (m, 6H); 13C NMR
(125 MHz CDCl3 δ) 157.2 (d, J = 26.5 Hz), 149.3, 139.9 (d, J =
234.9 Hz), 123.6 (d, J = 34.1 Hz), 80.9 (d, J = 13.1 Hz), 63.4 (d,
J= 163.8 Hz), 63.3 (d, J= 6.3 Hz), 62.7 (d, J= 6.3 Hz), 45.5, 35.5,
16.33, 16.30; HRMS-EI (m/z) [M]þ calcd for C12H19FN3O6P
351.0996, found 351.0998.
Reverse Transcriptase Inhibition Assay. The new synthesized
compounds 8a,b and 9a,b and the control compounds AZT
and tenofovir were activated through preincubation with a
crude extract prepared from phytohemagglutinin- and IL-2-
stimulated PBMCs from healthy donors negative for HIV and
hepatitis B and C viruses. For preparation of the crude extract,
1 ꢀ 106 PBMCs, previously stimulated with phytohemaggluti-
nin (2 μg/mL) and IL-2 (20 U/mL) for 72 h in RPMI medium
plus 20% FBS, were rinsed three times in cold phosphate-
buffered saline and then solubilized in lysis buffer on ice and
centrifuged at 10000g. Lysed extracts were incubated with the
compounds at different concentrations for 15 min on ice and
subsequently for 45 min at 30 °C. After incubation, the crude
extract-compound mixture was inactivated for 5 min at 95 °C.
The reverse transcriptase (RT) inhibition assay was performed
by using an RT assay kit (Roche), and the procedure for
assaying RT inhibition was performed as described in the kit
protocol. Briefly, the reaction mixture consists of template/
primer complex, 20-deoxy-nucleotide-50-triphosphates (dNTPs)
and RT enzyme in the lysis buffer with or without inhibitors.
After 1 h of incubation at 37 °C, the reaction mixture was
transferred to a streptavidine-coated microtiter plate (MTP).
The biotin-labeled dNTPs that are incorporated in the tem-
plate due to activity of RT were bound to streptavidine.
The unbound dNTPs were washed using wash buffer, and anti-
digoxigenin peroxidase (DIG-POD) was added in MTP. The
DIG-labeled dNTPs incorporated in the template was bound to
anti-DIG-POD antibody. The unbound anti-DIG-POD was
washed, and the peroxide substrate (ABST) was added to
the MTP. A colored reaction product was produced during
the cleavage of the substrate catalyzed by a peroxide enzyme.
Experimental Section
General Procedures for the Preparation of Truncated Phos-
phonated Carbocyclic 20-Oxa-30-aza Nucleosides 8 and 9. One-
Step Procedure. A solution of nitrone 1 (200 mg, 1.02 mmol) in
dry acetonitrile (20 mL) and vinyl nucleobases27 7a (156 mg,
1.02 mmol) or 7b (160 mg, 1.02 mmol) was put in a sealed tube
and irradiated under microwave conditions at 100 W, 90 °C, for
5 h. The removal of the solvent in vacuo afforded a crude
material which, after MPLC purification by using as eluent a
mixture of CHCl3/MeOH 99:1, gives the nucleosides 8 and 9.
Two-Step Procedure. A suspension of thymine (252 mg,
2 mmol) or 5-fluorouracil (260 mg, 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 solution of a mixture of isozaxolidines 10 and 11 (282 mg,
1 mmol) in dry acetonitrile (10 mL) and trimethylsilyl triflate
(72 μL, 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 carefully 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 1H NMR spectrum of
the crude reaction mixture shows the presence of β-anomers as
nearly exclusive adducts, while the R-anomers are present only
in traces. The residue was purified by MPLC on a silica gel
column using as eluent a mixture of CHCl3/MeOH 99:1 to
afford 8a with a 70% yield and 8b in 75% yield.
Diethyl [(10SR,40SR)-10-[5-Methyl-2,4-dioxo-3,4-dihydropyri-
mid-1(2H)-yl]-30-methyl-20-oxa-30-azacyclopent-40-yl]phosphonate
8a: sticky foam (243 mg, 70% yield by the two-step procedure;
1
40 mg, 12% yield by the one-step procedure); H NMR (700
MHz, CDCl3) δ 9.08 (s, NH, 1H), 7.75 (br q, J = 0.7 Hz, CHd,
1H), 6.23 (dd, J = 7.5 and 3.3 Hz, H-C10, 1H), 4.23-4.10 (m,
4H), 3.20 (dddd, J = 13.8, 9.0, 7.5, and 4.8 Hz, HR-C50, 1H), 3.01
(ddd, J = 9.6, 9.0, and 2.7 Hz, H-C40, 1H), 2.97 (s, 3H), 2.70
(dddd, J = 17.1, 13.8, 9.6, and 3.3 Hz, Hβ-C50, 1H), 1.95 (d, J =
0.7 Hz, 3H), 1.34 (t, J = 7.0 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H); 13
C
NMR (176 MHz, CDCl3) δ 164.2 (C4), 150.9 (C2), 136.6 (C6),
110.8 (C5), 82.3 (d, J = 10.6 Hz, C10), 64.2 (d, J = 167.3 Hz, C40),
63.6 (d, J = 7.0 Hz), 63.0 (d, J = 7.0 Hz), 46.2 (CH3N), 41.5 (d,
J = 3.5 Hz, C50), 16.8 (d, J = 5.2 Hz), 16.7 (d, J = 5.3 Hz), 13.0
(CH3-CHd); 31P NMR (121.5 MHz, CDCl3, δ) 21.44; HRMS-EI
(m/z) [M]þ calcd for C13H22N3O6P 347.1246, found 347.1242.
Diethyl [(10SR,40RS)-10-[5-Methyl-2,4-dioxo-3,4-dihydropyri-
mid-1(2H)-yl]-30-methyl-20-oxa-30-azacyclopent-40-yl]phosphonate
9a:stickyfoam (200 mg, 58%yieldbythe one-stepprocedure);1H
NMR (700 MHz, CDCl3) δ 8.97 (s, NH, 1H), 7.27 (q, J = 0.95
Hz, CHd, 1H), 6.07 (dd, J = 7.7, 3.9 Hz, H-C10, 1H), 4.27-4.22
(m, 2H), 4.22-4.15 (m, 2H), 3.30-3.25 (very br m, H-C40, 1H),
3.10-3.00 (br m, Hβ-C50, 1H), 3.05 (s, 3H), 2.60 (dddd, J = 13.6,
(27) Dalpozzo, R.; De Nino, A.; Maiuolo, L.; Procopio, A.; Romeo, R.;
Sindona, G. Synthesis 2002, 172–174.
2804 J. Org. Chem. Vol. 75, No. 9, 2010

Piperno et al.
JOCArticle
The absorbance of the sample was determined at OD 405 nM
using microtiter plate ELISA reader. The resulting color inten-
sity is directly proportional to the actual RT activity. The
percentage inhibitory activity of RT inhibitors was calculated
by comparing to a sample that does not contain an inhibitor.
The percentage inhibition was calculated by formula: % inhibi-
tion =100 - [(OD 405 nm with inhibitor/OD 405 nm without
inhibitor) ꢀ 100]. Data represent mean values for three separate
experiments, and the variation among triplicate samples was less
than 10%. Compounds 8ab completely inhibit (100% inhibition
rate) the RT activity at 1 ( 0.1 nM. No inhibition of reverse
transcriptase activity was observed at the higher concentration
tested (1000 nM) for compounds 9ab.
Solution; Promega). The assay was performed by seeding 1 ꢀ 104
MOLT-3 cells in 100 μL in the presence or absence of the different
compounds at different concentrations ranging from 1 μM to
1 mM in RPMI medium supplemented with 5% FBS. A 20-μL
portion of Cell Titer 96 Aqueous One Solution reagent was added
directly to culture wells at the end of the culture period and
incubated for 1-4 h, and then absorbance was read at 490 nm.
Each condition was analyzed in triplicate.
Acknowledgment. This work was partially supported by
CINMPIS and MIUR (PRIN).
Determination of Cytotoxycity of the Compounds. The cyto-
toxicity of the compounds on cells MOLT-3 was evaluated
by MTS assay. Inhibition of cell metabolic activity revealed
by reduction of the oxidative burst was detected through for-
mazan product formation using a commercial colorimetric kit
(MTS [3,4-(5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium salt], Cell Titer 96 Aqueous One
Supporting Information Available: General information
and for all new compounds 8a,b and 9a,b; proton and carbon
spectra; supplementary phosphorus NMR and 2D NOESY
spectra for compounds 9a and 9b. Tables of computational
Cartesian coordinates, energies, and TS frequencies. This
material is available free of charge via the Internet at http://
pubs.acs.org.
J. Org. Chem. Vol. 75, No. 9, 2010 2805