Synthesis of 2′-deoxy-1′-homo-N-nucleosides with anti-influenza activity by catalytic methyltrioxorhenium (MTO)/H2O2 oxyfunctionalization

Article abstract of DOI:10.1002/chem.201201285

This paper describes a new route for the synthesis of 1′-homo-N- nucleoside derivatives by means of either methyltrioxorhenium (MTO) or supported MTO catalysts, with H₂O₂ as the primary oxidant. Under these selective conditions, the oxyfunctionalization of the heterocyclic ring and the N heteroatom oxidation were operative processes, regardless of the type of substrate used, that is, purine or pyrimidine derivatives. In addition, the oxidation of 1′-homo-N-thionucleosides, showed the occurrence of site-specific oxidative nucleophilic substitutions of the heterocyclic ring. The MTO/H₂O₂ system showed, in general, high reactivity under both homogeneous and heterogeneous conditions, affording the final products with high conversion values of substrates and from medium to high yields. Many of the novel 1′-homo-N-nucleoside analogues were active against the influenza A virus, without any cytotoxic effects, retaining their activity in both protected and unprotected forms. Antiviral activity: Under either homogeneous or heterogeneous catalytic conditions, the high reactivity of the methyltrioxorhenium (MTO)/H₂O₂ oxidative system allowed the synthesis of new 1′-homo-N-nucleoside derivatives through a selective oxyfunctionalization of the heterocyclic ring and of the N-heteroatom moiety (see scheme). Many of the modified nucleosides have been shown to be active against the influenza A virus. Copyright

Full text of DOI:10.1002/chem.201201285

DOI: 10.1002/chem.201201285
Synthesis of 2’-Deoxy-1’-homo-N-nucleosides with Anti-Influenza Activity by
Catalytic Methyltrioxorhenium (MTO)/H2O2 Oxyfunctionalization
Raffaele Saladino,*[a] Veronica Neri,[a] Paola Checconi,[b, c] Ignacio Celestino,[b]
Lucia Nencioni,[b] Anna Teresa Palamara,[b, c] and Marcello Crucianelli*[d]
Abstract: This paper describes a new
route for the synthesis of 1’-homo-N-
nucleoside derivatives by means of
either methyltrioxorhenium (MTO) or
supported MTO catalysts, with H2O2 as
the primary oxidant. Under these selec-
tive conditions, the oxyfunctionaliza-
tion of the heterocyclic ring and the N
heteroatom oxidation were operative
processes, regardless of the type of sub-
strate used, that is, purine or pyrimi-
dine derivatives. In addition, the oxida-
tion of 1’-homo-N-thionucleosides,
showed the occurrence of site-specific
oxidative nucleophilic substitutions of
the heterocyclic ring. The MTO/H2O2
system showed, in general, high reac-
tivity under both homogeneous and
heterogeneous conditions, affording the
final products with high conversion
values of substrates and from medium
to high yields. Many of the novel 1’-
homo-N-nucleoside analogues were
active against the influenza A virus,
without any cytotoxic effects, retaining
their activity in both protected and un-
protected forms.
Keywords: antiviral agents · cataly-
sis · hydrogen peroxide · methyl-
trioxorhenium · nucleosides
Introduction
replacement of the furanose ring with a cyclopentane
moiety. Due to the absence of the natural N glycosidic
bond, these analogues possess greater metabolic stability to-
wards the hydrolytic activity of cellular hydrolases. The re-
sistance to enzymatic degradation is also the rational basis
for the preparation of 1’-homo-N-nucleosides (A and B),
bearing a methylene tether between the sugar moiety and
the nucleobase.[8]
The synthesis of nucleoside analogues has received a great
deal of attention in recent years mainly due to their high bi-
ological activity.[1] In principle, both modifications of the
sugar and nucleobase pharmacophores can afford active
compounds. These modifications include the synthesis of
therapeutically useful antiviral agents, such as azidothymi-
dine (AZT), dideoxycytidine (ddC), dideoxyinosine
(ddI),[2–4] acyclovir, penciclovir, famciclovir,[5] and lamivu-
dine.[6] To further increase the biological activity and selec-
tivity of nucleoside analogues, attention has been focused on
enzymatically stable derivatives, such as cyclopentyl nucleo-
sides (carbanucleosides),[7] derived from nucleosides by the
[a] Prof. Dr. R. Saladino, Dr. V. Neri
Department of Agrobiology and Agrochemistry
University of Tuscia, via S. Camillo de Lellis
I-01100 Viterbo (Italy)
1’-Homo-N-nucleosides are substrates for cellular kinases
and are able to pair with nucleosides by Watson–Crick inter-
actions without appreciable modifications of the structure of
the DNA (or RNA) molecule.[9]
Fax : (+39)0761-357242
E-mail: saladino@unitus.it
[b] Dr. P. Checconi, Dr. I. Celestino, Dr. L. Nencioni,
Prof. Dr. A. T. Palamara
1’-Homo-N-nucleosides are usually prepared by two main
methods.[8] In the first, the heterocyclic nucleobase reacts
with a preformed sugar derivative by nucleophilic displace-
ment of a leaving group at the C-6’ exocyclic position of the
pentofuranose ring.[10] In the second case, the nucleobase is
constructed by condensation reactions from a reactive C-1’
pentofuranosyl methylamino derivative.[11a,b] These methods
have been implemented with different degrees of success
due to the occurrence of complex multistep processes and
undesired side reactions. Further, among the a- and b-
anomeric nucleosides, the latter have been extensively stud-
Department of Public Health and Infectious Diseases
Institute Pasteur Cenci Bolognetti Foundation
Sapienza University of Rome, Rome (Italy)
[c] Dr. P. Checconi, Prof. Dr. A. T. Palamara
San Raffaele Pisana Scientific Institute for Research
Hospitalization and Health Care, Rome (Italy)
[d] Dr. M. Crucianelli
Department of Physical and Chemical Sciences
University of LꢀAquila
via Vetoio, I-67100 Coppito (AQ) (Italy)
Fax : (+39)0862-433753
E-mail: marcello.crucianelli@univaq.it
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FULL PAPER
ied due to their natural occurrence and their numerous bio-
logical activities, whereas the a-nucleosides have received
little attention.[11c–e]
report the possibility of using both of these procedures for
the selective oxyfunctionalization of 2’-deoxy-1’-homo-N-nu-
cleosides and 2’-deoxy-1’-homo-N-thionucleosides to yield
novel derivatives characterized by anti-influenza activity. To
design environmentally friendly procedures available for in-
dustrial applications, the reactions were performed under
homogeneous and heterogeneous conditions by using cata-
lysts based on the immobilization of MTO on low-cost and
easily available matrices, including the polystyrene and poly-
vinyl pyridine resins.
As an alternative, the one-pot modification of the hetero-
cyclic nucleobase in preformed nucleoside analogues has
been evaluated. In this case, the oxidative methods show
some advantages, such as mild experimental conditions and
the possibility to operate under catalytic conditions. Methyl-
ACHTUNGTRENNUNGtriACHTUNGTRENNUNGoxoACHTUNGTRENNUNG
rhenium (MTO)[12] is able to catalyze the selective
oxyfunctionalization of both purine and pyrimidine nucleo-
bases and nucleosides, including their sulfur derivatives in
which the amide and ureido moieties are replaced by the
corresponding thioamide and thioureido groups. MTO
shows a high efficiency in the activation of hydrogen perox-
ide and urea hydrogen peroxide adduct (UHP) for a large
panel of oxidative transformations through formation of two
Results and Discussion
Synthesis of starting materials: 2’-Deoxy-1’-homo-N-nucleo-
sides 2–5 and 2’-deoxy-1’-homo-N-thionucleosides 6–8: A
panel of 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-N-nucleosides
reactive
intermediates,
either
the
monoperoxo
[MeRe(O2)O2] or the bis
G
and
3’,5’-di-O-benzyl-2’-deoxy-1’-homo-N-thionucleosides,
complexes.[13] Pyrimidine nucleobases, such as uracil and
thymine, are oxidized by the MTO/H2O2 system to corre-
sponding C-5/C-6 oxirane derivatives, followed by a nucleo-
philic ring-opening reaction with formation of the corre-
sponding diols. The epoxides can be stabilized when the re-
action is performed in the presence of urea to buffer the
Lewis and Brønsted acidity of MTO. In the case of purine
nucleobases the reaction proceeds mainly with the forma-
tion of the corresponding N-oxides or 8-oxa-derivatives.
On the other hand, when one or more oxygen atoms in
the nucleobase are replaced by sulfur, a new method of re-
action, called the “oxidative nucleophilic substitution proc-
ess”, operates. Under these experimental conditions the re-
action proceeds through sulfonic and sulfenic acids as reac-
tive intermediates, which can be replaced as leaving groups
by nucleophiles present in the reaction medium. Herein, we
including 3’,5’-di-O-benzyl-2’-deoxy-1’-homoadenine 2, 3’,5’-
di-O-benzyl-2’-deoxy-1’-homocytidine 3, 3’,5’-di-O-benzyl-2’-
deoxy-1’-homothymidine 4, 3’,5’-di-O-benzyl-2’-deoxy-1’-
homo
ACHTUNGTRENuNUGN ridine 5, and 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-6-
thioinosine 6, were initially selected as starting compounds.
AHCTUNGTRENNUNG
The synthesis of compounds 2–6 was carried out by reaction
between the appropriate nucleobase (adenine, cytosine, thy-
mine, uracil and 6-mercaptopurine, respectively) and the 2’-
deoxyribose C-6’ mesyl derivative 1, which was prepared as
previously reported by Hossain and co-workers from 2’-de-
oxyribose (not shown) through a multistep procedure.[14a]
The reactions were performed in DMF at 908C in the pres-
ence of NaH (for adenine and 6-mercaptopurine), CsCO3
(for cytosine), and K2CO3 (for thymine and uracil), to afford
compounds 2–6 in 67, 71, 82, 85, and 83% yields, respective-
ly (Scheme 1).
Scheme 1. Synthesis of 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-N-nucleosides 2–5 and 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-6-thioinosine 6.
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R. Saladino, M. Crucianelli et al.
During the synthesis of com-
pound 6 a significant amount
of the S-alkylation product (ca.
15% yield) was obtained as a
byproduct due to the signifi-
cant nucleophilicity of the
thiol group in the 6-mercapto-
purine (this byproduct was not
further investigated because it
was not interesting for the pur-
poses of this work). 3’,5’-Di-O-
benzyl-2’-deoxy-1’-homo-4-
thio
benzyl-2’-deoxy-1’-homo-4-
thiouridine 8 were synthesized
ACHTUNGTRENNUNGthymidine 7 and 3’,5’-di-O-
ACHTUNGTRENNUNG
by starting from the corre-
sponding 3’,5’-di-O-benzyl-2’-
deoxy-1’-homo-N-nucleosides
4 and 5 by a modification of
the procedure previously de-
veloped by Fox and co-work-
ers for simple nucleoside de-
rivatives.[15] Briefly, compounds
4 and 5 (0.5 mmol) were dis-
solved in dioxane (2.0 mL) and
treated with a small excess of
P2S5 (0.6 mmol) at reflux to
afford 7 and 8 in 85 and 90%
yield, respectively (Scheme 2).
Scheme 3. Preparation of heterogeneous MTO-based catalysts I–V.
oxide (PVPN; obtained by selective oxidation of PVP with
meta-chloroperoxybenzoic acid, m-CPBA) with MTO.[16] PS/
MTO catalyst V was synthesized after the addition of MTO
to a solution of polystyrene in THF by slow evaporation of
the solvent.[17]
Oxidation of 2’-deoxy-1’-homo-N-nucleosides 2–4 and 2’-
deoxy-1’-homo-N-thionucleosides 6–8: Initially we studied
the oxidation of 2’-deoxy-1’-homo-N-nucleosides 2–4. Treat-
ment of 3’,5’-di-O-benzyl-2’-deoxy-1’-homoadenine
2
(0.1 mmol) with MTO (2% w/w) and H2O2 (0.2 equiv) in
EtOH (1.0 mL) at 508C afforded the 3’,5’-di-O-benzyl-2’-
deoxy-1’-homoadenine N(1)-oxide 9 as the only recovered
product in 77% conversion and 97% yield (Table 1, entry 1;
for the purine ring numbering, see Scheme 1). Unless other-
wise specified, the yields of the oxidative transformations
are calculated on the basis of converted substrate. This
result is in accordance with our data on the oxidation of a
large panel of adenine cyclic and alicyclic nucleoside deriva-
tives including 2’-deoxyadenosine, in which case N(1)-oxide
derivatives were obtained with high selectivity and yield.[18]
Adenine N(1)-oxide derivatives are useful intermediates in
the synthesis of 2-deuterioadenines by the “fission and re-
closure” procedure[19] and are easily transformed into 4-ami-
noimidazole-4-carboxamidooxime derivatives by simple
treatment with an alkali.[20] Furthermore, adenine N(1)-
oxide derivatives show significant antiviral activity against
human respiratory syncytial virus (RSV).[21]
Scheme 2. Synthesis of 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-thiothymi-
dine 7 and 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-thiouridine 8.
Preparation of heterogeneous MTO-based catalysts PVP2–
25%/MTO I–II, PVPN2–25%/MTO III–IV, and PS/MTO V:
In this study, we used two families of heterogeneous rheni-
um catalysts based on the immobilization of MTO on low
cost and easily available organic resins like poly(4-vinylpyri-
dine) (PVP) and polystyrene (PS; Scheme 3).
In the first case, the rhenium atom is coordinated by the
pyridine groups on the resin, whereas a physical entrapment
into microcapsules operates with polystyrene. In particular,
PVP2–25%/MTO catalysts I and II (I: n=2%, II: n=25%)
were prepared from PVP 2 and 25% cross-linked with di-
vinyl benzene, respectively, by treatment of the resin with
an alcoholic solution of MTO. In a similar way, PVPN2–
25%/MTO catalysts III and IV (III: n=2%, IV: n=25%)
were prepared by treatment of poly(4-vinylpyridine)-N-
When the reaction was repeated under similar experimen-
tal conditions but in a different solvent, such as CH3COOH
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Synthesis of 2’-Deoxy-1’-homo-N-nucleosides
FULL PAPER
Table 1. Oxidation of 3’,5’-di-O-benzyl-2’-deoxy-1’-homoadenine 2.
tion grade of a polymer on the reactivity and selectivity of
the catalyst.[25a,b] In the case of the oxidation with microen-
capsulated catalyst V, lower conversion was observed even
at 808C (Table 1, entry 8).
The oxidation of 3’,5’-di-O-benzyl-2’-deoxy-1’-homocyti-
dine 3 with MTO (2% w/w) in EtOH (1.0 mL) at 508C af-
forded 3’,5’-di-O-benzyl-2’-deoxy-1’-homocytidine N(3)-
oxide 11 as the main reaction product in 42% yield, as well
as 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-5-hydroxycytidine 12
in 21% yield (Table 2, entry 1).
Table 2. Oxidation of 3’,5’-di-O-benzyl-2’-deoxy-1’-homocytidine 3.
Solvent
EtOH
Catalyst
MTO
T
[8C]
Product(s) Conv. Yield
[%]
[%][a]
1
2
3
4
5
6
7
8
50
50
50
80
80
9
77
87
47
65
72
64
79
59
97
55 (27)
85
90
96
87
95
91
CH3COOH MTO
9 (10)
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
PVP2%/MTO I
PVP2%/MTO I
PVP25%/MTO II
PVPN2%/MTO III 80
PVPN25%/MTO IV 80
9
9
9
9
9
9
PS2%/MTO V
80
[a] Unless otherwise specified, the yields of the reactions described above
are calculated on the basis of converted substrate.
Catalyst
Product(s)
Conv.
[%]
Yield
[%][a]
1
2
3
4
5
6
MTO
11 (12)
11 (12)
11 (12)
11 (12)
11 (12)
11 (12)
>90
>90
87
>90
>90
90
42 (21)[b]
57 (28)
PVP2%/MTO I
PVP25%/MTO II
PVPN2%/MTO III
PVPN25%/MTO IV
PS2%/MTO V
(1.0 mL), that is, a medium able to increase the reactivity of
MTO,[22] a greater conversion was observed (87%) with for-
mation of compound 9 as the main reaction product (55%
yield) as well as a small amount of 3’,5’-di-O-benzyl-2’-
deoxy-1’-homo-7H-8-oxoadenine N(1)-oxide 10, derived
from a further oxidation of 9 at the C(8) position of the
purine ring (Table 1, entry 2).
64 (22)
27 (65)[c]
24 (69)[c]
38 (23)[b]
[a] Unless otherwise specified, the yields of the reactions described above
are calculated on the basis of converted substrate. [b] A low mass balance
was observed in this case, probably due to the formation of over-oxida-
tion products that were not isolated. [c] The opposite selectivity was ob-
served in comparison with PVP catalysts.
8-Oxoadenine and adenosine derivatives are usually pro-
duced by over-oxidation of the purine ring through initial
formation and rearrangement of reactive oxyranyl inter-
mediates.[23] They have been detected during oxidative
damage of DNA, in neoplastic livers of fish and in urine
samples of humans.[24] The oxidation was then studied under
heterogeneous conditions with catalysts I–V in EtOH, which
was shown to act as the most selective reaction solvent. Irre-
spective to experimental conditions, the N(1)-oxide deriva-
tive 9 was obtained as the only recovered product apart
from unreacted substrate, with a high yield and from moder-
ate to high conversion (Table 1, entries 3–8). As a general
reaction pattern, catalysts I–V were less reactive than MTO,
probably due to the presence of a kinetic barrier for the
substrate to approach the supported rhenium complexes.
For this reason, better results were obtained when perform-
ing the reaction at 808C (Table 1, entry 3 vs. 4). Concerning
the poly(4-vinylpyridine) catalysts I and II, an enhancement
of the value of the reticulation grade of the polymer (that is
2 vs. 25%) increased both the reactivity and selectivity of
the oxidation (Table 1, entry 4 vs. 5). Similar behavior was
observed with the poly(4-vinylpyridine-N-oxide) catalysts
III and IV (Table 1, entry 6 vs. 7). These data are in accord-
ance with our previous findings on the effect of the reticula-
The high value of conversion of substrate and the low
mass balance (that is, how much of the converted substrate
is transformed into reaction products) observed during the
oxidation of 3 suggests the formation of over-oxidation
products. Cytidine residues in nucleic acids are usually more
reactive than their uridine or thymidine counterparts.[26] Cyt-
ACTHNGUTRENNUGiCAHUTNGTRENNdNUG ine N-oxide derivatives are obtained by oxidation of the
natural nucleoside or nucleotide with different stoichiomet-
ric oxidants including m-CPBA,[27] terephthalic acid,[28] and
hydrogen peroxide (H2O2).[29] To the best of our knowledge
the oxidation with MTO is the first of the reported catalytic
procedures so far described for the synthesis of these deriva-
tives. Cytidine N-oxides are able to inhibit RNase[30] and can
undergo the Dimroth rearrangement, affording the corre-
sponding uridines.[31]
5-Hydroxy cytidine derivatives are usually produced in
the cell by radical damage.[32] As is known from the litera-
ture, the mono- or bisACTHNUGRTENUNG(peroxo) MTO active intermediates
transfer an oxygen atom to the substrate through a concert-
ed mechanism.[33] As a consequence, the synthesis of 12 can
reasonably be derived from the initial formation of a 5,6-di-
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R. Saladino, M. Crucianelli et al.
Table 3. Oxidation of 3’,5’-di-O-benzyl-1’-homothymidine 4.
hydroxy intermediate (not isolated under our experimental
conditions), followed by elimination of water. 5-Hydroxy-2’-
deoxycytidine derivatives have been used for the prepara-
tion of immunomodulatory oligonucleotides[34] and are in-
hibitors of histamine release.[35] When the oxidation of 3 was
performed with heterogeneous catalysts I–IV, compounds 11
and 12 were obtained with yields ranging from moderate to
high (Table 2, entries 2–5). The higher value of the mass bal-
ance observed under heterogeneous conditions with respect
to MTO suggests that the reactivity and selectivity of the
catalyst might be tuned by the properties of the polymeric
support. Concerning the selectivity of the oxidation, a differ-
ent result (expressed as a ratio between 11 and 12) was ob-
served depending on the type of PVP resin employed. In
particular, whereas 11 was synthesized in highest yield with
catalysts I and II, compound 12 became the major product
with III and IV (Table 2, entries 2 and 3 vs. 4 and 5). These
results are in accordance with our previous data on the oxi-
dation of d-tocopherylquinone, in which case a different
ratio between isomeric d-tocopherylquinones was obtained
depending on the nature of the PVP resin.[22] Microencapsu-
lated catalyst V showed a performance similar to that of ho-
mogeneous MTO (Table 2, entry 6).
Catalyst
Product(s)
Conv.
[%]
Yield
[%][a,b]
1
2
3
4
5
6
MTO
13 (14)
13
13
13
13
>90
>90
>90
>90
>90
>90
40 (10)
65
69
62
65
PVP2%/MTO I
PVP25%/MTO II
PVPN2%/MTO III
PVPN25%/MTO IV
PS2%/MTO V
13 (14)
37 (11)
[a] Unless otherwise specified, the yields of the reactions described above
are calculated on the basis of converted substrate. [b] A low mass balance
was observed in all cases, probably due to the formation of over-oxida-
tion products that were not isolated.
The oxidation of 3’,5’-di-O-benzyl-1’-homothymidine 4 af-
forded a mixture of chromatographically separable 3’,5’-di-
O-benzyl-1’-homo-5,6-oxiranyl-5,6-dihydrothymidine 13 and
3’,5’-di-O-benzyl-1’-homo-5,6-dihydroxy-5,6-dihydrothymi-
dine 14 (as a mixture of stereoisomers) in 40 and 10% yield,
respectively (Table 3, entry 1).
During our studies on the oxidation of simple thymine
and thymidine derivatives with MTO we showed that the se-
lectivity towards the synthesis of epoxides can be increased
by performing the reaction in the presence of the urea hy-
drogen peroxide adduct (UHP), in which case, urea is able
to buffer the acidity of MTO.[36] The pyridinyl moiety in
PVP resins acts in a similar way. Thus, when the oxidation
was repeated with heterogeneous catalysts I–IV, the epoxide
13 was obtained in appreciable yield, as the only recovered
product (Table 3, entries 2–5). In these cases, no appreciable
difference in product yield was observed depending on the
degree of crosslinking of the resin. Again, the microencapsu-
lated catalyst V showed a performance similar to that of ho-
mogeneous MTO (Table 3, entry 6).
1’-homo-4-thiouridine 8, we studied the oxidative functional-
ization by using EtOH as both reaction solvent and nucleo-
phile. The oxidation of 6 (0.1 mmol) with MTO (2% w/w)
and H2O2 (0.2 equiv) in EtOH (1.0 mL) at 508C afforded
3’,5’-di-O-benzyl-2’-deoxy-1’-homoinosine 15 as the only re-
covered product, in acceptable yield (Table 4, entry 1). In
accordance with results on the oxidation of 2-mercapto-
4(3H)quinazolinone derivatives, the reaction probably pro-
Table 4. Oxidation of 3’,5’-di-O-benzyl-2’-deoxy-1’-homothioinosine 6.
The oxidation of thiopyrimidine and thiopurine nucleo-
bases and nucleosides in the presence of oxygen and nitro-
gen-centered nucleophiles is a synthetic alternative for site-
specific nucleophilic substitutions of the heterocyclic ring,
and for the formation of disufides and/or for triggering de-
sulfuration processes.[37] In particular, the thioketo moieties
at the C-4 position of the pyrimidine ring, and at the C-6
and C-8 positions of the purine ring, are sites for selective
nucleophilic substitutions. Instead, thioketo moieties at the
C-2 position of both pyrimidine and purine rings are sites of
desulfuration or for the formation of disulfides.[38]
Catalyst
Product(s)
Conv.
[%]
Yield
[%][a]
1
2
3
4
5
6
MTO
15
>90
85
81
80
83
76
PVP2%/MTO I
PVP25%/MTO II
PVPN2%/MTO III
PVPN25%/MTO IV
PS2%/MTO V
15 (16)
15 (16)
15 (16)
15 (16)
15
65 (7)
74 (5)
66 (4)
79 (7)
75
With the aim to apply this procedure to 3’,5’-di-O-benzyl-
2’-deoxy-1’-homo-6-thioinosine 6, 3’,5’-di-O-benzyl-2’-deoxy-
1’-homo-4-thiothymidine 7, and 3’,5’-di-O-benzyl-2’-deoxy-
>90
[a] Unless otherwise specified, the yields of the reactions described above
are calculated on the basis of converted substrate.
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Synthesis of 2’-Deoxy-1’-homo-N-nucleosides
FULL PAPER
ceeded through formation of a reactive sulfinic or persulfin-
ic acid intermediate (not shown), followed by a desulfura-
tion process.[39a]
linking of the resin (Table 5, entries 2–5), with PVPN25%/
MTO being the best catalytic system. Once more, the higher
value of the mass balance observed under heterogeneous
conditions with respect to MTO suggests the role played by
the chemical and physical properties of the polymeric sup-
ports (Table 5, entries 2–6 vs. entry 1).[25]
Comparable results were observed during the oxidation of
3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-thiouridine 8 (Table 5)
to afford 3’,5’-di-O-benzyl-2’-deoxy-1’-homouridine 5 and
With catalysts I–IV, compound 15 was obtained as the
main reaction product, as well as a small amount of the nu-
cleophilic substitution product, namely 3’,5’-di-O-benzyl-2’-
deoxy-1’-homo-(6-O-ethyl)inosine 16 (Table 4, entries 2–5).
Usually the replacement of the mercapto group by nitrogen
nucleophiles occurs in high yield.[38] The low efficiency in
the synthesis of 16 may be due to the low nucleophilic
power of ethanol, although other factors related to the spe-
cific structural properties of 1’-homo-N-nucleosides, cannot
be completely ruled out. Again, the microencapsulated cata-
lyst V showed a performance similar to that of homogene-
ous MTO (Table 4, entry 6).
3’,5’-di-O-benzyl-2’-deoxy-1’-homo-(4-O-ethyl)uridine
18
(Table 5, entry 7). This trend was also confirmed during the
oxidation with I–IV in which the increase in the reticulation
grade of polymeric supports afforded better yields of both
oxidation products 5 and 18, with compounds II and IV
acting as the best catalytic systems (Table 5, entries 8–11).
The catalyst V showed, similarly to MTO, a great activity,
probably caused by the formation of not isolated over-oxida-
tion products (Table 5, entry 12 versus 7). Notably, the high
conversion values observed in the oxidation of thionucleo-
sides 7 and 8 were obtained under mild experimental condi-
tions, at room temperature. A similar chemoselectivity was
observed in the oxidation of 2-thiouridine to 4-pyrimidinone
nucleoside, by an aqueous solution of H2O2, during the
study of a model reaction mimicking the mechanism in-
volved in the damage of tRNA under oxidative stress condi-
tions.[39b–c]
A different behavior was observed in the oxidation of
3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-thiothymidine
7
and
3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-thiouridine 8 (Table 5).
Treatment of 7 with MTO in ethanol at room temperature
afforded a mixture of 3’,5’-di-O-benzyl-2’-deoxy-1’-homothy-
midine 4 and 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-O-ethyl-
5-methyl uridine 17 in comparable yields (Table 5, entry 1).
Similar results were obtained with heterogeneous catalysts
I–V. In the case of the PVP and PVPN families, the yield of
17 increased slightly when increasing the degree of cross-
Regarding the stability and recyclability of our heteroge-
neous catalysts, previous studies demonstrated the possibili-
ty of recovering and reusing poly(vinylpyridine) and poly-
styrene MTO-based catalysts for successive runs.[25a,b] As
shown in Table 6, the immobilized PVP2%/MTO I and
Table 5. Catalytic oxyfunctionalization of 2’-deoxy-1’-homo-N-thionu-
cleosides 7 and 8.[a]
Table 6. Reusability of PVP2%/MTO I and PVP25%/MTO II systems, in
the oxidation of 3’,5’-di-O-benzyl-2’-deoxy-1’-homoadenine 2, at T=
808C, in ethanol.
Run
Catalyst
Product
Conv.
[%]
Yield
[%][a]
1
2
3
4
PVP2%/MTO I
PVP2%/MTO I
PVP2%/MTO I
PVP2%/MTO I
9
9
9
9
65
67
63
60
90
87
85
84
Catalyst
Product(s)
Conv.
[%]
Yield
[%][b]
1
2
3
4
PVP25%/MTO II
PVP25%/MTO II
PVP25%/MTO II
PVP25%/MTO II
9
9
9
9
72
73
70
70
96
94
90
89
1
2
3
4
5
6
7
8
9
MTO
17 (4)
17 (4)
17 (4)
17 (4)
17 (4)
17 (4)
18 (5)
18 (5)
18 (5)
18 (5)
18 (5)
18 (5)
>90
90
89
90
90
>90
>95
87
92
93
32 (29)[c]
40 (37)
47 (41)
51 (39)
53 (40)
51 (37)
23 (48)[c]
35 (41)
42 (46)
39 (42)
41 (46)
29 (39)
PVP2%/MTO I
PVP25%/MTO II
PVPN2%/MTO III
PVPN25%/MTO IV
PS2%/MTO V
MTO
PVP2%/MTO I
PVP25%/MTO II
PVPN2%/MTO III
PVPN25%/MTO IV
PS2%/MTO V
[a] Unless otherwise specified, the yields of the reactions described above
are calculated on the basis of converted substrate.
PVP25%/MTO II catalysts, selected as representative exam-
ples, retained significant activity in the oxidation of 2 after
three recycling steps (runs 1–4), confirming their stability
under the reaction conditions previously described.
10
11
12
>95
>95
Debenzylation reaction: Products 4, 5, 9, 11–15, 17, and 18,
obtained in appreciable yield, were deprotected (compounds
19–28) by a typical procedure. Briefly, a mixture of the ap-
propriate substrate (0.1 mmol) and 10% palladium on
carbon (10 mg) in ethanol (5.0 mL) was stirred under a hy-
[a] Reactions performed at room temperature (RT). [b] Unless otherwise
specified, the yields of the reactions described above are calculated on
the basis of converted substrate. [c] A low mass balance was observed,
probably due to the formation of over-oxidation products that were not
isolated.
Chem. Eur. J. 2013, 19, 2392 – 2404
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2397

R. Saladino, M. Crucianelli et al.
vir, are inhibitors of the viral glycoprotein Neuraminidase
(NA).[42] However, the efficacy of these antiviral compounds
is often limited by toxicity and the almost inevitable selec-
tion of drug-resistant viral mutants.[44] Thus, new antivirals
are needed to efficiently combat influenza viruses. Several
authors have proposed different targets of viral structure
and its life-cycle that could be inhibited by novel com-
pounds. A potential target is the viral RdRp, which is char-
acterized by several unique enzymatic properties, making it
ideal for the development of specific antiviral agents.
In fact, inhibitors of the influenza polymerase activity
have already been described, and they have the potential to
become new antiviral drugs if proven safe and efficacious in
humans.[45] Indeed, T-705 (favipiravir) is a pyrazine deriva-
tive (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) with anti-
viral activity in in vitro and in vivo models.[46] On entering
the cell, the compound is initially converted to its mono-
phosphate by phosphoribosyl transferase, then to its triphos-
phate by cellular kinases and, in this form, it inhibits the
RdRp.[47] Finally, a rising trend of different approaches can
be observed that focus on cellular factors or pathways that
are essential for viral replication,[48] and that can be inhibit-
ed by natural or synthetic compounds.[22,49]
In order to evaluate a potential antiviral effect of novel
products on influenza A/PR8/H1N1 virus replication, mono-
layers of MDCK cells were infected with PR8 and, after
viral challenge, were treated with different concentrations
(ranging from 10 to 100 mgmLÀ1; Figure 1) of each com-
pound. After 24 h post infection (p.i.), supernatants from in-
fected cells were harvested, and viral titer was evaluated by
measuring the hemagglutinin units (HAU). Table 7 shows
the required inhibitory concentration (IC) of all the com-
pounds tested to reduce virus yield by 50% (IC50). Several
compounds were able to reduce viral replication; however,
the treatment with compounds 2, 6, 12, 15, 17, 19, 20, and 23
(Table 7, entries 1, 5, 11, 14, 16, 18, 19, and 22) was able to
inhibit the influenza virus in a dose-dependent manner (as
shown in Figure 1). For these compounds the IC50 was less
than 50 mgmLÀ1 at a range of 54.5–186 mm. Note that cell
morphology and viability were unaffected by treatment with
the tested compounds, except for compounds 15 and 20 at
high doses (80 and 100 mgmLÀ1, respectively), leading to the
exclusion of the possibility that the reduction in viral repli-
cation was due to their inhibitorꢀs cytotoxic effects.
drogen atmosphere for 20 h at room temperature. The reac-
tion mixture was filtered through a pad of Celite and the fil-
trate was concentrated to afford the desired product, in
quantitative yield.[40] In the case of compounds 9 and 11,
due to the known deoxygenation process operative under
hydrogenolysis conditions,[41] the reduction of N-oxide moi-
eties became a side reaction; in addition, the reductive ring-
opening of the epoxide moiety to the corresponding alcohol
was observed during the deprotection of compound 13.
Biological activity tests: Influenza A viruses continue to rep-
resent a severe threat worldwide, causing large epidemics
and pandemics responsible for thousands of deaths and hos-
pitalizations every year.[42] The influenza A virus is an envel-
oped virus belonging to the Orthomyxoviridae family, char-
acterized by a segmented single-stranded RNA genome that
encodes eleven proteins. Within the envelope, the eight viral
RNA segments, associated with the nucleoprotein (NP) and
the viral RNA-dependent RNA polymerase (RdRp) com-
plex, which includes polymerase basic 1 and 2 (PB1 and
PB2) and polymerase acid (PA), form helical ribonucleopro-
tein capsids (vRNPS). After infection, the vRNPS are trans-
ported to the host-cell nucleus, where they undergo tran-
scription and replication through the viral RdRp. This is
characterized by two enzymatic activities, that is, the RNA
replicative and the endonuclease (which cuts the host cell
mRNA at approximately 10–16 nucleotides from the 5’-cap).
The capped oligonucleotides are used as primers for initiat-
ing the transcription and subsequent translation processes.
In a late phase of the replication cycle, vRNP complexes are
exported into the cytosol to be assembled with the other
structural proteins and packaged into progeny virions.[43]
Several antiviral compounds have been developed against
the influenza virus. Among them, Amantadine and Riman-
tadine have a type-specific inhibitory effect on influenza A,
but not on influenza B, viruses.[42] These agents block the
ion-channel activity of the viral Matrix (M2) protein, which
is mainly required for virus uncoating. The other licensed
antiviral agents, that is, Oseltamivir, Zanamivir and Perami-
As a general trend, derivatives 2, 17, and 15 retain their
activity after removal of the benzyl groups, showing an ac-
tivity that is slightly higher for those protected (Table 7, en-
tries 1, 16, and 14 vs. 18, 19, and 22). The only exception to
this pattern is 12 which, after deprotection, loses activity
(Table 7, entry 11 vs. 21). Among 1’-homo-N-thionucleo-
sides, compound 6 was the only active compound (Table 7,
entry 5).
With the aim of verifying the possible mechanism(s) of
action of the new compounds, we selected two couples of re-
lated 1’-homo-N-nucleosides (that is, the protected/depro-
tected pairs) that showed the highest antiviral activity in the
same value range, namely compounds 2 and 19, and 15 and
2398
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Chem. Eur. J. 2013, 19, 2392 – 2404

Synthesis of 2’-Deoxy-1’-homo-N-nucleosides
FULL PAPER
Figure 1. Different concentrations (range 10–100 mgmLÀ1) of each compound were added to MDCK cells after PR8 infection (0.0001 m.o.i.). Then, 24 h
after infection, the virus released from the cells was measured in the supernatant as HAUmLÀ1 (see the Experimental Section). Viral yields are ex-
pressed as percentages of those recorded for control infected cells.
20. These compounds were evaluated in their capacity of in-
hibiting the RNA-dependent RNA polymerase (RdRP; EC
2.7.7.48), an enzyme that catalyzes the replication of RNA
from an RNA template. As in the case of other nucleoside
analogues,[46] the inhibition of RdRP in infected cells is a
crucial step for the viral replication. For this reason, experi-
ments of mRNA transcription by means of real-time PCR
(RT-PCR) were performed. Cells were infected with PR8
and treated with compounds 19 and 20, or with the parent,
protected derivatives 2 and 15. After 4 and 6 h p.i., the cells
were recovered and used for the analysis of mRNA expres-
sion of viral proteins NP, M, and HA. As shown in Figure 2,
with respect to untreated cells, compounds 19 and 20 signifi-
cantly inhibited mRNA expression (about 80% reduction)
of the three viral proteins. This reduction was detectable
until 6 h p.i., which indicated that the antiviral effect of 19
and 20 was due to direct inhibition of RdRp activity. By
contrast, no inhibition of mRNA expression was measured
in cells treated with 2 and 15, which showed a small increase
in mRNA levels at 4 h p.i. On the basis of this data, it is
Chem. Eur. J. 2013, 19, 2392 – 2404
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2399

R. Saladino, M. Crucianelli et al.
Table 7. Antiviral activity of nucleoside analogues against the influenza
A virus.
and they do not seem to generate resistant strains, probably
due to their cellular target.[51]
Compound
IC50
[mgmLÀ1
Compound
IC50
[mgmLÀ1
]
Different mechanisms of action have been proposed for
Ribavirin: indirect mechanisms, including IMPDH inhibi-
tion, and direct mechanisms, including inhibition of RNA
capping activity, inhibition of viral polymerases, and in-
creased mutation frequency through incorporation of Riba-
virin into newly synthesized genomes leading to error catas-
trophe.[52] Interestingly, the reduction in the cellular GTP
pools is the primary mechanism of antiviral activity for my-
G
]
R
1
2
3
4
5
6
7
8
9
10
11
12
13
14
2
3
4
5
6
7
8
9
10
11
12
13
14
15
39.9
57.4
70.3
108.2
41.5
219.4
n.a.[a]
n.a.
n.a.
80
47.6
n.a.
n.a.
15
16
17
18
19
20
21
22
23
24
25
26
27
16
17
18
19
20
21
22
23
24
25
26
27
28
n.a.
33.3
n.a.
49.3
25.2
n.a.
67.3
39.9
n.a.
n.a.
n.a.
70.4
94.2
cophenolic acid,
a potent uncompetitive inhibitor of
IMPDH, that does not need to be metabolically activated.[53]
At present, for the benzylated derivatives, it is not possi-
ble to define a specific post-transcriptional mechanism of
action. Among the post-transcriptional mechanisms descri-
bed for the antiviral activity of nucleoside derivatives that
cannot be phosphorylated (as in the case of benzylated 1’-
homo-N-nucleosides), the allosteric interference with re-
verse transcription by altering either the conformation or
mobility of the enzyme[54a] or redox changes in the virus life-
cycle are reported.[54b,49a,c] Studies are currently ongoing in
our laboratories to confirm these hypotheses.
24.3
[a] n.a.=not active.
Conclusion
In this study, novel synthetic pathways have been developed
for the preparation of modified 1’-homo-N-nucleoside deriv-
atives by means of catalytic oxidative procedures. Either
MTO or MTO-supported catalysts were employed in the
presence of H2O2 as the primary oxidant, overall obtaining
high conversion values of substrates and from acceptable to
high yields of products. Irrespective of the type of 1’-homo-
N-nucleoside, that is, purine or pyrimidine derivatives, the
oxyfunctionalization of the heterocyclic ring and the N-het-
eroatom oxidation were operative processes. In particular,
pyrimidine 1’-homo-N-nucleoside derivatives were both oxi-
dized on the C5–C6 double bond and on the N(3) atom,
whereas purine derivatives were mainly functionalized at
the C8 and N(1) positions. Furthermore, the oxidation of 1’-
homo-N-thionucleosides confirmed the occurrence of site-
specific oxidative nucleophilic substitutions of the heterocy-
clic ring, even if simple desulfurization processes were also
observed, in significant yield. It is worth noting that MTO-
supported catalysts showed a performance similar to that of
MTO, providing the opportunity to recycle the catalyst for
most successive transformations, and thus reducing the cost
and environmental impact of the process. Moreover, the
higher value of the mass balance observed in some cases
under heterogeneous conditions, with respect to MTO, sug-
gests the specific role played by the chemical and physical
properties of polymeric supports in affecting the selectivity
of the oxidation. For example, these results are in agreement
with the benign effect exerted by the poly(4-vinylpyridine)
resins on the Brçnsted and Lewis acidity of MTO, showing
an ability to modulate the reactivity of the metal by prevent-
ing undesired over-oxidation or hydrolytic side reac-
Figure 2. MDCK cells were infected with PR8 (0.01 m.o.i.) and treated
with deprotected (19, 20) or with protected (2, 15) compounds. After 4
and 6 h p.i., infected cells were recovered and a pellet was used for the
real-time polymerase chain reaction (RT-PCR) analysis of HA, NP, and
M mRNA levels (see the Experimental Section). mRNA levels are ex-
pressed as a ratio between treated and untreated infected cells.
clear that the debenzylated and benzylated family of com-
pounds have different mechanisms of action, the first ones
by direct inhibition of RdRp, and the second ones by means
of processes at post-transcriptional levels.
The mechanism of action described for compounds 19 and
20 is in accordance with one of the multiple mechanisms of
Ribavirin, a synthetic guanosine derivative effective against
several RNA viruses, including influenza virus, which we
used as a reference. Some authors report the IC50 of this
compound as ranging from 14 to 63.5 mm.[47,50] Several of the
studied compounds show an activity comparable with that
of Ribavirin (Table 7). Ribavirin and its prodrug viramidine
are active against both human and avian influenza viruses
2400
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Chem. Eur. J. 2013, 19, 2392 – 2404

Synthesis of 2’-Deoxy-1’-homo-N-nucleosides
FULL PAPER
m; H-2’), 1.83 (3H, s; CH3), 1.76–1.62 ppm (1H, m; H-2’’); 13C NMR
(100.6 MHz, CDCl3): d=164.40 (C-4), 151.26 (C-2), 141.77 (C-6), 137.60
(Ar-C), 128.20, 127.50 (Ar-CH), 109.47 (C-5), 83.47, 80.16, 76.98 (C-1’, C-
3’, C-4’), 73.17, 70.86, 70.43 (2Ar-CH2, C-5’), 50.11 (C-6’), 34.53 (C-2’),
12.02 ppm (CH3); MS (EI): m/z: 436.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homouridine 5: 1H NMR (400 MHz,
CDCl3): 9.97 (1H, brs; NH), 7.32–7.20 (11H, m; Ar-H, H-6), 5.54 (1H,
d, J=15.8 Hz; H-5), 4.48–4.45 (4H, m; 2CH2), 4.41–4.29 (1H, m; H-1’),
4.25–3.91 (4H, m; H-3’, H-4’, H-6’, H-6’’), 3.46–3.41 (2H, m; H-5’, H-5’’),
2.18–2.02 (1H, m; H-2’), 1.88–1.58 ppm (1H, m; H-2’’); 13C NMR
(100.6 MHz, CDCl3): d=164.10 (C-4), 151.25 (C-2), 145.99 (C-6), 137.53
(Ar-C), 128.27, 127.59 (Ar-CH), 101.14 (C-5), 83.52, 80.42, 76.83 (C-1’, C-
3’, C-4’), 73.23, 70.98, 70.40 (2Ar-CH2, C-5’), 50.31 (C-6’), 34.59 ppm (C-
2’); MS (EI): m/z: 422.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-6-thioinosine 6: 1H NMR (400 MHz,
CDCl3): d=8.69–8.66 (1H, m; H-2), 8.26–8.24 (1H, m; H-8), 7.28–7.25
(10H, m; Ar-H), 4.56–4.02 (7H, m; 3CH2, CH), 3.83–3.44 (4H, m; 2CH,
CH2), 2.43–1.79 ppm (2H, m; CH2); 13C NMR (100.6 MHz, CDCl3): d=
151.44 (C-2), 141.90 (C-8), 137.77 (Ar-C), 128.28, 127.57 (Ar-CH), 83.67
(CH), 82.77 (CH), 80.93 (CH), 77.39 (CH), 73.32 (CH2), 71.02 (CH2),
70.86 (CH2), 37.07 (CH2), 32.62 ppm (CH2); MS (EI): m/z: 462.
tions.[16,25b] Among the novel products, several showed anti-
influenza activity without any cytotoxic effects. These com-
pounds were evaluated in their capacity of inhibiting the
RNA-dependent RNA polymerase, an enzyme that catalyz-
es the replication of RNA from an RNA template. On the
basis of these data, it is clear that the debenzylated and pro-
tected family of compounds act with different mechanisms
of action, the first ones by direct inhibition of RdRp, the
second ones by processes at post-transcriptional levels. To
the best of our knowledge, this is the first report describing
the catalytic synthesis of modified 1’-homo-N-nucleosides
with anti-influenza activity.
Experimental Section
General methods: All commercial products were of the highest grade
available and were used as such. The starting substrate 2’-deoxyribose C-
General procedure for the synthesis of 3’,5’-di-O-benzyl-2’-deoxy-1’-
homo-N-thionucleosides 7 and 8: 1’-Homonucleoside 4 (or 5; 0.5 mmol)
was dissolved in dioxane (2.0 mL) and treated with a small excess of P2S5
(0.6 mmol) at reflux for 3 h. At the end of the reaction the solvent was
removed under high vacuum and the crude was purified by preparative
TLC (eluent: CH2Cl2/CH3OH 9.5:0.5) to afford 3’,5’-di-O-benzyl-1’-
homo-4-thiothymidine 7 and 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-4-thiouri-
dine 8 in 85 and 90% yield, respectively.
6’ mesyl derivative 1 was prepared according to the literature pro
ACHTUNGTRENNUNG
ACHTUNGERTNcNUNG e-
dure.[14a] NMR spectra were recorded on a Varian Mercury 400 (1H,
400 MHz) spectrometer. For 1H and 13C NMR spectroscopic data, d
values of chemical shifts are reported in ppm. Mass spectra were per-
formed on a GC/LCMS Varian spectrometer. Chromatographic purifica-
tions were performed on columns packed with silica gel, 230–400 mesh,
for a flash technique.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-4-thiothymidine
7:
1H NMR
Cell cultures: Madin Darby canine kidney (MDCK) cells were grown in
RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), glu-
tamine (0.3 mgmLÀ1), penicillin (100 UmLÀ1), and streptomycin
(100 mgmLÀ1). Cell viability was estimated by Trypan blue (0.02%) ex-
clusion. All reagents were purchased from Invitrogen (Milan, Italy).
(400 MHz, CDCl3): d=10.78 (1H, brs; NH), 7.30–7.19 (10H, m; Ar-H),
7.15 (1H, s; H-6), 4.50–4.40 (4H, m; 2CH2), 4.38–3.65 (5H, m; H-1’, H-
3’, H-4’, H-6’, H-6’’), 3.48–3.44 (2H, m; H-5’, H-5’’), 2.38–1.60 (2H, m; H-
2’, H-2’’), 2.01 ppm (3H, s; CH3); 13C NMR (100.6 MHz, CDCl3): d=
190.71 (C-4), 149.16 (C-2), 138.79 (C-6), 137.90 (Ar-C), 128.49, 127.80
(Ar-CH), 118.51 (C-5), 83.80, 80.38, 76.91 (C-1’, C-3’, C-4’), 73.48, 71.18,
70.63 (2Ar-CH2, C-5’), 51.04 (C-6’), 34.87 (C-2’), 17.01 ppm (CH3); MS
(EI): m/z: 452.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-4-thiouridine 8: 1H NMR (400 MHz,
CDCl3): d=9.70 (1H, brs; NH), 7.39–7.22 (10H, m; Ar-H), 7.09 (1H, d,
J=14.88 Hz; H-6), 6.25–6.20 (1H, m; H-5), 4.48–4.41 (4H, m; 2CH2),
4.40–3.62 (5H, m; H-1’, H-3’, H-4’, H-6’, H-6’’), 3.45–3.43 (2H, m; H-5’,
H-5’’), 2.18–1.58 ppm (2H, m; H-2’, H-2’’); 13C NMR (100.6 MHz,
CDCl3): d=189.84 (C-4), 148.54 (C-2), 141.04 (C-6), 137.70 (Ar-C),
128.41, 127.63 (Ar-CH), 112.34 (C-5), 83.72, 80.18, 76.58 (C-1’, C-3’, C-
4’), 73.39, 71.16, 70.41 (2Ar-CH2, C-5’), 51.11 (C-6’), 34.80 ppm (C-2’);
MS (EI): m/z: 438.
General procedure for the synthesis of 3’,5’-di-O-benzyl-2’-deoxy-1’-
homo-N-nucleosides 2–5 and 3’,5’-di-O-benzyl-2’-deoxy-1’-homo-6-thio-
ACHTUNGTRENNUNGinosine 6: The appropriate nucleobase (adenine, cytosine, thymine, uracil,
and 6-mercaptopurine, respectively) was added to a solution of 2’-deoxy-
ribose C-6’ mesyl derivative 1[14a] in dry DMF (5.0 mL) in the presence of
NaH (for adenine and 6-mercaptopurine), CsCO3 (cytosine), and K2CO3
(thymine and uracil), and the reaction was heated at 908C for 6 h. The
reaction mixture was diluted with water (5.0 mL) and extracted with
EtOAc (2ꢂ10 mL). Then the organic layer was dried on anhydrous
Na2SO4. After removal of the solvent under high vacuum the crude was
purified by flash chromatography (CH2Cl2/MeOH 95:5) to afford com-
pounds 2–6 in 67, 71, 82, 85, and 83% yield, respectively. NMR spectro-
scopic data (not shown) of 2’-deoxyribose C-6’ mesyl derivative 1 are in
total agreement with data previously published.[14]
Preparation of heterogeneous MTO-based catalysts I–V: Poly(4-vinylpyr-
idine)/MTO (PVP2%/MTO I, PVP25%/MTO II, PVPN2%/MTO III,
and PVPN25%/MTO IV) and polystyrene/MTO (PS2/MTO V) catalysts
were prepared as previously reported.[16,17] In summary, MTO (77 mg,
0.3 mmol) was added to a suspension of the appropriate resin (600 mg) in
ethanol (4 mL) or tetrahydrofuran in the case of polystyrene. The mix-
ture was stirred for 1 h by using a magnetic stirrer. Coacervates were
found to envelop the solid core dispersed in the medium and hexane
(5 mL) was added to harden the capsule walls. The solvent was removed
by filtration and the solid residue was washed with ethyl acetate and fi-
nally dried under high vacuum. In every case, MTO was completely in-
cluded into the polymer. This result was confirmed by spectroscopic anal-
ysis of the residue obtained after evaporation of the organic layers. The
catalysts were used without any further purification.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homoadenine 2: 1H NMR (400 MHz,
CDCl3): d=8.31 (1H, s; H-2), 7.90 (1H, s; H-8), 7.33–7.20 (10H, m; Ar-
H), 6.59 (2H, brs; NH2), 4.46–4.22 (7H, m; 3CH2, CH), 4.12–3.85 (2H,
m; 2CH), 3.43–3.27 (2H, m; CH2), 2.28–1.48 ppm (2H, m; CH2);
13C NMR (100.6 MHz, CDCl3): d=155.94 (C-6), 152.97 (C-2), 150.31 (C-
4), 141.93 (C-8), 138.04 (Ar-C), 128.49, 127.72 (Ar-CH), 119.33 (C-5),
83.88 (CH), 80.64 (CH), 76.61 (CH), 73.53 (CH2), 71.54 (CH2), 70.73
(CH2), 47.60 (CH2), 35.00 ppm (CH2); MS (EI): m/z: 445.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homocytidine 3: 1H NMR (400 MHz,
CDCl3): d=7.36–7.22 (11H, m; Ar-H, H-6), 5.73–5.64 (1H, m; H-5),
4.50–4.35 (5H, m; 2CH2, H-1’), 4.19–3.90 (3H, m; H-3’, H-4’, H-6’), 3.77–
3.66 (1H, m; H-6’’), 3.43–3.33 (2H, m; H-5’, H-5’’), 2.32–2.04 (1H, m; H-
2’), 1.85–1.60 ppm (1H, m; H-2’’); 13C NMR (100.6 MHz, CDCl3): d=
166.06 (C-4), 156.79 (C-2), 146.56 (C-6), 137.81 (Ar-C), 128.30, 127.55
(Ar-CH), 93.98 (C-5), 82.75, 80.45, 77.11 (C-1’, C-3’, C-4’), 73.20, 70.90,
70.57 (2Ar-CH2, C-5’), 52.00 (C-6’), 34.55 ppm (C-2’); MS (EI): m/z: 421.
General procedure for the catalytic oxidation of 2’-deoxy-1’-homo-N-nu-
cleosides 2–4 and 2’-deoxy-1’-homo-N-thionucleosides 6–8: H2O2
(2.0 equiv) was added to a flask containing the selected nucleoside deriv-
ative (0.2 mmol) dissolved in ethanol (1.5 mL), followed by an appropri-
ate amount of catalyst corresponding to 1.0% w/w of MTO with respect
to substrate, and the mixture was heated at a selected temperature under
stirring, for 8.0 h. The progress of oxidation was monitored by GCMS
3’,5’-Di-O-benzyl-2’-deoxy-1’-homothymidine 4: 1H NMR (400 MHz,
CDCl3): d=9.84 (1H, brs; NH), 7.38–7.24 (10H, m; Ar-H), 7.12 (1H, s;
H-6), 4.50–4.45 (4H, m; 2CH2), 4.42–4.35 (1H, m; H-1’), 4.25–3.90 (4H,
m; H-3’, H-4’, H-6’, H-6’’), 3.46–3.36 (2H, m; H-5’, H-5’’), 2.36–2.08 (1H,
Chem. Eur. J. 2013, 19, 2392 – 2404
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
2401

R. Saladino, M. Crucianelli et al.
analysis on microsamples of reaction mixture (1.0 mL), withdrawn period-
ically. At the end of the reaction, the catalyst was recovered by filtration
and washed with a small amount of EtOH. A small amount of MnO2
(2.0 mg) was added to eliminate the excess of primary oxidant. The com-
position of the reaction mixture was found to be unchanged after the ad-
dition of MnO2. The suspension was filtered once again and the filtrate
dried over anhydrous Na2SO4. After evaporation of the solvent, the
crude mixture was purified by flash chromatography. The identity of
products was confirmed by 1H and 13C NMR spectroscopic analyses.
Spectra, when necessary, were compared with those of authentic com-
pounds.
CDCl3): d=162.18 (C-6), 152.03 (C-H), 149.80 (C-H), 148.93 (C), 137.47
(C), 128.39, 127.77 (Ar-CH), 122.44 (C), 83.99 (CH), 82.98 (CH), 80.19
(CH), 73.33 (CH2), 71.38 (CH2), 70.49 (CH2), 57.53 (CH2), 37.83 ppm
(CH2); MS (EI): m/z: 446.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-(6-O-ethyl)-inosine 16: 1H NMR
(400 MHz, CDCl3): d=8.38 (1H, s; CH), 7.56 (1H, s; CH), 7.40–7.20
(10H, m; Ar-H), 4.60–4.55 (2H, m; CH2), 4.53–4.46 (4H, m; 2CH2),
4.09–4.02 (2H, m; CH2), 3.92–3.90 (2H, m; 2CH), 3.51–3.32 (2H, m;
CH2), 2.14–1.72 (2H, m; CH2), 1.44–1.40 ppm (3H, m; CH3); 13C NMR
(100.6 MHz, CDCl3): d=161.74 (C), 151.13 (CH), 149.74 (C), 143.26
(CH), 138.91 (C), 138.04 (C), 128.72, 128.0, 127.40 (Ar-CH), 121.01 (C),
83.89 (CH), 83.04 (CH), 75.41 (CH), 73.01 (CH2), 70.92 (CH2), 68.77
(CH2), 61.75 (CH2), 47.76 (CH2), 38.93 (CH2), 14.28 ppm (CH3); MS
(EI): m/z: 474.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homoadenine-N(1)-oxide
9:
1H NMR
(400 MHz, CDCl3): d=8.64 (1H, s; H-2), 7.98 (1H, s; H-8), 7.29–7.18
(10H, m; Ar-H), 4.46–4.21 (7H, m; 3CH2, CH), 4.17–3.88 (2H, m;
2CH), 3.43–3.30 (2H, m; CH2), 2.30–1.52 ppm (2H, m; CH2); 13C NMR
(100.6 MHz, CDCl3): d=148.19 (C), 143.97 (CH), 143.13 (C), 137.72 (Ar-
C), 128.40, 127.60 (Ar-CH), 118.26 (C), 83.87 (CH), 83.31 (CH), 76.57
(CH), 73.43 (CH2), 71.44 (CH2), 70.49 (CH2), 47.63 (CH2), 34.89 ppm
(CH2); MS (EI): m/z: 461.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-4-O-ethyl-5-methyluridine
17:
1H NMR (400 MHz, CDCl3): d=7.40–7.10 (11H, m; Ar, CH), 4.60–4.40
(4H, m; 2CH2), 4.42–4.25 (2H, m; CH2), 4.20–3.90 (3H, m; 3CH), 3.80–
3.60 (2H, m; CH2), 3.20–3.10 (2H, m; CH2), 2.10–1.60 (2H, m; CH2),
1.82 (3H, s; CH3), 1.35–1.31 ppm (3H, m; CH3); 13C NMR (100.6 MHz,
CDCl3): d=162.82 (C), 154.10 (C), 147.61 (CH), 138.07 (Ar-C), 128.67,
128.10, 127.20 (Ar-CH), 110.81 (C), 83.72 (CH), 81.51 (CH), 76.20 (CH),
74.07 (CH2), 70.82 (CH2), 68.27 (CH2), 62.17 (CH2), 55.01 (CH2), 37.69
(CH2), 14.10 (CH3), 13.70 ppm (CH3); MS (EI): m/z: 464.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-(7H)-8-oxoadenine N(1)-oxide 10:
1H NMR (400 MHz, CDCl3): d=8.22 (1H, s; H-2), 7.93 (1H, brs; NH),
7.29–7.18 (10H, m; Ar-H), 4.46–3.90 (9H, m; 3CH2, 3CH), 3.59–3.33
(2H, m; CH2), 2.32–1.62 ppm (2H, m; CH2); 13C NMR (100.6 MHz,
CDCl3): d=155.41 (C), 149.11 (CH), 139.28 (C), 137.93 (C), 137.15 (C),
136.0 (Ar-C), 128.37, 127.82, 127.50 (Ar-CH), 110.83 (CH), 107.46 (C),
80.57 (CH), 75.28 (CH), 71.52 (CH2), 70.54 (CH2), 46.51 (CH2),
35.48 ppm (CH2); MS (EI): m/z: 477.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homocytidine N(3)-oxide 11: 1H NMR
(400 MHz, CDCl3): d=7.37–7.24 (11H, m; Ar-H, H-6), 5.70–5.59 (1H,
m; H-5), 4.52–4.37 (5H, m; 2CH2, H-1’), 4.25–3.96 (3H, m; H-3’, H-4’,
H-6’), 3.76–3.63 (1H, m; H-6’’), 3.47–3.35 (2H, m; H-5’, H-5’’), 2.31–2.06,
1.87–1.63 ppm (2H, m; H-2’, H-2’’); 13C NMR (100.6 MHz, CDCl3): d=
165.05 (C-4), 155.92 (C-2), 147.38 (C-6), 137.90 (Ar-C), 128.42, 127.74
(Ar-CH), 94.13 (C-5), 82.99, 80.62, 76.93 (C-1’, C-3’, C-4’), 73.41, 71.08,
70.67 ppm (2Ar-CH2, C-5’), 53.28 (C-6’), 34.70 ppm (C-2’); MS (EI): m/z:
437.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-4-O-ethyl-uridine
18:
1H NMR
(400 MHz, CDCl3): d=7.48 (1H, d, J=14.4 Hz; H-6), 7.34–7.22 (10H, m;
Ar-H), 5.68 (1H, d, J=14.4 Hz; H-5), 4.53–4.29 (6H, m; 3CH2), 4.22–
3.68 (5H, m; 3CH, CH2), 3.43–3.36 (2H, m; CH2), 2.27–1.62 (2H, m;
CH2), 1.34–1.30 ppm (3H, m; CH3); 13C NMR (100.6 MHz, CDCl3): d=
171.38 (C), 148.36 (CH), 137.86 (Ar-C), 128.45, 127.65 (Ar-CH), 94.93
(CH), 83.59 (CH), 80.26 (CH), 76.59 (CH), 73.37 (CH2), 71.15 (CH2),
70.61 (CH2), 62.95 (CH2), 52.09 (CH2), 34.68 (CH2), 13.68 ppm (CH3);
MS (EI): m/z: 450.
General procedure for the debenzylation reaction: Palladium (10 mg,
10%) on carbon suspension was added to a flask containing the appropri-
ate substrate (0.1 mmol) dissolved in ethanol (5.0 mL) and the mixture
was stirred under a hydrogen atmosphere for 20 h at room temperature.
After filtration through a Celite pad, the filtrate was concentrated to
afford the desired product in quantitative yield.
2’-Deoxy-1’-homoadenosine 19:[14a] 1H NMR (400 MHz, [D6]DMSO): d=
8.18 (1H, s; H-2), 8.08 (1H, s; H-8), 7.20 (2H, brs; NH2), 4.95 (1H, d;
3’OH), 4.76 (1H, t; 5’OH), 4.38–4.32 (1H, m; H-1’), 4.28 (1H, d; H-6’),
4.18 (1H, d; H-6“), 4.19–3.95 (1H, m; H-3’), 3.66–3.42 (1H, m; H-4’),
3.28 (1H, d; H-5’), 3.19 (1H, d; H-5”), 1.83-1.78 (1H, m; H-2’), 1.68–
1.49 ppm (1H, m; H2“); 13C NMR (100.6 MHz, [D6]DMSO): d=156.1
(C-6), 152.5 (C-2), 149.8 (C-4), 141.7 (C-8), 118.6 (C-5), 87.5 (CH), 76.2
(CH), 71.6 (CH), 62.1 (CH2), 47.0 (CH2), 37.5 ppm (CH2); MS (EI): m/z:
265.
2’-Deoxy-1’-homoinosine 20: 1H NMR (400 MHz, D2O): d=8.08 (1H, s;
CH), 7.45 (1H, s; CH), 4.61–4.52 (1H, m; CH), 3.97–3.95 (2H, m; 2CH),
3.90–3.87 (2H, m; CH2), 3.80–3.61 (2H, m; CH2), 2.28–1.86 ppm (2H, m;
CH2); 13C NMR (100.6 MHz, D2O): d=156.43 (C), 152.40 (C), 145.0
(CH), 139.68 (CH), 125.0 (C), 85.89 (CH), 73.60 (CH), 70.33 (CH), 61.25
(CH2), 46.75 (CH2), 41.20 ppm (CH2); MS (EI): m/z: 266.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-5-hydroxycytidine
12:
1H NMR
(400 MHz, CDCl3): d=7.36 (1H, s; CH), 7.28–7.00 (10H, m; Ar-H),
4.67–4.56 (4H, m; CH2), 4.53–4.50 (1H, m; CH), 4.50–4.45 (2H, m;
CH2), 4.20–4.41 (2H, m; CH), 4.0–4.2 (2H, m; CH2), 3.42–3.61 (2H, m;
CH2), 1.87–2.19 ppm (2H, m; CH2); 13C NMR (100.6 MHz, CDCl3): d=
157.17 (C), 153.02 (C), 137.92 (Ar-C), 137.89 (C), 129.62 (Ar-CH), 128.72
(Ar-CH), 127.61 (Ar-CH), 116.84 (CH), 82.86 (CH), 80.92 (CH), 78.30
(CH), 71.38 (CH2), 71.00 (CH2), 70.90 (CH2), 62.28 (CH2), 35.86 ppm
(CH2); MS (EI): m/z: 437.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-5,6-oxiranyl-5,6-dihydrothymidine 13:
1H NMR (400 MHz, CDCl3): d=10.69 (1H, s; NH), 7.31–7.27 (10H, m;
Ar-H), 4.58–4.25 (5H, m; 2CH2, CH), 4.20–4.00 (1H, m; CH), 3.95–3.80
(2H, m; 2CH), 3.80–3.60 (2H, m; CH2), 3.51–3.32 (2H, m; CH2), 2.20–
2.05 (1H, m; H-2’), 1.82 (3H, s; CH3), 1.70–1.45 ppm (1H, m; H-2’’);
13C NMR (100.6 MHz, CDCl3): d=171.43 (C), 154.11 (C), 138.04 (Ar-C),
128.67, 128.04, 127.40 (Ar-CH), 89.17 (CH), 83.89 (CH), 83.20 (CH),
77.49 (CH), 73.10 (CH2), 70.92 (CH2), 68.77 (CH2), 63.7 (C), 47.07 (CH2),
37.87 (CH2), 16.29 ppm (CH3); MS (EI): m/z: 452.
2’-Deoxy-1’-homo-5-hydroxycytidine 21: 1H NMR (400 MHz, D2O): d=
7.60 (1H, s; CH), 4.26–4.19 (1H, m; CH), 4.10–3.90 (2H, m; 2CH), 3.87–
3.58 (4H, m; 2CH2), 2.25–1.80 ppm (2H, m; CH2); 13C NMR
(100.6 MHz, D2O): d=158.0 (C), 156.18 (C), 137.03 (C), 116.0 (CH), 85.0
(CH), 81.0 (CH), 70.50 (CH), 61.35 (CH2), 56.0 (CH2), 41.0 ppm (CH2);
MS (EI): m/z: 257.
2’-Deoxy-1’-homocytidine 22:[14a] 1H NMR (400 MHz, [D6]DMSO): d=
7.55 (1H, d; H-6), 5.64 (1H, d; H-5) 4.92 (1H, brs; 5’OH), 4.63 (1H,
brs; 3’OH), 4.24–4.15 (1H, m; H-1’), 4.12–3.96 (1H, m; H-3’), 3.81 (1H,
d; H-6’), 3.63–3.50 (2H, m; H4’, H6“), 3.30 (1H, d; H5’), 3.25 (1H, d;
H5’’), 1.78–1.71 (1H, m; H2’), 1.66–1.59 ppm (1H, m; H2”); 13C NMR
(100.6 MHz, [D6]DMSO): d=166.2 (C-4), 156.7 (C-2), 147.6 (C-6), 93.4
(C-5), 87.6 (CH), 76.3 (CH), 72.0 (CH), 62.5 (CH2), 52.7 (CH2), 37.7 ppm
(CH2); MS (EI): m/z: 241.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homo-5,6-dihydroxy-5,6-dihydrothymidine
14: H NMR (400 MHz, CDCl3): d=7.31–7.27 (10H, m; Ar-H), 5.69–5.49
1
(1H, m; CH), 4.51–4.47 (2H, m; CH2), 4.53–4.46 (2H, m; CH2), 4.31–
4.22 (1H, m; CH), 4.00–3.90 (2H, m; 2CH), 3.51–3.32 (2H, m; CH2),
3.62–3.49 (2H, m; CH2), 2.12–1.61 (2H, m; CH2), 1.20 ppm (3H, s; CH3);
13C NMR (100.6 MHz, CDCl3): d=172.22 (C), 155.49 (C), 138.0 (Ar-C),
128.72, 128.00, 127.40 (Ar-CH), 84.91 (CH), 83.83 (CH), 83.2 (CH), 77.51
(CH), 73.16 (C), 73.0 (CH2), 70.87 (CH2), 68.77 (CH2), 47.23 (CH2), 37.87
(CH2), 16.65 ppm (CH3); MS (EI): m/z: 470.
3’,5’-Di-O-benzyl-2’-deoxy-1’-homoinosine 15: 1H NMR (400 MHz,
CDCl3): d=9.16 (1H, s; H-2), 8.75 (1H, s; H-8), 7.34–7.13 (10H, m; Ar-
H), 4.77–4.56 (1H, m; H1’), 4.56–4.32 (4H, m; 2CH2), 3.99–3.82 (3H, m;
CH2, CH), 3.70–3.52 (1H, m; CH), 3.26–3.00 (2H, m; CH2), 2.35–2.15
(1H,
m
H2’), 2.01–1.72 ppm (1H, m; H2’’); 13C NMR (100.6 MHz,
2402
www.chemeurj.org
ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 2392 – 2404

Synthesis of 2’-Deoxy-1’-homo-N-nucleosides
FULL PAPER
2’-Deoxy-1’-homo-4-O-ethyl-thymidine 23: 1H NMR (400 MHz, D2O):
d=7.49 (1H, s; CH), 4.26–4.19 (1H, m; CH), 4.17–4.10 (2H, m; CH2),
3.97–3.95 (2H, m; CH), 3.80–3.61 (4H, m; 2CH2), 2.23–1.81 (2H, m;
CH2), 1.60 (3H, s; CH3), 1.34–1.30 ppm (3H, m; CH3); 13C NMR
(100.6 MHz, D2O): d=163.10 (C), 155.81 (C), 147.60 (CH), 110.18 (C),
85.02 (CH), 75.20 (CH), 70.46 (CH), 61.38 (CH2), 61.21 (CH2), 55.38
(CH2), 40.29 (CH2), 14.09 (CH3), 13.77 ppm (CH3); MS (EI): m/z: 284.
5’-TCACCAGGTCCAGACACAATA-3’ and reverse (5’AAGCAGA-
CAAATCACTCCACC-3’).
Acknowledgements
2’-Deoxy-1’-homo-4-O-ethyl-uridine 24: 1H NMR (400 MHz, D2O): d=
7.62–7.58 (1H, m; CH), 6.22-6.17 (1H, m; CH), 4.23–4.18 (1H, m; CH),
3.97–3.91 (2H, m; 2CH), 3.83–3.58 (4H, m; 2CH2), 2.20–1.79 (2H, m;
CH2), 1.39–1.35 ppm (3H, m; CH3); 13C NMR (100.6 MHz, D2O): d=
172.0 (C), 155.90 (C), 142.0 (CH), 91.38 (CH), 85.0 (CH), 73.20 (CH),
70.56 (CH), 61.30 (CH2), 54.0 (CH2), 40.19 (CH2), 13.60 ppm (CH3); MS
(EI): m/z: 270.
This work was partially supported by the Italian Ministry of Instruction,
University and Research (Projects PON01_01802 and FIRB internazio-
nale-RBIN06E9Z8) and by grants from the Istituto Superiore di Sanitꢃ-
2012. MIUR PRIN 2008 is also acknowledged. We also thank Dr. D. Li-
mongi for her technical assistance.
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[2] H. Mitsuya, K. J. Weinhold, P. A. Furnam, M. H. St. Clair, S. N.
Lehrman, R. C. Gallo, D. Bolognesi, D. W. Barry, S. Broder, Proc.
Natl. Acad. Sci. USA 1985, 82, 7096–7100.
[3] a) H. Mitsuya, S. Broder, Proc. Natl. Acad. Sci. USA 1986, 83, 1911–
1915; b) R. F. Schinazi, Perspect. Drug Discovery Des. 1993, 1, 151–
180; c) J. J. Lipsky, Lancet 1996, 348, 800–803.
2’-Deoxy-1’-homo-5-hydroxy-5,6-dihydrothymidine
25:
1H NMR
(400 MHz, D2O): d=4.20–4.08 (2H, m; 2CH), 4.00–3.95 (2H, m; 2CH),
3.80–3.61 (2H, m; CH2), 3.54–3.32 (3H, m; CH2, CH), 2.22–1.81 (2H, m;
CH2), 1.26 ppm (3H, s; CH3); 13C NMR (100.6 MHz, D2O): d=170.22
(C), 148.02 (C), 85.23 (CH), 73.37 (CH), 70.60 (CH), 69.70.(C), 61.35
(CH2), 53.20 (CH2), 51.05 (CH2), 40.47 (CH2), 21.60 ppm (CH3); MS
(EI): m/z: 274.
2’-Deoxy-1’-homo-5,6-dihydroxy-5,6-dihydrothymidine
26:
1H NMR
(400 MHz, D2O): d=5.63–5.57 (1H, m; CH), 4.23–4.18 (1H, m; CH),
4.00–3.90 (2H, m; 2CH), 3.80–3.60 (4H, m; 2CH2), 2.20–1.79 (2H, m;
CH2), 1.20 ppm (3H, s; CH3); 13C NMR (100.6 MHz, D2O): d=172.06
(C), 155.03 (C), 85.81 (CH), 84.08 (CH), 76.02 (CH), 73.10 (C), 70.50
(CH), 61.22 (CH2), 47.08 (CH2), 40.51 (CH2), 16.60 ppm (CH3); MS (EI):
m/z: 290.
2’-Deoxy-1’-homothymidine 27:[14a] 1H NMR (400 MHz, D2O): d=7.34–
7.30 (1H, m; CH), 4.23–4.16 (1H, m; CH), 4.10–3.95 (2H, m; 2CH),
3.93–3.56 (4H, m; 2CH2), 2.30–1.79 (2H, m; CH2), 1.70 ppm (3H, s;
CH3); 13C NMR (100.6 MHz, D2O): d=164.27 (C), 150.75 (C), 142.03
(CH), 107.08 (C), 84.39 (CH), 76.06 (CH), 70.46 (CH), 61.30 (CH2), 53.17
(CH2), 40.01 (CH2), 11.85 ppm (CH3); MS (EI): m/z: 256.
2’-Deoxy-1’-homouridine 28: 1H NMR (400 MHz, D2O): d=7.42–7.38
(1H, m; CH), 5.51–5.45 (1H, m; CH), 4.27–4.21 (1H, m; CH), 4.10–3.94
(2H, m; 2CH), 3.92–3.55 (4H, m; 2CH2), 2.29–1.77 (2H, m; CH2);
13C NMR (100.6 MHz, D2O): d=163.96 (C), 150.82 (C), 146.51 (CH),
101.02 (CH), 86.10 (CH), 75.24 (CH), 70.45 (CH), 61.31 (CH2), 52.86
(CH2), 41.15 ppm (CH2); MS (EI): m/z: 242.
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Biological tests: Virus production, infection, and titration: Influenza virus
A/Puerto Rico/8/34 H1N1 (PR8 virus) was grown in the allantoic cavities
of 10-day-old embryonated chicken eggs. After 48 h at 378C, the allantoic
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of allantoic fluid from uninfected eggs. Virus production was determined
in the supernatants of infected cells 24 h post infection (p.i.), by measur-
ing the hemagglutinin units (HAU), with human type 0 Rh+ erythro-
cytes. For the evaluation of antiviral activity, stock solutions of com-
pounds dissolved in DMSO were diluted in RPMI to final concentrations
of 40, 60, 80, and 100 mgmLÀ1. Compounds were added after the adsorp-
tion period and maintained in the culture media until the end of the ex-
periments. The highest DMSO concentration present in the culture
medium was 0.2%. Control cells were treated with DMSO alone at the
same concentration present in the test substance being evaluated.
Real-time PCR assay: RNA was extracted from infected cells with a
RNeasy Mini kit (Qiagen). RNA (1 mg) was used for retrotranscription
with the iScriptcDNA Synthesis kit (BioRad), qPCR was performed in
duplicate by using the following primers set:
HA forward (5’-TGTATAGGCTACCATGCGAAC-3’) and reverse (5’-
TTCCGTTGTGGCTGTCTTC-3’); NP forward (5’-CGTCCCAAGG-
CACCAAAC-3’) and reverse (5’-AATCGTCCAATTCCACCAATC-3’);
M forward (5’-GCAAGCGATGAGAACCATTGG-3’) and reverse (5’-
GCGGCAATAGCGAGAGGATC-3’); and housekeeping 18S forward
Chem. Eur. J. 2013, 19, 2392 – 2404
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www.chemeurj.org
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Received: April 16, 2012
Revised: October 15, 2012
Published online: December 6, 2012
2404
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ꢁ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 2392 – 2404