Synthesis and antiviral evaluation of α-l-2′- deoxythreofuranosyl nucleosides

Article abstract of DOI:10.1016/j.ejmech.2011.05.036

The synthesis of a series of α-l-2′-deoxythreofuranosyl nucleosides featuring the nucleobases A, T, C and U is described in seven steps from 1,2-O-isopropyledene-α-l-threose, involving a Vorbru?ggen coupling and a Barton-McCombie deoxygenation protocol as the key steps. All analogues, including a phosphoramidate nucleoside phosphate prodrug of the T analogue, were evaluated against a broad panel of different viruses but found inactive, while also lacking notable cellular toxicity. The thymidine analogue showed inhibition to mitochondrial thymidine kinase-2 (TK-2), herpes simplex virus type 1 (HSV-1) TK, varicella-zoster virus (VZV) TK and Mycobacterium tuberculosis thymidylate kinase.

Full text of DOI:10.1016/j.ejmech.2011.05.036

European Journal of Medicinal Chemistry 46 (2011) 3704e3713
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral evaluation of
a-L
-20-deoxythreofuranosyl nucleosides
,
Kiran S. Toti a, Marco Derudas b, Christopher McGuigan b, Jan Balzarini c, Serge Van Calenbergh a
*
a Laboratory for Medicinal Chemistry (FFW), UGent, Harelbekestraat 72, 9000 Gent, Belgium
b Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3NB, UK
c Rega Institute for Medical Research, Katholieke Universiteit Leuven, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
-20-deoxythreofuranosyl nucleosides featuring the nucleobases A, T, C and
-threose, involving a Vorbrüggen coupling
Article history:
The synthesis of a series of a-L
U is described in seven steps from 1,2-O-isopropyledene-a-L
Received 7 April 2011
Received in revised form
13 May 2011
Accepted 13 May 2011
Available online 20 May 2011
and a Barton-McCombie deoxygenation protocol as the key steps. All analogues, including a phosphor-
amidate nucleoside phosphate prodrug of the T analogue, were evaluated against a broad panel of
different viruses but found inactive, while also lacking notable cellular toxicity. The thymidine analogue
showed inhibition to mitochondrial thymidine kinase-2 (TK-2), herpes simplex virus type 1 (HSV-1) TK,
varicella-zoster virus (VZV) TK and Mycobacterium tuberculosis thymidylate kinase.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Nucleoside analogs
Antivirals
Enzyme inhibitors
Prodrugs
1. Introduction
moiety at the 30-position, which mimicks the monophosphate ester
of a 30-hydroxymethyl substituent.
The human immunodeficiency virus (HIV) epidemic has
fostered research on antiviral drugs both in academia and industry
[1]. Although preventive vaccines are available for a number of viral
diseases, the chances of successfully developing a safe and effective
vaccine for HIV and hepatitis C virus (HCV) in the near future looks
very uncertain [2]. Structural modifications on either the sugar or
base moiety of natural nucleosides has resulted in a series of 24
nucleoside or nucleotide analogues licensed as drugs against
various viral infections, such as herpes simplex virus (HSV), human
cytomegalovirus (HCMV), varicella-zoster virus (VZV), HIV-1,
human hepatitis B virus (HBV) and HCV [3e5]. Despite this
achievement, continued efforts towards the discovery of new
nucleoside-based antivirals are required to overcome common
problems in antiviral chemotherapy, such as toxicity, adverse
effects, drugedrug interactions and, last but not least, the devel-
opment of drug resistance.
These nucleoside analogues require two additional phosphory-
lation steps by cellular kinases before they are incorporated in the
viral genome and lead to chain termination of the proviral DNA
strand during the reverse transcription process.
By synthesizing and evaluating the isomeric analogues of
PMDTA, the importance of the relative (cis) and absolute orienta-
tion of the substituents at the 10 and 30-positions has been
demonstrated to be crucial for antiviral activity [6b]. Also, it was
shown that homologation of the 30-O-phosphonomethyl group of
Ia-b to a phosphonoethyl was at the expense of the biological
activity [6c]. Most likely, this loss of activity results from the poor
acceptance of these homologues as substrates by kinases required
for conversion to their metabolic diphosphate form. Noteworthy, in
2000 Eschenmoser and coworkers showed that (L)-a-threofur-
anosyl oligonucleotides (TNAs) containing vicinally connected
(30 / 20) phosphodiester bridges undergo informational base
pairing in antiparallel strand orientation and are capable of cross-
pairing with RNA and DNA [7].
Inspired by the promising anti-HIV-1/HIV-2 activity of Ia-b, we
decided to explore the synthesis and the antiviral properties of the
corresponding non-phosphonylated 2-deoxythreose-based nucle-
oside analogues II, characterized by a four-carbon-only car-
bohydrate part. We expected that due to the absence of the
40-hydroxymethyl group, the secondary 30eOH could become
accessible for phosphorylation by cellular nucleoside kinases and
Recently, Herdewijn and coworkers showed that selected L-2-
deoxythreose nucleoside phosphonates (I; Fig. 1) selectively
inhibit HIV without affecting human DNA synthesis [6]. PMDTA (Ia)
and PMDTT (Ib) lack a hydroxymethyl group at the 40-position of
the furanose ring, but instead have a phosphonomethyl ether
* Corresponding author. Tel.: þ32 9 264 81 24; fax: þ32 9 264 81 46.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.036

K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
3705
A Vorbrüggen coupling reaction of 4 and 5 with the preformed
silyl-protected nucleobases exclusively gave the -nucleosides
O
B
B
P
O
HO
a
O
O
O
O
6e11 due to anchimeric assistance from the 2-O-acetyl group [10].
The benzyl-protected thymine- and uracil-nucleosides 6 and 7
were isolated in considerably better yields than the PMB protected
analogues, due to instability of the PMB-protecting group under
Vorbrüggen conditions. Moreover, the reaction between PMB-
protected glycon 5 with silylated N6-benzoyladenine and N4-ace-
tylcytosine failed to produce the desired coupling products in
acceptable yields, which forced us to restrict further investigations
to the benzyl-protected nucleosides. Under normal coupling
conditions, the yield of the adenine coupling product 10 was also
very low, possibly due to the formation of other isomers [11].
However, prolonged reaction time at elevated temperature (50 ꢀC)
afforded the thermodynamically favoured isomer 10 in an accept-
able yield of 42%. Under the normal coupling conditions the cyti-
dine analogue 11 was obtained in 70% yield.
Ia B = adenin-9-yl
(PMDTA)
IIa B = adenin-9-yl (25)
IIb B = thymin-1-yl (26)
IIc B = uracil-1-yl (27)
IId B = cytosin-1-yl (29)
Ib B = thymin-1-yl
(PMDTT)
Fig. 1. Structures of active L-2-deoxythreose nucleoside phosphonates (I) and the
nucleoside surrogates of this study (II).
anticipated IIa-d to have a superior bioavailability compared to the
phosphonates. Remarkably, the synthesis of such
a-L
-20-deoxy-
threofuranosyl analogues (exhibiting the 1’R, 30R configuration) has
not been reported before.
2. Results and discussion
The 20-O-acetyl group of 6 and 7 was removed by treatment with
7N ammonia in methanol. Similar treatment of 8 and 9 led to the
formation of small amounts of 20,30-dihydroxy analogues (10e15%).
Lowering the ammonia concentration to 1N allowed to obtain the
desired compounds 14 and 15 in highyields. Stirringcompound 10 at
room temperature in a 7N solution of ammonia in methanol for 2
days removed both the 20-O-acetyl and the N(6)-benzoyl group to
yield 16. Likewise, 11 was deprotected to the dideacetylated
compound 17, which was selectively N-acylated to 18 with one
equivalent of acetic anhydride in dimethylformamide [12].
2.1. Chemical synthesis
The synthesis of target compounds started from 1,2-O-iso-
propyledene-a-L-threose 1 (Scheme 1), which was synthesized in
multi-gram scale following literature procedures [8,9]. The
hydroxyl group of 1 was protected using either benzyl bromide or
4-methoxybenzyl (PMB) chloride to give compounds 2 and 3 in
excellent yields. A benzyl group is a safe and versatile protecting
group for carbohydrates, but may require harsh deprotection
conditions. We also explored a more sensitive PMB-protecting
group, which may be removed under alternative conditions. Next,
deprotection of the isopropylidene group upon treatment with 80%
aq. acetic acid followed by acetylation afforded the key precursors 4
and 5 in good yield.
Deoxygenation of the 20-hydroxy group was realized following
a two step Barton-McCombie procedure [13]. The xanthate was
formed by reacting the 20eOH group of 12e16 and 18 with p-tolyl
chlorothionocarbonate in the presence of 4-dimethylaminopyridine.
B
O
O
O
O
OAc
OAc
c
O
a / b
O
d / e
O
O
RO
OAc
HO
RO
RO
1
2: R = Bn
4: R = Bn
6: R = Bn, B = T
7: R = Bn, B = U
3: R = PMB
5: R = PMB
8: R = PMB, B = T
9: R = PMB, B = U
10: R = Bn, B = ABz
11: R = Bn, B = CAc
B
B
B
O
O
O
f / g
i
j / k / l
RO
OH
RO
HO
12: R = Bn, B = T
13: R = Bn, B = U
14: R = PMB, B = T
15: R = PMB, B = U
16: R = Bn, B = A
17: R = Bn, B = C
18: R = Bn, B = CAc
19: R = Bn, B = T
20: R = Bn, B = U
21: R = PMB, B = T
22: R = PMB, B = U
23: R = Bn, B = A
24: R = Bn, B = CAc
25: B = T
26: B = U
27: B = A
28: B = CAc
f
29: B = C
h
Scheme 1. Synthesis of a
-L-20-deoxythreofuranosyl nucleoside analogues 25e27 and 29. Reagents and conditions: (a) benzyl bromide, NaH, DMF, 0 ꢀC / RT, 4 h, 87%; (b) p-
methoxybenzyl chloride, NaH, DMF, ꢁ20 ꢀC / RT, 4 h, 84%; (c) i. 80% aq. CH3COOH, 80 ꢀC, 8 h; ii. Ac2O, pyridine, RT, 4 h, 63e81%; (d) i. nucleobase, pyridine, TMSCl, HMDS, 135 ꢀC,
overnight; ii. silylated base, 4/5, TMSOTf, 1,2-dichloroethane, 0 ꢀC / RT, 3e5 h, 45e87%; (e, for 10) i. nucleobase, pyridine, TMSCl, HMDS, 135 ꢀC, overnight; ii. silylated base, 4/5,
TMSOTf, 1,2-dichloroethane, 50 ꢀC, 24 h, 42%; (f) 7N NH3-MeOH, RT, 4e6 h, 81e95%; (g, for 14e15) 1N NH3-MeOH, RT, 5 h, 78e90%; (h) Ac2O, DMF, RT, 24 h, 78%; (i) i. DMAP, O-(p-
tolyl) chlorothionoformate, CH3CN, 0 ꢀC / RT, 4 h; ii. Bu3SnH, AIBN, toluene, 100 ꢀC, 2e4 h, 47e86%; (j) Pd-C/H2, MeOH, RT, 3e8 h, 40e89%; (k, on 21 and 22) CAN, CH3CN :H2O,
0
ꢀC / RT, 1 h, 78e84%; (l, for 28) DDQ, CH2Cl2, 50 ꢀC, 72 h, 11%.

3706
K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
O
N
The intermediate formed was subjected to heating with tribu-
tyltinhydride and azoisobisbutyronitrile in toluene to give the
20-deoxygenated compounds 19e24 in moderate to good yields
(47e86%).
NH
CH3
CH3
O
a
O
P
Cl
O
P
Cl
O
+
+
O
O
H2N
Bn
Bn
O
Cl
O
N
H
O
OH
O
All target compounds were obtained after debenzylation.
However, optimal debenzylation procedures were distinct for each
analogue. The 20-deoxythreose thymine and uracil analogues 25
and 26 could be obtained by catalytic hydrogenation of 19 and 20 in
good albeit variable yields, possibly due to catalyst poisoning by the
residual sulphur from the previous deoxygenation reaction.
Desulphurization with hydrogen over Raney-Nickel prior to
palladium-catalysed debenzylation improved the reproducibility of
this step. Alternatively, CAN-mediated deprotection [14] of 21 and
22 afforded 25 and 26, respectively. The adenine analogue 27 was
obtained by a palladium-catalysed hydrogenation reaction in
moderate yields.
31
32
33
25
O
NH
CH3
NH
BnO
N
O
b
O O
P
O
O
O
34
Scheme 3. Synthesis of the ProTide of 25. Reagents and conditions: (a) anhydrous TEA,
anhydrous DCM, ꢁ78 ꢀC for 30 min, rt, 2 h; (b) 1.0 M tert-butyl magnesium chloride in
THF, anhydrous THF, rt, 24 h.
The cytosine analogue 29 was obtained via two different routes.
Afteranunsuccessfulattempttohydrogenate24, the benzyl group was
removed by treatment with an excess of dichlorodicyanoquinone
(DDQ) in dichloromethane at 50 ꢀC in a sealed reaction vial for 3 days
[15]. This method gave 28 in a disappointing yield of 11%. Hence, we
also explored the transformation of the uridine analogue 26 to
compound 29 (Scheme 2) [16]. This was realized by protecting the 30-
hydroxy group of 26 to the corresponding acetate 30, which was
subsequently treated with phosphorous oxychloride and 1,2,4-
triazole, bubbling ammonia gas and a 7N solution of ammonia in
methanol.
To access the phosphoramidate pro-drug the coupling between
the phenyldichlorophosphate (31) and L-alanine benzyl ester
tosylate (32) has been performed in the presence of Et3N (2 eq)
giving the desired product (33) as an oil, which was used in the
following step as a crude (Scheme 3). The final coupling of the
nucleoside 25 has been performed using an excess of the phos-
phorochloridate (33) (3 eq.) in the presence of tBuMgCl (3 eq.)
following the procedure reported by Uchiyama [17] and extensively
used for the synthesis of the ProTides [18]. The desired compound
34 was obtained as a mixture of two diastereoisomers confirmed by
the presence of two peaks in the 31P NMR (2.92, 2.27).
nucleoside analogues, a nucleoside monophosphate prodrug was
prepared from compound 25 (Scheme 3). Such prodrugs may
intracellularly release the free monophosphorylated form accord-
ing to a multistep process lined-out in Scheme 4. However, also the
phosphoramidate 34 failed to show activity against HIV-1, HIV-2,
HSV-1, HSV-2, vaccinia virus, VSV and HSV-1 (TKꢁ).
2.3. Enzymatic study of the ProTide using carboxypeptidase
Y enzyme
The first step of the bioactivation of the phosphoramidate Pro-
Tide moiety involves the hydrolysis of the ester moiety which is
supposed to be mediated by a carboxypeptidase-type enzyme
(Scheme 4). An enzymatic study using carboxypeptidase Y enzyme
has been performed in order to understand whether compound 34
may be metabolized under these conditions.
The experiment was performed by incubating compound 34
with carboxypeptidase Y in acetone-d6 and trizma buffer (pH ¼ 7.6)
following its conversion by 31P NMR. The spectra (Fig. 2) showed
a fast conversion of one of the diastereoisomers 34 (dP ¼ 2.38) to the
compound 37 (dP ¼ 6.25) through the intermediate 35 (dP ¼ 3.66).
The other diastereoisomer (dP ¼ 2.24) was slower converted and it
was still detectable after 14 h. This experiment showed that 34 is
partially converted to the metabolite 37.
2.2. Pharmacological activity
None of the final nucleoside analogues showed cytotoxicity at
2.4. Interaction of test compounds with nucleoside kinases
100
mg/mL. No anti-HIV-1 and anti-HIV-2 activity was observed in
human T-lymphocyte (CEM) cells (EC50 > 100
m
g/mL). Further-
Interestingly, upon evaluation of their capacity to inhibit
thymidine (1 mM) phosphorylation by recombinant purified human
cytosolic TK-1, human mitochondrial TK-2, HSV-1 TK, VZV TK, and
Drosophila melanogaster (Dm) dNK (Table 1), compound 25 proved
inhibitory toTK-2, HSV-1 TK, and VZV TK at an IC50 ranging between
more, no significant activity could be detected against the following
viruses: human cytomegalovirus (HEL), VZV (HEL); influenza A
(H1N1 & H3N2) and B virus (MDCK), feline corona & feline
herpes virus (CRFK); herpes simplex virus type 1 (HSV-1), HSV-2,
vesicular stomatitis virus (VSV) and vaccinia virus (HEL); Cox-
sackie virus B4 and respiratory syncytial virus (HeLa); para-
Influenza virus-3, reovirus-3, Sindbis virus and Punta Toro virus
(Vero). Since the lack of antiviral activity could be due to the
inability of virally-induced and/or cellular enzymes to activate the
14 and 91
with a Ki of 18
in detail. Whereas 75% of the natural substrate thymidine (100
m
g/mL and Mycobacterium tuberculosis thymidylate kinase
M. Therefore, compound 25 was investigated more
M)
m
m
was converted to its 50-mono- (and 50-di)phosphate within 5 min
upon exposure to HSV-1 TK, no signs of any conversion of
compound 25 to its phosphorylated derivative was observed after
60 min of incubation with the enzyme. Therefore, we may assume
that these nucleosides purely act as enzyme inhibitors, rather than
acting as alternative substrates. LineweavereBurk kinetic analysis
revealed a non-competitive mechanism of inhibitory action of
compound 25 against HSV-1 TK using dThd as the natural substrate
(Fig. 3). The inhibitory potential of some of the test compounds
O
N
O
N
NH2
N
NH
NH
a
b
O
O
N
O
O
O
O
HO
AcO
HO
opens perspectives for using this L-a
-20-deoxythreofuranosyl ring
26
30
29
as a scaffold for optimizing these enzyme inhibitory activities.
In conclusion, we have developed a practical and straight-
forward procedure for the synthesis of
Scheme 2. An improved synthesis of the cytosine analogue 29. Reagents and condi-
tions: (a) Acetic anhydride, DMAP, DMF, RT, 24 h, 97%; (b) i. 1,2,4-triazole, POCl3,
pyridine, RT, 4 h; ii. NH3, 2 h; iii. NH3-MeOH, RT, 5 h, 36%.
a series of a-L
-20-

K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
3707
O
O
N
O
N
NH
NH
NH
CH3
CH3
NH
CH3
NH
O
N
O
BnO
O
O
NH
O O P
a
O
b
O
P
O
O P
O
O
O
O
O
O
O
O
O
34
36
35
c
a. Carboxypeptidase type enzyme;
b. Spontaneous;
O
N
O
N
c. Spontaneous;
d. Phosphoramidase type enzyme.
NH
NH
CH3
O
O
O
NH
O P
O
O
O P
O
O
O
O
d
O
O
38
37
Scheme 4. Putative mechanism of bioactivation of the ProTide.
deoxythreofuranosyl nucleosides containing four natural nucleo-
bases. None of the analogues displayed significant antiviral activity
or cytotoxicity. In an effort to bypass the first activation (phos-
phorylation) step of the nucleoside analogues, a phosphoramidate
nucleoside phosphate prodrug was synthesized for compound 25,
but this prodrug still lacked any measurable antiviral activity.
However, the thymine analogue showed encouraging inhibitory
activity towards a number of thymidine kinases, which may be
a new starting point towards more potent and selective nucleoside
kinase inhibitors.
reactions were carried out under argon atmosphere. Precoated
Merck silica-gel F254 plates were used for TLC, spots were exam-
ined under ultraviolet light at 254 nm and further visualized by
sulphuric acid-anisaldehyde spray. Column chromatography was
performed on silica gel (63e200 mm, 60 Å, Biosolve, Valkenswaard,
The Netherlands). NMR spectra were determined using a Varian
Mercury 300 MHz or a Bruker Avance 500 MHz spectrometer.
Chemical shifts are given in ppm (d) relative to the residual solvent
signals or TMS as internal standard. Exact mass measurements
were performed on a Waters LCT PremierXETMTime of flight (TOF)
mass spectrometer equipped with a standard electrospray ioniza-
tion (ESI) and modular LockSpray TM interface. Samples were
3. Experimental section
infused in a CH3CN/water (1:1) mixture at 10 mL/min.
3.1. Chemical synthesis
3.1.1. (3aR,6S,6aR)-6-(benzyloxy)-tetrahydro-2,2-dimethylfuro[2,3-
d][1,3]dioxole (2)
All reagents were from standard commercial sources and of
analytic grade. Dry solvents were obtained directly from commer-
cial sources and stored on molecular sieves. Moisture sensitive
To a solution of compound 1 (2.3 g, 14.36 mmol) in dry DMF
(50 mL) cooled at 0 ꢀC under inert atmosphere, NaH (60% in mineral
Fig. 2. Carboxypeptidase-mediated cleavage of compound 34, monitorated by 31P NMR.

3708
K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
Table 1
3.1.3. (3R,4S)-4-(benzyloxy)-tetrahydro-2, 3-furanyl diacetate (4)
Nucleoside kinase activity (IC50 in
m
g/mL).
HSV-1 TK
A solution of 2 (2.0 g, 8 mmol) in 80% aq. acetic acid (40 mL) was
stirred at 80 ꢀC for 8 h. The reaction mixture was evaporated to give
the crude intermediate as syrup. This syrup was dissolved in pyri-
dine (30 mL) and treated with acetic anhydride (7.5 mL, 80 mmol).
The solution was stirred at room temperature for 4h. The solvent
was removed under vacuum and the resulting residue was purified
by silica-gel column chromatography (20% EtOAc-hexanes) to yield
TK-1
TK-2
VZV TK
Dm dNK
25
26
27
29
>200
>200
>200
>200
91 ꢂ 40
60 ꢂ 41
>200
14 ꢂ 3
>200
>200
>200
26 ꢂ 9
>200
>200
>200
ꢃ200
>200
>200
>200
>200
4 (1.9 g, 81%) as a low melting solid.
(CDCl3, 300 MHz): 1.98, 2.00, 2.02, 2.03 (s’s, 6H), 3.78e4.29 (m,
a,b
anomeric ratio 3:2. 1H NMR
oil, 0.86 g, 21.54 mmol) was added portionwise and the mixture
was stirred for 10 min. Benzyl bromide (2.56 mL, 21.54 mmol) was
added dropwise to the above suspension, which was allowed to
come to room temperature and stirred for an additional 4 h. The
reaction mixture was quenched by addition of methanol (1 mL) and
the volatile solvents were evaporated under reduced pressure. The
residue was partitioned between water-ethyl acetate (1:3, 300 mL).
The organic layer was separated, washed with water, brine and
dried over anhydrous MgSO4. After evaporation, the crude product
was purified by silica-gel chromatography (15% EtOAc-hexanes) to
afford compound 2 (3.1 g, 87%) as a colourless oil. 1H NMR(CDCl3,
d
3H), 4.43e4.71 (m, 2H), 5.13e5.19 (m, 1H), 6.06e6.32 (s & d,
J ¼ 4.51 Hz, 1H), 7.20e7.32 (m, 5H). ESI-HRMS for [C15H18O6 þ K]þ
calcd, 333.0735; found, 333.0738.
3.1.4. (3R,4S)-4-(4-methoxybenzyloxy)-tetrahydro-2,3-furanyl
diacetate (5)
Employing the procedure described above on 2.2 g of compound
3 resulted in 1.6 g (63% yield) of compound 5 as a white solid.
a,b
anomeric ratio 1:1. 1H NMR (CDCl3, 300 MHz):
d
2.04, 2.07, 2.08,
2.09 (s’s, 6H), 3.79 (s, 3H), 3.82e4.32 (m, 3H), 4.42e4.68 (m, 2H),
5.17e5.24 (m, 1H), 6.11, 6.37 (2d’s, J ¼ 4.49, 2.39 Hz, 1H), 6.83e6.91
(m, 2H), 7.20e7.30 (m, 2H). ESI-HRMS for [C16H20O7 þ Na]þ calcd,
347.1101; found, 347.1112.
300 MHz):
4.61 (d, J ¼ 3.77 Hz, 1H), 5.96 (d, J ¼ 3.76 Hz, 1H), 7.27e7.39 (m, 5H).
13C NMR (CDCl3, 75 MHz):
26.15, 26.71, 70.27, 71.09, 81.81, 82.84,
d 1.32 (s, 3H), 1.47 (s, 3H), 4.01e4.05 (m, 3H), 4.57 (s, 2H),
d
105.44, 111.46, 127.59, 127.80, 128.41, 137.32. ESI-HRMS for
[C14H18O4 þ NH4]þ calcd, 268.1549; found, 268.1565.
3.1.5. General condition for Vorbrüggen coupling reaction
The nucleobase (protected in case of adenine and cytosine)
(1.2 eq.) was suspended in hexamethyldisilazane (50 eq.) contain-
ing trimethylsilyl chloride (0.7 eq.) and pyridine (10 eq.). The
mixture was heated at reflux overnight. After cooling, the reaction
mixture was evaporated and dried under high vacuum. The sily-
lated nucleobase and the diacetate compound 4 or 5 (1 eq.) were
dissolved in dry 1,2-dichloroethane (7 mL/mmol), and trime-
thylsilyl triflate (1.2 eq.) was added dropwise at 0 ꢀC. The clear
solution was stirred for 4 h at room temperature. The reaction
mixture was diluted with dichloromethane (50 mL/mmol) and
washed with saturated aqueous NaHCO3. The organic layer was
dried over anhydrous MgSO4 and evaporated. Purification of the
residue by silica-gel column chromatography (1% MeOH-
3.1.2. (3aR,6S,6aR)-6-(4-methoxybenzyloxy)-tetrahydro-2,2-
dimethylfuro[2,3-d][1,3]dioxole (3)
Compound 1 (1.5 g, 9.4 mmol) was reacted with NaH (60% in
mineral oil, 0.45 g, 11.24 mmol) and p-methoxybenzyl chloride
(1.5 mL, 11.24 mmol) as described for synthesis of 2 to afford
compound 3 (2.2 g, 84%) as a white low melting solid. 1H NMR
(CDCl3, 300 MHz): d 1.31 (s, 3H), 1.46 (s, 3H), 3.78 (s, 3H), 3.98e4.02
(m, 3H), 4.48 (s, 3H), 4.58 (d, J ¼ 3.76 Hz, 1H), 5.94 (d, J ¼ 3.76 Hz,
1H), 6.87 (d, J ¼ 8.72 Hz, 2H), 7.24 (d, J ¼ 8.77 Hz, 2H). 13C NMR
(CDCl3, 75 MHz):
d 26.15, 26.71, 55.16, 70.28, 70.78, 81.45, 82.87,
105.42, 111.42, 113.82, 129.27, 129.34, 159, 30. ESI-HRMS for
[C15H20O5 þ K]þ calcd, 319.0942; found, 319.0937.
Fig. 3. LineweavereBurk plot for compound 25.

K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
3709
dichloromethane) afforded the pure coupling product as a white
foam.
(2R,3R,4S)-4-(benzyloxy)-tetrahydro-2-(3,4-dihydro-5-methyl-
2,4-dioxopyrimidin-1(2H)-yl)furan-3-yl acetate (6). Following the
general reaction conditions a Vorbrüggen coupling between 4 and
thymine afforded the title compound (1.07 g, 87%) 1H NMR (CDCl3,
(d, J ¼ 3.76 Hz, 1H), 4.31 (dd, J ¼ 10.34, 3.89 Hz, 1H), 4.4e4.52 (m,
3H), 5.48 (s, 1H), 6.71 (s, 1H), 7.00e7.10 (m, 5H), 7.31e7.50 (m, 3H),
8.08 (s, 1H), 8.12e8.19 (m, 2H), 8.59 (s, 1H). 13C NMR (CDCl3,
75 MHz):
d 21.03, 72.12, 76.07, 79.94, 80.49, 93.07, 127.81, 128.08,
128.16, 128.22, 128.56, 128.74, 129.89, 132.32, 136.84, 137.81, 142.01,
143.61, 148.71, 169.68, 175.13. ESI-HRMS for [C25H23N5O5 þ H]þ
calcd, 474.1772; found, 474.1698.
300 MHz):
d
1.72 (d, J ¼ 1.19 Hz, 3H), 2.12 (s, 3H), 4.00e4.1 (m, 2H),
4.32 (app-dm, J ¼ 8.75 Hz, 1H), 4.63 (app-dd, J ¼ 14.68, 11.48 Hz,
(2R,3R,4S)-4-(benzyloxy)-tetrahydro-2-(4-(acetylamino)-2-
2H), 5.22 (s, 1H), 6.08 (d, J ¼ 1.36 Hz, 1H), 7.22e7.34 (m, 5H), 7.37 (d,
oxopyrimidin-1(2H)-yl)furan-3-yl acetate (11).
A general Vor-
J ¼ 1.22 Hz, 1H), 9.42 (brs, 1H). 13C NMR (CDCl3, 75 MHz):
d
12.57,
brüggen coupling condition between N4-acetylcytosine and
21.03, 71.81, 73.74, 79.93, 80.88, 89.41, 110.94, 128.17, 128.47, 128.83,
136.41, 136.89, 150.69, 164.23, 169.90. ESI-HRMS for [C18H20N2O6
þ H]þ calcd, 361.1400; found, 361.1399.
compound 4 (295 mg, 1.0 mmol) yielded compound 11 (270 mg,
70%). 1H NMR (CDCl3, 300 MHz):
d 2.11 (s, 3H), 2.28 (s, 3H), 4.01 (d,
J ¼ 3.77 Hz,1H), 4.20 (dd, J ¼ 10.31, 3.86 Hz,1H), 4.35 (d, J ¼ 10.25 Hz,
1H), 4.97 (app-q, J ¼ 13.94 Hz, 2H), 5.28 (s, 1H), 5.34 (s, 1H), 6.05 (s,
1H), 7.14e7.20 (m, 2H), 7.25e7.32 (m, 3H), 7.34 (d, J ¼ 7.61 Hz, 1H),
7.89 (d, J ¼ 7.57 Hz, 1H), 10.34 (brs, 1H). 13C NMR (CDCl3, 75 MHz):
(2R,3R,4S)-4-(benzyloxy)-tetrahydro-2-(3,4-dihydro-2,4-
dioxopyrimidin-1(2H)-yl)furan-3-yl acetate (7). Vorbrüggen
coupling between 4 (500 mg, 1.7 mmol) and uracil (230 mg,
2.04 mmol) afforded 481 mg of the uridine analogue 7 (82% yield).
d
21.10, 25.10, 71.67, 75.25, 79.15, 80.15, 91.22, 96.48, 128.21, 128.37,
1H NMR (CDCl3, 300 MHz):
d
2.07 (s, 3H), 3.96 (d, J ¼ 3.78 Hz, 1H),
128.77, 136.84, 145.07, 155.25, 163.53, 169.70, 171.44. ESI-HRMS for
4.02 (dd, J ¼ 10.25, 3.81 Hz,1H), 4.24 (d, J ¼ 10.28 Hz,1H), 4.56 (app-
q, J ¼ 11.76 Hz, 2H), 5.17 (s, 1H), 5.55 (dd, J ¼ 8.19, 1.77 Hz, 1H), 5.96
(d, J ¼ 1.18 Hz, 1H), 7.17e7.32 (m, 5H), 7.48 (d, J ¼ 8.20 Hz, 1H), 8.93
[C19H21N3O6 þ H]þ calcd, 388.1503; found, 388.1509.
1-((2R,3R,4S)-4-(benzyloxy)-tetrahydro-3-hydroxyfuran-2-yl)-
5-methylpyrimidine-2,4(1H,3H)-dione (12). The 20-O-acetylated
nucleoside 6 (580 mg, 1.6 mmol) was treated with 7N methanolic
ammonia solution (15 mL) at room temperature overnight. Evap-
oration yielded a residue which was purified by column chroma-
tography (2% MeOH-CH2Cl2) to afford compound 12 (420 mg, 82%)
(brs, 1H). 13C NMR (CDCl3, 75 MHz):
d 21.03, 71.88, 74.27, 79.72,
80.52, 89.82, 102.23, 128.28, 128.54, 128.87, 136.80, 140.58, 150.39,
163.39, 169.79. ESI-HRMS for [C17H18N2O6 þ H]þ calcd, 347.1238;
found 347.1246.
(2R,3R,4S)-4-(4-methoxybenzyloxy)-tetrahydro-2-(3,4-dihydro-
5-methyl-2,4-dioxopyrimidin-1(2H)-yl)furan-3-yl acetate (8). Vor-
brüggen coupling between 5 (600 mg, 1.85 mmol) and thymine
yielded 420 mg of the title compound (58%). 1H NMR (CDCl3,
as a white foam. 1H NMR (CDCl3, 300 MHz):
d
1.67 (d, J ¼ 0.75 Hz,
3H), 4.07 (s, 1H), 4.30 (s, 2H), 4.42 (s, 3H), 5.59 (brs, 1H), 7.78 (s, 1H),
7.06e7.24 (m, 5H), 7.30 (d, J ¼ 1.14 Hz, 1H), 10.60 (brs, 1H). 13C NMR
(CDCl3, 75 MHz):
d 12.24, 71.45, 74.97, 78.10, 82.20, 93.54, 109.38,
300 MHz):
d
1.68 (d, J ¼ 1.21 Hz, 3H), 2.07 (s, 3H), 3.73 (s, 3H), 3.98 (dd,
127.59, 127.96, 128.45, 136.75, 136.92, 150.96, 164.73. ESI-HRMS for
J ¼ 14.17, 3.72 Hz, 2H), 4.14e4.25 (m, 1H), 4.50 (app-q, J ¼ 14.14 Hz,
2H), 5.15 (m,1H), 6.00 (d, J ¼ 1.34 Hz,1H), 6.80 (d, J ¼ 8.72 Hz, 2H), 7.14
(d, J ¼ 8.78 Hz, 2H), 7.31 (d, J ¼ 1.24 Hz, 1H), 8.76 (brs, 1H). 13C NMR
[C16H18N2O5eH]ꢁ calcd, 317.1143; found 317.1147.
3.1.7. 1-((2R,3R,4S)-4-(benzyloxy)-tetrahydro-3-hydroxyfuran-2-
yl)pyrimidine-2,4(1H,3H)-dione (13)
(CDCl3, 75 MHz):
d 12.60, 21.05, 55.54, 71.48, 73.85, 79.87, 80.53,
89.45, 110.83, 114.21, 128.95, 129.88, 136.50, 150.48, 159.84, 163.97,
169.87. ESI-HRMS for [C19H22N2O7 þ H]þ calcd, 391.15; found,
391.1521.
Following the procedure described for 12, compound 7 (475 mg,
1.37 mmol) was hydrolysed to afford compound 13 (340 mg, 81%)
as a white foam. 1H NMR (CDCl3, 300 MHz):
d 4.05 (s, 1H), 4.28 (s,
(2R,3R,4S)-4-(4-methoxybenzyloxy)-tetrahydro-2-(3,4-dihydro-
2,4-dioxopyrimidin-1(2H)-yl)furan-3-yl acetate (9). Vorbrüggen
couplingbetween 5 (325 mg,1.0mmol) and uracilyielded compound
2H), 4.41 (s, 1H), 4.43 (s, 2H), 5.54 (dd, J ¼ 8.10, 1.81 Hz, 1H), 5.73 (s,
1H), 7.10e7.30 (m, 5H), 7.48 (d, J ¼ 8.16 Hz, 1H), 10.52 (brs, 1H). 13C
NMR (CDCl3, 75 MHz):
d 71.85, 75.31, 82.29, 94.00, 101.32, 128.01,
9 (180 mg, 47%).1H NMR (CDCl3, 300 MHz):
d
2.12 (s, 3H), 3.79 (s, 3H),
128.37, 128.79, 137.11, 140.94, 151.30, 164.38. ESI-HRMS for
4.00 (d, J ¼ 3.73 Hz, 1H), 4.07 (dd, J ¼ 10.24, 3.81 Hz, 1H), 4.27 (d,
J ¼ 10.23 Hz,1H), 4.55 (app-q, J ¼ 12.73 Hz, 2H), 5.23 (s,1H), 5.62 (dd,
J ¼ 8.21,1.47 Hz,1H), 6.02 (d, J ¼ 1.03 Hz,1H), 6.86 (d, J ¼ 8.81 Hz, 2H),
7.19 (d, J ¼ 8.79 Hz, 2H), 7.54 (d, J ¼ 8.21 Hz, 1H), 9.36 (brm, 1H). 13C
[C15H16N2O5 þ H]þ calcd, 305.1132; found, 305.1132.
1-((2R,3R,4S)-4-(4-methoxybenzyloxy)-tetrahydro-3-
hydroxyfuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (14).
Compound 8 (400 mg, 1.02 mmol) was treated with 1N meth-
anolic ammonia solution (10 mL) at room temperature for 6 h.
Evaporation yielded a residue which was purified by column
chromatography (2% MeOH-CH2Cl2) to afford compound 14
NMR (CDCl3, 75 MHz): d 21.00, 55.50, 71.42, 74.32, 79.61, 80.15, 89.76,
102.16, 114.20, 128.91, 129.99, 140.68, 150.71, 159.80, 164.03, 169.85.
ESI-HRMS for [C18H20N2O7 þ H]þ calcd, 377.1343; found 377.1369.
(280 mg, 78%) as a white foam. 1H NMR (CDCl3, 300 MHz):
d 1.69
3.1.6. (2R,3R,4S)-2-(6-(benzamido)-9H-purin-9-yl)-4-(benzyloxy)-
tetrahydrofuran-3-yl acetate (10)
(d, J ¼ 1.1 Hz, 3H), 3.71 (s, 3H), 4.03 (m, 1H), 4.27 (d, J ¼ 1.84 Hz,
2H), 4.35 (s, 2H), 4.40 (s, 1H), 5.76 (s, 1H), 6.76 (d, J ¼ 8.72 Hz,
2H), 7.03 (d, J ¼ 8.70 Hz, 2H), 7.29 (d, J ¼ 1.21 Hz, 1H),10.47 (brs,
To a suspension of N6-benzoyladenine (1.0 g, 4.08 mmol) in
hexamethyldisilazane (35 mL, 0.17 mol) was added trimethylsilyl
chloride (0.3 mL, 2.38 mmol) and pyridine (2.7 mL, 34 mmol). The
mixture was heated at reflux overnight. After cooling it was evap-
orated and dried under high vacuum. The silylated nucleobase
and the diacetate 4 (1.0 g, 3.4 mmol) were dissolved in dry
1,2-dichloroethane (20 mL) and trimethylsilyl triflate (0.74 mL,
4.08 mmol) was added dropwise at 0 ꢀC. The clear solution was
stirred at 50 ꢀC for 24 h. After dilution with dichloromethane
(250 mL), the reaction mixture was washed with saturated aqueous
NaHCO3. The organic layer was dried over anhydrous MgSO4 and
evaporated. Purification of the residue by silica-gel flash column
chromatography (60% EtOAc e hexanes) afforded pure 10 (0.68 g,
1H). 13C NMR (CDCl3, 75 MHz):
d 12.57, 55.54, 71.41, 75.15, 78.48,
82.24, 93.77, 109.64, 114.15, 129.30, 129.57, 137.02, 151.20, 159.67,
164.92. ESI-HRMS for [C17H20N2O6 þ H]þ calcd, 349.1394; found,
349.1389.
1-((2R,3R,4S)-4-(4-methoxybenzyloxy)-tetrahydro-3-
hydroxyfuran-2-yl)pyrimidine-2,4(1H,3H)-dione (15). The ester
moiety of 9 (180 mg, 0.48 mmol) was hydrolysed as described in the
preparation of 14 to give compound 15 (145 mg, 90%) as a white
foam. 1H NMR (CDCl3, 300 MHz):
d 3.77 (s, 3H), 4.08e4.12 (m, 1H),
4.26e4.36 (m, 2H), 4.42 (s, 2H), 4.46 (s, 1H), 5.55 (brs, 1H), 5.60 (dd,
J ¼ 8.14, 1.74 Hz, 1H), 5.79 (s, 1H), 6.83 (d, J ¼ 8.77 Hz, 2H), 7.11 (d,
J ¼ 8.65 Hz, 2H), 7.53 (d, J ¼ 8.19 Hz, 1H), 10.74 (brs, 1H). 13C NMR
42%) as white foam. 1H NMR (CDCl3, 300 MHz):
d
2.08 (s, 3H), 4.04
(CDCl3, 75 MHz): d 55.52, 71.50, 75.44, 78.24, 82.05, 93.93, 101.26,

3710
K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
114.17, 129.28, 129.69, 141.05, 151.40, 159.71, 164.51. ESI-HRMS for
purified by column chromatography (30e40% EtOAc e hexanes) to
yield the 20-deoxy compound 19e24 as a foam.
[C16H18N2O6 þ H]þ calcd, 335.1238; found, 335.1255.
3.1.8. (2R,3R,4S)-2-(6-amino-9H-purin-9-yl)-4-(benzyloxy)-tetrahyd-
rofuran-3-ol (16)
3.1.12. 1-((2R,4R)-4-(benzyloxy)-tetrahydrofuran-2-yl)-5-methylp-
yrimidine-2,4(1H,3H)-dione (19)
Nucleoside 10 (680 mg, 1.43 mmol) was treated with 7 N
methanolic ammonia solution (20 mL) at room temperature for 2
days. Evaporation yielded a residue which was purified by column
chromatography (4% MeOH-CH2Cl2) to afford compound 16
(450 mg, 95%) as a white foam. 1H NMR (DMSO-D6, 300 MHz):
Deoxygenation of 12 (420 mg, 1.32 mmol) rendered 330 mg of
compound 19 in 82% yield. 1H NMR (CDCl3, 300 MHz):
d 1.69
(d, J ¼ 1.19 Hz, 3H), 2.19 (ddd, J ¼ 15.07, 1.19, 0.88 Hz, 1H),
(ddd, J ¼ 15.11, 7.94, 5.45 Hz, 1H), 3.82 (dd, J ¼ 10.27, 3.60 Hz, 1H),
4.19 (dd, J ¼ 5.47, 3.66 Hz, 1H), 4.30 (dd, J ¼ 10.33, 1.50 Hz, 1H), 4.42
(app-dd, J ¼ 15.15, 11.27 Hz, 2H), 6.17 (dd, J ¼ 7.88, 2.17 Hz, 1H),
7.16e7.32 (m, 5H), 7.47 (d, J ¼ 1.22 Hz, 1H), 8.67 (brs, 1H). 13C NMR
d
4.10e4.15 (m, 1H), 4.19 (dd, J ¼ 9.75, 4.72 Hz, 1H), 4.29 (dd,
J ¼ 9.72, 2.61 Hz, 1H), 4.57 (s, 2H), 4.66e4.71 (m, 1H), 5.95 (d,
J ¼ 2.28 Hz, 1H), 6.02 (d, J ¼ 4.52 Hz, 1H), 7.20e7.40 (m, 7H), 8.16 (s,
(CDCl3, 75 MHz):
d 12.60, 38.49, 71.41, 74.51, 77.58, 85.48, 110.35,
2H). 13C NMR (DMSO-D6, 75 MHz):
d
75.82, 78.18, 82.47, 87.32,
127.90, 128.32, 128.82, 136.98, 137.28, 150.76, 164.10. ESI-HRMS for
95.55, 123.17, 131.76, 131.96, 132.43, 141.20, 143.53, 152.62, 156.47,
159.74. ESI-HRMS for [C16H18N5O3 þ H]þ calcd, 328.1404; found,
328.1419.
[C16H18N2O4 þ H]þ calcd, 303.1339; found, 303.1342.
3.1.13. 1-((2R,4R)-4-(benzyloxy)-tetrahydrofuran-2-yl)pyrimidine-
2,4(1H,3H)-dione (20)
3.1.9. 4-amino-1-((2R,3R,4S)-4-(benzyloxy)-tetrahydro-3-hydroxyfu-
ran-2-yl)pyrimidin-2(1H)-one (17)
Deoxygenation of 13 (306 mg, 1.0 mmol) rendered 250 mg of
compound 20 in 86% yield. 1H NMR (CDCl3, 300 MHz):
d 2.23 (dm,
The 20-O-acetylated nucleoside 11 (210 mg, 0.54 mmol) was
treated with 7 N methanolic ammonia solution (15 mL) at room
temperature overnight. Evaporation yielded a residue which was
purified by column chromatography (2% MeOH-CH2Cl2) to afford
140 mg of compound 17 (85% yield) as a white foam. 1H NMR
J ¼ 15.11 Hz, 1H), 2.39 (ddd, J ¼ 15.11, 7.56, 5.31 Hz, 1H), 3.85 (dd,
J ¼ 10.25, 3.67 Hz, 1H), 4.17 (app-t, J ¼ 4.40 Hz, 1H), 4.28
(dd, J ¼ 10.24, 1.83 Hz, 1H), 4.41 (app-dd, J ¼ 13.75, 11.63 Hz, 2H),
5.55 (dd, J ¼ 8.16, 1.73 Hz, 1H), 6.12 (dd, J ¼ 7.54, 2.00 Hz, 1H),
7.14e7.34 (m, 5H), 7.61 (d, J ¼ 8.16 Hz, 1H), 8.86 (brs, 1H). 13C NMR
(DMSO-D6, 300 MHz):
d
3.94 (app-d, J ¼ 3.98 Hz, 1H), 4.10 (dd,
(CDCl3, 75 MHz): d 38.56, 71.39, 74.99, 77.28, 86.15, 101.69, 127.96,
J ¼ 10.08, 4.06 Hz, 1H), 4.18 (d, J ¼ 4.28 Hz, 1H), 4.28 (d, J ¼ 10.08 Hz,
1H), 4.47 (app-q, J ¼ 11.70 Hz, 2H), 5.60 (d, J ¼ 7.44 Hz, 1H), 5.68
(d, J ¼ 1.08 Hz, 1H), 5.75 (s, 1H), 5.80 (d, J ¼ 4.35 Hz, 1H), 7.00 (brd,
J ¼ 23.36 Hz, 2H), 7.19e7.26 (m, 2H), 7.26e7.35 (m, 3H), 7.53 (d,
128.36, 128,85, 137.20, 141. 09, 150.70, 163.63. ESI-HRMS for
[C15H16N2O4 þ H]þ calcd, 289.1183; found, 289.1183.
3.1.14. 1-((2R,4R)-4-(4-methoxybenzyloxy)-tetrahydrofuran-2-yl)-
5-methylpyrimidine-2,4(1H,3H)-dione (21)
J ¼ 7.41 Hz, 1H). 13C NMR (DMSO-D6, 75 MHz):
d 55.60, 70.91, 73.60,
78.12, 83.64, 93.06, 93.53, 128.18, 128.34, 128.86, 138.44, 142.12,
155.94, 166.44. ESI-HRMS for [C15H17N3O4 þ H]þ calcd, 304.1292;
found, 304.1294.
Deoxygenation reaction of 14 (280 mg, 0.81 mmol) rendered
200 mg of compound 21 in 75% yield. 1H NMR (CDCl3, 300 MHz):
d
1.70 (d, J ¼ 1.20 Hz, 3H), 2.16 (ddd, J ¼ 15.09, 2.04, 1.05 Hz, 1H),
2.40 (ddd, J ¼ 15.1, 7.93, 5.52 Hz,1H), 3.73 (s, 3H), 3.80 (dd, J ¼ 10.22,
3.66 Hz,1H), 4.17 (app-t, J ¼ 4.46 Hz,1H), 4.28 (dd, J ¼ 10.26,1.34 Hz,
1H), 4.35 (dd, J ¼ 14.60,11.00 Hz, 2H), 6.16 (dd, J ¼ 7.90, 2.20 Hz,1H),
6.80 (d, J ¼ 8.74 Hz, 2H), 7.12 (d, J ¼ 8.74 Hz, 2H), 7.46 (d, J ¼ 1.23 Hz,
3.1.10. N-(1-((2R,3R,4S)-4-(benzyloxy)-tetrahydro-3-hydroxyfu-
ran-2-yl)-1,2-dihydro-2-oxopyrimidin-4-yl)acetamide (18)
To a solution of compound 17 (180 mg, 0.59 mmol) in dry DMF
(1.5 mL) was added acetic anhydride (56
mL, 0.59 mmol) and the
1H), 8.69 (brs, 1H). 13C NMR (CDCl3, 75 MHz):
d 12.65, 38.44, 55.55,
mixture was stirred at room temperature for 24 h. The residue
obtained after distillation of the volatiles under reduced pressure,
was purified by silica-gel column chromatography to give 18
71.06, 74.57, 85.50, 110.28, 114.21, 129.37, 129.56, 137.04, 150.79,
159.71, 164.16. ESI-HRMS for [C17H20N2O5 þ H]þ calcd, 333.1445;
found, 333.1461.
(160 mg, 78%) as a white foam. 1H NMR (CDCl3, 300 MHz):
d 2.2 (s,
3H), 4.00e4.04 (m, 1H), 4.23 (dd, J ¼ 9.79, 1.48 Hz, 1H), 4.29 (dd,
J ¼ 9.93, 3.88 Hz,1H), 4.39 (s, 1H), 4.42 (s,1H), 4.85 (brs, 1H), 5.73 (d,
J ¼ 0.75 Hz, 1H), 7.05e7.11 (m, 2H), 7.17e7.25 (m, 3H), 7.31
(d, J ¼ 7.50 Hz, 1H), 7.88 (d, J ¼ 7.50 Hz, 1H), 9.36 (brs, 1H). 13C NMR
3.1.15. 1-((2R,4R)-4-(4-methoxybenzyloxy)-tetrahydrofuran-2-yl)
pyrimidine-2,4(1H,3H)-dione (22)
Deoxygenation of 15 (100 mg, 0.3 mmol) rendered 70 mg of
compound 22 in 74% yield. 1H NMR (CDCl3, 300 MHz):
d 2.21 (app-
(CDCl3, 75 MHz):
d
25.14, 71.68, 74.99, 78.65, 82.67, 94.89, 96.26,
dm, J ¼ 15.15 Hz, 1H), 2.37 (ddd, J ¼ 15.03, 7.47, 5.28 Hz, 1H), 3.73 (s,
3H), 3.83 (dd, J ¼ 10.20, 3.66 Hz, 1H), 4.25 (dd, J ¼ 10.14, 1.17 Hz, 1H),
4.33 (d, J ¼ 1.40 Hz, 2H), 5.56 (d, J ¼ 8.13 Hz, 1H), 6.11 (dd, J ¼ 7.54,
1.96 Hz, 1H), 6.80 (d, J ¼ 8.70 Hz, 2H), 7.09 (d, J ¼ 8.70 Hz, 2H), 7.61
127.96, 128.17, 128.69, 137.25, 145.33, 156.17, 163.04, 171.17, 177.08.
ESI-HRMS for [C17H19N3O5 þ H]þ calcd, 346.1397; found, 346.1400.
3.1.11. Barton e McCombie deoxygenation procedure for the
synthesis of compounds 19e24
(d, J ¼ 8.13 Hz, 1H), 9.21 (brs, 1H). 13C NMR (CDCl3, 75 MHz):
d 38.46,
55.53, 71.00, 75.06, 76.97, 86.18, 101.67, 114.22, 129.28, 129.63,
141,24, 150.96, 159.70. ESI-HRMS for [C16H18N2O5 þ H]þ calcd,
319.1294; found, 319.1295.
To an ice-cold solution of compound 12e18 (1 eq.) and DMAP
(2 eq.) in CH3CN (25 mL/mmol) was gradually added p-tolyl
chlorothionocarbonate (1.2 equivalents) at 0 ꢀC and stirring was
continued at room temperature. After 2e4 h the solvent was
removed in vacuo and the residue was dissolved in ethyl acetate.
The solution was washed twice with water, dried over anhydrous
MgSO4, filtered, and evaporated in vacuo to give the corresponding
xanthate as yellow solid/syrup. The latter was dissolved in toluene
(50 mL/mmol), to which azobisisobutyronitrile (AIBN, 2 equivalent)
was added. Tri-n-butyltinhydride (2.5 equivalent) was added to this
mixture at 60e70 ꢀC, which was further stirred for 2e4 h at
95e100 ꢀC. The solvent was removed in vacuo and the residue was
3.1.16. 9-((2R,4R)-4-(benzyloxy)-tetrahydrofuran-2-yl)-9H-purin-
6-amine (23)
Deoxygenation of 16 (450 mg, 1.38 mmol) rendered 200 mg of
compound 23 in 47% yield. 1H NMR (DMSO-D6, 300 MHz):
d 2.59
(dm, J ¼ 14.60 Hz, 1H), 2.70 (ddd, J ¼ 14.60, 7.61, 5.84 Hz, 1H), 4.00
(dd, J ¼ 9.88, 4.45 Hz, 1H), 4.25 (app-dt, J ¼ 10.05, 1.30 Hz, 1H), 4.41
(m, 1H), 4.54 (app-d, J ¼ 2.42, 2H), 6.34 (dd, J ¼ 7.55, 2.64 Hz, 1H),
7.24 (brs, 2H), 7.26e7.38 (m, 5H), 8.24 (s, 1H), 8.22 (s, 1H). 13C NMR
(DMSO-D6, 75 MHz):
d 37.32, 70.30, 73.21, 77.47, 82.78, 118.52,

K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
3711
127.43, 127.53, 128.18, 137.87, 138.79, 149. 04, 152. 47, 155.85. ESI-
2.70, 1.54 Hz, 1H), 2.68 (ddd, J ¼ 14.42, 8.24, 6.10 Hz, 1H), 3.91 (dd,
J ¼ 9.30, 4.10 Hz, 1H), 3.97 (ddd, J ¼ 9.32, 2.10, 1.06 Hz, 1H), 4.48 (m,
1H), 5.79 (d, J ¼ 4.35 Hz, 1H), 6.28 (dd, J ¼ 8.27, 2.56 Hz, 1H), 7.28 (s,
HRMS for [C16H17N5O2 þ H]þ calcd, 312.1455; found, 312.1469.
3.1.17. N-(1-((2R,4R)-4-(benzyloxy)-tetrahydrofuran-2-yl)-1,2-
dihydro-2-oxopyrimidin-4-yl)acetamide (24)
2H), 8.15 (s, 1H), 8.36 (s, 1H). 13C NMR (DMSO-D6, 75 MHz):
d 40.86,
69.95, 76.98, 83.83,119.53, 140.40, 149.53, 153.06,156.73. ESI-HRMS
Deoxygenation of 18 (190 mg, 0.55 mmol) rendered 100 mg of
for [C9H11N5O2 þ H]þ calcd, 222.0986; found, 222.0938.
compound 24 in 56% yield. 1H NMR (CDCl3, 300 MHz):
d 2.20 (s, 3H),
2.35e2.44 (m, 2H), 3.98 (dd, J ¼ 10.12, 3.72 Hz, 1H), 4.13e4.18 (m,
1H), 4.31 (s, 2H), 4.34 (dd, J ¼ 10.20, 0.96 Hz, 1H), 6.09 (dd, J ¼ 6.08,
2.31 Hz, 1H), 7.06e7.12 (m, 2H), 7.21 (d, J ¼ 8.44 Hz, 1H), 7.20e7.28
(m, 3H), 7.91 (d, J ¼ 7.50 Hz, 1H), 9.46 (brs, 1H). 13C NMR (CDCl3,
3.1.21. N-(1,2-dihydro-1-((2R,4R)-tetrahydro-4-hydroxyfuran-2-
yl)-2-oxopyrimidin-4-yl)acetamide (28)
2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ, 770 mg, 3.4
mmol) was added to a stirring solution of compound 24 (112 mg,
0.34 mmol) in dry dichloromethane (5.0 mL). The mixture was
heated to 50 ꢀC for 2 days in a sealed tube. Then it was sequentially
treated with NaHCO3 (2.0 g), water (0.25 mL) and 20 mL of 1:1
CH2Cl2-MeOH. After addition of anhydrous MgSO4 the resulting
slurry was filtered. The filtrate was concentrated and purified by
column chromatography (4e6% MeOH-CH2Cl2) to afford 9 mg of
75 MHz):
d 25.06, 38.46, 71.08, 75.89, 77.20, 88.21, 96.20, 127.92,
128.19, 128.76, 137.23, 145.37, 155.54, 163.35, 171.57. ESI-HRMS for
[C17H19N3O4 þ H]þ calcd, 330.1448; found, 330.1463.
Deprotection method A. A solution of benzyl-protected nucleo-
side in MeOH (40 mL/mmol) was hydrogenated at atmospheric
pressure for 5e24 h in the presence of 10% Pd/C (150 mg/mmol).
The catalyst was removed by filtration over a Celite path and the
filtrate was concentrated in vacuo. The residue was purified by
column chromatography (3% MeOH-CH2Cl2) to isolate the desired
product as a white amorphous solid.
compound 28 (11% yield). 1H NMR (CDCl3, 300 MHz):
d 2.14 (s, 3H),
2.37 (ddd, J ¼ 14.90, 7.02, 4.53 Hz, 1H), 2.61 (d, J ¼ 14.90 Hz, 1H),
4.02 (dd, J ¼ 9.80, 3.46 Hz, 1H), 4.28 (dd, J ¼ 9.78, 1.14 Hz, 1H), 4.52
(t, J ¼ 3.86 Hz, 1H), 5.94 (d, J ¼ 6.56 Hz, 1H), 7.32 (d, J ¼ 7.50 Hz, 1H),
8.03 (d, J ¼ 7.48 Hz, 1H), 9.39 (brs, 1H). 13C NMR (CDCl3, 75 MHz):
Deprotection method B. To
a solution of benzyl-protected
nucleoside (1 eq.) in an acetonitrile-water (5:1) mixture (20 mL/
mmol) at 0 ꢀC ceric ammonium nitrate (CAN, 2.5 equivalents) was
added and stirring was continued for 1 h. The reaction mixture was
treated with solid NaHCO3 (2.0 g/mmol) followed by 25% MeOH-
CH2Cl2 (50 mL/mmol). The mixture was filtered over celite and
washed with 25% MeOH-CH2Cl2 (100 mL/mmol). The filtrate was
concentrated in vacuum, and the residue was purified by column
chromatography (3% MeOH-CH2Cl2) to isolate the final product as
a white amorphous solid.
d 25.12, 41.49, 70.28, 78.87, 89.48, 95.73, 146.47, 155.43, 162.11,
170.95. ESI-HRMS for [C10H13N3O4 þ H]þ calcd, 240.0979; found,
240.0982 & [2M þ H]þ 479.1936.
3.1.22. 4-amino-1-((2R,4R)-tetrahydro-4-hydroxyfuran-2-yl)
pyrimidin-2(1H)-one (29)
Method 1. Applying the reaction conditions described for 12 on
compound 28 (18 mg, 75 mmol) gave 10 mg of compound 29 (67%
yield) as an amorphous solid.
Method 2. To a stirring mixture of 1,2,4-triazole (223 mg,
3.1.18. 1-((2R,4R)-tetrahydro-4-hydroxyfuran-2-yl)-5-
3.24 mmol) in 5 mL of pyridine POCl3 (75 mL, 0.81 mmol) was
methylpyrimidine-2,4(1H,3H)-dione (25)
added dropwise at room temperature. After 10 min compound 30
(65 mg, 0.27 mmol) was added and stirring was continued for an
additional 3 h. After that time the reaction was bubbled with
ammonia gas for 2 h. Since TLC indicated that the deacetylation
was not completed, the volatiles were removed and the residue
treated with 7N methanolic ammonia (5 mL) for 5 h. After
evaporation the residue was purified by flash column chroma-
tography (6% MeOH-CH2Cl2) to give 19 mg of the title compound
Employing deprotection method A, 30 mg of title compound
was obtained in 75% yield from 19 (57 mg, 0.19 mmol), while
method B afforded 80 mg of the same compound from 21 (150 mg,
0.45 mmol) in 84% yield. 1H NMR (DMSO-D6, 300 MHz):
d 1.77
(d, J ¼ 1.16 Hz, 3H), 1.87 (ddd, J ¼ 14.49, 2.89, 1.52 Hz, 1H), 2.46 (ddd,
J ¼ 14.05, 8.07, 5.59 Hz, 1H), 3.77 (dd, J ¼ 9.37, 3.72 Hz, 1H), 3.98 (dt,
J ¼ 9.36, 1.41 Hz, 1H), 4.36 (brm, 1H), 5.29 (d, J ¼ 2.68 Hz, 1H), 6.05
(dd, J ¼ 8.13, 2.72 Hz,1H), 7.76 (d, J ¼ 1.21 Hz,1H), 11.23 (brs, 1H). 13C
29 in 36% yield. 1H NMR (DMSO-D6, 300 MHz):
d 1.82 (ddd,
NMR (DMSO-D6, 75 MHz):
d
13.06, 40.75, 69.54, 77.04, 85.32,
J ¼ 14.28, 1.40, 1.10 Hz, 1H), 2.40 (ddd, J ¼ 14.06, 7.87, 5.59 Hz, 1H),
3.81 (dd, J ¼ 9.35, 3.74Hz, 1H), 3.97 (dt, J ¼ 9.31, 1.28 Hz, 1H), 4.33
(m, 1H), 5.16 (d, J ¼ 2.76 Hz, 1H), 5.69 (d, J ¼ 7.42 Hz, 1H), 5.99 (dd,
J ¼ 7.83, 2.43 Hz, 1H), 7.08 (brd, J ¼ 15.32 Hz, 2H), 7.75 (d,
109.20, 137.79, 151.21, 164.54. ESI-HRMS for [C9H12N2O4 ꢁ H]ꢁ
calcd, 211.0724; found, 211.0732.
3.1.19. 1-((2R,4R)-tetrahydro-4-hydroxyfuran-2-yl)pyrimidine-
2,4(1H,3H)-dione (26)
Employing deprotection method A, 43 mg of the title compound
was obtained in 89% yield from 20 (70 mg, 0.24 mmol), while
compound 22 (70 mg, 0.22 mmol) was converted to 34 mg of the
title compound (78% yield) using method B. 1H NMR (DMSO-D6,
J ¼ 7.41 Hz, 1H). 13C NMR (DMSO-D6, 75 MHz):
d 41.48, 69.70,
77.26, 86.52, 93.85, 142.46, 155.98, 166.37. ESI-HRMS for
[C8H11N3O3 þ H]þ calcd, 198.0873; found, 198.0876.
3.1.23. (3R,5R)-tetrahydro-5-(3,4-dihydro-2,4-dioxopyrimidin-
1(2H)-yl)furan-3-yl acetate (30)
300 MHz):
d
1.87 (ddd, J ¼ 14.53, 2.91, 1.88 Hz, 1H), 2.43 (ddd,
To a stirring solution of compound 26 (55 mg, 0.277 mmol) and
4-dimethyl aminopyridine (DMAP, 50 mg, 0.41 mmol) in 2 mL of
DMF acetic anhydride (31 mL, 0.33 mmol) was added. After stirring
J ¼ 14.45, 8.00, 5.48 Hz, 1H), 3.78 (dd, J ¼ 9.43, 3.66 Hz, 1H), 3.97
(app-dt, J ¼ 9.46, 1.25 Hz,1H), 4.34 (t, J ¼ 4.23 Hz,1H), 5.27 (brs, 1H),
5.60 (d, J ¼ 8.05 Hz, 1H), 6.01 (dd, J ¼ 8.00, 2.23 Hz, 1H), 7.85 (d,
for 24 h, the solvent was evaporated under vacuum and the residue
J ¼ 8.11 Hz, 1H), 11.19 (brs, 1H). 13C NMR (CDCl3, 75 MHz):
d
41.64,
was purified by column chromatography to isolate compound 30
70.08, 78.56, 87.97, 100.61, 141.97, 150.95, 165.38. ESI-HRMS for
(65 mg, 97%). 1H NMR (CDCl3, 300 MHz):
d 1.96 (s, 3H), 2.22 (ddd,
[C8H10N2O4 þ H]þ calcd, 199.0713; found, 199.072.
J ¼ 15.48, 2.74, 1.76 Hz, 1H), 2.58 (ddd, J ¼ 15.49, 7.24, 5.81 Hz, 1H),
4.04 (dd, J ¼ 10.95, 3.87 Hz, 1H), 4.21 (ddd, J ¼ 10.95, 1.60, 0.69 Hz,
1H), 5.31 (app-tt, J ¼ 4.80, 0.84 Hz,1H), 5.68 (d, J ¼ 8.18 Hz,1H), 6.07
(dd, J ¼ 7.21, 1.94 Hz, 1H), 7.45 (d, J ¼ 8.18 Hz, 1H), 9.20 (brs, 1H). 13C
3.1.20. (3R,5R)-5-(6-amino-9H-purin-9-yl)-tetrahydrofuran-3-ol
(27)
Using method A with catalytic amount of acetic acid compound
23 (100 mg, 0.32 mmol) was converted to 29 mg of compound 27 in
NMR (CDCl3, 75 MHz): d 21.24, 39.13, 72.88, 75.39, 86.61, 101.63,
139.77, 150.53, 163.72, 170.19. ESI-HRMS for [C10H12N2O5 þ H]þ
40% yield. 1H NMR (DMSO-D6, 300 MHz):
d
2.29 (ddd, J ¼ 14.44,
calcd, 241.0819; found, 241.0830.

3712
K.S. Toti et al. / European Journal of Medicinal Chemistry 46 (2011) 3704e3713
3.1.24. Phenyl-(benzoxy-
L
-alaninyl)-phosphorochloridate (33)
(vesicular stomatitis virus, Coxsackie virus B4, and respiratory
syncytial virus) cell cultures. Confluent cell cultures in microtiter
96-well plates were inoculated with 100 cell culture inhibitory
dose-50 (CCID50) of virus (1 CCID50 being the virus dose to infect 50%
of the cell cultures). Aftera 1 h virus adsorptionperiod, residualvirus
was removed, and the cell cultures wereincubated in the presence of
To a stirred solution of phenyldichlorophosphate 31 (0.30 mL,
2.00 mmol), -alanine benzyl ester tosylate 32 (0.43 g, 2.00 mmol) in
L
anhydrous DCM (15 mL), was added dropwise under an Ar atmo-
sphere at ꢁ78 ꢀC anhydrous TEA (0.56 mL, 4.00 mmol). Following
the addition the reaction mixture was stirred at ꢁ78 ꢀC for 30 min,
then at room temperature for 2 h. Formation of the desired
compound was monitored by 31P NMR. After this period the solvent
was removed under reduced pressure and the residue triturated
with anhydrous diethyl ether. The precipitate was filtered under
nitrogen and the solution was concentrated to give a yellow oil
varying concentrations (200, 40, 8, . mM) of the test compounds.
Viral cytopathicity was recorded as soon as it reached completion in
the control virus-infected cell cultures that were not treated with
the test compounds. The methodology of the anti-HIV assays was as
follows: human CEM (3 ꢄ105 cells/cm3) cells were infected with 100
(87%, 0.62 g). 1H NMR (CDCl3, 500 MHz):
OCH2Ph), 5.15e5.13 (2H, m, OCH2Ph), 4.18e4.13 (1H, m, CHNH),
1.46e1.44 (3H, m, CH3). 31P NMR (CDCl3, 202 MHz):
7.86, 7.52.
d
7.33e7.28 (10H, m, PhO,
CCID50 of HIV-1(IIIB) or HIV-2(ROD)/mL and seeded in 200 mL wells
of a microtiter plate containing appropriate dilutions of the test
compounds. After 4 days of incubation at 37 ꢀC, HIV-induced CEM
giant cell formation was examined microscopically.
d
3.1.25. 1-((2R,4R)-tetrahydro-4-hydroxyfuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione -[ phenyl-(benzoxy-L-
3.3. Enzymatic assay
alaninyl)] phosphate (34)
To a solution of 25 (0.093 g, 0.44 mmol) in anhydrous THF
(10 mL) was added 1.0 M solution of tert-butyl magnesium chloride
in THF (0.88 mL, 0.88 mmol) and the reaction mixture was stirred
under an Ar atmosphere for 30 min. After this period, a solution of
33 (0.31 g, 0.88 mmol) in anhydrous THF (5 mL) was added drop-
wise and the reaction mixture was stirred at room temperature for
18 h. Then, 1.0 M solution of tert-butyl magnesium chloride in THF
(0.44 mL, 0.44 mmol) and a solution of 33 (0.15 g, 0.44 mmol) in
anhydrous THF (3 mL) was added and the stirring was continued for
further 5 h. After this period, the solvent was removed and the
residue was purified by column chromatography, gradient elution
of DCM/MeOH ¼ 98/2 then 96/4 to give a white solid (33%, 0.077 g).
Compound 34 (5.5 mg) was dissolved in d6-acetone (150 mL),
and Trizma buffer (pH 7.6) (300 mL) was added. The resulting cloudy
solution was placed in a NMR tube and a 31P NMR experiment at
25 ꢀC was recorded as the blank experiment. Then a solution of
carboxypeptidase Y (0.1 mg) in Trizma buffer (150 mL) was added
and a 31P NMR experiment was performed recording the spectrum
every 5 min.
3.4. Nucleoside kinase assay using [CH3-3H]dThd as the natural
substrate
The activity of recombinant thymidine kinase 1 (TK-1), TK-2,
herpes simplex virus-1 (HSV-1) TK, varicella-zoster virus (VZV)
TK, and the multifunctional deoxynucleoside kinase (dNK) of D.
melanogaster and the 50% inhibitory concentration of the test
1H NMR (MeOD, 500 MHz):
d
7.52 (0.5H, d, J ¼ 1.2 Hz, H-6 of one
diastereoisomer), 7.47 (0.5H, d, J ¼ 1.2 Hz, H-6 of one diastereo-
isomer), 7.38e7.30 (7H, m, PhO, OCH2Ph), 7.22e7.16 (2H, m, PhO,
OCH2Ph), 7.12e7.10 (1H, m, PhO, OCH2Ph), 6.10 (0.5H, dd, J ¼ 7.8 Hz,
2.3 Hz, H-10), 6.06 (0.5H, dd, J ¼ 7.5 Hz, 1.8 Hz, H-10), 5.21e5.17 (1H,
m, H-30), 5.16, 5.15 (2H, 2s, OCH2Ph), 4.45 (0.5H, dd, J ¼ 10.8 Hz,
1.7 Hz, H-40 of one diastereoisomer), 4.37 (0.5H, dd, J ¼ 10.8 Hz,
1.7 Hz, H-40 of one diastereoisomer), 4.03e3.92 (2H, m, H-40,
CHCH3), 2.63e2.55 (1H, m, H-20), 2.32 (0.5H, d, J ¼ 15.5 Hz, H-20 of
one diastereoisomer), 2.24 (0.5H, d, J ¼ 15.5 Hz, H-20 of one dia-
stereoisomer), 1.83 (1.5H, d, J ¼ 1.1 Hz, 5eCH3 of one diastereo-
isomer), 1.82 (1.5H, d, J ¼ 1.1 Hz, 5eCH3 of one diastereoisomer),
1.36 (1.5H, d, J ¼ 0.9 Hz, CHCH3 of one diastereoisomer), 1.34 (1.5H,
d, J ¼ 1.0 Hz, CHCH3 of one diastereoisomer). 13C NMR (MeOD,
compounds were assayed in a 50
mL reaction mixture containing
50 mM Tris/HCl, pH 8.0, 2.5 mM MgCl2, 10 mM dithiothreitol,
0.5 mM CHAPS, 3 mg/mL bovine serum albumin, 2.5 mM ATP, 1 mM
[methyl-3H]dThd, and enzyme. The samples were incubated at
37 ꢀC for 30 min in the presence or absence of different concen-
trations (5-fold dilutions) of the test compounds. At this time point,
the enzyme reaction still proceeded linearly. Aliquots of 45 mL of the
reaction mixtures were spotted on Whatman DE-81 filter paper
disks (Whatman, Clifton, NJ). The filters were washed three times
for 5 min each in 1 mM ammonium formate, once for 1 min in
water, and once for 5 min in ethanol. The radioactivity was deter-
mined by scintillation counting.
125 MHz):
d
12.71 (5eCH3), 20.16 (d, JCeP ¼ 7.5 Hz, CHCH3), 20.33 (d,
JCeP ¼ 6.5 Hz, CHCH3), 40.51 (d, JCeP ¼ 5.6 Hz, C-20), 40.73 (d,
JCeP ¼ 3.9 Hz, C-20), 51.60, 51.84 (2s, CHCH3), 68.00, 68.04 (2s,
OCH2Ph), 76.34 (d, JCeP ¼ 4.0 Hz, C-40), 76.79 (d, JCeP ¼ 5.5 Hz, C-40),
77.95 (d, JCeP ¼ 5.2 Hz, C-30), 77.99 (d, JCeP ¼ 4.9 Hz, C-30), 86.95,
87.47 (2s, C-10), 110.70, 111.03 (2s, C-5), 121.09, 121.14, 121.18, 126.26,
126.29, 129.41, 129.43, 129.64, 129.65, 130.82, 130.92 (PhO,
OCH2Ph), 137.29 (‘ipso’ OCH2Ph), 137.77, 137.80 (2s, C-6), 152.03,
152.07, 152.12, 152.22, 152.31 (C-2, (‘ipso’ OPh), 166.49, 166.55 (2s,
C-4), 174.49 (d, JCeP ¼ 4.6, COOCH2Ph), 174.91 (d, JCeP ¼ 4.1,
Acknowledgements
The authors thank the K.U.Leuven for financial support (GOA 10/
014). We thank, Helene Munier-Lehmann, Laboratoire de Chimie
Structurale des Macromolecules (URACNRS 2185), Institut Pasteur
for providing TMPKmt data, and Mrs. Lizette van Berckelaer and Mr.
Izet Karalic for excellent technical assistance.
COOCH2Ph). 31P NMR (MeOD, 202 MHz):
d 2.92, 2.27. ES-
Appendix. Supplementary material
MS ¼ 528.16 (M run on negative mode). HPLC ¼ H2O/AcCN from 95/
5 to 0/100 in 30 min ¼ retention time 13.87, 14.19 min.
The supplementary data associated with this article can be
found in the on-line version at doi:10.1016/j.ejmech.2011.05.036.
3.2. Antiviral assays
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vaccinia virus and vesicular stomatitis virus], Vero (parainfluenza-3,
reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), or HeLa
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