Synthesis and evaluation of 5′-modified thymidines and 5-hydroxymethyl-2′-deoxyuridines as Mycobacterium tuberculosis thymidylate kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.10.018

We report the synthesis of 5′-modified thymidines (16, 18, 21, 23) and 5,5′-bis-substituted 2′-deoxyuridine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 5′-modified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of protecting groups for the 5-CH₂OH moiety of 2′-deoxyuridines is described that enables to introduce the desired 5′-modification.

Full text of DOI:10.1016/j.bmc.2012.10.018

Bioorganic & Medicinal Chemistry 21 (2013) 257–268
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of 50-modified thymidines and
5-hydroxymethyl-20-deoxyuridines as Mycobacterium tuberculosis thymidylate
kinase inhibitors
Kiran S. Toti a, Frederick Verbeke a, Martijn D.P. Risseeuw a, Vladimir Frecer b, Hélène Munier-Lehmann c,d
,
Serge Van Calenbergh a,
⇑
a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences (FFW), Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium
b Department of Physical Chemistry of Drugs, Faculty of Pharmacy, Comenius University, 83232 Bratislava, Slovakia
c Institut Pasteur, Unité de Chimie et Biocatalyse, 28 rue du Dr Roux, 75724 Paris Cedex 15, France
d CNRS UMR3523, 28 rue du Dr Roux, France
a r t i c l e i n f o
a b s t r a c t
We report the synthesis of 50-modified thymidines (16, 18, 21, 23) and 5,50-bis-substituted 20-deoxyuri-
dine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis
(TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 50-mod-
ified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of
protecting groups for the 5-CH2OH moiety of 20-deoxyuridines is described that enables to introduce
the desired 50-modification.
Article history:
Received 23 August 2012
Revised 17 October 2012
Accepted 18 October 2012
Available online 5 November 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
Thymidine monophosphate kinase
Inhibitor
Nucleoside analogues
5-Hydroxymethyl pyrimidines
Phosphate bioisosteres
1. Introduction
stead of substrate.5 Mainly based on inhibitory data reported by
our group, Frecer et al. recently reported on the combinatorial de-
Tuberculosis (TB) is responsible for more than 2 million human
deaths annually. The emergence of multi-drug and extremely drug
resistant TB strains asks for new drugs preferably acting on yet
unaddressed targets.1 The knowledge of the TB genome should en-
able to reveal new metabolic pathways and targets, which may fuel
the discovery process of antituberculosis drugs.2 Phosphorylation
of nucleosides is a prerequisite for DNA synthesis and the enzymes
in charge are crucial for cell growth. Thymidine monophosphate
kinase is situated at the junction of the de novo and salvage path-
ways for thymidine triphosphate (dTTP) synthesis. TMPK is the last
specific enzyme in these pathways and has been recently validated
in vivo as an antibacterial target.3 We have focused our effort on
the TMPK of Mycobacterium tuberculosis (TMPKmt) for generating
anti-TB drugs. Previously, we designed a number of promising
TMPKmt inhibitors such as the bicyclic inhibitor 1 and the 50-thio-
sign and structure-based in silico screening of a virtual focused li-
brary of bicyclic thymidine analogs in an effort to identify more
potent TMPKmt inhibitors.6 In their study they used the three
dimensional structure of TMPKmt complexed with 5-hydroxy-
methyl-dUMP (3) to develop a QSAR model, to parameterize a tar-
get-specific scoring function for TMPKmt and to select virtual hits
which display the highest predicted binding to the target. Com-
pound 4 emerged as one of the best analogues. The binding of 4
to TMPKmt is predicted to be similar to that of 1. Beside the fact
that it is a carba-nucleoside contains a sulfondiamide group replac-
ing the thiourethane moiety of the condensed ring of 1, 4 differs
from 1 by the presence of a 5-CH2OH group, known to possibly
make an extra hydrogen bond with a water molecule (W12) or
with the side chain of Ser99, and a 50-(N-methylsulfamoyl)methyl
moiety. The latter fragment, which replaces the phosphate moiety
present in TMP, was anticipated to favorably interact Arg95,
Arg153 and especially with the magnesium ion. Alternative phos-
phate isosters suggested in the Frecer publication are a 5-tetra-
zolylmethyl and methylsulfonylmethyl group. In this pilot study
we decided to explore the importance of the proposed 5-CH2OH
and 50-modifications for TMPKmt inhibition.
urea-substituted
we showed that hydroxylation of the 5-methyl group of the TMP
substrate (3) resulted in a moderately potent TMPKmt inhibitor in-
a
-thymidine analogue 2 (Fig. 1).4 Furthermore,
⇑
Corresponding author. Tel.: +32 9 264 81 24; fax: +32 9 264 81 46.
E-mail address: serge.vancalenbergh@ugent.be (S. Van Calenbergh).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.018

258
K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
proper Wittig–Horner reagents to give 14, 17 and 19 in moderate
O
N
H
N
H
N
yields. Methylsulfone 16 was obtained by Pd catalyzed hydrogena-
tion of 14, followed by the removal of TBDMS group. The conju-
NH
O
S
12
HO
O
gated double bond in 17 was reduced using NaBH4/NiCl2 and
Cl
N
S
O
O
OH
the product was desilylated using TBAF affording 18. Conjugate
reduction of nitrile 19 was achieved with NaBH4 in a pyridine–
methanol mixture at elevated temperature.13 Deprotection of 20
afforded the 60-nitrile analogue 21 in 76% yield. A cycloaddition be-
tween nitrile 20 and TMSN3 in the presence of dibutyltinoxide gave
22 in moderate yield.14 Even though the conversion of 22 to 23 was
complete (by LCMS), the isolated yield is lowered by the elaborate
purification necessary to isolate the product.15
Hydroxymethylation at position 5 of dU (24) of pyrimidine is
reported. However, selective protection of this hydroxyl group ap-
pears difficult.16 An attempt to hydroxymethylate the 5-position of
25 failed, possibly due to the low solubility of the substrate in for-
malin-triethylamine system and the lability of the TBDMS groups
under the harsh reaction conditions. Hence, we decided to intro-
duce the 5-CH2OH group after modifying the 50-position in 20-
deoxyuridine (Scheme 3). As a model substrate for this strategy,
compound 29 was synthesized smoothly using similar transforma-
tions as before. Unfortunately, reaction of 29 with formaldehyde at
elevated temperature was very slow with only traces of the desired
product formed after several days, while under microwave condi-
tions, the starting material degraded.
CF3
NH
O
S
HN
: Kiexp = 3.5 µM
: Kiexp = 0.17 µM
2
1
O
O
HO
NH
HO
O
NH
H
N
N
O
S
P
O
O
N
O
O
O
O
O
HN
S
O
NH
OH
O
: Kiexp = 110 µM
: Kipre = 0.29 µM
4
3
Figure 1. Selected promising TMPKmt inhibitors designed previously in our
laboratory (1, 2, and 3)4,5 or predicted by a QSAR model (4).6 Kiexp and Kpi re indicate
experimental and predicted Ki values, respectively. The Km value for dTMP is
4.5 lM.
Because of this setback, we decided to start from compound 31,
obtained via the benzylic bromide oxidation as reported by Grover
et al.10 (Scheme 4). Selective primary desilylation of 31 gave 32. At-
tempted successive Dess–Martin and Wittig–Horner reactions
involving two different Horner reagents failed to give the desired
products.
At this point we chose to look for a suitable protecting group for
the 5-hydroxymethyl moiety that is compatible with the reaction
conditions used to modify the 50-position and can also be removed
under mild conditions. Towards this end 5-hydroxymethyl deriva-
tive 33 was synthesized according to a literature procedure.18
BOM derivatization of 33 to 34 proceeded in 37% yield
(Scheme 5). Since the desired product also features a benzylic hy-
droxyl group, we ran a model hydrogenation reaction on 34 with
palladium as catalyst. Unsurprisingly, the reaction mainly afforded
thymidine 11, besides minor amounts (24%) of 12. Next, the use of
a 2-(trimethylsilyl)ethoxymethyl (SEM) protecting group, known
2. Results and discussion
2.1. Chemistry
We envisioned synthesizing the target molecules using a
Wittig–Horner reaction to introduce the desired 50-modifications
as a key step. For this purpose two non-commercial Horner
reagents were self-made (Scheme 1). Diethyl((methylsulfonyl)
methyl)phosphonate 6 was prepared according to known proce-
dures,7 while Horner reagent 9 was obtained by reacting half an
equivalent of chlorodiethylphosphate with bis-lithiated 8.8,9
30-Silylated thymidine 12 was produced by a selective cleavage
of the primary TBDMS group on 11.10,11
To synthesize the 50-modified thymidines 16, 18, 21 and 23
(Scheme 2), the 50-OH group of 12 was oxidized using Dess–Martin
periodinane and the resulting aldehyde 13 was reacted with the
O
P
O
O
P
O
O
S
O
Ref. 7
S
O
O
5
6
O
P
O
O
S
O
O
S
O
O
S
O
a
Ref. 8
NH
O
NH
Cl
7
8
9
O
N
O
N
O
N
NH
NH
NH
HO
TBSO
HO
O
O
O
Ref. 10
b
O
O
O
OH
10
OTBS
OTBS
11
12
Scheme 1. Synthesis of Horner reagents 6 and 9, and intermediate 14. Reagents and conditions: (a) THF, BuLi, ꢀ78 °C, 1 h, diethylchlorophosphate (0.5 equiv), ꢀ78 °C ? 0 °C,
1 h, 65%; (b) THF, pyridine, HF-pyridine, 0 °C, 1 h, 0 °C ? rt, 1 h, 36%.

K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
259
O
N
O
O
N
NH
NH
d
O
NH
O
S
O
c
O
S
O
S
O
O
O
N
O
O
O
O
OTBS
14
OTBS
15
OH
16
b
O
N
O
O
N
O
N
NH
NH
NH
NH
CN
a
g
h
i
12
O
O
N
O
O
O
NC
NC
O
O
O
O
OTBS
OTBS
OTBS
20
OH
21
13
19
e
j
O
N
O
N
O
N
O
N
NH
S
NH
NH
NH
NH
O
O
k
f
O
S
NH
O
N
N
O
O
O
O
O
O
O
O
N
N
NH
NH
N
N
OTBS
OH
18
OTBS
OH
23
17
22
Scheme 2. Synthesis of the 50-modified thymidines. Reagents and conditions: (a) CH2Cl2, Dess–Martin periodinane, rt, 4 h; (b) THF, 6, BuLi, ꢀ78 °C, 15 min, ꢀ78 °C, 1 h, rt,
18 h, 20% from 12; (c) H2, Pd-C, MeOH, 4 h, rt; (d) THF, TBAF, 40 °C, 15 h, 20% over two steps; (e) THF, 9, BuLi, ꢀ78 °C, 1 h, ꢀ78 °C, 1 h, rt, 18 h, 48% from 12; (f) (i) MeOH-THF
(5:1), NiCl2, NaBH4, 0 °C, 1 h; (ii) THF, TBAF, 40 °C, 1 h, 12% over two steps; (g) THF, cyanomethyltriphenylphosphonium chloride, BuLi, 0 °C, 30 min, 0 °C ? rt, 18 h, 57% from
12; (h) pyridine-MeOH (3:1), NaBH4, 120 °C, 4 h, 53%; (i) THF, TBAF, rt, 4 h, 76%; (j) toluene, TMSN3, Bu2SnO, 110 °C, 4 h, 32%; (k) THF, TBAF, rt, 4 h, 34%.
O
N
O
N
O
N
O
N
NH
NH
NH
NH
CN
HO
TBSO
HO
O
O
O
O
a
b
Ref. 17
O
O
O
O
OH
24
OTBS
OTBS
OTBS
25
26
27
c
O
N
O
N
O
N
NH
NH
NH
HO
O
O
O
NC
NC
NC
e
d
O
O
O
OH
OH
OTBS
30
29
28
Scheme 3. Attempted hydroxymethylation route to 30. Reagents and conditions: (a) THF, pyridine, HF-pyridine, 0 °C, 1 h, 0 °C ? rt, 1 h, 53%; (b) (i) CH2Cl2, Dess–Martin
periodinane, rt, 4 h; (ii) THF, cyanomethyltriphenylphosphonium chloride, BuLi, 0 °C, 30 min, 0 °C ? rt, 18 h, 51% over two steps; (c) H2, Pd-C, MeOH, 4 h, rt; (d) THF, TBAF, rt,
4 h, 73%; (e) paraformaldehyde, H2O, TEA, 100 °C, 3 days or paraformaldehyde, H2O, TEA, continuous irradiation of microwave from rt ? 150 °C in 3 min, 10 min at 150 °C.
(See above mentioned reference for further information).
to be cleavable under mild conditions, was investigated. Com-
pound 33 was converted to 35 in moderate yield. Selective removal
of the primary TBS gave 36, which on DMP oxidation followed by
Horner reaction gave 37. Unfortunately, attempts to remove both
SEM groups with TBAF in anhydrous THF at elevated temperature
failed.
Attempts to protect 33 as 4-methoxyphenol (PMP) ether under
the Mitsunobu conditions led to degradation of the starting mate-
rial. Conditions used to protect 33 with two 4-methoxybenzyl
(PMB) groups afforded 38 only in low yields (13%), mainly because
of simultaneous formation of compounds with three PMB & one
TBS group and four PMB groups, leaving these protection methods
unattractive.
These problems finally led us to protect the 5-CH2OH group of
33 as pivaloate ester in good yield (Scheme 6). A series of routine
transformations was used to convert 39 into the acrylonitrile 41
in acceptable yields. Several conditions were evaluated to reduce
the double bond in 41, including H2–Pd–C, NaBH4 in pyridine

260
K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
O
N
O
O
N
NH
AcO
TBSO
NH
HO
TBSO
NH
HO
O
N
O
O
Ref. 10
Ref. 18
O
O
O
OH
OTBS
OTBS
10
31
33
a
O
N
O
N
O
N
AcO
NH
AcO
HO
NH
AcO
NH
O
O S
O
O
O
NC
c
b
O
O
O
OTBDMS
OTBS
OTBS
32
Scheme 4. Attempted 50-derivatization of acetyl protected 20-deoxy-5-hydroxymethyluridine. Reagents and conditions: (a) anhyd THF, pyridine, HF-pyridine, 0 °C, 1 h,
0 °C ? rt, 1 h, 33%; (b) (i) anhyd CH2Cl2, Dess–Martin periodinane, rt, 4 h; (ii) anhyd THF, 6, BuLi, ꢀ78 °C, 15 min, ꢀ78 °C, 1 h, rt; (c) (i) anhyd CH2Cl2, Dess–Martin
periodinane, rt, 4 h; (ii) anhyd THF, cyanomethyltriphenylphosphonium chloride, BuLi, 0 °C, 30 min, 0 °C ? rt.
O
O
N
O
N
O
NH
PMBO
TBSO
NPMB
O
TBSO
O
O
O
OTBS
OTBS
38
g
h
O
N
O
N
O
N
O
N
HO
TBSO
NH
BOMO
TBSO
NBOM
O
NH
NH
TBSO
HO
O
O
O
a
O
O
b
O
O
+
OTBS
OTBS
OTBS
OTBS
33
34
11
12
c
SEMO
OH
O
O
N
O
N
O
N
O
N
NH
SEMO
TBSO
NSEM
O
SEMO
HO
NSEM
O
NSEM
O
O
S
O
O S O
O
d
e
f
O
O
O
OTBS
OTBS
OTBS
OH
35
36
37
Scheme 5. Screening viable protecting group for 5-hydroxymethyl-20-deoxyuridine. Reagents and conditions: (a) DMF, DIPEA, BOM-Cl, 0 °C ? rt, 16 h, 37%; (b) MeOH, H2, Pd-
C; (c) CH2Cl2, DIPEA, SEM-Cl, 40 °C, 6 h, 50%; (d) THF, pyridine, HF-pyridine, 0 °C, 1 h, 0 °C ? rt, 1 h, 38%; (e) (i) CH2Cl2, Dess–Martin periodinane, rt, 4 h; (ii) THF, 6, BuLi,
ꢀ78 °C, 15 min, ꢀ78 °C, 1 h, rt, 16 h, 29% over two steps; (f) THF, TBAF,
D; (g) THF, DEAD, triphenylphosphine, PMP, 0 °C ? rt; (h) DMF, PMB-Cl, NaH, 0 °C ? rt, 4 h, 13%.
and NaBH4–NiCl2 in MeOH–THF. Unfortunately, all these methods
led to deoxygenation of the 5-CH2OH group and formation of a thy-
mine base as a predominant side product. Attempts to remove the
pivaloyl prior from 41 with tetrabutylammmonium hydroxide
gave undesired product 42. Hydrogenation using platinum on car-
bon afforded 43 in 70% yield. Subsequent removal of the TBDMS
group with TBAF and the pivaloyl group with sodium methoxide
gave 30 in excellent yield. Azidotrimethylsilane mediated cycload-
dition of nitrile 43 gave 45 in 74% yield. Removal of the remaining
protecting groups with NaOMe in methanol and NH4F in methanol
allowed obtaining 47 in excellent yield. For the desilylation NH4F
proved to be superior to TBAF with regard to purification of the fi-
nal product.
2.2. Biological results
The capacity of compounds 16, 18, 21, 23, 30 and 47 to inhibit
TMPKmt was assessed via a spectrophotometric binding assay (Ki)
as previously described.4 Compound 21 (Ki = 48
lM) and 23
(Ki = 70
lM) showed the highest activity, while compounds 16

K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
261
O
N
O
N
O
N
HO
TBSO
NH
PivO
TBSO
NH
PivO
HO
NH
O
O
O
O
O
O
a
b
OTBS
33
OTBS
39
OTBS
40
c
OPiv
O
N
O
N
O
N
O
N
NH
NH
NH
PivO
PivO
NH
PivO
CN
O
O
O
O
NC
NC
O
O
O
O
f
e
d
NC
OH
OH
OTBS
OTBS
44
42
43
41
g
h
O
N
O
N
O
N
O
N
HO
NH
PivO
NH
HO
NH
HO
NH
O
N
O
N
O
N
O
NC
i
j
O
N
O
N
O
N
O
NH
NH
NH
N
N
N
OH
30
OTBS
OTBS
OH
47
45
46
Scheme 6. Synthesis of the 50-modified 20-deoxy-5-hydroxymethyluridine analogs 30 and 47. Reagents and conditions: (a) pyridine, DMAP, Piv-Cl, rt, 18 h,74%; (b) THF,
pyridine, HF-pyridine, 0 °C, 1 h, 0 °C ? rt, 1 h, 59%; (c) (i) CH2Cl2, Dess–Martin periodinane, rt, 4 h; (ii) anhyd THF, cyanomethyltriphenylphosphonium chloride, ꢀ78 °C, BuLi,
30 min, ꢀ78 °C ? rt, 18 h, 82% over two steps; (d) THF, Bu4NOH, rt, 5 h, 40%; (e) MeOH, H2, Pt-C, rt, 4 h, 70%; (f) THF, TBAF, rt, 4 h, 90%; (g) 0.5 M NaOMe in MeOH, rt, 3 h, 95%;
(h) toluene, TMSN3, Bu2SnO, 110 °C, 4 h, 74%; (i) 0.5 M NaOMe in MeOH, rt, 3 h, 92%; (j) NH4F, MeOH, 50 °C, 2 days, 87%.
(340
ingly, compounds 30 and 47, in which the favorable 50-modifica-
tions were combined with 5-CH2OH modification of the
nucleobase, failed to inhibit the enzyme at the highest concentra-
tion tested (2.6 and 2.4 mM, respectively). Clearly, introduction of
the 5-hydroxymethyl group jeopardized the binding of 50-modified
analogues to TMPKmt.
l
M) and 18 (240
l
M) were comparatively less active. Surpris-
inhibitors 23 and 47 at the active site of TMPKmt (Fig. 2) shows
that the 50-tetrazolylmethyl moiety is firmly anchored in a polar
cavity formed by the charged residues Asp9, Arg160, Asp163, and
Glu166 and coordinated by the Mg2+ ion and a structural water
molecule. However, the thymidine ring shows a higher degree of
deviation between the two inhibitors, while the 5-CH2OH group
of 47 does not seem to reach up to an ideal stabilizing position.
As shown on Figure 2, the oxygen of the 5-CH2OH group is oriented
mainly by the interactions with the Arg74 and Phe70 residues into
a position where it directs its H atom towards the hydrogen of Cb of
Phe36 with a relatively short distance of only 1.85 Å (red line in
Fig. 2). This leads to an electrostatic repulsion, which may be
responsible for the observed weak inhibitory potency of 47. In
the crystal structure of TMPKmt co-crystallyzed with 5-CH2OH
dUMP5 the 5-hydroxymethyl group of dUMP is stabilized by
hydrogen bonding interactions with the guanidine group of
Arg74 and nitrogen atom of Pro37. Still, this does not exclude that
a
2.3. Discussion and conclusions
In short, a small series of 50-modified thymidine analogues was
synthesized and evaluated as TMPKmt inhibitors. The analogues in
which the 50-hydroxyl group was replaced by an acetonitrile or a
50-tetrazolylmethyl moiety proved capable of inhibiting the target
enzyme with substantial affinity.
Hence we developed a synthetic route that allowed combining
these 50-modifications with a hydroxymethyl moiety at position
5 of the pyrimidine base. Given the recent interest in the phenom-
enon of hydroxymethylation of pyrimidine bases in mammalian
genomes, the successful synthetic strategy, featuring pivaloyl
protection of the 5-CH2OH group prior to 50-modification, may find
future application in the synthesis of other 5-hydroxymethyl-20-
deoxyuridine tool compounds.
a 20,30-
a-fused cyclic sulfuryldiamide substituent may show syner-
gistic inhibitory effects with a 5-CH2OH group, since conforma-
tional restriction by a fused ring may very well change the
topology of the molecular interactions with the enzyme.
3. Experimental
It is rather unfortunate that enzyme assays indicated that com-
bination of both modifications did not reinforce the TMPKmt bind-
ing affinity and led to very weak inhibitors. A possible explanation
of the observed drop in the inhibitory potencies of the 5-CH2OH
substituted compounds may be related to an elevated steric strain
in the bisubstituted analogs. A superposition of the models of
All reagents were from standard commercial sources and of
analytical grade. Dry solvents were obtained directly from com-
mercial sources and stored on molecular sieves. All reactions were
carried out under argon atmosphere unless specified otherwise.
Precoated Merck silica-gel F254 plates were used for TLC; spots

262
K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
Figure 2. Comparison of structure and interactions of models of 23 (dark blue) and 47 (light blue) superimposed at the active site of TMPKmt. Side chains of selected active
site residues are shown in stick representations. Majority of hydrogen atoms were omitted for better clarity. Structures of the enzyme-inhibitor complexes were obtained by
flexible docking and refinement of the model ligands into the crystal structure of TMPKmt co-crystallyzed with 5-CH2OH dUMP (PDB entry code 1MRS5). For method details
see Ref. 6
were examined under ultraviolet light at 254 nm and further visu-
alized by sulphuric acid–anisaldehyde spray. Column chromatog-
raphy was performed on silica gel (200–400 mm, 60 Å). NMR
spectra were recorded on a Varian Mercury 300 MHz spectrometer.
Chemical shifts are given in ppm (d), calibrated to the residual sol-
vent signals or TMS. Exact mass measurements were performed on
a Waters LCT PremierXETM time of flight (TOF) mass spectrometer
equipped with a standard electrospray ionization (ESI) and modu-
lar LockSpray TM interface. Samples were infused in a CH3CN/
water (1:1 v/v) mixture at 10 mL/min. The microwave reactions
were carried out in Milestone MicroSYNTH Advanced Microwave
Synthesis Lab station, equipped with 2 ꢁ 800 W magnetrons
(effective maximum output 1500 W pulsed/continuous), an optical
fiber temperature sensor, a pressure sensor, under continues
power mode in a closed PTFE vessel. A temperature of 25 3 °C
is referred to as ‘room temperature, rt’ throughout the manuscript.
NMR signals of sugar protons and carbons are indicated with a
prime, and signals of base protons and carbons are given without
a prime.
3.0.2. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-5-methylpyrimidine-
2,4(1H,3H)-dione (12)
Compound 11 (9.71 g, 20.64 mmol) and anhydrous THF
(106.4 mL) was placed in a Teflon flask under inert condition and
cooled to 0 °C. In a separate polypropylene flask, anhydrous THF
(79.8 mL) and anhydrous pyridine (31.9 mL) were placed under in-
ert atomsphere; to this at 0 °C ꢂ70%HF in pyridine (28.7 mL) was
added drop wise. The chilled THF-Py–HF.Py mixture was added
dropwise to a flask containing compound 11 also at 0 °C and stirred
at this temperature for 1 h. The cold bath was removed and reac-
tion continued at room temperature for 45 min. The reaction mix-
ture was poured to an ice-cold solution of NaHCO3 (100 g in
500 mL) with vigorous stirring. The compound was extracted in
ethyl acetate (3 ꢁ 150 mL), washed with brine and dried over an-
hyd Na2SO4. The residue after evaporation was subjected to flash
column chromatography (20–50% EtOAc in hexanes) to obtain
compound 12 as white foam (2.615 g, 36%). 1H NMR (300 MHz,
CDCl3) d ppm 0.07 (s, 6H, SiMe2), 0.88 (s, 9H, tBu), 1.88 (s, 3H, 5-
Me), 2.20 (ddd, J = 13.25, 6.37, 3.81 Hz, 1H, 20-H), 2.32 (dt,
J = 13.40, 6.63 Hz, 1H, 20-H), 2.98–3.13 (m, 1H, 50-OH), 3.68–3.80
(m, 1H, 50-H), 3.85–3.96 (m, 2H, 50 & 40-H), 4.48 (dt, J = 6.44,
3.51 Hz, 1H, 30-H), 6.16 (t, J = 6.74 Hz, 1H, 10-H), 7.35–7.48 (m,
1H, 6-H), 9.31 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3) d ppm
ꢀ4.66 (SiMe-C), ꢀ4.50 (SiMe-C), 12.69 (5-Me-C), 18.15 (tBu-tC),
25.92 (tBu-C), 40.76 (20-C), 62.11 (50-C), 71.81 (30-C), 86.87 (10-C),
87.84 (40-C), 111.13 (5-C), 137.25 (6-C), 150.67 (2-C), 164.26 (4-
C). ESI-HRMS for [C16H28N2O5Si+H]+ Calcd, 357.1846. Found,
357.1847.
3.0.1. Diethyl ((N-methylsulfamoyl)methyl)phosphonate (9)
To a solution of 8 (1.0 g, 9.1 mmol) in anhydrous THF (40.0 mL)
at ꢀ78 °C was added n-BuLi (1.6 M, 11.5 mL, 18.3 mmol) dropwise
under argon atmosphere. The mixture was stirred for an hour at
ꢀ78 °C and diethyl clorophosphate (0.67 mL, 4.6 mmol) was added
slowly. The reaction mixture was stirred at 0 °C for an hour. The
reaction was stopped by adding satd NH4Cl solution (10 mL) fol-
lowed by extraction with dichloromethane (3 ꢁ 50 mL). The com-
bined organic layers were dried over anhyd Na2SO4. The
desiccant was filtered off and the filtrate was concentrated. The
residue was purified by flash column chromatography (50–80%
EtOAc in hexanes) to afford unreacted starting material (450 mg)
and product 9 as colourless oil (800 mg, 65% based on recovered
starting material). 1H NMR (300 MHz, CDCl3) d ppm 1.36 (td,
J = 7.2, 0.9 Hz, 6H, OCH2Me), 2.37 (d, J = 5.4 Hz, 3H, NMe), 3.60 (d,
J = 16.2 Hz, 2H, SCH2P), 4.22 (qt, J = 6.9 Hz, 0.9 Hz, 4H, OCH2Me),
5.26 (q, J = 5.1 Hz, NH). 13C NMR (75 MHz, CDCl3) d ppm 16.14 (d,
J = 6 Hz, OCH2CH3), 29.59 (NCH3), 46.90 (d, J = 138.5 Hz, PCH2S),
63.53 (d, J = 6.1 Hz, OCH2CH3). 31P NMR (121 MHz, CDCl3) d ppm
13.52. ESI-HRMS for [C6H16NO5PS+H]+ Calcd, 246.0565. Found,
246.0548.
3.0.3. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-((E)-2-
(methylsulfonyl)vinyl)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (14)
To a solution of Dess–Martin reagent (1.28 g, 3 mmol) in anhy-
drous dichloromethane (5 mL) at 0 °C under argon atmosphere was
added compound 12 (713 mg, 2 mmol) in Dichloromethane (5 mL)
and the reaction continued at room temperature. After 4 h satu-
rated NaHCO3 solution (10 mL) was added and the product ex-
tracted in dichloromethane (3 ꢁ 20 mL). The organic layers were
combined, washed with brine, dried over anhyd Na2SO4, filtered.

K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
263
The filtrate was evaporated and dried under high vacuum to give
aldehyde 13 as white foamy residue which was used without puri-
fication for Horner–Wittig reaction.
109.97 (5-C), 136.17 (6-C), 150.47 (2-C), 163.68 (4-C). ESI-HRMS
for [C12H18N2O6S+H]+ Calcd, 319.0964. Found 319.0963.
In a separate flask, diethyl((methylsulfonyl)methyl)phospho-
nate 6 (553 mg, 2.4 mmol) was dissolved in anhydrous THF
(20 mL) under argon atmosphere. The mixture was brought to
ꢀ78 °C and n-BuLi (1.6 M in hexanes, 1.38 mL, 2.2 mmol) was
added slowly and stirred for 15 min. The solution of the above
aldehyde 13 (2 mmol) in anhydrous THF (15 mL) was added drop-
wise. The flask was kept at ꢀ78 °C for 1 h and at room temperature
overnight (18 h). Water (10 mL) was added and the product ex-
tracted in ethyl acetate (3 ꢁ 50 mL). The combined organic layers
were washed with brine, dried over anhyd Na2SO4, filtered and fil-
trate was evaporated. The residue was purified by flash column
chromatography (20–50% EtOAc in hexanes) to afford 14 as white
solid (175 mg, 20%). 1H NMR (300 MHz, CDCl3) d ppm 0.03 (s, 3H,
SiMe), 0.04 (s, 3H, SiMe), 0.84 (s, 9H, t-Bu), 1.89 (d, J = 1.17 Hz,
3H, 5-Me), 2.16–2.24 (m, 2H, 20-H), 2.91 (s, 3H, SO2Me), 4.25 (dt,
J = 6.37, 4.87 Hz, 1H, 30-H), 4.33–4.43 (m, 1H, 40-H), 6.22 (t,
J = 6.74 Hz, 1H, 10-H) 6.62 (dd, J = 15.08, 1.90 Hz, 1H, 60-H), 6.93
(dd, J = 14.94, 4.10 Hz, 1H, 50-H), 6.96 (d, J = 1.17 Hz, 1H, 6-H),
8.39 (s, 1H, NH). 13C NMR (75 MHz, CDCl3) d ppm -4.78 (SiMe-C),
ꢀ4.66 (SiMe-C), 12.64 (5Me-C), 17.90 (tBu-tC), 25.63 (tBuMe-C),
39.46 (20-C), 42.68 (SO2Me–C), 74.66 (30-C), 84.05 (40-C), 85.56
(10-C), 111.93 (5-C), 130.75 (60-C), 135.28 (6-C), 142.90 (50-C),
150.00 (2-C), 163.14 (4-C). ESI-HRMS for [C18H30N2O6SSi+H]+
Calcd, 431.1672. Found, 431.1519.
3.0.6. (E)-2-((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)-N-methylethenesulfonamide (17)
Compound 12 (100 mg, 0.28 mmol) was reacted in similar fash-
ion as described for 14 to get the aldehyde 13. In a separate flask, to
the solution of diethyl ((N-methylsulfamoyl)methyl)phosphonate
11 (83 mg, 0.36 mmol) in anhydrous THF (3 mL) at ꢀ78 °C under
inert atmosphere was added n-BuLi (1.6 M in hexanes, 0.39 mL,
0.62 mmol) drop wise and stirred for 1 h. A solution of above alde-
hyde 13 (0.28 mmol) in THF (3 mL) was added and the mixture was
kept at ꢀ78 °C for 1 h then allowed to stir at room temperature
overnight (18 h). Water (5 mL) was added and the reaction mixture
was extracted with ethyl acetate (3 ꢁ 25 mL). The combined or-
ganic layer was washed with brine, dried over anhyd Na2SO4, fil-
tered, and evaporated. The residue was purified by preperative
TLC (50% EtOAc in toluene) to obtain 17 as a white foam (60 mg,
48%). 1H NMR (300 MHz, CDCl3): d 0.10 (s, 3H, SiMe), 0.11 (s, 3H,
SiMe), 0.91 (s, 9H, t-BuSi), 1.96 (d, J = 1.2 Hz, 3H, 5-Me), 2.23–
2.29 (m, 2H, 20-H), 2.76 (s, 3H, NHMe), 4.28–4.33 (m, 1H, 40-H),
4.41 (td, J = 4.8, 1.2 Hz, 1H, 30-H), 6.30 (t, J = 6.9 Hz, 1H, 10H), 6.47
(dd, J = 15.3, 1.5 Hz, 1H, 60-H), 6.82 (dd, J = 15.3, 4.5 Hz, 1H, 50-H),
7.04 (d, J = 1.2 Hz, 1H, 6-H) ESI-HRMS for [C18H31N3O6SSi+H]+
Calcd, 446.1781. Found, 446.1773.
3.0.4. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(2-
(methylsulfonyl)ethyl)tetrahydrofuran-2-yl)-5-methyl-
pyrimidine-2,4(1H,3H)-dione (15)
3.0.7. 2-((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetra-
hydrofuran-2-yl)-N-methylethanesulfonamide (18)
To a solution of 14 (175 mg, 0.41 mmol) in ethyl acetate
(10.0 mL) was added Pd-C (10% Pd, ꢂ50% H2O, 100 mg) and stirred
with purging H2 gas through the reaction mixture at room temper-
ature for 4 h. The catalyst was filtered over celite and filtrate was
evaporated. The residue was dried under high vacuum to afford
practically pure compound 15 (175 mg) which was used as such
for the next step. 1H NMR (300 MHz, CDCl3) d ppm 0.02 (s, 3H,
SiMe), 0.03 (s, 3H, SiMe), 0.83 (s, 9H, tBu), 1.89 (d, J = 1.17 Hz,
3H, 5-Me), 1.92–2.07 (m, 2H, 50-H), 2.14–2.30 (m, 2H, 20-H), 2.88
(s, 3H, SO2Me), 3.04 (ddd, J = 13.7, 11.1, 5.7 Hz, 1H, 60-H), 3.13–
3.25 (m, 1H, 60-H), 3.75 (dt, J = 10.40, 3.88 Hz, 1H, 40-H), 4.08–
4.12 (m, 1H, 30-H), 6.09 (t, J = 6.88 Hz, 1H, 10-H), 6.99 (d,
J = 1.46 Hz, 1H, 6-H), 8.08 (br s, 1H, NH). ESI-HRMS for
[C18H32N2O6SSi+H]+ Calcd, 433.1829. Found, 433.0574.
To a solution of 17 (60 mg, 0.135 mmol) in 5:1 MeOH-THF
(1.5 mL) at 0 °C was added NiCl.6H2O (16 mg, 0.075 mmol) and
NaBH4 (10.2 mg, 0.27 mmol). The reaction mixture was stirred for
1 h at 0 °C and the solvent was evaporated. The residue suspended
in ethyl acetate, filtrated over celite, the filtrate was evaporated. The
residue obtained was dissolved in THF (1.5 mL) and was added TBAF
(80 lL, 0.268 mmol). The reaction mixture was stirred at 40 °C for
1 h. The solvent was evaporated to dryness and the residue was
purified by flash column chromatography (3–7% MeOH in CH2Cl2)
to afford 18 (5 mg, 12%) as white solid. 1H NMR (300 MHz, DMSO-
d6) d ppm 1.80 (d, J = 0.88 Hz, 3H, 5-Me), 1.91 (td, J = 9.15, 4.54 Hz,
1H, 50-H), 1.96–2.10 (m, 2H, 20 & 50-H), 2.24 (dt, J = 13.84, 6.99 Hz,
1H, 20-H), 2.57 (d, J = 4.98 Hz, 3H, N-Me), 3.07 (dt, J = 9.81,
5.64 Hz, 2H, 60-H), 3.73 (dt, J = 8.42, 4.43 Hz, 1H, 40-H), 4.11 (dd,
J = 6.74, 4.10 Hz, 1H, 30-H), 5.34 (d, J = 4.39 Hz, 1H, 30-OH), 6.14 (t,
J = 6.88 Hz, 1H, 10-H), 6.92 (q, J = 4.98 Hz, 1H, MeNH) 7.42 (d,
J = 1.17 Hz, 1H, 6-H), 11.30 (br s, 1H, NH). 13C NMR (75 MHz,
DMSO-d6) d ppm 11.96 (5-Me-C), 27.01 (50-C), 28.47 (NMe-C),
38.04 (20-C), 46.01 (60-C), 72.73 (30-C), 83.29 (10-C), 83.62 (40-C),
109.84 (5-C), 136.00 (6-C), 150.33 (2-C), 163. 55 (4-C). ESI-HRMS
for [C12H19N3O6S+H]+ Calcd, 334.1073. Found, 334.1085.
3.0.5. 1-((2R,4S,5R)-4-hydroxy-5-(2-(methylsulfonyl)ethyl)tet-
rahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (16)
To a solution of compound 15 (175 mg, 0.41 mmol) in anhy-
drous THF (3.0 mL) was added tetrabutylammonium fluoride
(TBAF, 0.14 mL, 0.49 mmol) dropwise. The flask was heated to
40 °C overnight. The volatile materials were evaporated under high
vacuum and the residue was purified by flash column chromatog-
raphy (3% MeOH in CH2Cl2). The product was purified further by
crystallization/triturating in methanol to afford compound 16 as
a colorless needles (25 mg, 20%). 1H NMR (300 MHz, DMSO-d6) d
ppm 1.80 (d, J = 1.17 Hz, 3H, 5-Me), 1.87–2.00 (m, 1H, 50-H),
2.00–2.15 (m, 2H, 20 & 50-H), 2.25 (dt, J = 13.84, 6.99 Hz, 1H, 20-
H), 3.00 (s, 3H, SO2Me), 3.19 (t, J = 8.05 Hz, 2H, 60-H), 3.72 (dt,
J = 8.57, 4.36 Hz, 1H, 40-H), 4.08–4.18 (m, 1H, 30-H), 5.34 (d,
J = 4.39 Hz, 1H, 30-OH), 6.15 (t, J = 7.03 Hz, 1H, 10-H), 7.44 (d,
J = 1.17 Hz, 1H, 6-H), 11.30 (s, 1H, NH). 13C NMR (75 MHz, DMSO-
3.0.8. (E)-3-((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetra-
19
hydrofuran-2-yl)acrylonitrile (19)
Compound 12 (1.22 g, 3.43 mmol) was reacted in similar fash-
ion as described for 14 to get the aldehyde 13. In a separate flask,
to
a solution of cyanomethyltriphenylphosphonium chloride
(3.48 g, 10.3 mmol) in anhydrous THF (35 mL) at 0 °C under argon
atmosphere was added drop wise n-BuLi (1.6 M in hexanes, 6.4 mL,
10.3 mmol) and stirred for 30 min. To this ylide at 0 °C was added
slowly a solution of above aldehyde 13 (3.43 mmol) in anhydrous
THF (15 mL) and stirred at room temperature overnight. The
d6)
d
ppm 12.06 (5-Me-C), 25.78 (50-C), 38.05 (20-C), 40.15
(SO2Me-C), 50.39 (60-C), 72.84 (30-C), 83.46 (10-C), 83.83 (40-C),

264
K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
reaction was quenched with water (30 mL) and extracted with
ethyl acetate (3 ꢁ 100 mL). Combined organic layer was washed
with brine, dried over anhyd Na2SO4, filtered and evaporated.
The residue obtained was purified by flash column chromatogra-
phy (15–30% EtOAc in hexanes) to afford 19 as a light yellow foam
(740.4 mg, 57%). 1H NMR (300 MHz, CDCl3) d ppm 0.09 (s, 3H,
SiMe), 0.10 (s, 3H, SiMe), 0.90 (s, 9H, tBu), 1.96 (d, J = 1.17 Hz,
3H, 5-Me), 2.27–2.36 (m, 2H, 20-H), 4.27–4.35 (m, 2H, 30-H and
40-H), 5.67 (dd, J = 16.26, 1.61 Hz, 1H, 60-H), 6.19 (t, J = 6.59 Hz,
1H, 10-H), 6.80 (dd, J = 16.11, 4.69 Hz, 1H, 50-H), 7.01 (d,
J = 1.17 Hz, 1H, 6-H), 9.46 (s, 1H, NH). 13C NMR (75 MHz, CDCl3) d
ppm ꢀ4.82 (SiMe-C), ꢀ4.63 (SiMe-C), 12.65 (5-Me-C), 17.89 (tBu-
tC), 25.63 (tBu-C), 39.79 (20-C) 74.69 (30-C), 84.96 (40-C), 86.14
(10-C), 101.00 (60-C), 111.74 (5-C), 116.56 (70-C), 135.74 (6-C)
149.95 (50-C), 150.25 (2-C), 163.80 (4-C). ESI-HRMS for
[C18H27N3O4Si+H]+ Calcd, 378.1849. Found, 378.1852.
16 mg) and azidotrimethylsilane (TMSN3, 158 lL, 1.6 mmol) under
argon atmosphere. The reaction vessel was sealed with septum and
stirred at 110 °C for 4 h. The volatiles were evaporated under re-
duced pressure and the residue was purified by flash column chro-
matography (2% AcOH/ EtOAc) to afford 22 as a white foam (43 mg,
32%). 1H NMR (300 MHz, DMSO-d6) d ppm 0.00 (s, 6H, SiMe2), 0.78
(s, 9H, t-Bu), 1.73 (d, J = 0.88 Hz, 3H, 5-Me), 1.86–2.07 (m, 3H, 20-H
and 50-H), 2.21 (dt, J = 13.62, 6.96 Hz, 1H, 20-H), 2.80–2.96 (m, 2H,
60-H), 3.61 (dt, J = 8.42, 4.43 Hz, 1H, 40-H), 4.20 (dt, J = 6.59, 4.03 Hz,
1H, 30-H), 6.04 (t, J = 6.88 Hz, 1H, 10-H), 7.33 (d, J = 1.17 Hz, 1H, 6-
H), 11.23 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) d ppm
ꢀ4.89 (SiMe-C), ꢀ4.72 (SiMe-C), 12.10 (5-Me-C), 17.61 (tBu-tC),
19.63 (60-C), 25.65 (tBu-C), 30.42 (50-C), 38.60 (20-C), 74.35 (30-C),
83.46 (10-C), 84.44 (40-C), 109.89 (5-C), 136.21 (6-C), 150.39 (2-
C), 155.69 (70-C) 163.66 (4-C). ESI-HRMS for [C18H30N6O4SiꢀH]ꢀ
Calcd, 421.2020; Found, 421.1249.
3.0.9. 3-((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(5-
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)tetrahydrofuran-2-yl)propanenitrile (20)
3.0.12. 1-((2R,4S,5R)-5-(2-(1H-tetrazol-5-yl)ethyl)-4-
hydroxytetrahydrofuran-2-yl)-5-methylpyrimidine-2,4(1H,3H)-
dione (23)
To a solution of 19 (443 mg, 1.18 mmol) in 3:1 anhydrous pyr-
idine–MeOH (2 mL) was added NaBH4 (45 mg, 1.18 mmol) and the
reaction mixture was stirred at 120 °C under inert atmosphere for
4 h. The reaction mixture was evaporated and the residue was
purified by flash column chromatography (30–50% EtOAc in hex-
anes) to afford 20 as white foam (240 mg, 54%). 1H NMR
(300 MHz, CDCl3) d ppm 0.09 (s, 3H, SiMe), 0.10 (s, 3H, SiMe),
0.90 (s, 9H, t-Bu), 1.84–1.93 (m, 1H, 50-H), 1.95 (d, J = 1.46 Hz,
3H, 5-Me), 2.02–2.16 (m, 1H, 50-H), 2.27 (t, J = 6.44 Hz, 2H, 20-H),
2.41–2.62 (m, 2H, 60-H), 3.80 (ddd, J = 9.30, 5.35, 3.81 Hz, 1H, 40-
H), 4.12–4.20 (m, 1H, 30-H), 6.08 (t, J = 6.59 Hz, 1H, 10-H), 7.05 (d,
J = 1.17 Hz, 1H, 6-H), 8.44 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3)
d ppm ꢀ4.81 (SiMe-C), ꢀ4.53 (SiMe-C), 12.62 (5-Me-C), 14.22 (60-
C), 17.91 (tBu-tC), 25.68 (tBu-C), 28.94 (50-C), 40.15 (20-C), 74.43
(30-C), 84.04 (40-C), 85.60 (10-C), 111.35 (5-C), 119.11 (70-C),
135.87 (6-C), 149.86 (2-C), 163.32 (6-C). ESI-HRMS for
[C18H29N3O4Si+H]+ Calcd, 380.2006. Found, 380.2015.
Compound 22 (40 mg, 0.1 mmol) was dissolved in anhydrous
THF (2 mL) and added TBAF (0.5 mL, 0.5 mmol) at room tempera-
ture. After 4 h, methanol (2 mL) was added, followed by CaCO3
(1 g) with vigorous stirring. After 10 min was added Dowex resin
(H+ form, 1 g) portion wise. The mixture was stirred for 30 min
and filtered over a pad of celite. The filtrate was concentrated and
purified by flash column chromatography (5–8% MeOH + 2% AcOH
in CH2Cl2) to afford title compound 23 as a white solid (10 mg,
34%). 1H NMR (300 MHz, DMSO-d6) d ppm 1.80 (d, J = 1.17 Hz, 3H,
5-Me), 1.89–2.14 (m, 3H, 20-H and 50-H), 2.14–2.33 (m, 1H, 20-H),
2.87–3.06 (m, 2H, 60-H), 3.66 (dt, J = 8.57, 4.36 Hz, 1H, 40-H), 4.05–
4.17 (m, 1H, 30-H), 5.30 (br s, 1H, 30-OH), 6.14 (t, J = 6.88 Hz, 1H,
10-H), 7.39 (d, J = 0.88 Hz, 1H, 6-H), 11.30 (s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6) d ppm 11.99 (5-Me-C), 19.39 (60-C), 30.56 (50-
C), 38.20 (20-C), 72.73 (30-C), 83.18 (10-C), 84.30 (40-C), 109.79 (5-
C), 135.94 (6-C), 150.33 (2-C), 155.07 (70-C), 163.56 (4-C). ESI-HRMS
for [C12H16N6O4ꢀH]ꢀ Calcd, 307.1155. Found, 307.0597.
3.0.10. 3-((2R,3S,5R)-3-hydroxy-5-(5-methyl-2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)propanenitrile (21)
3.0.13. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-
(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-
dione (26)
To the stirring solution of compound 20 (100 mg, 0.26 mmol) in
anhydrous THF (2 mL) was added tetrabutylammonium fluoride
(TBAF, 1 M in THF, 0.5 mL, 0.5 mmol) under inert atmosphere and
stirred at room temperature for 4 h. The reaction mixture was evap-
orated under vacuum and the residue was purified by flash column
chromatography (EtOAc) to afford product 21 as a white solid
(53 mg, 76%). 1H NMR (300 MHz, DMSO-d6) d ppm 1.80 (d,
J = 1.17 Hz, 3H, 5-Me), 1.83–1.98 (m, 2H, 50-H), 2.05 (ddd, J = 13.55,
6.52, 3.95 Hz, 1H, 20-H), 2.22 (dt, J = 13.77, 6.88 Hz, 1H, 20-H), 2.52–
2.62 (m, 2H, 60-H), 3.63–3.74 (m, 1H, 40-H), 4.04–4.17 (m, 1H, 50-
H), 5.34 (d, J = 4.39 Hz, 1H, 30-OH), 6.15 (t, J = 6.88 Hz, 1H, 10-H),
7.44 (d, J = 1.17 Hz, 1H, 6-H), 11.30 (br s, 1H, NH). 13C NMR
(75 MHz, DMSO-d6) d ppm 11.96 (5-Me-C), 13.20 (60-C), 28.37
(50-C),v 38.01 (20-C), 72.50 (30-C), 83.42 (10-C), 83.92 (40-C), 109.75
(5-C), 120.30 (70-C), 136.13 (6-C), 150.33 (2-C), 163.56 (4-C). ESI-
HRMS for [C12H15N3O4ꢀH]ꢀ Calcd, 264.099. Found, 264.0617.
Removal of primary TBS group using the procedure described
for compound 12, compound 25 (1.8 g, 4 mmol) rendered 26
(600 mg, 53%) as a white solid. 1H NMR (300 MHz, CDCl3) d ppm
0.09 (s, 6H, SiMe), 0.90 (s, 9H, tBu), 2.24–2.32 (m, 2H, 20-H) 2.50
(br s, 1H, 50-OH), 3.69–3.82 (m, 1H, 50-H), 3.87–4.00 (m, 2H, 40-H
and 50-H), 4.43–4.55 (m, 1H, 30-H), 5.74 (dd, J = 8.20, 2.05 Hz, 1H,
5-H), 6.18 (t, J = 6.59 Hz, 1H, 10-H), 7.65 (d, J = 8.20 Hz, 1H, 6-H),
8.89 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3) d ppm ꢀ4.64
(SiMe-C), ꢀ4.47 (SiMe-C), 18.18 (tBu-tC), 25.93 (tBu-C), 41.09 (20-
C), 62.09 (50-C), 71.64 (30-C), 87.02 (10-C), 87.82 (40-C), 102.71 (5-
C), 141.31 (6-C), 150.42 (2-C), 163.40 (4-C). ESI-HRMS for
[C15H26N2O5Si+H]+ Calcd, 343.1689. Found, 343.1682.
3.0.14. (E)-3-((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)acrylonitrile (27)
3.0.11. 1-((2R,4S,5R)-5-(2-(1H-tetrazol-5-yl)ethyl)-4-((tert-
Butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-5-
methylpyrimidine-2,4(1H,3H)-dione (22)
Following the oxidation and Wittig reaction procedure de-
scribed for compound 19, compound 26 (600 mg, 1.75 mmol) ren-
dered 27 (325 mg, 51%) as a white solid. 1H NMR (300 MHz, CDCl3)
d ppm 0.10 (s, 6H, SiMe2), 0.90 (s, 9H, tBu), 2.19–2.43 (m, 2H, 20-H),
4.24–4.30 (m, 1H, 30-H), 4.30–4.37 (m, 1H, 40-H), 5.67 (dd, J = 16.40,
1.76 Hz, 1H, 60-H), 5.81 (dd, J = 8.20, 2.34 Hz, 1H, 5-H), 6.19 (t,
Compound 20 (120 mg, 0.32 mmol) was dessolved in anhy-
drous toluene (5 mL), to this was added dibutyltin oxide (Bu2SnO,

K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
265
J = 6.44 Hz, 1H, 10-H), 6.77 (dd, J = 16.11, 4.69 Hz, 1H, 50-H), 7.21 (d,
J = 8.20 Hz, 1H, 6-H), 8.65 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3)
d ppm ꢀ4.82 (SiMe-C), ꢀ4.63 (SiMe-C), 17.90 (tBu-tC), 25.63 (tBu-
C), 40.06 (20-C), 74.59 (30-C), 85.04 (40-C), 86.30 (10-C), 101.19 (60-
C), 103.21 (5-C), 116.42 (70-C), 139.83 (6-C), 149.61 (50-C), 149.82
(2-C), 162.71 (4-C). ESI-HRMS for [C17H25N3O4Si+CH3CN+H]+ Calcd,
405.1958. Found, 405.1955.
160
l
L, 0.92 mmol) at 0 °C under inert atmosphere was added drop
L, 0.68 mmol)
wise benzyloxymethyl chloride (ꢂ75% BOMCl, 94
l
and stirred at room temperature overnight. Saturated aq.NH4Cl
(5 mL) was added to quench the reaction. The products were ex-
tracted in ethyl acetate (50 mL), organic layer washed with water,
brine, dried over anhyd Na2SO4 and evaporated. The residue was
purified by flash column chromatography (5–10% EtOAc in hex-
anes) to yield 34 (80 mg, 36%). 1H NMR (300 MHz, CDCl3) d ppm
0.08–0.12 (m, 12H, SiMe), 0.88–0.94 (m, 18H, tBu), 1.97 (ddd,
J = 13.34, 7.64, 5.97 Hz, 1H, 20-H), 2.30 (ddd, J = 13.16, 5.74,
2.44 Hz, 1H, 20-H), 3.76 (dd, J = 11.40, 3.08 Hz, 1H, 50-H), 3.82 (dd,
J = 11.22, 3.26 Hz, 1H, 50-H), 3.96 (q, J = 3.08 Hz, 1H, 40-H), 4.34–
4.47 (m, 3H, 30-H & 5-CH2), 4.65 (s, 2H, OCH2Ph), 4.71 (s, 2H,
OCH2Ph), 4.80–4.87 (m, 2H, OCH2O), 5.50 (s, 2H, NCH2O), 6.31
(dd, J = 7.87, 5.70 Hz, 1H, 10-H), 7.22–7.41 (m, 10H, Ar), 7.65 (s,
1H, 6-H). 13C NMR (75 MHz, CDCl3) d ppm ꢀ5.50, ꢀ5.40, ꢀ4.84,
ꢀ4.65 (SiMe-C), 17.98, 18.39 (tBu-tC), 25.73, 25.93 (tBu-C), 41.35
(10-C), 62.99 (5-CH2-C), 63.07 (50-C), 69.39 (Bn-C), 70.45 (NCH2O–
C), 72.24 (30-C), 72.28 (Bn-C), 85.95 (10-C), 87.94 (40-C), 94.36
(OCH2O–C), 110.72 (5-C) 126.97, 127.61, 127.69, 127.92, 128.26,
128.39, 128.56 (Ar-C), 136.92 (6-C), 137.70, 137.93 (Ar-C), 150.80
(2-C), 162.24 (4-C). ESI-HRMS for [C38H58N2O8Si2+H]+ Calcd,
727.381. Found, 727.3805.
3.0.15. 3-((2R,3S,5R)-3-((tert-Butyldimethylsilyl)oxy)-5-(2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)propanenitrile (28)
Following the reaction procedure described for compound 15 but
using methanol as solvent, compound 27 (55 mg, 0.15 mmol) gave
28 as a white solid (50 mg, 89%). 1H NMR (300 MHz, CDCl3) d ppm
0.09 (d, 6H, SiMe2), 0.90 (s, 9H, tBu), 1.89 (dddd, J = 13.84, 9.45,
7.47, 6.15 Hz, 1H, 50-H), 2.03–2.15 (m, 1H, 50-H), 2.19–2.38 (m, 2H,
20-H), 2.42–2.62 (m, 2H, 60-H), 3.82 (ddd, J = 9.37, 5.42, 3.66 Hz,
1H, 40-H), 4.1
d 4
(dt, J = 7.03, 5.42 Hz, 1H, 30-H), 5.77 (d,
J = 7.91 Hz, 1H, 5-H), 6.08 (t, J = 6.59 Hz, 1H, 10-H), 7.26 (d,
J = 8.20 Hz, 1H, 6-H), 8.40 (br s, 1H, NH). 13C NMR (75 MHz, CDCl3)
d ppm ꢀ4.83 (SiMe-C), ꢀ4.54 (SiMe-C), 14.25 (60-C), 17.90 (tBu-
tC), 25.67 (tBu-C), 28.98 (50-C), 40.40 (20-C), 74.37 (30-C), 84.20 (40-
C), 85.95 (10-C), 102.83 (5-C), 119.01 (70-C), 140.08 (6-C), 149.84
(2-C), 162.84 (4-C). ESI-HRMS for [C17H27N3O4Si+H]+ Calcd,
366.1849. Found, 366.1842.
3.0.19. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-5-(((2-
(trimethylsilyl)ethoxy)methoxy)methyl)-3-((2-
(trimethylsilyl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione
(35)
3.0.16. 3-((2R,3S,5R)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-
yl)-3-hydroxytetrahydrofuran-2-yl)propanenitrile (29)
Following the reaction procedure described for compound 21,
compound 28 (50 mg, 0.14 mmol) rendered 29 (25 mg, 73%) as a
white solid. 1H NMR (300 MHz, CD3OD) d ppm 1.89–2.14 (m, 2H,
50-H), 2.20–2.37 (m, 2H, 20-H), 2.48–2.69 (m, 2H, 60-H), 3.85 (dt,
J = 9.23, 4.47 Hz, 1H, 40-H), 4.13–4.25 (m, 1H, 30-H), 5.71 (d,
J = 7.91 Hz, 1H, 5-H), 6.18 (t, J = 6.74 Hz, 1H, 10-H), 7.62 (d,
J = 8.20 Hz, 1H, 6-H). 13C NMR (75 MHz, CD3OD) d ppm 14.48 (60-C),
30.32 (50-C), 40.21 (20-C), 74.68 (30-C), 85.87 (40-C), 86.71 (10-C),
103.04 (5-C), 120.79 (70-C), 142.57 (6-C), 152.07 (2-C), 166.15 (4-C).
ESI-HRMS for [C11H13N3O4ꢀH]ꢀ Calcd, 250.0828. Found, 250.0833.
Under an inert condition compound 33 (486 mg, 1 mmol) was
dissolved in anhydrous CH2Cl2 (3 mL) and anhydrous DIPEA
(0.5 mL, 3 mmol) and added ethyltrimethysilylchloromethyl ether
(SEMCl, 0.44 ml, 2.5 mmol). The reaction was continued at 40 °C
for 6 h. The reaction mixture was partitioned between brine
(5 mL) and ethyl acetate (20 mL). The organic layer was separated
and dried over anhyd Na2SO4. The residue after evaporation of or-
ganic phase was purified by flash column chromatography (5%
EtOAc in hexanes) to afford compound 35 (370 mg, 50%) as a glass.
1H NMR (300 MHz, CDCl3) d ppm 0.00 (s, 9H, SiMe3-SEM), 0.03 (s,
9H, SiMe3-SEM), 0.08 (s, 3H, SiMe-TBS), 0.09 (s, 3H, SiMe-TBS), 0.11
(s, 6H, SiMe2-TBS), 0.90 (s, 9H, tBu), 0.92 (s, 9H, tBu), 0.94–1.02 (m,
4H, Me3SiCH2), 2.00 (ddd, J = 15.3 Hz,7.8 Hz, 1.8 Hz, 1H, 20-H), 2.31
(ddd, J = 13.2 Hz, 5.7 Hz, 2.4 Hz, 1H, 20-H), 3.60–3.73 (m, 4H,
OCH2CH2SiMe3), 3.76 (dd, J = 11.10, 3.15 Hz, 1H, 50-H), 3.82 (dd,
J = 11.29, 3.33 Hz, 1H, 50-H), 3.96 (q, J = 2.96 Hz, 1H, 40-H), 4.31
(d, J = 12.24 Hz, 1H, 5-CH2), 4.37 (d, J = 12.0 Hz, 1H, 5-CH2), 4.37–
4.42 (m, 1H, 30-H), 4.74 (s, 2H, OCH2O), 5.40 (s, 2H, NCH2O), 6.32
(1H, dd, J = 7.8 Hz, 5.7 Hz, 10-H), 7.65 (s, 1H, 6-H). 13C NMR
(75 MHz, CDCl3): d ppm -5.50, -5.38, -4.84, -4.67 (TBSSiMe–C),
ꢀ1.44, ꢀ1.38 (SEMSiMe3–C), 17.97 (t-Bu-tC), 18.07, 18.11 (SEMS-
iCH2–C); 18.40 (t-Bu-tC), 25.72, 25.95 (tBu-C), 41.34 (20-C), 62.96
(5-CH2O–C), 62.99 (50-C), 65.28, 67.49 (SEMCH2O–C), 70.22
(NCH2O–C), 72.43 (30-C), 86.11 (10-C), 88.06 (40-C), 94.94
(OCH2O–C), 111.08 (5-C), 136.71 (6-C), 150.99 (2-C), 162.34 (4-
C). ESI-HRMS for [C34H70N2O8Si4+H]+ Calcd, 747.4287. Found,
747.4144.
3.0.17. (1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl acetate (32)
Following the procedure described for the synthesis of 12, com-
pound 31 (1 g, 1.94 mmol) rendered 32 (265 mg, 33%) as a white
foam.
1H NMR (300 MHz, CDCl3) d ppm 0.09 (s, 6H, SiMe), 0.90 (s, 9H,
tBu), 2.06 (s, 3H, OAc), 2.18–2.40 (m, 2H, 20-H), 3.77 (dd, J = 12.7,
3.2 Hz,1H, 50-H), 3.88–4.04 (m, 2H, 50 & 40-H), 4.52 (dt, J = 5.80,
3.70 Hz, 1H, 30-H), 4.79–4.97 (m, 2H, 5-CH2O), 6.24 (t, J = 6.49 Hz,
1H, 10-H), 8.11 (s, 1H, 6-H), 9.02 (s, 1H, NH). 13C NMR (75 MHz,
CDCl3) d ppm ꢀ4.90, ꢀ4.74 (SiMe-C), 17.94 (tBu-tC), 21.14 (Ac-C),
25.69 (tBu-C), 41.36 (10-C), 58.82 (5-CH2O–C), 61.82 (50-C), 71.66
(30-C), 86.49 (10-C), 88.12 (40-C), 109.02 (5-C), 142.86 (6-C),
149.94 (2-C), 162.58 (4-C), 171.96 (Ac-C@ O). ESI-HRMS for
[C18H30N2O7Si+H]+ Calcd, 415.1901. Found, 415.1904.
3.0.20. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-5-(((2-
(trimethylsilyl)ethoxy)methoxy)methyl)-3-((2-
(trimethylsilyl)ethoxy)methyl)pyrimidine-2,4(1H,3H)-dione
(36)
3.0.18. 5-(((Benzyloxy)methoxy)methyl)-3-((benzyloxy)-
methyl)-1-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-
yl)pyrimidine-2,4(1H,3H)-dione (34)
To a mixture of compound 33 (150 mg, 0.31 mmol) in anhy-
drous DMF (2.5 mL) and anhydrous diisopropylethylamine (DIPEA,
Following the procedure described for the synthesis of 12, com-
pound 35 (350 mg, 0.48 mmol) gave compound 36 as a glass

266
K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
(114 mg, 38%). 1H NMR (300 MHz, CDCl3) d ppm 0.00 (s, 9H, SiMe3-
SEM), 0.03 (s, 9H, SiMe3-SEM), 0.09 (s, 6H, SiMe-TBS), 0.90 (s, 9H,
tBu); 0.92–1.00 (m, 4H, SEMSiCH2), 2.22–2.41 (m, 2H, 20-H), 2.58
(t, J = 4.8 Hz, 1H, 50-OH), 3.60–3.72 (m, 4H, SEMCH2O), 3.72–3.80
(m, 1H, 50-H), 3.87–3.93 (m, 1H, 50-H), 3.93–3.98 (m, 1H, 40-H),
4.39 (s, 2H, 5-CH2O), 4.51 (1H, dt, J = 6.6, 3.81 Hz, 30-H), 4.73 (s,
2H, OCH2O), 5.39 (s, 2H, NCH2O), 6.20 (t, J = 6.44 Hz, 1H, 10-H),
7.75 (s, 1H, 6-H). 13C NMR (75 MHz, CDCl3) d ppm ꢀ4.86, ꢀ4.69
(TBSSiMe–C), ꢀ1.43, ꢀ1.42 (SEMSiMe3–C), 17.95 (tBu-tC), 18.12
(SEMSiCH2–C), 25.71 (tBu-C), 40.91 (20-C), 62.04 (50-C), 62.27 (5-
CH2O–C), 65.56, 67.58 (SEMCH2O–C), 70.03 (NCH2O–C), 71.68 (30-
C), 87.80 (10-C), 87.88 (40-C), 93.96 (OCH2O–C), 110.24 (5-C),
138.05 (6-C), 150.86 (2-C), 162.03 (4-C). ESI-HRMS for
[C28H56N2O8Si3+H]+ Calcd, 633.3423. Found, 633.3451.
3.0.23. (1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2,4-
dioxo-1,2,3,4-tetrahydropyrimidin-5-yl)methyl pivalate (39)
To a solution of 33 (2.0 g, 4.1 mmol) in anhydrous pyridine
(20 mL) and DMAP (100 mg) under inert atmosphere was added
pivaloyl chloride (0.76 mL, 6.16 mmol) and the reaction mixture
was stirred at room temperature overnight (18 h). Pyridine was
evaporated under reduced pressure and the residue was purified
by flash column chromatography (10–25% EtOAc in hexanes) to af-
ford 39 as a white solid (2.0 g, 74%). 1H NMR (300 MHz, CDCl3) d
ppm 0.09 (s, 3H, SiMe), 0.09 (s, 2H, SiMe), 0.10 (s, 6H, SiMe2),
0.90 (d, 9H, Si-tBu), 0.91 (d, 9H, Si-tBu), 1.19 (s, 9H, Piv-tBu), 2.01
(ddd, J = 13.40, 7.69, 5.86 Hz, 1H, 20-H), 2.32 (ddd, J = 13.18, 5.86,
2.64 Hz, 1H, 20-H), 3.73–3.85 (m, 2H, 50-H), 3.96 (q, J = 3.22 Hz,
1H, 40-H), 4.40 (dt, J = 5.49, 2.67 Hz, 1H, 30-H), 4.80 (s, 2H, 5-CH2),
6.28 (dd, J = 7.91, 5.86 Hz, 1H, 10-H), 7.78 (s, 1H, 6-H), 8.28 (s, 1H,
NH). 13C NMR (75 MHz, CDCl3) d ppm ꢀ5.42 (SiMe-C), -5.38
(SiMe-C), ꢀ4.83 (SiMe-C), ꢀ4.65 (SiMe-C), 18.01 (Si-tBu-tC),
18.42 (Si-tBu-tC), 25.74 (Si-tBu-C), 25.93 (Si-tBu-C), 27.15 (Piv-
tBu-C), 38.79 (Piv-tBu-tC), 41.39 (20-C), 59.07 (5-CH2-C), 63.11
(50-C), 72.36 (30-C), 85.51 (10-C), 88.09 (40-C), 109.62 (5-C), 140.25
(6-C), 149.76 (2-C), 161.86 (4-C), 178.23 (Piv-C@ O–C). ESI-HRMS
for [C27H50N2O7Si2+H]+ Calcd, 571.3235. Found, 471.3242.
3.0.21. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-((E)-2-
(methylsulfonyl)vinyl)tetrahydrofuran-2-yl)-5-(((2-(trimethyl-
silyl)ethoxy)methoxy)methyl)-3-((2-(trimethylsilyl)ethoxy)-
methyl)pyrimidine-2,4(1H,3H)-dione (37)
Following the procedure described for the synthesis of 14, com-
pound 36 (113 mg, 0.18 mmol) rendered 37 as white solid (33 mg,
29%). 1H NMR (300 MHz, CDCl3) d ppm 0.00 (s, 9H, SEM SiMe3),
0.02 (s, 9H, SEM SiMe3), 0.10 (s, 3H, TBSSiMe), 0.10 (s, 3H, TBSSiM-
e), 0.90 (s, 9H, tBu), 0.92–1.02 (s, 4H, SEMSiCH2), 2.15–2.40 (m, 2H,
20-C), 2.97 (s, 3H, SO2Me), 3.58–3.75 (m, 4H, SEMCH2O), 4.33 (dt,
J = 6.4, 4.7 Hz, 1H, 40-H), 4.40 (s, 2H, 5-CH2O), 4.47 (td, J = 4.7,
1.8 Hz, 1H, 30-H), 4.73 (s, 2H, OCH2O), 5.39 (s, 2H, NCH2O), 6.33
(t, J = 6.6 Hz, 1H, 10-H), 6.69 (dd, J = 15.3 Hz, 1.8 Hz, 1H, 60-H),
7.00 (1H, dd, J = 15 Hz, 4.2 Hz, 50-H), 7.34 (s, 1H, 6-H). ESI-HRMS
for [C30H58N2O9SSi3+Na]+ Calcd, 729.3069. Found, 729.1077.
3.0.24. (1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-
(hydroxymethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl pivalate (40)
Following the procedure described for the synthesis of 12, com-
pound 39 (2.0 g, 3.05 mmol) gave compound 40 as a white solid
(822 mg, 59%). 1H NMR (300 MHz, CDCl3) d ppm 0.09 (s, 6H, SiMe),
0.90 (s, 9H,TBS-tBu), 1.18 (s, 9H, Piv-tBu), 2.19–2.38 (m, 2H, 20-H),
2.94 (t, J = 5.32 Hz, 1H, 50-OH), 3.70–3.81 (m, 1H, 50-H), 3.90–3.99
(m, 2H, 50 & 40-H), 4.52 (dt, J = 6.25, 3.54 Hz, 1H, 30-H), 4.83–4.94
(m, 2H, 5-CH2O), 6.23 (t, J = 6.67 Hz, 1H, 10-H), 8.08 (s, 1H, 6-H),
8.78 (s, 1H, NH). 13C NMR (75 MHz, CDCl3) d ppm ꢀ4.86, ꢀ4.70
(SiMe-C), 17.98 (TBS tBu-tC), 25.73 (tBu-C), 27.04 (tBu-C), 38.97
(piv tBu-tC), 41.42 (20-C), 58.63 (5-CH2O-C), 62.06 (50-C), 71.91
(30-C), 86.60 (10-C), 88.29 (40-C), 109.26 (5-C), 142.71 (6-C),
149.70 (2-C), 161.95 (4-C), 179.70 (PivCO-C). ESI-HRMS for
[C21H36N2O7Si+H]+ Calcd, 457.2370. Found, 457.2414.
3.0.22. 1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(((tert-
butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-3-(4-
methoxybenzyl)-5-(((4-methoxybenzyl)oxy)methyl)-
pyrimidine-2,4(1H,3H)-dione (38)
To a stirring solution of 33 (200 mg, 0.41 mmol) in anhydrous
DMF (2 mL) at 0 °C under argon atmosphere was added 4-
methoxybenzyl chloride (PMBCl, 167 lL, 1.23 mmol) followed
by NaH (60% in mineral oil, 50 mg, 1.23 mmol) portion wise
and stirred at room temperature for 4 h. Ethyl acetate (20 mL)
and saturated aq.NH4Cl (10 mL) was added. The aqueous layer
was extracted with ethyl acetate (3 ꢁ 20 mL) and the combined
organic layers were dried over anhyd Na2SO4, filtered, evaporated.
The residue was purified by flash column chromatography (10%
EtOAc in hexanes) to afford the title compound 38 as a white
foam (40 mg, 13%). 1H NMR (300 MHz, CDCl3) d ppm 0.05 (s,
3H, SiMe), 0.06 (s, 6H, SiMe), 0.07 (s, 3H, SiMe), 0.88 (s, 9H,
tBu), 0.89 (s, 9H, tBu), 1.97 (ddd, J = 13.40, 7.69, 6.15 Hz, 1H, 20-
H), 2.26 (ddd, J = 13.18, 5.71, 2.49 Hz, 1H, 20-H), 3.72–3.76 (m,
1H, 50-H), 3.77 (s, 3H, PMB-OMe), 3.79 (s, 2H, PMB-OMe), 3.92
(q, J = 3.22 Hz, 1H, 50-H), 4.18–4.33 (m, 2H, 5-CH2O,) 4.37 (dt,
J = 5.64, 2.60 Hz, 1H, 30-H), 4.52 (s, 2H, PMB OCH2), 4.95–5.13
(m, 2H, PMB NCH2), 6.32 (dd, J = 7.91, 5.56 Hz, 1H, 10-H), 6.82
(d, J = 8.79 Hz, 2H, Ar), 6.86 (d, J = 8.49 Hz, 2H, Ar), 7.26 (d,
J = 8.49 Hz, 2H, Ar), 7.44 (d, J = 8.79 Hz, 2H, Ar), 7.59 (s, 1H, 6-
H). 13C NMR (75 MHz, CDCl3) d ppm ꢀ5.51, ꢀ5.43, ꢀ4.84, ꢀ4.66
(SiMe-C), 17.98, 18.39 (tBu-tC), 25.74, 25.94 (tBu-C), 41.17 (20-
C), 43.88 (NCH2–C), 55.23, 55.25 (OMe–C), 63.01 (50-C), 65.06
(5-CH2O–C), 72.30 (30-C), 72.81 (PMBCH2O–C), 85.83 (10-C),
87.80 (40-C), 111.19 (5-C), 113.69, 113.80, 129.05, 129.51,
130.06, 130.73 (Ar-C) 135.99 (6-C), 150.80 (2-C), 159.04, 159.27
(Ar-C), 162.23 (2-C). ESI-HRMS for [C38H58N2O8Si2+H]+ Calcd,
727.3810. Found, 727.3797.
3.0.25. (1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(2-
cyanovinyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl pivalate (41)
Following the similar procedure to synthesize 13, the reaction
of compound 40 (0.8 g, 1.75 mmol), Dess–Martin reagent (0.48 M
in CH2Cl2, 4.4 mL, 2.1 mmol) at room temperature for 7 h gave
aldehyde as white foamy residue.
In a separate flask, to a solution of cyanomethyltriphenylphos-
phonium chloride (1.78 g, 5.25 mmol) in anhydrous THF (30 mL)
at ꢀ78 °C under argon condition was added drop wise n-BuLi
(1.6 M in hexanes, 3.3 mL, 5.25 mmol) and stirred for 30 min. To
this ylide at ꢀ78 °C was added slowly a solution of above aldehyde
in anhydrous THF (5 mL) and stirred at room temperature over-
night. Following the similar workup procedure described for com-
pound 19, afforded 41 as a white solid (E-isomer–450 mg and E + Z
mixture–240 mg, 82%). Data for E-isomer: 1H NMR (300 MHz,
CDCl3) d ppm 0.10 (s, 3H, SiMe), 0.11 (s, 3H, SiMe), 0.90 (s, 9H,
TBS tBu), 1.20 (s, 9H, Piv tBu), 2.21–2.42 (m, 2H, 20-H), 4.25–4.38
(m, 2H, 30 & 40-H), 4.81–4.95 (m, 2H, 5-CH2O), 5.72 (dd, J = 16.40,
1.76 Hz, 1H, 60-H), 6.24 (t, J = 6.44 Hz, 1H, 10-H), 6.79 (dd,
J = 16.26, 4.54 Hz, 1H, 50-H), 7.54 (s, 1H, 6-H), 9.34 (br s, 1H, NH).

K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
267
13C NMR (75 MHz, CDCl3) d ppm ꢀ4.84, ꢀ4.66 (SiMe-C), 17.89 (TBS
tBu-tC), 25.63 (tBu-C), 27.12 (tBu-C), 38.91 (Piv tBu-tC), 40.10 (20-
C), 58.36 (5-CH2O-C), 74.53 (30-C), 85.12 (40-C), 86.07 (10-C), 101.41
(60-C), 110.38 (70-C), 116.49 (5-C), 140.93 (6-C), 149.39 (50-C),
149.70 (2-C), 162.32 (4-C), 178.84 (Piv CO–C). ESI-HRMS for
[C23H35N3O6Si+H]+ Calcd, 478.2373. Found, 478.2383.
151.87 (2-C), 164.85 (4-C), 180.09 (Piv CO-C). ESI-HRMS for
[C17H23N3O6 + H]+ Calcd, 366.1665. Found, 366.1631.
3.0.29. 3-((2R,3S,5R)-3-hydroxy-5-(5-(hydroxymethyl)-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-
yl)propanenitrile (30)
3.0.26. (1-((2R,4S)-5-(2-cyanoethylidene)-4-
hydroxytetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl pivalate (42)
A solution of 0.5 M NaOMe in methanol (0.7 mL, 0.342 mmol)
was added to compound 44 (25 mg, 0.068 mmol) and stirred at
room temperature for 3 h. Acetic acid (30 lL, 0.5 mmol) was added
and the solvents were evaporated. The residue was purified by
flash column chromatography (4–8% MeOH in CH2Cl2) to afford
30 as a white solid (19 mg, 95%). 1H NMR (300 MHz, DMSO-d6) d
ppm 1.76–1.90 (m, 1H, 50-H), 1.90–2.03 (m, 1H, 50-H), 2.06–2.25
(m, 2H, 20-H), 2.53–2.68 (m, 2H, 60-H), 3.72 (dt, J = 8.49, 4.54 Hz,
1H, 40-H), 4.05–4.13 (m, 1H, 30-H), 4.16 (s, 2H, 5-CH2O), 4.97 (br
s, 1H, 5-MeOH), 5.39 (br s, 1H, 30-OH), 6.16 (t, J = 6.74 Hz, 1H, 10-
H), 7.42 (s, 1H, 6-H), 11.34 (br s, 1H, NH). 13C NMR (75 MHz,
DMSO-d6) d ppm 13.21 (60-C), 28.53 (50-C), 38.36 (20-C), 55.70 (5-
CH2O–C), 72.53 (30-C), 83.87 (10-C), 83.97 (40-C), 114.46 (5-C),
120.17 (70-C), 136.38 (6-C), 150.13 (2-C), 162.42 (4-C). ESI-HRMS
for [C12H15N3O5+HCOOꢀ]ꢀ Calcd, 326.0988. Found, 326.0985.
To a solution of compound 41 (65 mg, 0.136 mmol) in THF
(1 mL) was added tetrabutylammonium hydroxide (Bu4N+OHꢀ,
40% in H2O, 186 mg, 0.286 mmol) and stirred at room temperature
for 5 h. Volatiles were evaporated under reduced pressure and the
residue was purified by flash column chromatography (40–70%
EtOAc in hexanes) to afford 42 as a white powder (20 mg, 40%).
1H NMR (300 MHz, CDCl3) d ppm 1.19 (s, 9H, tBu), 2.20 (d,
J = 3.51 Hz, 1H, 30-OH), 2.31 (dt, J = 13.84, 6.70 Hz, 1H, 20-H), 2.56
(ddd, J = 14.06, 6.44, 2.64 Hz, 1H, 20-H), 3.17 (dd, J = 7.03, 1.17 Hz,
2H, 60-H), 4.77 (t, J = 7.03 Hz, 1H, 50-H), 4.83–4.92 (m, 3H, 30-H &
5-CH2O-H), 6.62 (t, J = 6.74 Hz, 1H, 10-H), 7.38 (s, 1H, 6-H), 8.46 (s,
1H, NH). 13C NMR (75 MHz, CDCl3) d ppm 13.70 (6-C), 27.10 (tBu-
C) 38.90 (tBu-tC), 39.22 (20-C), 58.48 (5-CH2O-C), 70.25 (30-C),
87.13 (10-C), 89.96 (50-C), 111.06 (5-C), 117.77 (70-C), 139.55 (6-
C), 149.40 (2-C), 159.39 (4-C), 161.42 (40-C), 178.68 (Piv CO-C).
ESI-HRMS for [C17H21N3O6+H]+ Calcd, 364.1509. Found, 364.1455.
3.0.30. (1-((2R,4S,5R)-5-(2-(1H-tetrazol-5-yl)ethyl)-4-((tert-
Butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl pivalate (45)
Following the procedure described for the synthesis of 22, com-
pound 43 (80 mg, 0.17 mmol) rendered 45 as white solid (74 mg,
74%). 1H NMR (300 MHz, CDCl3) d ppm 0.08 (s, 6H, SiMe), 0.89 (s,
9H, TBS tBu), 1.24 (s, 9H, Piv tBu), 1.74–1.91 (m, 1H, 50-H), 2.04–
2.17 (m, 1H, 20-H), 2.19–2.33 (m, 1H, 50-H), 2.39 (ddd, J = 13.55,
5.93, 3.95 Hz, 1H, 20-H), 3.13–3.28 (m, 2H, 60-H), 4.03 (dt, J = 10.69,
3.00 Hz, 1H, 40-H), 4.10–4.18 (m, 1H, 30-H), 4.93–5.08 (m, 2H, 5-
CH2O), 6.21 (t, J = 6.30 Hz, 1H, 10-H), 7.79 (s, 1H, 6-H), 8.99 (br s,
1H, NH). 13C NMR (75 MHz, CDCl3) d ppm ꢀ4.83, ꢀ4.66 (SiMe-C),
17.95 (TBS tBu-tC), 20.40 (60-C), 25.68 (tBu-C), 27.12 (tBu-C),
33.34 (50-C), 39.38 (Piv tBu-tC), 40.56 (20-C), 59.14 (5-CH2O–C),
75.13 (30-C), 86.66 (40-C), 87.00 (10-C), 109.27 (5-C), 142.20 (6-C),
149.73 (2-C), 155.28 (70-C), 162.27 (4-C), 182.48 (Piv CO-C). ESI-
HRMS for [C23H38N6O6SiꢀH]ꢀ Calcd, 521.2544. Found, 521.2551.
3.0.27. (1-((2R,4S,5R)-4-((tert-Butyldimethylsilyl)oxy)-5-(2-
cyanoethyl)tetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl pivalate (43)
To a solution of 41 (240 mg, 0.5 mmol) in ethylacetate (10 mL)
was added 5%Pt-C (250 mg) and stream of hydrogen gas was bub-
bled through the reaction mixture for 4 h. The catalyst was filtered
off and the filtrate was concentrated. The residue was purified by
flash column chromatography (20–40% EtOAc in hexanes) to afford
43 as a white solid (170 mg, 70%). 1H NMR (300 MHz, CDCl3) d ppm
0.09 (s, 3H, SiMe), 0.10 (s, 3H, SiMe), 0.90 (s, 9H, TBS tBu), 1.20 (s,
9H, Piv tBu), 1.94 (dddd, J = 13.88, 9.63, 7.54, 6.15 Hz, 1H, 50-H),
2.02–2.17 (m, 1H, 50-H), 2.23 (ddd, J = 13.40, 7.25, 5.71 Hz, 1H, 20-
H), 2.34 (ddd, J = 13.77, 7.03, 5.86 Hz, 1H, 20-H), 2.43–2.65 (m,
2H, 60-H), 3.85 (ddd, J = 9.37, 5.42, 3.66 Hz, 1H, 40-H), 4.16 (dt,
J = 11.13, 5.57 Hz, 1H, 30-H), 4.88 (s, 2H, 5-CH2O), 6.12 (dd,
J = 6.74, 5.86 Hz, 1H, 10-H), 7.55 (s, 1H, 60-H), 9.13 (br s, 1H, NH).
13C NMR (75 MHz, CDCl3) d ppm ꢀ4.88, ꢀ4.61 (SiMe-C), 14.22
(60-C), 17.86 (TBS tBu-tC), 25.64 (tBu-C), 27.09 (tBu-C), 28.96 (50-
C), 38.86 (Piv tBu-tC), 40.48 (20-C), 58.38 (5-CH2O–C), 74.27 (30-
C), 84.26 (40-C), 85.84 (10-C), 109.99 (5-C), 118.95 (70-C), 141.07
(6-C), 149.64 (2-C), 162.27 (4-C), 178.77 (Piv CO-C). ESI-HRMS for
[C23H37N3O6Si+H]+ Calcd, 480.2530. Found, 480.2535.
3.0.31. 1-((2R,4S,5R)-5-(2-(1H-tetrazol-5-yl)ethyl)-4-((tert-
butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-5-
(hydroxymethyl)pyrimidine-2,4(1H,3H)-dione (46)
Following the procedure described for the synthesis of 30, com-
pound 45 (90 mg, 0.17 mmol) after chromatography (5–7% MeOH
+ 0.5% HCOOH in CH2Cl2) rendered 46 as white solid (70 mg,
92%). 1H NMR (300 MHz, CD3OD) d ppm 0.11 (s, 6H, SiMe), 0.91
(s, 9H, tBu), 2.00–2.12 (m, 1H, 50-H), 2.12–2.38 (m, 3H, 20 & 50-H),
2.99–3.19 (m, 2H, 60-H), 3.82 (dt, J = 9.37, 4.10 Hz, 1H, 40-H), 4.31
(dt, J = 6.00, 4.32 Hz, 1H, 30-H), 4.35 (d, J = 1.17 Hz, 2H, 5-CH2O),
6.20 (t, J = 6.74 Hz, 1H, 10-H), 7.53 (s, 1H, 6-H). 13C NMR (75 MHz,
CD3OD) d ppm ꢀ4.70, ꢀ4.50 (SiMe-C), 18.80 (tBu-tC), 21.38 (60-
C), 26.25 (tBu-C), 32.52 (50-C), 40.87 (20-C), 57.80 (5-CH2O–C),
76.34 (30-C), 86.61 (10-C), 87.00 (40-C), 115.70 (5-C), 138.70 (6-C),
152.12 (2-C), 158.26 (70-C), 165.00 (4-C). ESI-HRMS for
[C18H30N6O5SiꢀH]ꢀ Calcd, 437.1969. Found, 437.1982.
3.0.28. (1-((2R,4S,5R)-5-(2-cyanoethyl)-4-
hydroxytetrahydrofuran-2-yl)-2,4-dioxo-1,2,3,4-
tetrahydropyrimidin-5-yl)methyl pivalate (44)
Following the synthetic procedure described for compound 21,
compound 43 (80 mg, 0.17 mmol) afforded product 44 as a white
solid (55 mg, 90%). 1H NMR (300 MHz, CD3OD) d ppm 1.18 (s, 9H,
tBu), 1.92–2.06 (m, 1H, 50-H) 2.06–2.16 (m, 1H, 50-H), 2.29 (dd,
J = 6.59, 5.71 Hz, 2H, 20-H), 2.52–2.71 (m, 2H, 60-H), 3.87 (dt,
J = 9.01, 4.43 Hz, 1H, 40-H), 4.17–4.24 (m, 1H, 30-H), 4.84 (s, 2H,
5-CH2O), 6.20 (t, J = 6.59 Hz, 1H, 10-H), 7.78 (s,1H, 6-H). 13C NMR
(75 MHz, CD3OD) d ppm 14.51 (60-C), 27.53 (tBu-C), 30.37(50-C),
39.90 (tBu-tC), 40.30 (20-C), 60.22 (5-CH2O), 74.70 (30-C), 86.05
(40-C), 86.85 (10-C), 110.70 (5-C), 120.82 (70-C), 143.01 (6-C),
3.0.32. 1-((2R,4S,5R)-5-(2-(1H-tetrazol-5-yl)ethyl)-4-
hydroxytetrahydrofuran-2-yl)-5-(hydroxymethyl)pyrimidine-
2,4(1H,3H)-dione (47)
In a polypropylene vessel, to a solution of compound 46 (70 mg,
0.16 mmol) in methanol (10 mL) was added NH4F (120 mg,

268
K. S. Toti et al. / Bioorg. Med. Chem. 21 (2013) 257–268
Guler, S. Y.; Hentemann, M. F.; Joseph-McCarthy, D.; Kawatkar, S.; Kutschke, A.;
Loch, J. T.; McKenzie, A. R.; Pradeepan, S.; Prasad, S.; Martínez-Botella, G. ACS
Chem. Biol. 2012.
3.2 mmol) and stirred at 50 °C for 2 days. Dichloromethane (10 mL)
was added and filtered. The filtrate was concentrated and the res-
idue purified by flash column chromatography (8–12% MeOH +
0.5% HCOOH in CH2Cl2) to afford 47 as a white solid (45 mg,
87%).1H NMR (300 MHz, DMSO-d6) d ppm 1.88–2.01 (m, 1H, 50-
H), 2.01–2.24 (m, 3H, 50 & 20-H), 2.86–3.05 (m, 2H, 60-H), 3.65–
3.75 (m, 1H, 40-H), 4.05–4.13 (m, 1H, 30-H), 4.16 (d, J = 0.88 Hz,
2H, 5-CH2O), 6.15 (t, J = 6.74 Hz, 1H, 10-H), 7.44 (s, 1H, 6-H),
11.35 (br s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) d ppm 19.65
(60-C), 30.89 (50-C), 38.63 (20-C), 55.69 (5-CH2–C), 72.77 (30-C),
83.60 (10-C), 84.50 (40-C), 114.48 (5-C), 136.18 (6-C), 150.14 (2-
C), 155.96 (70-C), 162.42 (4-C). ESI-HRMS for [C12H16N6O5ꢀH]ꢀ
Calcd, 323.1104. Found, 323.1234.
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Supplementary data
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