Synthesis and biological evaluation of branched and conformationally restricted analogs of the anticancer compounds 3′-C-ethynyluridine (EUrd) and 3′-C-ethynylcytidine (ECyd)

Article abstract of DOI:10.1016/j.bmc.2005.01.029

The synthesis of branched and conformationally restricted analogs of the anticancer nucleosides 3′-C-ethynyluridine (EUrd) and 3′-C- ethynylcytidine (ECyd) is presented. Molecular modeling and ¹H NMR coupling constant analysis revealed that the furanose rings of all analogs except the LNA analog are conformationally biased towards South conformation, and are thus mimicking the structure of ECyd. All target nucleosides were devoid of anti-HIV or anticancer activity.

Full text of DOI:10.1016/j.bmc.2005.01.029

Bioorganic & Medicinal Chemistry 13 (2005) 2597–2621
Synthesis and biological evaluation of branched and
conformationally restricted analogs of the anticancer compounds
30-C-ethynyluridine (EUrd) and 30-C-ethynylcytidine (ECyd)
Patrick J. Hrdlicka,a Nicolai K. Andersen,a Jan S. Jepsen,b Flemming G. Hansen,a
Kim F. Haselmann,c Claus Nielsend and Jesper Wengela,*
aNucleic Acid Center,ꢀ Department of Chemistry, University of Southern Denmark, DK-5230Odense M, Denmark
bDanish Cancer Society, Institute of Cancer Biology, Department of Tumor Endocrinology, DK-2100 Copenhagen, Denmark
cMS-Laboratory, Department of Chemistry, University of Southern Denmark, DK-5320Odense M, Denmark
dRetrovirus Laboratory, Department of Virology, State Serum Institut, DK-2300 Copenhagen, Denmark
Received 24 September 2004; revised 13 January 2005; accepted 18 January 2005
Abstract—The synthesis of branched and conformationally restricted analogs of the anticancer nucleosides 30-C-ethynyluridine
1
(EUrd) and 30-C-ethynylcytidine (ECyd) is presented. Molecular modeling and H NMRcoupling constant analysis revealed that
the furanose rings of all analogs except the LNA analog are conformationally biased towards South conformation, and are thus
mimicking the structure of ECyd. All target nucleosides were devoid of anti-HIV or anticancer activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1.Introduction
HO
B
HO
B
HO
B
O
O
O
30-C-Ethynyluridine (EUrd, 1) and its cytidine analog
(ECyd, 2) (Fig. 1) display excellent broad-spectrum anti-
tumor activities in vitro as well as in tumor models.1
EUrd and ECyd undergo phosphorylation catalyzed
by uridine–cytidine kinase (EC 2.7.1.48, UCK) to give
the corresponding 50-monophosphates, which are fur-
ther phosphorylated by nucleotide kinases to the phar-
macologically active triphosphates. The triphosphates
of EUrd and ECyd competitively inhibit human RNA
polymerases I, II, III, and thereby DNA templated
RNA synthesis, which leads to cell apoptosis presum-
ably via RNase L catalyzed rRNA fragmentation.2–4
Both nucleosides are distributed very selectively into tu-
mor tissue,5,6 which accounts for the absence of severe
toxicities in nude rat models,2,7 and renders them as
promising agents in the therapy of solid human cancers.
O
OH
O
OH OH
OH
1 B = uracil-1-yl
2 B = cytosin-1-yl
3
4
HO
B
HO
U
O
HO
HO
B
O
O
O
O
OH
OH OH
OH
5 B = uracil-1-yl
6 B = cytosin-1-yl
8 B = uracil-1-yl
9 B = cytosin-1-yl
7
CH3
HO
HO
HO
B
B
O
O
OH OH
OH OH
Keywords: Bicyclic nucleosides; Branched nucleosides; EUrd; TAS-1-
06; LNA.
10 B = uracil-1-yl
11 B = cytosin-1-yl
12 B = uracil-1-yl
13 B = cytosin-1-yl
*
Corresponding author. Tel.: +45 65502510; fax: +45 66158780;
e-mail: jwe@chem.sdu.dk
ꢀ A research center funded by the Danish National Research Foun-
dation for studies on nucleic acid chemical biology.
Figure 1. EUrd 1, ECyd 2, conformationally restricted nucleosides 3–4
and target nucleosides 5–13.
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.01.029

2598
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
ECyd, which is more potent than EUrd, is currently in
Phase I clinical trials.
solution.11 Most enzymes involved in nucleoside metab-
olism and nucleotide polymerization have strict confor-
mational preferences for the furanose ring of their
substrates. Conformational restriction of the furanose
ring has been a successful strategy to probe for confor-
mational preferences of such enzymes, and has revealed
that activating kinases and the final target enzymes may
have antipodal conformational requirements for the
furanose ring.14–17 30-O,40-C-methylene ribonucleosides
(3, Fig. 1)18 and 20-O,40-C-Methylene ribonucleosides
(locked nucleic acid nucleosides, i.e., LNA nucleosides)
(4, Fig. 1),19 represent two classes of conformationally
restricted nucleosides where the furanose ring is fixed
in South and North conformations, respectively.
Several ECyd-analogs have been synthesized, which has
clarified the importance of the nucleobase,1 the C30-sub-
stituent,8–10 the configurational requirements at the
C20- and C30-positions,8 and the 40-oxo function11 for
anticancer activity. The major conclusion from these
structure–activity relationship (SAR) studies is that
UCK has a very strict substrate specificity, which has
precluded development of even more potent analogs.
While EUrd/ECyd are sufficiently phosphorylated, ana-
logs carrying bulkier C30-substituents such as ethyl,
ethenyl, or cyclopropyl are not.8 30-Deoxy and xylo-
EUrd/ECyd-analogs are also inactive implying that
ribo-configuration of the furanose moiety is important
for UCK-catalyzed phosphorylation.8
A set of EUrd/ECyd analogs with the furanose rings re-
stricted in S-type or N-type sugar conformations may
elucidate the substrate preference of UCK. Further-
more, if UCK, nucleotide kinases and RNA polymerase
have similar preferences for the sugar conformation of
their substrates, the favorable entropic gain arising from
preorganization of the furanose ring, may potentially
lead to stronger binding and increased anticancer activ-
ity. We therefore chose to evaluate 30-O,40-C-methylene-
linked bicyclic EUrd/ECyd analogs 5–6 and LNA–EUrd
analog 7 (Fig. 1). Furthermore, since no SARstudies
have evaluated the effect of additional branching at the
C-40- and C-50-position on anticancer activity, we ad-
dressed this shortcoming by synthesizing and evaluating
40-C-hydroxymethyl EUrd/ECyd-analogs 8–9, 50-C-
hydroxymethyl EUrd/ECyd-analogs 10–11 and 50-C-
methyl EUrd/ECyd-analogs 12–13 (Fig. 1).
The furanose ring of nucleosides in solution normally
adopts a number of conformations, which can be
described by the phase angle P in the pseudorotational
cycle (Fig. 2).12 In solid state, however, nucleosides
typically cluster in two antipodal domains centered
around P = 0ꢁ (North) and P = 180ꢁ (South).13 The
furanose ring of ECyd is known to adopt a South con-
formation in crystalline state (P = 182ꢁ) as well as in
3′
HO
B
O
2′
3T2
2.Synthesis
North
2.1. Synthesis of key intermediate 20 toward 40-C-hydr-
oxymethyl, 30-O,40-C-methylene-linked, and LNA-type
EUrd/ECyd analogs
3E
0o
Triol 20 was identified as a suitable divergence point to-
ward the bicyclic nucleosides 5–7 and the 4-C-hydroxy-
methyl nucleosides 8–9 (Scheme 1). The starting
material for synthesis of triol 20, 4-C-hydroxymethyl
furanose 14,20 was conveniently obtained in four steps
from inexpensive 1,2;5,6-di-O-isopropylidene-a-D-glu-
cose without purification of the intermediates in 64%
overall yield. Protection of the hydroxyl groups of fura-
nose 14 as tert-butyldimethylsilyl (TBDMS) ethers
furnished furanose 15 in 71% yield. Subsequent
debenzylation of 15 by catalytic hydrogenation using
H2 and Pd(OH)2/C in ethanol afforded alcohol 16 in
76% yield along with a by-product which NMR(results
not shown) and MALDI-MS (m/z 357 [M+Na]+) con-
firmed to arise from the cleavage of one of the TBDMS
groups.21 Prolonged exposure (>36 h) of furanose 15 to
debenzylation conditions resulted in undesired cleavage
of the benzyl group and both TBDMS groups to furnish
the known triol 17,22 verifying the recently reported
solvent-dependent lability of TBDMS O-ethers under
Pd/C-catalyzed hydrogenation conditions.23,24 Triol
17,22 more conveniently obtained from 1,2;5,6-di-O-iso-
propylidene-a-D-glucose in three steps without purifica-
270o
90o
West
East
180o
2E
South
2T3
2′
O
HO
B
3′
Figure 2. Pseudorotational cycle of the furanose ring in nucleosides.

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2599
OBn
OBn
HO
HO
TBDMSO
TBDMSO
O
O
a
O
O
O
O
O
O
14
15
OH
b
HO
HO
OH
O
TBDMSO
c
O
TBDMSO
O
O
17
16
d
TMS
O
TBDMSO
TBDMSO
TBDMSO
TBDMSO
O
e
f
O
O
O
O
OH
O
18
19
H
HO
HO
O
O
O
O
O
OH
O
OH
21
O
20
Scheme 1. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt, 71%; (b) H2, 20% Pd(OH)2/C, EtOH, rt, 76%; (c) TBDMSCl, imidazole,
DMF, 0 ꢁC to rt, 71%; (d) Dess–Martin periodinane, CH2Cl2, 0 ꢁC to rt, 97% or TEMPO, BAIB, CH2Cl2, rt, 91%; (e) TMSC„CH, n-BuLi, THF,
ꢀ78 ꢁC, 90%; (f) TBAF, THF, rt, 74%.
tion of intermediates (64% overall yield), was converted
to furanose 16 in 71% yield by selective TBDMS protec-
tion of the primary hydroxyl groups. Oxidation of fura-
nose 16 to 3-ulose 18 was accomplished with Dess–
Martin periodinane25 or with 2,2,6,6-tetramethyl-1-piper-
idinyloxyl (TEMPO)26 and [(diacetoxy)iodo]benzene
(BAIB) as co-oxidant in excellent yields (97% and
prior to glycosylation, and triol 20 was therefore selec-
tively mesylated to afford bis-sulfonic ester 22 in 77%
yield (Scheme 2). Cleavage of the 1,2–O-isopropylidene
group of 22 by treatment with aqueous trifluoroacetic
acid (TFA) furnished the anomeric triol, which upon
treatment with acetic anhydride and DMAP at elevated
temperatures resulted in a complicated reaction mixture
from which glycosyl donor 23 was isolated in relatively
low yield (<50%). Instead, the crude triol was peracety-
lated using acetic anhydride and catalytic trimethylsilyl
triflate (TMSOTf).30 Interestingly, the choice of solvent
was critical for the outcome of this reaction. Desired gly-
cosyl donor 23 was not obtained when the reaction was
carried out in acetonitrile, a solvent recommended for
this type of reaction,30 whereas excellent yield (88%,
from 22) of 23 was observed when the reaction was
carried out in dichloromethane. Subsequent one-pot
reactions of glycosyl donor 23 with uracil or 4-N-
benzoylcytosine, N,O-bis(trimethylsilyl)acetamide (BSA)
and TMSOTf,31 stereoselectively afforded the N1-
linked b-nucleosides via anchimeric assistance of
the O2-acetyl group in 74% yield for both 24 and 25.
Treatment of nucleosides 24 and 25 with saturated
methanolic ammonia resulted in tandem deacylation
and selective 30-O,40-C ring closure. Subsequent nucleo-
philic substitution of the remaining C50-mesylate group
91%,
respectively).
Nucleophilic
addition
of
LiC„CTMS (prepared in situ from trimethylsilylacetyl-
ene and n-butyllithium in THF) to 3-ulose 18, occurred
from the sterically less hindered b face to exclusively give
ribo-configured furanose 19 in 90% yield.27 Desilylation
of 19 with tetrabutylammonium fluoride (TBAF) fur-
nished the key intermediate 20 in 74% yield.
Attempts to shorten the synthetic route by subjecting
the known hydroxy aldehyde 2128 to typical crossed
aldol condensation and crossed Cannizzaro reduction
conditions (37% aq formaldehyde, 2 M NaOH, 1,4-
dioxane),20 failed.
2.2. Synthesis of 30-O,40-C-methylene-linked EUrd/ECyd
analogs 5 and 6
Inspired by the improved synthetic route to LNA,29 we
decided to install methanesulfonate groups at C50/C500

2600
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
MsO
MsO
MsO
MsO
O
O
a
b,c
OAc
20
O
OAc OAc
OH
O
23
22
B
B
MsO
MsO
HO
O
O
d
e-g
OAc OAc
O
OH
5 B = uracil-1-yl
6 B = cytosin-1-yl
24 B = uracil-1-yl
25 B = 4-N-benzoylcytosin-1-yl
Scheme 2. Reagents and conditions: (a) MsCl, pyridine, 0 ꢁC to rt, 77%; (b) 80% aq TFA, 0 ꢁC; (c) Ac2O, TMSOTf, CH2Cl2, 0 ꢁC, 88% (two steps);
(d) uracil/4-N-benzoylcytosine, BSA, TMSOTf, 1,2-dichloroethane, reflux, 74% for 24, 74% for 25; (e) satd NH3/MeOH, rt; (f) NaOBz, DMF,
110 ꢁC; (g) satd NH3/MeOH, rt, 62% for 5 (three steps), 26% for 6 (three steps).
with sodium benzoate in DMF afforded benzoates,
which were deacylated with saturated methanolic
ammonia to furnish the bicyclic nucleosides 5 and 6 in
modest yields (62% of 5 from 24 and 26% of 6 from
25, respectively).
Selective 30-O,40-C ring closure as observed during syn-
thesis of 5 and 6 rather than 20-O,40-C ring closure
(which would furnish LNA analogs), has also been ob-
served during synthesis of 30-O,40-C-methyleneribonu-
cleosides.18 This selectivity has been proposed to arise
from preorganization of the nucleosides in a South con-
formation, by which the 30-OH group is in closer prox-
imity to the leaving group at the C500-position than the
20-OH group.
Evidence for the proposed dioxabicyclo[3.2.0]heptane
skeleton of 5 and 6 was obtained by 1H NMR
(DMSO-d6) experiments. The signal of the 20-OH group
of nucleoside 6 appeared at 5.87 ppm as an exchange-
able doublet. Furthermore, the signal of the 50-OH
group appeared as an exchangeable triplet at
4.93 ppm, which coupled to H-50 (3.70 ppm, doublet).
The oxetane ring protons H-500 appeared as two doublets
at 4.29 and 4.70 ppm. Also, key NOE contacts between
H-50B0 =H-10 (7%) and H20/H-6 (12%) verified the dioxabi-
cyclo[3.2.0]heptane skeleton and b-configuration of 6,
respectively. Similar conclusions were made from 1H
NMR- and NOE-experiments of nucleoside 5.
2.3. Synthesis of EUrd–LNA-type analog 7
Based on the observations during synthesis of 5 and 6, it
was obvious that synthesis of EUrd–LNA-analog 7
(Scheme 3) would require a suitable protection of the
O3-hydroxyl group of triol 20 to prevent undesired 30-
Scheme 3. Reagents and conditions: (a) DMTCl, DMAP, pyridine, rt;
O,40-C ring closure at a later stage. Protection of the
(b) NaH, BnBr, Bu4I, THF; (c) 80% aq AcOH, rt, 55% (three steps);
O3-hydroxyl group as a benzyl ether was chosen since
this protecting group has been successfully employed
at a similar stage in the synthesis of LNA29 and it was
(d) MsCl, pyridine, rt; (e) 80% aq TFA, rt; (f) Ac2O, pyridine, rt, 89%
(three steps); (g) uracil, BSA, TMSOTf, CH3CN, 50 ꢁC, 88%; (h) 2 M
NaOH, 1,4-dioxane–H2O (2:1, v/v), rt, 94%; (i) NaOBz, DMF, 110–
therefore expected to be fully compatible with the subse-
quent synthetic steps. Furthermore, deprotection of
140 ꢁC; (j) satd NH3/MeOH, rt, 62% (two steps); (k) BCl3, CH2Cl2,
hexanes, ꢀ78 ꢁC to rt, 63%. U = uracil-1-yl.

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2601
benzyl ethers is known to be orthogonal with the alkyne
functionality in related systems.28 The primary hydroxyl
groups of triol 20 were therefore transiently protected as
the 4,40-dimethoxytrityl (DMT) ethers and subsequent
O3-benzylation furnished fully protected furanose 26.
Deprotection of the DMT ethers with acetic acid affor-
ded furanose 27 in 55% yield over three steps.
a secondary hydroxyl group (e.g., a 20-OH group) were
observed.
2.4. Synthesis of 40-C-hydroxymethyl EUrd/ECyd
analogs 8 and 9
Selective acetylation of triol 20 was followed by 1,2-O-
isopropylidene cleavage of resulting alcohol 3 by treat-
ment with aqueous trifluoroacetic acid (Scheme 4).
The crude triol was peracetylated using acetic anhydride
and catalytic DMAP to give the glycosyl donor 34 in
55% yield from 20. Elevated temperatures were required
to facilitate acetylation of the tertiary hydroxyl group.
Reaction of glycosyl donor 34 with persilylated uracil
Attempts to shorten the route to furanose 27 by subject-
ing known O3-benzylated aldehyde 2828 to tandem
crossed aldol condensation and crossed Cannizzaro
reduction conditions, failed. This is quite surprising
since the corresponding reaction with a C3-allyl group
is known to occur satisfactorily.32
or cytosine under modified Vorbruggen conditions,31
¨
Permesylation of furanose 27 followed by isopropylid-
ene cleavage and peracetylation using standard condi-
tions furnished glycosyl donor 29 in 89% yield over
three steps. One-pot reaction of glycosyl donor 29 with
stereoselectively afforded b-nucleosides 35 and 36 in
77% and 54% yield, respectively. Subsequent treatment
of nucleosides 35 and 36 with saturated methanolic
ammonia gave 40-C-hydroxymethyl EUrd/ECyd analogs
8 and 9 in 97% and 75% yield, respectively.
31
uracil, BSA and TMSOTf exclusively gave b-nucleo-
side 30 in 88% yield. Treatment of nucleoside 30 with
saturated methanolic ammonia resulted merely in O20-
deacylation (results not shown) whereas subjecting 30
to aqueous sodium hydroxide in 1,4-dioxane,29 resulted
in tandem deacetylation and ring closure to give LNA-
type derivative 31 in excellent 94% yield. Nucleophilic
displacement of the remaining mesylate group with so-
dium benzoate required harsh conditions (140 ꢁC,
3 days), which resulted in partial decomposition as seen
by the formation of tars. The crude benzoate was
reacted with saturated methanolic ammonia to give
nucleoside 32 in 62% yield over two steps. Final
debenzylation of nucleoside 32 with boron trichloride
afforded EUrd–LNA-type analog 7 in 63% yield without
affecting the alkyne functionality.
The relative configuration of the nucleobase and 30-C-
ethynyl group was verified by single crystal X-ray dif-
fraction studies of the 40-C-hydroxymethyl nucleosides
8 and 9 (Fig. 3). The absolute configuration follows
from the stereochemically pure starting materials and
the applied synthetic route (Schemes 1 and 4).
2.5. Synthesis of 50-C-hydroxymethyl EUrd/ECyd
analogs 10 and 11
Pyridinium dichromate (PDC) oxidation of commer-
cially available 1,2;5,6-di-O-isopropylidene-a-D-glucose
37 to the known 3-ulose34 followed by nucleophilic addi-
tion of LiC„CTMS formed in situ, furnished alcohol 38
in 73% yield over two steps (Scheme 5). Desilylation of
alcohol 37 with TBAF to afford known furanose 3935 in
76% yield confirmed the exclusive formation of allo-con-
figured alcohol 38 during acetylide addition. Selective
5,6-O-isopropylidene cleavage of furanose 38 afforded
crystalline triol 40 (82% yield), which was enough
reactive to undergo peracetylation with excess acetic
anhydride and catalytic DMAP at room temperature
The dioxabicyclo[2.2.1]heptane skeleton of LNA–EUrd-
analog 7 was ascertained by 1H NMRexperiments. The
signals of H-10 and H-20 appeared as singlets, which is
a
typical observation for the locked dioxabicy-
clo[2.2.1]heptane skeleton of LNA.29,33 The signals of
the 30-OH and 50-OH groups appeared as an exchange-
able singlet and triplet, respectively, while no signals for
Figure 3. Molecular structures (ORTEP-plots) of 40-C-hydroxymethyl nucleosides 8 and 9.

2602
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
AcO
O
a
20
AcO
O
B
OH
33
O
AcO
AcO
RO
RO
O
O
b,c
d
OAc
OAc OAc
OR OR
35 R = Ac, B = uracil-1-yl
36 R = Ac, B = cytosin-1-yl
34
e
e
8 R = H, B = uracil-1-yl
9 R = H, B = cytosin-1-yl
Scheme 4. Reagents and conditions: (a) Ac2O, pyridine, 0 ꢁC to rt; (b) 80% aq TFA, 0 ꢁC; (c) Ac2O, DMAP, pyridine, 100 ꢁC, 55% (three steps, from
20); (d) uracil/cytosine, BSA, TMSOTf, 1,2-dichloroethane, reflux, 77% for 35, 54% for 36; (e) NH3/MeOH, rt, 97% for 8, 75% for 9.
TMS
O
O
O
O
O
O
OH
O
O
O
a,b
c
O
O
O
O
OH
O
OH
39
O
37
38
d
R
TMS
AcO
AcO
HO
HO
O
O
i
O
O
OH
O
OH
O
40
44 R = TMS
45 R = H
f
e
g,h
R
AcO
AcO
AcO
AcO
RO
RO
B
O
O
O
OAc
g,h
j
O
OAc OAc
OAc
O
OR OR
43
46 R = Ac, B = uracil-1-yl
47 R = Ac, B = cytosin-1-yl
10 R = H, B = uracil-1-yl
11 R = H, B = cytosin-1-yl
k
41 R = TMS
42 R = H
f
k
˚
Scheme 5. Reagents and conditions: (a) PDC, AcOH, 3 A molecular sieves powder, CH2Cl2, 0 ꢁC to rt; (b) TMSC„CH, n-BuLi, THF, ꢀ78 ꢁC, 73%
(two steps); (c) TBAF, THF, rt, 76%; (d) 80% aq AcOH, rt, 82%; (e) Ac2O, DMAP, pyridine, rt, 64%; (f) TBAF, AcOH, THF, rt, 82% for 42, 92% for
45; (g) 80% aq TFA, 0 ꢁC; (h) Ac2O, DMAP, pyridine, rt, 61% from 42 (two steps), 82% from 45 (two steps); (i) Ac2O, pyridine, 0 ꢁC to rt, 70%; (j)
uracil/cytosine, BSA, TMSOTf, CH3CN, reflux, 46% for 46, 52% for 47; (k) satd NH3/MeOH, rt, 69% for 10, 54% for 11.
yielding furanose 41 in 64% yield. Desilylation of fura-
nose 41 using a mixture of TBAF and acetic acid gave
furanose 42 in 82% yield. In absence of acetic acid, polar
byproducts (presumably deacylated products) were formed
resulting in lower yields. Cleavage of the remaining
isopropylidene group of furanose 42 with trifluoro-

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2603
acetic acid was followed by peracetylation to give glycosyl
donor 43 in 61% yield over two steps. In an alternative
route to glycosyl donor 43, triol 40 was selectively
diacetylated to give alcohol 44 in 70% yield, which on
desilylation (using the same conditions as for 41) gave
furanose 45 in 92% yield. Conversion of furanose 45
to glycosyl donor 43 (using the same conditions as for
42) proceeded in 82% yield over two steps. This alterna-
tive route to glycosyl donor 43 was more efficient (53%
yield of 43 from 40) than the initial route (33% yield
of 43 from 40). Glycosylation of donor 43 with persily-
lated uracil or cytosine stereoselectively afforded pro-
tected b-nucleosides 46 and 47 in moderate yield (46%
and 52%, respectively) which on deacylation with satu-
rated methanolic ammonia furnished target nucleosides
10 and 11 in 69% and 54% yield, respectively.
from 1,2;5,6-di-O-isopropylidene-a-D-glucose in four
steps, was protected as a TBDMS ether using standard
procedures to give furanose 49 in 91% yield, which on
debenzylation by catalytic hydrogenation afforded alco-
hol 50 in 86% yield. PDC oxidation of the O3-hydroxyl
group of 50 gave the 3-ulose intermediate, which was re-
acted directly with LiC„CTMS formed in situ to stere-
oselectively give allo-configured furanose 51 in excellent
90% yield over two steps.37 Standard desilylation of
furanose 51 afforded key intermediate 52 in 98% yield.
Efforts were made to shorten the synthetic route to key
intermediate 52. Known triol 53,28 easily obtained from
1,2;5,6-di-O-isopropylidene-a-D-glucose in three steps,
was selectively tosylated in 57% yield to afford furanose
54, which subsequently was subjected to typical condi-
tions for mild reductive displacement of primary tosyl-
ate groups. However, neither treatment of furanose 54
with tetrabutylammonium borohydride38 in toluene at
room temperature or reflux, or sodium borohydride in
DMF afforded key intermediate 52, perhaps due to
instability of the alkyne functionality under these
conditions.
2.6. Synthesis of 50-C-methyl EUrd/ECyd analogs 12
and 13
Retrosynthetic analysis identified diol 52 as the key
intermediate in the synthesis of 50-C-methyl EUrd/ECyd
analogs 12 and 13 (Scheme 6). Alcohol 48,36 obtained
TMS
OR2
R1O
TBDMSO
O
O
c,d
e
O
O
O
OH
O
48 R1 = H, R2 = Bn
51
a
b
49 R1 = TBDMS, R2 = Bn
50 R1 = TBDMS, R2 = H
RO
HO
HO
O
O
O
O
OH
O
OH
O
52
53 R = H
54 R = Ts
f
g-i
B
AcO
RO
O
O
j
OAc
OAc OAc
OR OR
55
56 R = Ac, B = uracil-1-yl
k
k
57 R = Ac, B = 4-N-benzoylcytosin-1-yl
12 R = H, B = uracil-1-yl
13 R = H, B = cytosin-1-yl
˚
Scheme 6. Reagents and conditions: (a) TBDMSCl, imidazole, CH2Cl2, rt, 91%; (b) H2, Pd(OH)2/C, EtOAc, 86%; (c) PDC, AcOH, 3 A molecular
sieves powder, CH2Cl2, rt; (d) TMSC„CH, n-BuLi, THF, ꢀ78 ꢁC, 90% (two steps); (e) TBAF, THF, rt, 98%; (f) TsCl, pyridine, ꢀ50 ꢁC to rt, 57%;
(g) Ac2O, DMAP, pyridine, rt; (h) 80% aq TFA, rt; (i) Ac2O, DMAP, pyridine, rt, 72% (three steps); (j) uracil/4-N-benzoylcytosine, BSA, TMSOTf,
CH3N, reflux, 79% for 56, 75% for 57; (k) satd NH3/MeOH, rt, 78% for 12, 64% for 13.

2604
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
Diez–Altona–Donders equation as implemented within
the MESTRE-J program.42 Very good agreement between
0
0
predicted JH1 –H2 values and experimental values indi-
cates that solution conformations of nucleosides 2, 5,
7, 8, 10, and 12 closely match the lowest energy struc-
tures from molecular modeling.
As anticipated, the 30-O,40-C-methylene-linkage of
nucleoside 5 restricts the sugar ring in a South confor-
mation (2T1) similar to ECyd 2. However, a flatter ring
pucker (Dmm = ꢀ 7ꢁ) than in ECyd is observed. Also
unsurprisingly, LNA-type EUrd-analog 7 is locked in
a
North conformation with an extreme pucker
(P = 24ꢁ, mm = 55ꢁ). Introduction of a hydroxymethyl
or methyl group at C-50 as in nucleosides 10 and 12,
does not seem to influence the sugar conformation,
whereas introduction of a C40-hydroxymethyl group as
in 8 results in a more pronounced South conformation
Figure 4. Molecular structure (ORTEP-plot) of 50-C-methyl nucleo-
side 12.
2
(P = 181ꢁ, T3), which also is reflected experimentally
0
0
in a larger observed value of JH1 –H2 for 8 as compared
to ECyd, 10 and 12. Single crystal X-ray diffraction
studies of the 40-C-hydroxymethyl nucleosides 8 and 9
(Fig. 3) and 50-C-methyl nucleoside 12 (Fig. 4) show that
the furanose ring also adopts a South conformation in
solid state (P = 168ꢁ, 169ꢁ, and 170ꢁ for 8, 9, and 12,
respectively).
Key intermediate 52 was converted to glycosyl donor 55
by a three-step sequence involving diacetylation, isopro-
pylidene cleavage, and peracetylation (72% yield).
Glycosylation of 53 with persilylated uracil or 4-N-
benzoylcytosine under modified Vorbruggen condi-
¨
tions,31 gave b-nucleosides 56 and 57 in 79% and 75%
yield, respectively. Deprotection of nucleosides 56 and
57 with saturated methanolic ammonia afforded the tar-
get nucleosides 12 and 13 in 78% and 64% yield,
respectively.
4.Biological evaluation
Reference nucleosides 1–2, target nucleosides 5–13 and
acylated derivatives 35, 36, 46, 47, 56, and 57 were eval-
uated for antiviral activity against HIV-1 in MT-4 cells
as previously described.43 All compounds were inactive
against HIV-1 at the highest tested concentration of
100 lM. EUrd and ECyd displayed significant cytotox-
icity (CD50 = 0.03 and 0.02 lM, respectively)44 whereas
LNA-type EUrd analog 7 displayed marginal cytotoxic-
ity (CD50 = >30 lM). All other compounds were non-
toxic toward MT-4 cells. EUrd, ECyd, and nucleosides
5–13 were evaluated against human adenocarcinoma
breast cancer (MCF-7) and prostate cancer (PC-3) cell
lines as previously described.45 As expected, EUrd and
ECyd displayed very potent activities in vitro with
IC50 values46 of 2.5 and 2.2 nM, respectively (MCF-7)
and 1.7 and 0.15 nM, respectively (PC-3), whereas
nucleosides 5–13 were inactive at the highest tested con-
centration of 25 lM.
Single crystal X-ray diffraction study of the 50-C-methyl
nucleoside 12 (Fig. 4), verified the relative configuration
of the nucleobase and 30-C-ethynyl group. The absolute
configuration follows from the stereochemically pure
starting materials and the applied synthetic route
(Scheme 6).
3.Conformational analysis
Target nucleosides 5, 7, 8, 10, and 12 (Fig. 1) and ECyd
2 were subjected to Monte Carlo based conformational
39
searches using the AMBERforce–field and general-
ized born/surface area solvation model40 as implemented
in the MACROMODEL V7.2 suite.41 Values of the pseudo-
rotational phase angle P, maximal puckering amplitude
m
the lowest energy structures are listed in Table 1.
m and glycosidic torsion angle v (O40–C10–N1–C2)13 of
3
0
0
JH1 –H2 coupling constants were estimated from the ob-
Although the branched nucleosides 8–13 adopt furanose
conformations similar to those of EUrd and ECyd in
served torsion angles / (H10–C10–C20–H20) by using the
0
0
Table 1. Pseudorotational parameters, torsion angles and predicted JH1 –H2 values of lowest energy structures from molecular modeling and observed
0
0
experimental JH1 –H2 values
0
0
0
0
0
0
0
0
0
0
Compounds
P (ꢁ)
mm (ꢁ)
vO4 –C1 –N1–C2 (ꢁ)
/
(ꢁ)
Jpre;H1 –H2 (Hz)
Jexp;H1 –H2 (Hz)
H1 –C1 –C2 –H2
5
139
24
32
55
35
39
39
39
ꢀ163
ꢀ176
ꢀ164
ꢀ164
ꢀ164
ꢀ162
161
86
9
1
8
9
9
9
7.7
ꢁ0
8.2
7.0
7.0
6.6a
7
8
10
181
142
144
138
157
168
168
168
12
ECyd
a Data from Ref. 1.

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2605
solution, the substituents at the C-40 or C-50-position
may impose unfavorable steric clashes leading to (1)
decreased recognition by UCK or nucleoside kinases,
resulting in insufficient phosphorylation or (2) lack of
recognition of triphosphates by RNA polymerase. The
lack of anticancer activity of conformationally restricted
nucleosides 5–7 may, in addition to the aforementioned
reasons, arise from antipodal conformational require-
ments for furanose conformation of UCK, nucleotide
kinases and RNA polymerase.
by disappearance of peaks on D2O addition. Assign-
ments of NMRspectra were based on 2D spectra (HET-
COR, COSY) and follow standard carbohydrate/
nucleoside nomenclature. The carbon atom of C4 sub-
stituents is numbered C-50 in furanose derivatives and
C-500 in nucleoside derivatives. Similar conventions ap-
ply for the corresponding hydrogen atoms. Assignments
of C-5/C-50 and H-5/H-50 in furanose derivatives and C-
50/C-500 and H-50/H-500 in monocyclic nucleoside deriva-
tives may be interchanged. Quaternary carbons were not
assigned in 13C NMR. Traces of solvents in NMR spec-
tra were identified by reference to published data.48
MALDI-HRMS were recorded in positive ion mode
on a IonSpec Fourier transform mass spectrometer.
UV spectra were recorded at room temperature on a
Shimadzu UV-160A spectrophotometer in the range
230–500 nm, using a quartz cell with a 1 cm path length.
The pH was adjusted by addition of concentrated aque-
ous HCl or NaOH. The concentrations of the aqueous
solutions of compounds were adjusted to give absorp-
tion peaks in the linear range. Elemental analyses were
obtained from the Microanalytical Department, Univer-
sity of Copenhagen.
5.Experimental
5.1. General experimental section
All solvents and reagents were obtained from commer-
cial suppliers and used without further purification un-
less stated otherwise. Reference compounds EUrd and
ECyd were obtained via a previously published route.45
All solvents used for chromatography were of technical
grade and used without further purification except
CH2Cl2, which was distilled prior to use. Petroleum
ether of the distillation range 60–80 ꢁC was used. Sol-
vents for use in reactions were of analytical grade.
Anhydrous DMF and pyridine were used directly as ob-
tained from commercial suppliers. Acetonitrile was
5.2. Synthesis of 3-O-benzyl-4-C-hydroxymethyl-1,2-O-
isopropylidene-b-L-threo-pentofuranose (14) from diace-
tone-a-D-glucose without purification of intermediates
˚
dried through storage over activated 3 A molecular
sieves. Dichloromethane, 1,2-dichloroethane and tolu-
ene for use in anhydrous reactions were dried through
A suspension of 1,2;5,6-di-O-isopropylidene-a-D-gluco-
furanose (40.29 g, 0.15 mol) in anhydrous THF
(100 mL) was added dropwise over 30 min to an ice-cold
suspension of NaH (60% suspension in mineral oil,
9.60 g, 0.24 mol) in anhydrous THF (30 mL). After
ended addition, benzyl bromide (22.4 mL, 0.19 mol),
and tetrabutylammonium iodide (4.00 g, 10.8 mmol)
were added and the reaction mixture stirred for 6 h at
rt. The reaction mixture was cooled to 0 ꢁC and H2O
(75 mL) added. The separated aqueous phase was ex-
tracted with CH2Cl2 (3 · 100 mL), and the combined
organic phase evaporated to dryness to give the crude
O3-benzylated furanose, which was directly dissolved
in 80% aqueous acetic acid (300 mL). After stirring for
22 h, the reaction mixture was washed with petroleum
ether (4 · 100 mL) to remove remaining benzyl bromide
from the previous step. The aqueous phase was evapo-
rated to dryness and coevaporated with absolute
EtOH/toluene (3 · 100 mL, 1:1 v/v). The resulting resi-
due was taken up in CH2Cl2 (200 mL), washed with satd
aq NaHCO3 (2 · 75 mL) and the organic phase evapo-
rated to dryness to leave the crude diol, which was used
in the next step without further purification. To an ice-
cold solution of the crude diol in THF/H2O (500 mL,
1:1 v/v) was added sodium periodate (32.90 g,
0.15 mol) and the reaction mixture stirred at 0 ꢁC for
2 h, whereupon insoluble solids were filtered off and
washed with ether (500 mL). Combined filtrates were
collected and the phases separated. The aqueous phase
was extracted with CH2Cl2 (3 · 150 mL) and the com-
bined organic phase evaporated to dryness. The result-
ing crude aldehyde was used immediately in the next
reaction without further purification. To a solution of
the crude aldehyde in 1,4-dioxane (150 mL) was added
formaldehyde (37% solution in H2O, 40 mL, 0.54 mol)
˚
storage over activated 4 A molecular sieves. THF was
dried either by distillation from sodium/benzophenone
˚
or by storing over activated 4 A molecular sieves. Water
content of anhydrous solvents was checked by Karl–
Fischer apparatus. Reactions were conducted under an
atmosphere of argon when anhydrous solvents were
used. All reactions were monitored by thin-layer chro-
matography (TLC) using silica gel coated plates with
fluorescence indicator (SiO2-60, F-254), which were
visualized (a) under UV light, (b) by dipping in 5%
concd sulfuric acid in absolute ethanol (v/v) followed
by heating, or (c) by dipping in a solution of molyb-
dato–phosphoric acid (12.5 g/L) and cerium(IV)sulfate
(5 g/L) in 3% concd sulfuric acid in water (v/v) followed
by heating. Dry column vacuum chromatography47 and
silica gel column chromatography using moderate pres-
sure (pressure ball) were performed with Silica gel 60
(particle size 0.040–0.063 mm, Merck). Evaporation of
solvents was carried out under reduced pressure with a
temperature not exceeding 50 ꢁC unless stated other-
wise. After column chromatography, appropriate frac-
tions were pooled, evaporated, and dried at high
vacuum for at least 12 h to give obtained products in
high purity (>95%), unless stated otherwise. In absence
of elemental analysis, 1H NMRand/or 13C NMRascer-
tained sample purity. No corrections in yields were
1
made for solvent of crystallization. H NMRand 13C
NMRspectra were recorded at 300 and 75.5 MHz,
respectively. Chemical shifts are reported in parts per
million (ppm) relative to tetramethylsilane or deuterated
solvent as the internal standard (dH: CDCl3 7.26 ppm,
DMSO-d6 2.50 ppm; dC: CDCl3 77.00 ppm, DMSO-d6
39.43 ppm). Exchangeable (ex) protons were detected

2606
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
and 2 M NaOH (150 mL, 0.30 mol). The reaction mix-
ture was stirred for 42 h at rt whereupon it was diluted
with ether (200 mL) and saturated with NaCl (s). The
separated aqueous phase was extracted with CH2Cl2
(4 · 100 mL) and the combined organic phase evapo-
rated to dryness and purified by silica gel column chro-
matography (0–65% EtOAc in petroleum ether, v/v) to
afford the known furanose 1420 (30.39 g, 64% from
was concentrated to near dryness, diluted with H2O
(100 mL) and extracted with EtOAc (3 · 100 mL). The
combined organic phase was washed with satd aq NaH-
CO3 (2 · 50 mL) and brine (2 · 50 mL), dried (MgSO4),
filtered, and evaporated. Purification of the residue by
silica gel column chromatography (20% EtOAc in petro-
leum ether, v/v) afforded 16 (8.27 g, 71%) as a white
solid material. Rf = 0.3 (10% EtOAc in petroleum ether,
1,2;5,6-di-O-isopropylidene-a-D-glucofuranose) as
a
v/v); MALDI-HRMS m/z 471.2572 ([M+Na]+,
white solid material. 13C NMRdata were identical to
previously reported data.20
C21H44O6Si2ÆNa+: Calcd 471.2569); H NMR(CDCl ):
1
3
d
5.94 (d, 1H, J = 3.8 Hz, H-1), 4.57 (d, 1H,
J = 3.8 Hz, H-2), 4.29 (d, 1H, ex, J = 6.8 Hz, 3-OH),
4.20 (d, 1H, J = 6.8 Hz, H-3), 3.56–4.12 (m, 4H, H-5,
H-50), 1.53 (s, 3H, C(CH3)2), 1.39 (s, 3H, C(CH3)2),
0.89–0.90 (m, 18H, C(CH3)3), 0.05–0.11 (m, 6H,
Si(CH3)2); 13C NMR(CDCl 3): d 112.5, 105.1 (C-1),
89.3, 88.7 (C-2), 78.8 (C-3), 65.5 (C-5), 65.3 (C-50),
27.2 (C(CH3)2), 26.4 (C(CH3)2), 25.9 (C(CH3)3), 25.7
(C(CH3)3), 18.3, 18.1, ꢀ5.4 (Si(CH3)2), ꢀ5.45
(Si(CH3)2), ꢀ5.49 (Si(CH3)2), ꢀ5.7 (Si(CH3)2). Anal.
Calcd for C21H44O6Si2: C, 56.21; H, 9.88. Found: C,
56.24; H, 9.95.
5.3. 3-O-Benzyl-5-O-(tert-butyldimethylsilyl)-4-C-(tert-
butyldimethylsilyloxymethyl)-1,2-O-isopropylidene-b-L-
threo-pentofuranose (15)
To a stirred solution of diol 1420 (0.89 g, 2.87 mmol) in
anhydrous DMF (5 mL) at rt, was added imidazole
(1.16 g, 17.0 mmol) and TBDMSCl (1.36 g, 9.02 mmol).
After stirring at 35 ꢁC for 20 h, H2O (10 mL) was added
and the aqueous phase extracted with Et2O (2 · 40 mL).
The combined organic phase was washed with satd aq
NaHCO3 (2 · 25 mL) and brine (25 mL), dried
(MgSO4), filtered, and evaporated to dryness. Purifica-
tion by silica gel column chromatography (0–6% EtOAc
in petroleum ether, v/v) afforded furanose 15 (1.10 g,
71%) as a clear oil. Rf = 0.5 (10% EtOAc in petroleum
5.5. Synthesis of 4-C-(hydroxymethyl)-1,2-O-isopropy-
lidene-b-L-threo-pentofuranose (17) from diacetone-a-D-
glucose without purification of intermediates
ether, v/v); MALDI-HRMS m/z 561.3061 ([M+Na]+,
1,2;5,6-Di-O-isopropylidene-a-D-glucofuranose (80.00
g, 0.31 mol) was dissolved in 67% aqueous acetic acid
(1.0 L) and stirred for 15 h at rt, whereupon the reaction
mixture was evaporated to dryness and coevaporated
with absolute EtOH/toluene (3 · 300 mL, 1:1 v/v)
affording crude triol. To an ice-cold solution of the
crude triol in MeOH/H2O (1.0 L, 5:1 v/v) was added so-
dium periodate (75.65 g, 0.35 mol). After stirring for 2 h
at rt, ethylene glycol (10 mL) was added, and nonsoluble
residues filtered off, washed (MeOH) and the combined
organic phase evaporated to dryness. The residue was
partitioned between EtOAc (300 mL) and brine
(100 mL) and the separated aqueous phase extracted
with EtOAc (3 · 200 mL). The combined organic phase
was evaporated to dryness affording crude aldehyde,
which was directly dissolved in H2O (400 mL). To this,
formaldehyde (37% solution in H2O, 190 mL,
2.55 mol) and aqueous NaOH (1.0 M, 950 mL,
0.95 mol) were added and the reaction mixture stirred
at rt for 72 h. The mixture was neutralized with formic
acid, evaporated to dryness, and coevaporated with tol-
uene (3 · 200 mL). Purification of the residue by silica
gel column chromatography (80–100% EtOAc in petro-
leum ether, then 0–10% MeOH in EtOAc, v/v) afforded
17 (43.03 g, 64% from 1,2;5,6-di-O-isopropylidene-a-D-
glucofuranose) as a white solid material. 13C NMR
(DMSO-d6): d 111.2, 104.0, 90.1, 88.2, 75.7, 61.0, 27.0,
1
C28H50O6Si2ÆNa+: Calcd 561.3038); H NMR(CDCl ):
3
d 7.18–7.22 (m, 5H, Ph), 5.89 (d, 1H, J = 4.4 Hz, H-1),
4.65 (dd, 1H, J = 4.4 Hz, 2.2 Hz, H-2), 4.59 (d, 1H,
J = 11.5 Hz, CH2Ph), 4.40 (d, 1H, J = 11.5 Hz, CH2Ph),
4.00 (d, 1H, J = 2.2 Hz, H-3), 3.42–3.69 (m, 4H, H-5, H-
50), 1.43 (s, 3H, C(CH3)2), 1.25 (s, 3H, C(CH3)2), 0.75 (s,
9H, C(CH3)3), 0.74 (s, 9H, C(CH3)3), ꢀ0.10 (s, 6H,
Si(CH3)2), ꢀ0.11 (s, 3H, Si(CH3)2), ꢀ0.12 (s, 3H,
Si(CH3)2); 13C NMR(CDCl 3): d 137.9, 128.2 (Ph),
127.6 (Ph), 112.9, 105.0 (C-1), 89.6, 86.9 (C-2), 84.4
(C-3), 72.3, 63.6 (C-5), 63.2 (C-50), 27.8 (C(CH3)2),
27.3 (C(CH3)2), 25.9 (C(CH3)3), 18.4, 18.3, ꢀ5.4
(Si(CH3)2), ꢀ5.45 (Si(CH3)2), ꢀ5.47 (Si(CH3)2), ꢀ5.6
(Si(CH3)2). Anal. Calcd for C28H50O6Si2: C, 62.41; H,
9.35. Found: C, 62.18; H, 9.23.
5.4. 5-O-(tert-Butyldimethylsilyl)-4-C-(tert-butyldimeth-
ylsilyloxymethyl)-1,2-O-isopropylidene-b-L-threo-pento-
furanose (16)
Method A (from 15): To a stirred solution of pentofura-
nose 15 (26.61 g, 49.4 mmol) in absolute EtOH (50 mL)
was added 20% Pd(OH)2/C (2.76 g). The mixture was
evacuated with H2 several times. After stirring for 18 h
at rt, the mixture was filtered through a Celite pad,
which was washed with absolute EtOH. The combined
filtrate was evaporated and the residue purified by dry
column vacuum chromatography (2–5% EtOAc in
petroleum ether, v/v) to furnish furanose 16 (16.95 g,
76%) as a white solid material. Method B (from 17):
TBDMSCl (10.26 g, 68.1 mmol) was added to a stirred
mixture of triol 1722 (4.06 g, 18.4 mmol) in anhydrous
DMF (30 mL) at 0 ꢁC. Subsequently, imidazole
(9.66 g, 0.14 mol) was added in three portions over
30 min. After stirring for 72 h at rt, the reaction mixture
1
26.6. H NMR(DMSO- d6) data identical to previously
reported data.22
5.6. 5-O-(tert-Butyldimethylsilyl)-4-C-(tert-butyldimeth-
ylsilyloxymethyl)-1,2-O-isopropylidene-a-D-glycero-
pentofuranos-3-ulose (18)
Method A: Dess–Martin periodinane (231.5 mg,
0.55 mmol) was slowly added to furanose 16

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2607
(160.6 mg, 0.36 mmol) in anhydrous CH2Cl2 (2 mL) at
0 ꢁC. After allowing the reaction mixture to reach rt, it
was stirred for 21 h, whereupon it was evaporated to
dryness, resuspended in Et2O (8 mL), and filtered
through a short pad of Na2SO4. The pad was washed
with Et2O and the combined filtrate was stirred with
Na2S2O3 (1 g) in satd aq NaHCO3 (20 mL). The organic
phase was separated, dried (Na2SO4), filtered and evap-
orated to afford 3-ulose 18 (155.1 mg, 97%) as a clear oil
pure by NMR. Method B: To a stirred solution of fura-
nose 16 (8.67 g, 19.3 mmol) in CH2Cl2 (20 mL) was first
added 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO,
0.30 g, 1.92 mmol) and then [(diacetoxy)iodo]benzene
(BAIB, 8.00 g, 24.9 mmol) in three portions over
15 min. The reaction mixture was stirred at rt for 24 h
when CH2Cl2 (50 mL) and satd aq Na2S2O3 (15 mL)
were added. The separated aqueous phase was extracted
with CH2Cl2 (2 · 15 mL), and the combined organic
phase was dried (MgSO4), filtered, evaporated to dry-
ness and purified by silica gel column chromatography
(0–6% EtOAc in petroleum ether, v/v) to give 3-ulose
18 (7.84 g, 91%) as a clear oil. Rf = 0.5 (10% EtOAc in
petroleum ether, v/v); MALDI-HRMS m/z 469.2434
([M+Na]+, C21H42O6Si2ÆNa+: Calcd 469.2412); 1H
13C NMR(CDCl ): d 114.9, 105.4 (C-1), 105.1, 92.9,
3
89.9, 87.7 (C-2), 73.7, 66.7 (C-5), 63.1 (C-50), 27.4
(C(CH3)2), 26.8 (C(CH3)2), 26.1 (C(CH3)3), 26.0
(C(CH3)3), 18.4, 18.3, 0.00 (Si(CH3)3), ꢀ5.3 (Si(CH3)2),
ꢀ5.36 (Si(CH3)2), ꢀ5.38 (Si(CH3)2), ꢀ5.5 (Si(CH3)2).
Anal. Calcd for C26H52O6Si3: C, 57.31; H, 9.62. Found:
C, 57.33; H, 9.61.
5.8. 3-C-Ethynyl-4-C-(hydroxymethyl)-1,2-O-isopropyl-
idene-a-D-erythro-pentofuranose (20)
To a solution of furanose 19 (4.13 g, 7.58 mmol) in THF
(40 mL) was added tetrabutylammonium fluoride
(1.0 M solution in THF, 11.5 mL, 11.5 mmol). After
stirring for 3 h at rt, the reaction mixture was evapo-
rated to dryness, coevaporated several times with tolu-
ene and purified by dry column vacuum
chromatography (80–100% EtOAc in petroleum ether,
v/v) to give triol 20 (1.37 g, 74%) as a white solid mate-
rial. Rf = 0.2 (EtOAc); MALDI-HRMS m/z 267.0835
1
([M+Na]+, C11H16O6ÆNa+: Calcd 267.0839); H NMR
(DMSO-d6): d 5.80 (d, 1H, J = 4.4 Hz, H-1), 5.64 (s,
1H, ex, 3-OH), 4.67 (d, 1H, J = 4.4 Hz, H-2), 4.65 (t,
1H, ex, J = 5.6 Hz, 5-OH), 4.25 (t, 1H, ex, J = 5.6 Hz,
50-OH), 3.61–3.71 (m, 4H, H-5, H-50), 3.53 (s, 1H,
HC„C), 1.49 (s, 3H, CH3), 1.18 (s, 3H, CH3); 13C
NMR(DMSO- d6): d 113.3, 103.8 (C-1), 90.2, 87.0 (C-
2), 84.1, 76.7, 74.0, 63.2 (C-5), 60.4 (C-50), 27.1 (CH3),
26.5 (CH3). Anal. Calcd for C11H16O6: C, 54.09; H,
6.60. Found: C, 53.75; H, 6.73.
NMR(CDCl ): d 6.12 (d, 1H, J = 4.4 Hz, H-1), 4.32
3
(d, 1H, J = 4.4 Hz, H-2), 3.73–3.84 (m, 4H, H-5, H-50),
1.49 (s, 3H, C(CH3)2), 1.41 (s, 3H, C(CH3)2), 0.87 (s,
9H, C(CH3)3), 0.85 (s, 9H, C(CH3)3), 0.05 (2s, 6H,
Si(CH3)2), 0.02 (s, 6H, Si(CH3)2); 13C NMR(CDCl ):
3
d 210.2, 114.8, 102.8 (C-1), 90.5, 78.4 (C-2), 66.8 (C-5),
64.9 (C-50), 28.0 (C(CH3)2), 27.4 (C(CH3)2), 26.0
(C(CH3)3), 26.0 (C(CH3)3), 18.6, 18.3, ꢀ5.2 (Si(CH3)2),
ꢀ5.4 (Si(CH3)2), ꢀ5.6 (Si(CH3)2). Anal. Calcd for
C21H42O6Si2: C, 56.46; H, 9.48. Found: C, 56.43; H,
9.49.
5.9. 3-C-Ethynyl-1,2-O-isopropylidene-4-C-(methanesul-
fonoxymethyl)-5-O-methanesulfonyl-a-D-erythro-pento-
furanose (22)
Triol 20 (3.90 g, 16.0 mmol) was dried by coevaporation
with anhydrous pyridine, dissolved in anhydrous pyri-
dine (35 mL) and MsCl (2.5 mL, 32.0 mmol) added
dropwise over 5 min at 0 ꢁC. The reaction mixture was
warmed to rt and after stirring for 3 h the heterogeneous
orange reaction mixture was partitioned between EtOAc
(150 mL) and satd aq NaHCO3 (30 mL). The phases
were separated and the aqueous phase extracted with
EtOAc (4 · 50 mL). The combined organic phase was
evaporated to dryness and coevaporated several times
with anhydrous EtOH and toluene (1:1 v/v). The residue
was absorbed on Kieselguhr and purified by silica gel
column chromatography (25–70% EtOAc in petroleum
ether, v/v) to afford pentofuranose 22 (4.93 g, 77%) as
a white solid material. Rf = 0.5 (60% EtOAc in petro-
leum ether, v/v); MALDI-HRMS m/z 423.0395
([M+Na]+, C13H20O10S2ÆNa+: Calcd 423.0390); 1H
NMR(DMSO- d6): d 6.78 (s, 1H, ex, 3-OH), 5.87 (d,
1H, J = 3.7 Hz, H-1), 4.72 (d, 1H, J = 3.7 Hz, H-2),
4.40–4.59 (m, 2H, H-5), 4.32 (br s, 2H, H-50), 3.97 (s,
1H, HC„C), 3.23 (s, 3H, CH3SO2), 3.20 (s, 3H,
CH3SO2), 1.55 (s, 3H, C(CH3)2), 1.30 (s, 3H,
C(CH3)2); 13C NMR(DMSO- d6): d 113.3, 103.8 (C-1),
85.3 (C-2), 83.8, 81.3, 80.1, 75.5, 68.4 (C-50), 67.4 (C-
5), 36.8 (CH3SO2), 36.6 (CH3SO2), 25.80 (C(CH3)3),
25.76 (C(CH3)3). Anal. Calcd for C13H20O10S2: C,
38.99; H, 5.03; S, 16.02. Found: C, 39.29; H, 5.07; S,
15.62.
5.7. 5-O-(tert-Butyldimethylsilyl)-4-C-(tert-butyldimeth-
ylsilyloxymethyl)-1,2-O-isopropylidene-3-C-[2-(trimethyl-
silyl)ethynyl]-a-D-erythro-pentofuranose (19)
A solution of n-butyllithium (2.0 M in cyclohexane,
20.0 mL, 40.0 mmol) was added dropwise over 10 min
to trimethylsilylacetylene (6.5 mL, 46.0 mmol) in anhy-
drous THF (75 mL) at ꢀ78 ꢁC. After stirring for
30 min, a solution of 3-ulose 18 (8.20 g, 18.4 mmol) in
anhydrous THF (35 mL) was added dropwise over
30 min to the metal acetylide solution. Satd aq NH4Cl
(30 mL) was added 45 min after completed addition,
and the mixture was warmed to rt, concentrated to 1/4
volume and extracted with Et2O (2 · 100 mL). The com-
bined organic phase was washed with brine (2 · 75 mL),
dried (MgSO4), filtered, evaporated to dryness and the
resulting residue purified by silica gel column chroma-
tography (CH2Cl2) to afford furanose 19 (9.01 g, 90%)
as a white solid material. Rf = 0.3 (5% EtOAc in petro-
leum ether, v/v); MALDI-HRMS m/z 567.2964
([M+Na]+, C26H52O6Si3ÆNa+: Calcd 567.2980); 1H
NMR(CDCl ): d 5.94 (d, 1H, J = 4.9 Hz, H-1), 4.87
3
(d, 1H, J = 4.9 Hz, H-2), 3.76–4.11 (m, 4H, H-5, H-50),
3.75 (s, 1H, ex, 3-OH), 1.60 (s, 3H, C(CH3)2), 1.39 (s,
3H, C(CH3)2), 0.92 (s, 9H, C(CH3)3), 0.90 (s, 9H,
C(CH3)3), 0.15 (s, 12H, Si(CH3)2), 0.09 (s, 3H,
Si(CH3)3), 0.08 (s, 3H, Si(CH3)3), 0.06 (s, 3H, Si(CH3)3);

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P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
5.10. 1,2,3-Tri-O-acetyl-3-C-ethynyl-4-C-(methanesulf-
onoxy)methyl-5-O-methanesulfonyl-a,b-D-erythro-pentof-
uranose (23)
4.69 (2d, 2H, J = 10.6 Hz, H-50), 4.56–4.60 (d, 1H,
J = 11.0 Hz, H-500), 4.44–4.49 (d, 1H, J = 11.0 Hz,
H-500), 4.27 (s, 1H, HC„C), 3.30 (s, 6H, CH3SO2),
2.17 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO); 13C NMR
(DMSO-d6): d 168.2, 167.4, 162.5, 150.0, 139.5 (C-6),
103.2 (C-5), 86.4 (C-10), 84.0, 83.7, 76.2 (C-20), 75.9,
75.5, 67.8 (C-50), 65.5 (C-500), 36.8 (CH3SO2), 36.7
(CH3SO2), 20.5 (CH3CO), 20.1 (CH3CO). A trace
amount of CHCl3 was identified.
Bis-sulfonic ester 22 (3.18 g, 7.96 mmol) was dissolved in
ice-cold 80% aqueous TFA (40 mL). The reaction mix-
ture was allowed to warm up to rt and after stirring
for 90 min analytical TLC showed full conversion to
two polar products (Rf = 0.2 and 0.3, respectively, 80%
EtOAc in petroleum ether, v/v). The reaction mixture
was evaporated to dryness and the resulting residue
coevaporated with toluene (2 · 25 mL) to afford crude
anomeric triol as a yellow oil (3.2 g), which was used
in the next step without further purification. To a sus-
pension of crude triol in anhydrous CH2Cl2 (20 mL),
was added Ac2O (3.8 mL, 39.8 mmol) followed by drop-
wise addition of TMSOTf (140 lL, 0.80 mmol) over
10 min at 0 ꢁC. After stirring for 70 min at 0 ꢁC, MeOH
(5 mL) was added and the reaction mixture evaporated
to dryness. The residue was taken up in CH2Cl2
(50 mL) and washed with satd aq NaHCO3
(2 · 40 mL). The combined organic phase was evapo-
rated to dryness, and the resulting residue purified by sil-
ica gel column chromatography (0–5% MeOH in
CH2Cl2, v/v) to afford an anomeric mixture (ꢁ1:3 by
1H NMR) of glycosyl donor 23 (3.39 g, 88% over two
steps) as a white solid material. Data for anomeric mix-
ture: Rf = 0.7 (80% EtOAc in petroleum ether, v/v);
MALDI-HRMS m/z 509.0389 ([M+Na]+, C16H22O13S2Æ
Na+: Calcd 509.0394); 13C NMR(CDCl 3): d 168.9,
168.7, 168.2, 168.1, 167.8, 167.7, 97.9, 93.5, 86.0, 84.2,
80.30, 80.28, 79.3, 77.4, 77.3, 75.9, 75.5, 74.5, 68.8,
68.0, 66.1, 65.6, 38.1, 38.03, 37.99, 37.8, 21.1, 20.8,
20.6, 20.3. Anal. Calcd for C16H22O13S2Æ1/2 H2O: C,
38.79; H, 4.68; S, 12.94. Found: C, 38.62; H, 4.28; S,
12.58.
5.12. 1-[2,3-Di-O-acetyl-3-C-ethynyl-4-C-(methanesulf-
onoxymethyl)-5-O-methanesulfonyl-b-D-erythro-pento-
furanosyl]-4-N-benzoylcytosine (25)
4-N-Benzoylcytosine (0.58 g, 2.68 mmol) was dried by
coevaporation with 1,2-dichloroethane (2 · 15 mL),
and resuspended in 1,2-dichloroethane (10 mL). BSA
(1.3 mL, 5.35 mmol) was added and the heterogeneous
solution refluxed for 60 min. After cooling to rt, glycosyl
donor 23 (0.87 g, 1.78 mmol) dissolved in anhydrous
1,2-dichloroethane (10 mL) and TMSOTf (1.0 mL,
5.35 mmol) were added and the reaction mixture re-
fluxed for 24 h. Then satd aq NaHCO3 (20 mL) and
H2O (50 mL) were added, and the phases separated,
and the aqueous phase extracted with CH2Cl2
(4 · 30 mL). The combined organic phase was evapo-
rated to dryness and the resulting residue purified by sil-
ica gel column chromatography (0–4% MeOH in
CH2Cl2, v/v) to afford protected nucleoside 25 (0.85 g,
74%) as off-white foam, which was used in the next step
without further purification. Rf = 0.6 (10% MeOH in
CH2Cl2,
v/v);
MALDI-HRMS
m/z
664.0852
([M+Na]+, C25H27N3O13S2ÆNa+: Calcd 664.0878); 1H
NMR(DMSO- d6): d 11.39 (br s, 1H, ex, NH), 8.24 (d,
1H, J = 7.7 Hz, H-6), 8.00 (d, 2H, J = 8.1 Hz, Ph),
7.60–7.64 (t, 1H, J = 7.5 Hz, Ph), 7.44–7.55 (m, 3H, H-
5, Ph), 6.18 (d, 1H, J = 4.0 Hz, H-10), 5.82 (d, 1H,
J = 4.0 Hz, H-20), 4.76 (br s, 2H, H-50), 4.60–4.65 (d,
1H, J = 11.0 Hz, H-500), 4.50–4.54 (d, 1H, J = 11.0 Hz,
H-500), 4.29 (s, 1H, HC„C), 3.33 (s, 3H, CH3SO2),
3.31–3.32 (overlap with H2O, CH3SO2), 2.15 (s, 3H,
CH3CO), 2.11 (s, 3H, CH3CO); 13C NMR(DMSO-
d6): d 167.6, 167.4, 163.7, 153.9, 144.6 (C-6), 132.9,
132.8, 128.42, 128.36, 96.9 (C-5), 88.7 (C-10), 84.8,
84.3, 77.4 (C-20), 75.8, 75.6, 67.9 (C-500), 65.5 (C-50),
36.9 (CH3SO2), 20.4 (CH3CO), 20.1 (CH3CO). A trace
impurity of EtOAc was identified.
5.11. 1-[2,3-Di-O-acetyl-3-C-ethynyl-4-C-(methanesulf-
onoxymethyl)-5-O-methanesulfonyl-b-D-erythro-pento-
furanosyl]uracil (24)
Uracil (0.91 g, 8.15 mmol) was dried by coevaporation
with anhydrous 1,2-dichloroethane (2 · 25 mL), and
resuspended in anhydrous 1,2-dichloroethane (20 mL).
To this was added N,O-bis(trimethylsilyl)acetamide
(BSA, 3.4 mL, 13.6 mmol) and the reaction mixture
was refluxed for 1 h. After cooling the homogenous
solution to rt, glycosyl donor 23 (2.20 g, 4.53 mmol) dis-
solved in anhydrous 1,2-dichloroethane (20 mL) and
TMSOTf (2.5 mL, 13.6 mmol) were added. After reflux-
ing the reaction mixture for 26 h, satd aq NaHCO3
(30 mL) was added and the phases were separated.
The aqueous phase was extracted with CH2Cl2
(4 · 30 mL), the combined organic phases evaporated
to dryness and the residue purified by silica gel column
chromatography (0–4% MeOH in CHCl3, v/v) to afford
protected nucleoside 24 (1.80 g, 74%) as a white foam,
which was used in the next step without further purifica-
tion. Rf = 0.6 (10% MeOH in CH2Cl2, v/v); MALDI-
HRMS m/z 561.0430 ([M+Na]+, C18H22N2O13S2ÆNa+:
5.13. (1S,3R,4R,5S)-5-Ethynyl-4-hydroxy-1-hydroxy-
methyl-3-(uracil-1-yl)-2,6-dioxabicyclo[3.2.0]heptane (5)
Protected nucleoside 24 (0.78 g, 1.45 mmol) was dis-
solved in satd methanolic ammonia (80 mL) and stirred
in a sealed flask for 23 h whereupon analytic TLC
showed full conversion to a slightly more polar product
(Rf = 0.4; 10% MeOH in CH2Cl2, v/v). The reaction
mixture was evaporated to dryness and the residue
coevaporated several times with anhydrous EtOH to
give a crude bicyclic nucleoside (0.8 g), which was tenta-
tively assigned as (1R,3R,4R,5S)-5-ethynyl-4-hydroxy-1-
(methanesulfonoxy)methyl-3-(uracil-1-yl)-2,6-dioxabi-
cyclo[3.2.0]heptane (MALDI-HRMS m/z 381.0358
([M+Na]+, C13H14N2O8SÆNa+: Calcd 381.0363), and
1
Calcd 561.0456); H NMR(DMSO- d6): d 11.57 (s, 1H,
ex, NH), 7.74 (d, 1H, J = 8.1 Hz, H-6), 6.14 (d, 1H,
J = 5.1 Hz, H-10), 5.75–5.79 (m, 2H, H-20, H-5), 4.61–

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2609
used in the next step without further purification. To a
stirred solution of crude mesylate in anhydrous DMF
(20 mL) was added sodium benzoate (0.42 g,
2.89 mmol). After heating at 110 ꢁC for 19 h, analytical
TLC showed full conversion to a less polar product
(Rf = 0.8; 10% MeOH in CH2Cl2, v/v). Solids were
filtered off, washed (EtOAc), and the combined organic
filtrates evaporated to near dryness. The residue was
partitioned between H2O (25 mL) and CH2Cl2
(25 mL), the phases separated and the aqueous phase ex-
tracted with CH2Cl2 (4 · 25 mL). The combined organic
phase was evaporated to dryness and the residue puri-
fied on a short silica plug (0–5% MeOH in CH2Cl2,
v/v) to give a crude nucleoside (0.8 g) tentatively as-
signed as (1R,3R,4R,5S)-1-(benzoyloxy)methyl-5-ethynyl-
4-hydroxy-3-(uracil-1-yl)-2,6-dioxabicyclo[3.2.0]heptane
silica pad (0–20% MeOH in CH2Cl2, v/v) to afford
a crude nucleoside which was tentatively assigned
as
yl)-5-ethynyl-4-hydroxy-2,6-dioxabicyclo[3.2.0]heptane
(MALDI-HRMS m/z 406.1010
([M+Na]+,
(1R,3R,4R,5S)-1-(benzoyloxy)methyl-3-(cytosin-1-
C19H17N3O6ÆNa+: Calcd 406.1001). Crude bicyclic ben-
zoate (174 mg) was dissolved in satd methanolic ammo-
nia (10 mL) and after stirring in a sealed flask at rt for
18 h, the reaction mixture was evaporated to near dry-
ness, and was coevaporated several times with anhy-
drous EtOH. The resulting residue was taken up in
H2O (15 mL) and washed with CH2Cl2 (2 · 15 mL)
and the aqueous phase evaporated to dryness. The
resulting crude powder was purified by recrystallization
from i-PrOH and minimal H2O to give bicyclic nucleo-
side 6 (18.7 mg, 26% yield from 25, over three steps)
as white needles. Rf = 0.2 (20% MeOH in CH2Cl2, v/
v); mp (i-PrOH/H2O) > 220 ꢁC; UV kmax pH 1,
277 nm, kmax H2O, 238, 268 nm, kmax pH 11, 272 nm;
MALDI-HRMS m/z 302.0755 ([M+Na]+, C12H13N3O5Æ
(MALDI-HRMS
m/z
407.0850
([M+Na]+,
C19H16N2O7ÆNa+: Calcd 407.0869), which was dissolved
in satd methanolic ammonia (40 mL). After stirring in a
sealed flask for 46 h, the reaction mixture was evapo-
rated to dryness and coevaporated with anhydrous
EtOH (20 mL). The resulting residue was adsorbed on
Kieselguhr and purified by silica gel column chromatog-
raphy with (0–12% MeOH in CH2Cl2, v/v) to afford
bicyclic nucleoside 5 (0.25 g, 62% from 24, over three
steps) as an off-white solid material. Rf = 0.3 (20%
MeOH in CH2Cl2, v/v); UV kmax pH 1, 258 nm, kmax
H2O, 258 nm, kmax pH 11, 262 nm; MALDI-HRMS
m/z 303.0571 ([M+Na]+, C12H12N2O6ÆNa+: Calcd
1
Na+: Calcd 302.0747); H NMR(DMSO- d6) 7.68 (d,
1H, J = 7.7 Hz, H-6), 7.29 (s, 1H, ex, NH), 7.26 (s,
1H, ex, NH), 6.26 (d, 1H, J = 8.1 Hz, H-10), 5.87 (d,
1H, ex, J = 7.6 Hz, 20-OH), 5.78 (d, 1H, J = 7.3 Hz, H-
5), 4.93 (t, 1H, ex, J = 5.6 Hz, 50-OH), 4.70 (d, 1H,
J = 7.9 Hz, H-500), 4.29 (d, 1H, J = 7.9 Hz, H-500), 4.05
(m, 1H, H-20), 3.99 (s, 1H, HC„C), 3.70 (d, 2H,
J = 5.6 Hz, H-50); 13C NMR(DMSO- d6): d 165.4,
155.3, 142.0 (C-6), 94.9 (C-5), 86.4 (C-10), 85.5, 85.0,
81.8, 79.6, 77.4 (C-20), 75.9 (C-500), 60.8 (C-50).
1
303.0588); H NMR(DMSO- d6): d 11.32 (br s, 1H, ex,
NH), 7.79 (d, 1H, J = 8.1 Hz, H-6), 6.19 (d, 1H,
J = 7.7 Hz, H-10), 6.02 (d, 1H, ex, J = 7.0 Hz, 20-OH),
5.72 (d, 1H, J = 8.1 Hz, H-5), 5.01 (br t, 1H, ex,
J = 5.5 Hz, 50-OH), 4.72 (d, 1H, J = 8.1 Hz, H-500),
4.31 (d, 1H, J = 8.1 Hz, H-500), 4.06 (br t, 1H, H-20),
4.02 (s, 1H, HC„C), 3.72 (br s, 2H, H-50); 13C NMR
(DMSO-d6): d 162.8, 150.8, 141.0 (C-6), 102.6 (C-5),
86.0, 85.3 (C-10), 84.7, 82.0, 79.2, 77.2 (C-20), 75.9 (C-
500), 60.8 (C-50).
5.15. 3-O-Benzyl-5-O-(4,40-dimethoxytrityl)-4-C-(4,40-
dimethoxytrityloxy)methyl-3-C-ethynyl-1,2-O-isopropyl-
idene-a-D-erythro-pentofuranose (26)
Triol 20 (4.50 g, 18.4 mmol) was dried by coevaporation
with anhydrous pyridine (2 · 25 mL), dissolved in anhy-
drous pyridine (50 mL), and 4,40-dimethoxytrityl chlo-
ride (15.61 g, 46.1 mmol) and DMAP (2.25 g,
18.4 mmol) added. After stirring the reaction mixture
at rt for 44 h, additional 4,40-dimethoxytrityl chloride
(3.10 g, 9.15 mmol) and DMAP (0.45 g, 3.68 mmol)
were added. After stirring for further 23 h, MeOH
(20 mL) was added and the reaction mixture was evapo-
rated to almost dryness, coevaporated with toluene
(75 mL) and the residue taken up in EtOAc (250 mL).
The organic phase was washed with satd aq NaHCO3
(2 · 100 mL) and brine (100 mL) and evaporated to al-
most dryness and coevaporated with toluene
(2 · 100 mL). The resulting residue was purified by silica
gel column chromatography (0–39.5% EtOAc and 0.5%
pyridine in petroleum ether, v/v/v) to afford a crude light
yellow solid material, which was tentatively assigned as
5-O-(4,40-dimethoxytrityl)-4-C-(4,40-dimethoxytrityl-
oxy)methyl-3-C-ethynyl-1,2-O-isopropylidene-a-D-ery-
thro-pentofuranose (Rf = 0.4, 50% EtOAc in petroleum
ether, v/v; MALDI-HRMS m/z 871.3470 ([M+Na]+,
C53H52O10ÆNa+: Calcd 871.3453). Crude DMT-pro-
tected furanose (12.8 g), was coevaporated with toluene
(50 mL), dissolved in anhydrous THF (110 mL) and
added over 45 min at 0 ꢁC to a suspension of sodium
hydride in anhydrous THF (30 mL). After stirring the
reaction mixture for 10 min, benzyl bromide (1.9 mL,
5.14. (1S,3R,4R,5S)-3-(Cytosin-1-yl)-5-ethynyl-4-
hydroxy-1-hydroxymethyl-2,6-dioxabicyclo[3.2.0]heptane
(6)
Protected nucleoside 25 (164.8 mg, 0.26 mmol) was dis-
solved in satd methanolic ammonia (15 mL) and after
stirring in a sealed flask at rt for 42 h, analytical TLC
showed full conversion to a single product with lower
mobility (Rf = 0.2, 10% MeOH in CH2Cl2, v/v). The
reaction mixture was evaporated to dryness and coevap-
orated several times with anhydrous EtOH to give a
crude nucleoside, which tentatively was assigned as
(1R,3R,4R,5S)-3-(cytosin-1-yl)-5-ethynyl-4-hydroxy-1-
(methanesulfonoxy)methyl-2,6-dioxabicyclo[3.2.0]hep-
tane (MALDI-HRMS m/z 380.0519 ([M+Na]+,
C13H15N3O7SÆNa+: Calcd 380.0523). To a solution of
crude bicyclic mesylate (0.15 g) in anhydrous DMF
(5 mL), was added sodium benzoate (74 mg, 0.51 mmol)
and the reaction mixture was heated at 110 ꢁC for 14 h.
At this time analytical TLC showed complete conver-
sion into a product with higher mobility (Rf = 0.7,
20% MeOH in CH2Cl2, v/v). The dark reaction mixture
was evaporated to dryness and filtered through a short

2610
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
15.7 mmol) and tetrabutylammonium iodide (0.55 g,
1.50 mmol) were added. After stirring for 16 h at rt,
crushed ice (75 mL) was added, the reaction mixture
diluted with EtOAc (50 mL), the phases separated and
the organic phase washed with brine (2 · 75 mL). The
organic phase was evaporated to dryness affording crude
furanose 26 as a slightly red foam (16.2 g), which was
used in the next step without further purification. An ali-
quot was purified by silica gel column chromatography
(0–29.5% EtOAc and 0.5% pyridine in petroleum ether,
v/v/v). Rf = 0.2 (20% EtOAc in petroleum ether, v/v);
upon crushed ice (20 mL) was added. The reaction mix-
ture was evaporated to near dryness, coevaporated with
toluene (75 mL) and the residue taken up in EtOAc
(150 mL). The organic phase was washed with satd aq
NaHCO3 (2 · 50 mL), and then evaporated to dryness
and coevaporated with toluene (2 · 75 mL) to afford a
crude residue tentatively assigned as 3-O-benzyl-3-C-
ethynyl-1,2-O-isopropylidene-4-C-(methanesulfonoxy)-
methyl-5-O-methanesulfonyl-a-D-erythro-pentofuranose
(MALDI-HRMS m/z 513.0866 ([M+Na]+, C18H22O6Æ
Na+: Calcd 513.0860), which was directly dissolved in
ice-cold 80% aqueous TFA (50 mL) and stirred at 0 ꢁC
for 2 h. At this time analytical TLC showed full conver-
sion to one compound of lower mobility (Rf = 0.4, 90%
EtOAc in petroleum ether, v/v). Solvents were evapo-
rated off and the residue coevaporated with toluene
(2 · 50 mL) to afford a crude residue, which was tenta-
MALDI-HRMS m/z 961.3921 ([M+Na]+, C60H58O10Æ-
1
Na+: Calcd 961.3922); H NMR(CDCl ): d 6.62–7.42
3
(m, 31H, Ar), 6.07 (d, 1H, J = 4.4 Hz, H-1), 4.93 (d,
1H, J = 4.4 Hz, H-2), 4.79–4.85 (d, 1H, J = 11.4 Hz,
CH2Ph), 4.65–4.70 (d, 1H, J = 11.4 Hz, CH2Ph), 3.85–
3.89 (d, 1H, J = 8.8 Hz, H-5), 3.56–3.79 (m, 15H, H-5,
2 · H-50, 4 · CH3O), 2.14 (s, 1H, HC„C), 1.30 (s, 3H,
tively
assigned
as
3-O-benzyl-3-C-ethynyl-4-C-
C(CH3)2), 1.16 (s, 3H, C(CH3)2); 13C NMR(CDCl ):
(methanesulfonoxy)methyl-5-O-methanesulfonyl-a,b-D-
erythro-pentofuranose (MALDI-HRMS m/z 473.0531
([M+Na]+, C17H22O10S2ÆNa+: Calcd 473.0547) and used
in the next step without further purification. The crude
diol was dried by coevaporation with anhydrous pyri-
dine (25 mL) and dissolved in anhydrous pyridine
(25 mL). To this was added Ac2O (3.58 mL, 37.8 mmol)
and the reaction mixture was stirred at rt for 20 h where-
upon crushed ice (20 mL) was added. The reaction mix-
ture was evaporated to dryness, coevaporated with
toluene (50 mL) and the residue taken up in EtOAc
(150 mL). The organic phase was washed with satd aq
NaHCO3 (2 · 50 mL), and the organic phase evaporated
to near dryness and subsequently coevaporated with tol-
uene (2 · 50 mL). The resulting residue was purified by
silica gel column chromatography (30–40% EtOAc in
petroleum ether, v/v) to give an anomeric mixture (ratio
3
d 158.40, 158.36, 158.29, 158.28, 145.1, 144.7, 138.8,
136.4, 136.2, 136.1, 135.9, 130.8, 130.63, 130.58, 129.2,
129.0, 128.6, 128.4, 128.1, 127.8, 127.6, 127.1, 127.0,
126.6, 126.4, 125.4, 114.4, 113.09, 113.05, 112.9, 105.5,
91.0, 87.6, 87.0, 86.4, 81.9, 80.4, 78.2, 68.5, 65.8, 63.8,
55.3, 55.2, 27.2, 26.6.
5.16. 3-O-Benzyl-3-C-ethynyl-4-C-hydroxymethyl-1,2-O-
isopropylidene-a-D-erythro-pentofuranose (27)
Crude furanose 26 (16.2 g) was suspended in 80% aque-
ous acetic acid (200 mL) and stirred at rt for 23 h,
whereupon the reaction mixture was evaporated to near
dryness, and coevaporated with toluene (100 mL). The
residue was purified by silica gel column chromatogra-
phy (0–50% EtOAc in petroleum ether, v/v) to give diol
27 (3.38 g, 55% from 20, over three steps) as a yellow
solid material. Rf = 0.1 (10% AcOH, 18% EtOAc in
petroleum ether, v/v/v); MALDI-HRMS m/z 357.1305
1
ꢁ9:10 by H NMR) of glycosyl donor 29 (4.50 g, 89%
over three steps) as a yellow foam. Data for anomeric
mixture: Rf = 0.6 (90% EtOAc in petroleum ether, v/v);
MALDI-HRMS m/z 557.0732 ([M+Na]+, C21H26O12S2-
ÆNa+: Calcd 557.0758); 13C NMR(CDCl 3): d 169.3,
169.03, 169.00, 168.9, 137.2, 136.7, 128.6, 128.5, 128.2,
128.1, 127.8, 127.7, 98.2, 93.5, 86.0, 85.5, 82.2, 81.4,
80.4, 79.5, 79.4, 76.5, 76.2, 76.0, 70.3, 70.0, 68.2, 68.1,
67.11, 67.07, 37.84, 37.81, 37.7, 37.6, 21.1, 20.9, 20.7,
20.5. Anal. Calcd for C21H26O12S2Æ1/16 H2O: C, 47.09;
H, 4.92. Found: C, 46.77; H, 4.94.
1
([M+Na]+, C18H22O6ÆNa+: Calcd 357.1309); H NMR
(DMSO-d6): d 7.23–7.38 (m, 5H, Ph), 5.85 (d, 1H,
J = 4.0 Hz, H-1), 4.80–4.85 (m, 2H, H-2, CH2Ph), 4.69
(t, 1H, ex, J = 5.5 Hz, OH), 4.60–4.65 (d, 1H,
J = 11.3 Hz, CH2Ph), 4.05 (dd, 1H, ex, J = 6.6 Hz,
5.1 Hz, OH), 3.87–3.94 (m, 2H, HC„C, H-5), 3.70–
3.78 (m, 2H, H-50), 3.61 (dd, 1H, J = 10.6 Hz, 5.9 Hz,
H-5), 1.49 (s, 3H, CH3), 1.29 (s, 3H, CH3); 13C NMR
(DMSO-d6): d 138.3, 128.0 (Ph), 127.2 (Ph), 127.0
(Ph), 112.9, 103.4 (C-1), 89.1, 85.3 (C-2), 81.8, 81.5,
79.7, 67.7 (CH2Ph), 62.3 (C-50), 59.5 (C-5), 26.4
(C(CH3)3), 26.2 (C(CH3)3). Anal. Calcd for C18H22O6:
C, 64.66; H, 6.63. Found: C, 64.61; H, 6.62.
5.18. 1-[2–O-Acetyl-3-O-benzyl-3-C-ethynyl-4-C-
(methanesulfonoxymethyl)-5-O-methanesulfonyl-b-D-
erythro-pentofuranosyl]uracil (30)
Glycosyl donor 29 (2.50 g, 4.68 mmol) and uracil
(1.05 g, 9.35 mmol) were coevaporated with anhydrous
acetonitrile (50 mL) and suspended in anhydrous aceto-
nitrile (50 mL). To this was added BSA (4.6 mL,
18.7 mmol) and the solution was refluxed until becom-
ing homogenous (<1 h). After cooling the solution to
rt, TMSOTf (1.9 mL, 10.3 mmol) was added and the
reaction mixture heated at 50 ꢁC for 15 h. Since analyt-
ical TLC revealed the reaction only to occur sluggishly a
second portion of TMSOTf (1.0 mL, 5.53 mmol) was
added after cooling the reaction mixture to rt. After stir-
ring at 50 ꢁC for further 24 h, starting material was fully
5.17. 1,2-Di-O-acetyl-3-O-benzyl-3-C-ethynyl-4-C-
(methanesulfonoxy)methyl-5-O-methanesulfonyl-a,b-D-
erythro-pentofuranose (29)
Furanose 28 (3.16 g, 9.45 mmol) was dried by coevapo-
ration with anhydrous pyridine (2 · 25 mL) and dis-
solved in anhydrous pyridine (25 mL). MsCl (2.2 mL,
28.4 mmol) was added dropwise over 5 min and the mix-
ture stirred for 20 h at rt. Analytical TLC showed com-
plete conversion to one product with high mobility
(Rf = 0.6, 90% EtOAc in petroleum ether, v/v) where-

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2611
converted and the reaction mixture was poured into ice-
cold satd aq NaHCO3 (20 mL). The solvents were evap-
orated and the residue taken up in EtOAc (200 mL) and
H2O (100 mL). The phases were separated, and the
aqueous phase extracted with EtOAc (2 · 100 mL).
The combined organic phase was evaporated and the
resulting residue purified by silica gel column chroma-
tography (0–2% MeOH in CH2Cl2, v/v) to afford the
nucleoside 30 (2.41 g, 88%) as a white solid material.
Rf = 0.5 (5% MeOH in CH2Cl2, v/v); MALDI-HRMS
m/z 609.0835 ([M+Na]+, C23H26N2O12S2ÆNa+: Calcd
609.0819); 1H NMR(DMSO- d6): d 11.50 (s, 1H, ex,
NH), 7.82 (d, 1H, J = 8.1 Hz, H-6), 7.32–7.47 (m, 5H,
Ph), 6.10 (d, 1H, J = 7.7 Hz, H-10), 5.89 (d, 1H,
J = 7.7 Hz, H-20), 5.74 (d, 1H, J = 8.1 Hz, H-5),
4.85–4.93 (2d, 2H, H-50/H-500/CH2Ph), 4.71–4.76 (d,
1H, J = 10.5 Hz, H-50/H-500/CH2Ph), 4.59–4.64 (d, 1H,
J = 10.5 Hz, H-50/H-500/CH2Ph), 4.43–4.48 (d, 1H,
J = 10.3 Hz, H-50/H-500/CH2Ph), 4.32–4.37 (m, 2H,
HC„C, H-50/H-500/CH2Ph), 3.31 (s, 3H, CH3SO2),
3.17 (s, 3H, CH3SO2), 2.12 (s, 3H, CH3CO); 13C
NMR(DMSO- d6): d 169.2, 162.6, 150.5, 140.3 (C-6),
137.4, 128.3 (Ph), 127.82 (Ph), 127.79 (Ph), 102.7 (C-
5), 84.7, 84.6, 84.5, 78.1, 77.2 (C-20), 74.7, 69.9, 68.4,
65.9, 36.7 (CH3SO2), 36.6 (CH3SO2), 20.2 (CH3CO).
Anal. Calcd for C23H26N2O12S2Æ1/16 H2O: C, 47.00;
H, 4.48; N, 4.77; S, 10.91. Found: C, 46.67; H, 4.24;
N, 4.76; S, 10.70.
99.4 (C-5), 88.1, 88.0, 84.7, 79.2 (C-20), 77.8, 75.3, 72.7,
68.5, 66.0, 36.9 (CH3).
5.20. (1S,3R,4R,7S)-7-Benzyloxy-7-ethynyl-1-(hydroxy-
methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (32)
To a solution of LNA derivative 31 (0.33 g, 0.74 mmol)
in anhydrous DMF (10 mL) was added sodium benzo-
ate (213 mg, 1.48 mmol) and the mixture was stirred at
110 ꢁC for 48 h, whereupon analytical TLC revealed
approximately 40% conversion to a less polar product
(Rf = 0.6, EtOAc). Additional sodium benzoate
(250 mg, 1.73 mmol) was added and heating increased
to 140 ꢁC. After stirring for additional 24 h, reaction
mixture was cooled to rt and unsoluble residues were fil-
tered off and washed (EtOAc). The combined filtrates
were evaporated to near dryness and diluted with
EtOAc (25 mL) and brine (25 mL) and the phases sepa-
rated. Extraction of the aqueous phase with EtOAc
(2 · 25 mL) was complicated by formation of emulsions.
The combined organic phase was evaporated to dryness
to afford the crude benzoate (0.43 g, MALDI-HRMS m/
z
497.1325 ([M+Na]+, C26H22N2O7ÆNa+: Calcd
497.1325), which was immediately dissolved in satd
methanolic ammonia (10 mL) and stirred in a sealed
container at rt for 48 h. The reaction mixture was evap-
orated to dryness, coevaporated with absolute EtOH
(2 · 10 mL) and the resulting residue adsorbed on silica
gel and purified by silica gel column chromatography
(50–90% EtOAc in petroleum ether, v/v) to afford nucle-
oside 32 (170 mg, 62%, over two steps) as a white solid
material. Rf = 0.3 (EtOAc); MALDI-HRMS m/z
5.19. (1R,3R,4R,7S)-7-Benzyloxy-7-ethynyl-1-(methane-
sulfonoxy)methyl-3-(uracil-1-yl)-2,5-dioxabicy-
clo[2.2.1]heptane (31)
393.1061
([M+Na]+,
C19H18N2O6ÆNa+:
Calcd
To a stirred solution of nucleoside 30 (0.50 g, 0.85 mmol)
in 1,4-dioxane–H2O (3 mL, 2:1, v/v) was added 2 M
aqueous NaOH (3 mL, 6 mmol). Shortly after addition
(5 min), analytical TLC (10% MeOH in CH2Cl2, v/v) re-
vealed approximately 50% conversion to the O20-deacet-
ylated nucleoside (vide infra) having greater polarity
(Rf = 0.4, 10% MeOH in CHCl3, v/v) than starting mate-
rial 30. After stirring the reaction mixture for further 3 h
at rt, the O20-deacetylated nucleoside had fully disap-
peared to give a product having identical mobility as
the starting material 30. Hereupon the reaction mixture
was neutralized with satd aq NH4Cl and diluted with
EtOAc (25 mL) and H2O (25 mL). The phases were sep-
arated and the aqueous phase was extracted with EtOAc
(3 · 25 mL). The combined organic phase was evapo-
rated and the resulting residue purified by silica gel col-
umn chromatography (0–5% MeOH in CH2Cl2, v/v) to
afford LNA derivative 31 (0.36 g, 94%) as a white solid
material. Rf = 0.6 (10% MeOH in CHCl3, v/v); MAL-
DI-HRMS m/z 471.0820 ([M+Na]+, C20H20N2O8SÆNa+:
393.1057); 1H NMR(DMSO- d6): d 11.40 (s, 1H, ex,
NH), 7.71 (d, 1H, J = 8.2 Hz, H-6), 7.28–7.38 (m, 5H,
Ph), 5.58 (d, 1H, J = 8.2 Hz, H-5), 5.52 (s, 1H, H-10),
5.14 (t, 1H, ex, J = 5.9 Hz, 50–OH), 4.88 (s, 1H, H-20),
4.65–4.70 (d, 1H, J = 11.0 Hz, CH2Ph), 4.55–4.60 (d,
1H, J = 11.0 Hz, CH2Ph), 4.09–4.13 (d, 1H,
J = 8.1 Hz, H-500), 3.78–3.99 (m, 4H, HC„C, H-50,
H-500); 13C NMR(DMSO- d6): d 163.5, 150.1, 141.0,
137.5, 128.4, 127.8, 127.7, 99.8, 91.3, 87.8, 83.9, 79.4,
77.7, 76.5, 73.5, 68.4, 57.4.
5.21. (1S,3R,4R,7S)-7-Ethynyl-7-hydroxy-1-(hydroxy-
methyl-3-(uracil-1-yl)-2,5-dioxabicyclo[2.2.1]heptane (7)
Nucleoside 32 (92.3 mg, 0.25 mmol) was dried by coeva-
poration
with
anhydrous
1,2-dichloroethane
(2 · 10 mL), dissolved in anhydrous CH2Cl2 (5 mL)
and the solution cooled to ꢀ78 ꢁC. To this was added
BCl3 (1 M in hexane, 3.0 mL, 3.0 mmol) over 30 min.
After complete addition, the reaction mixture was al-
lowed to warm to rt and stirred for 48 h, when addi-
tional BCl3 (1 M in hexane, 1.0 mL, 1.0 mmol) was
added. After stirring the reaction mixture for further
24 h, H2O (1 mL) was added. The reaction mixture
was evaporated to near dryness, coevaporated with
absolute EtOH (2 · 10 mL) and the resulting residue ad-
sorbed on silica gel and purified by silica gel column
chromatography (0–7% MeOH in CHCl3, v/v) to afford
nucleoside 32 (43.8 mg, 63%) as a white solid material.
Rf = 0.4 (20% MeOH in CH2Cl2, v/v); UV kmax pH 1,
1
Calcd 471.0833); H NMR(DMSO- d6): d 11.44 (s, 1H,
ex, NH), 7.75 (d, 1H, J = 8.1 Hz, H-6), 7.29–7.37 (m,
5H, Ph), 5.56–5.60 (m, 2H, H-10, H-5), 4.99 (s, 1H, H-
20), 4.75–4.79 (d, 1H, J = 11.7 Hz, H-50/H-500/CH2Ph),
4.69–4.73 (d, 1H, J = 11.0 Hz, H-50/H-500/CH2Ph),
4.59–4.64 (m, 2H, H-50/H-500/CH2Ph), 4.15–4.18 (d, 1H,
J = 8.4 Hz, H-50/H-500/CH2Ph), 4.10 (s, 1H, HC„C),
4.00–4.04 (d, 1H, J = 8.4 Hz, H-50/H-500/CH2Ph), 3.29
(s, 3H, CH3); 13C NMR(DMSO- d6): d 163.3, 149.9,
140.7 (C-6), 137.0, 128.2 (Ph), 127.7 (Ph), 127.6 (Ph),

2612
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
263 nm, kmax H2O, 263 nm, kmax pH 11, 263 nm; MAL-
DI-HRMS m/z 303.0589 ([M+Na]+, C12H12N2O6ÆNa+:
Calcd 303.0588); H NMR(DMSO- d6): d 11.34 (br s,
mixture (ratio ꢂ1:3 by 1H NMR) of furanose 34
(4.08 g, 55% from 20) as a white solid material.
Rf = 0.7 (7% MeOH in CH2Cl2, v/v); MALDI-HRMS
m/z 437.1053 ([M+Na]+, C18H22O11ÆNa+: Calcd
437.1054); 13C NMR(DMSO- d6 main isomer): d
169.6, 169.5, 168.5, 168.2, 167.6, 97.4, 86.7, 81.6, 78.3,
77.0, 76.2, 63.5, 61.4, 20.7, 20.5, 20.4, 20.2, 20.2. Anal.
Calcd for C18H22O11 (anomeric mixture): C, 52.17; H,
5.35. Found: C, 52.10; H, 5.31.
1
1H, ex, NH), 7.64 (d, 1H, J = 8.2 Hz, H-6), 6.67 (s,
1H, ex, 30-OH), 5.55 (dd, 1H, J = 8.2, 2.2 Hz, H-5),
5.37 (s, 1H, H-10), 5.04 (t, 1H, ex, J = 5.7 Hz, 50-OH),
4.39 (s, 1H, H-20), 4.00–4.04 (d, 1H, J = 8.1 Hz, H-50),
3.80–3.90 (m, 3H, H-50, H-500), 3.65 (s, 1H, HC„C);
13C NMR(DMSO- d6): d 163.3, 150.0, 140.7 (C-6),
99.2 (C-5), 91.2, 87.5 (C-10), 81.8 (C-20), 81.1, 79.7,
72.7 (C-50), 71.2, 57.3 (C-500). CHCl3 was identified as
a trace impurity.
5.24. 1-[4-C-(Acetoxymethyl)-2,3,5-tri-O-acetyl-3-C-
ethynyl-b-D-erythro-pentofuranosyl]uracil (35)
5.22. 4-C-(Acetoxymethyl)-5-O-acetyl-3-C-ethynyl-1,2-
O-isopropylidene-a-D-erythro-pentofuranose (33)
To a suspension of uracil (97.4 mg, 0.87 mmol) in anhy-
drous 1,2-dichloroethane (8 mL) was added BSA
(0.32 mL, 1.30 mmol) and the suspension was heated
under reflux for 1 h. The resulting homogenous solution
was cooled to rt whereupon a solution of glycosyl donor
34 (200.0 mg, 0.48 mmol) in anhydrous 1,2-dichloroeth-
ane (6 mL) was added. TMSOTf (0.17 mL, 0.91 mmol)
was added and the mixture heated under reflux for 5 h.
After cooling to rt, the mixture was diluted with CHCl3
(15 mL), and satd aq NaHCO3 (5 mL) was added. The
separated aqueous phase was extracted with CHCl3
(2 · 15 mL), and the combined organic phase was dried
(MgSO4), filtered, and evaporated to dryness. The result-
ing residue was purified by silica gel column chromatog-
raphy (0–5% MeOH in CH2Cl2, v/v) to give nucleoside 35
(186.6 mg, 77%) as an off-white solid material. Rf = 0.7
(12% MeOH in CH2Cl2, v/v); UV kmax pH 1, 259 nm,
To a solution of triol 20 (4.41 g, 18.1 mmol) in anhy-
drous pyridine (150 mL) was added acetic anhydride
(5.0 mL, 52.9 mmol) at 0 ꢁC. After stirring for 60 h at
rt, crushed ice (50 mL) was added and the mixture evap-
orated to dryness. The residue was taken up in EtOAc
(100 mL), and the organic phase washed with satd aq
NaHCO3 (2 · 50 mL) and brine (30 mL). The organic
phase was evaporated to dryness and coevaporated sev-
eral times with toluene, affording alcohol 33 (5.96 g) as a
brown oil, which was used in the next step without puri-
fication. Purification of an aliquot by silica gel column
chromatography (6% MeOH in CH2Cl2, v/v) afforded
a pure sample of furanose 33 as a pale yellow oil.
Rf = 0.7 (12% MeOH in CH2Cl2, v/v); MALDI-HRMS
m/z 351.1055 ([M+Na]+, C15H20O8ÆNa+: Calcd
k
max H2O, 259 nm, kmax pH 11, 262 nm; MALDI-HRMS
1
351.1050); H NMR(DMSO- d6): d 6.38 (s, 1H, ex, 3–
m/z 489.1099 ([M+Na]+, C20H22N2O11ÆNa+: Calcd
1
OH), 5.81 (d, 1H, J = 4.4 Hz, H-1), 4.68 (d, 1H,
J = 4.4 Hz, H-2), 4.29–4.39 (m, 2H, H-5), 4.19 (s, 2H,
H-50), 3.78 (s, 1H, HC„C), 2.01 (s, 3H, CH3CO), 1.99
(s, 3H, CH3CO), 1.48 (s, 3H, C(CH3)2), 1.28 (s, 3H,
C(CH3)2); 13C NMR(DMSO- d6): d 169.9, 169.8,
113.0, 103.7 (C-1), 85.7 (C-2), 85.0, 82.2, 78.6, 75.0,
64.3 (C-5), 62.3 (C-50), 26.1 (C(CH3)2), 26.0 (C(CH3)2),
20.52 (CH3CO), 20.47 (CH3CO).
489.1116); H NMR(CDCl ): d 8.91 (br s, 1H, NH),
3
7.60 (d, 1H, J = 8.2 Hz, H-6), 6.23 (d, 1H, J = 4.4 Hz,
H-10), 5.81 (d, 1H, J = 8.2 Hz, H-5), 5.73 (d, 1H,
J = 4.4 Hz, H-20), 4.42–4.66 (m, 4H, H-50, H-500), 2.93
(s, 1H, HC„C), 2.14 (s, 3H, CH3), 2.13 (s, 3H, CH3),
2.11 (s, 3H, CH3), 2.10 (s, 3H, CH3); 13C NMR(CDCl 3):
d 170.1, 169.7, 168.3, 167.3, 162.3, 149.8, 138.8 (C-6),
103.8 (C-5), 86.8 (C-10), 85.6, 80.0, 77.7 (C-20), 76.8,
76.5, 63.9 (C-50), 60.7 (C-500), 20.7 (CH3), 20.4 (CH3).
Anal. Calcd for C20H22N2O11Æ2 H2O: C, 47.81; H, 4.41;
N, 5.58. Found: C, 47.69; H, 4.42, N, 5.43.
5.23. 4-C-(Acetoxymethyl)-1,2,3,5-tetra-O-acetyl-3-C-
ethynyl-a,b-D-erythro-pentofuranose (34)
Crude alcohol 33 (5.96 g) was dissolved in ice-cold 80%
aqueous TFA (55 mL). After stirring for 30 min at room
temperature analytical TLC showed full conversion to a
polar product (Rf = 0.7, 7% MeOH in CH2Cl2, v/v). The
brown reaction mixture was evaporated to dryness and
coevaporated several times with toluene and then anhy-
drous pyridine to give a residue, which was directly dis-
solved in anhydrous pyridine (200 mL). To this was
added acetic anhydride (12.0 mL, 0.13 mol) and DMAP
(0.33 g, 2.72 mmol) and after heating at 100 ꢁC for 12 h,
the dark reaction mixture was cooled to rt and crushed
ice (20 mL) added. The mixture was evaporated to dry-
ness and the residue coevaporated several times with tol-
uene. The residue was taken up in CH2Cl2 (200 mL) and
washed with satd aq NaHCO3 (2 · 50 mL) and brine
(50 mL). The organic phase was evaporated to near dry-
ness, coevaporated several times with toluene, and puri-
fied by dry column vacuum chromatography (60%
EtOAc in petroleum ether, v/v) to afford an anomeric
5.25. 1-[4-C-(Acetoxymethyl)-2,3,5-tri-O-acetyl-3-C-
ethynyl-b-D-erythro-pentofuranosyl]cytosine (36)
A suspension of cytosine (241.1 mg, 2.17 mmol) and
BSA (0.81 mL, 3.28 mmol) in anhydrous 1,2-dichloro-
ethane (20 mL) was heated under reflux for 1 h. The
homogenous solution was allowed to cool to rt where-
upon a solution of glycosyl donor 34 (0.50 g, 1.21 mmol)
in anhydrous 1,2-dichloroethane (15 mL) was added.
TMSOTf (0.41 mL, 2.27 mmol) was added dropwise
and the mixture heated under reflux for 48 h. After cool-
ing to rt, the mixture was diluted with CHCl3 (15 mL)
and satd aq NaHCO3 (10 mL). The separated aqueous
phase was extracted with CHCl3 (2 · 30 mL) and the
combined organic phase was dried (MgSO4), filtered
and evaporated. The resulting residue was purified by
silica gel column chromatography (0–7% MeOH in
CH2Cl2, v/v) to afford nucleoside 36 (0.31 g, 54%) as a
white solid material. Rf = 0.4 (12% MeOH in CH2Cl2,

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2613
v/v); UV kmax pH 1, 277 nm, kmax H2O, 269 nm, kmax
pH 11, 273 nm; MALDI-HRMS m/z 488.1252
([M+Na]+, C20H23N3O10ÆNa+: Calcd 488.1276); 1H
5.73 (d, 1H, J = 7.1 Hz, H-5), 5.61–5.62 (m, 2H, 2ex,
20–OH, 30–OH), 5.05 (t, 1H, ex, J = 5.7 Hz, 50-OH),
4.52 (t, 1H, ex, J = 5.7 Hz, 500-OH), 4.38–4.43 (m, 1H,
H-20), 3.65–3.80 (m, 2H, H-50), 3.47–3.57 (m, 2H,
H-500), 3.43 (s, 1H, HC„C); 13C NMR(DMSO- d6): d
165.3, 155.7, 142.1 (C-6), 94.3 (C-5), 88.2, 86.2 (C-10),
83.3, 77.9 (C-20), 76.0, 73.7, 63.4 (C-50), 61.7 (C-500).
Anal. Calcd for C12H15N3O6: C, 48.48; H, 5.09; N,
14.14. Found: C, 48.40; H, 5.01; N, 13.93.
NMR(CDCl ): d 7.63 (d, 1H, J = 7.7 Hz, H-6), 6.29
3
(d, 1H, J = 4.1 Hz, H-10), 5.89 (d, 1H, J = 7.7 Hz, H-
5), 5.76 (d, 1H, J = 4.1 Hz, H-20), 4.40–4.70 (m, 4H,
H-50, H-500), 2.98 (s, 1H, HC„C), 2.08–2.11 (m, 12H,
CH3); 13C NMR(CDCl ): d 170.1, 169.9, 168.1, 167.4,
3
165.8, 155.4, 140.0 (C-6), 96.2 (C-5), 87.8 (C-10), 85.5,
80.4, 78.3 (C-20), 76.9, 76.6, 64.0 (C-50), 60.9 (C-500),
20.8 (CH3), 20.5 (CH3). Anal. Calcd for
C20H23N3O10Æ1/2 H2O: C, 50.63; H, 5.10; N, 8.86.
Found: C, 50.83; H, 4.91; N, 8.83.
5.28. 1,2;5,6-Di-O-isopropylidene-3-C-[2-(trimethylsi-
lyl)ethynyl]-a-D-allo-hexofuranose (38)
1,2;5,6-Di-O-isopropylidene-a-D-gluco-furanose
37
5.26. 1-[3-C-Ethynyl-4-C-(hydroxymethyl)-b-D-erythro-
pentofuranosyl]uracil (8)
(15.00 g, 57.6 mmol) was dried by coevaporation with
toluene (2 · 75 mL) and resuspended in anhydrous
CH2Cl2 (300 mL). To this, PDC (28.04 g, 74.5 mmol)
˚
Protected nucleoside 35 (0.36 g, 0.76 mmol) was dis-
solved in satd methanolic ammonia (6 mL) and metha-
nol (14 mL) and the reaction mixture stirred for 21 h
in a sealed flask. The reaction mixture was evaporated
to dryness and coevaporated with absolute EtOH sev-
eral times to afford nucleoside 8 (0.22 g, 97%) as a white
solid material pure by NMR. An analytically pure sam-
ple of 8 was obtained by dissolving a sample of 8 in a
minimal amount of methanol. Several hours after addi-
tion of a few drops of toluene, white crystals of 8 were
obtained and isolated by filtration. Rf = 0.3 (20% MeOH
in CH2Cl2, v/v); UV kmax pH 1, 260 nm, kmax H2O,
261 nm, kmax pH 11, 263 nm; MALDI-HRMS m/z
and freshly activated 3 A molecular sieve powder (53 g)
were added. The suspension was cooled to 0 ꢁC and gla-
cial AcOH (5.9 mL, 0.10 mol) added dropwise over
10 min. After stirring the heterogeneous mixture at rt
for 16 h, the mixture was evaporated to dryness and
coevaporated with toluene (2 · 75 mL). The dark residue
was suspended in HPLC grade EtOAc (400 mL), and so-
lid material filtered off on a 5 cm silica pad, which was
washed with EtOAc (1.5 L). The solvent was evaporated
off and the crude ketone dried by coevaporation with tol-
uene (75 mL) and used in the next step without further
purification. The crude ketone (11.73 g) in anhydrous
THF (80 mL), was added dropwise over 40 min at
ꢀ78 ꢁC to a solution of n-butyllithium (33.1 mL, 2.0 M
in cyclohexane, 66.2 mmol) and (trimethylsilyl)acetylene
(10.7 mL, 75.6 mmol) in anhydrous THF (80 mL). After
ended addition, the reaction mixture was stirred for addi-
tional 15 min whereupon satd aq NH4Cl (25 mL) was
added and solvents were evaporated off. The residue
was taken up in H2O (50 mL) and the aqueous phase ex-
tracted with CH2Cl2 (2 · 100 mL). The combined organ-
ic phase was dried (MgSO4), and evaporated affording
furanose 38 (15.06 g, 73% over two steps) as an off-white
solid material, which was pure by NMR. Recrystalliza-
tion of a small sample from anhydrous EtOH afforded
an analytically pure sample as white crystals. Rf = 0.6
(3% MeOH in CH2Cl2, v/v); mp (abs EtOH) 114–
321.0691
([M+Na]+,
C12H14N2O7Na+:
Calcd
1
321.0693); H NMR(DMSO- d6): d 11.31 (br s, 1H, ex,
NH), 8.14 (d, 1H, J = 8.2 Hz, H-6), 5.86 (d, 1H,
J = 8.2 Hz, H-10), 5.79 (d, 1H, ex, J = 7.1 Hz, 20–OH),
5.71 (s, 1H, ex, 30–OH), 5.69 (d, 1H, J = 8.2 Hz, H-5),
5.16 (t, 1H, J = 4.1 Hz, ex, 50–OH), 4.58 (t, 1H, ex,
J = 5.8 Hz, 500-OH), 4.40–4.46 (m, 1H, H-20), 3.67–3.84
(m, 2H, H-50), 3.50–3.55 (m, 2H, H-500), 3.46 (s, 1H,
C„CH); 13C NMR(DMSO- d6): d 162.9, 151.0, 141.0
(C-6), 102.1 (C-5), 88.8, 84.8 (C-10), 83.0, 77.5 (C-20),
76.3, 73.6, 63.3, 61.7 (C-50, C-500). Anal. Calcd for
C12H14N2O7: C, 48.32; H, 4.73; N, 9.39. Found: C,
48.38; H, 4.66; N, 9.41.
5.27. 1-[3-C-Ethynyl-4-C-(hydroxymethyl)-b-D-erythro-
pentofuranosyl]cytosine (9)
115 ꢁC; MALDI-HRMS m/z 379.1562 ([M+Na+],
1
C17H28O6SiÆNa+: Calcd 379.1547); H NMR(CDCl ):
3
d 5.81 (d, 1H, J = 3.3 Hz, H-1), 4.56 (d, 1H, J = 3.3 Hz,
H-2), 4.40 (m, 1H, H-5), 4.13 (dd, 1H, J = 8.2 Hz,
6.2 Hz, H-6), 4.01 (dd, 1H, J = 8.2 Hz, 5.5 Hz, H-6),
3.87 (d, 1H, J = 7.1 Hz, H-4), 3.02 (s, 1H, ex, 3-OH),
1.58 (s, 3H, C(CH3)2), 1.44 (s, 3H, C(CH3)2), 1.36
(s, 6H, C(CH3)2), 0.18 (s, 9H, Si(CH3)3); 13C NMR
(CDCl3): d 113.6, 109.5, 104.2 (C-1), 101.6, 94.2, 83.9
(C-2), 81.4 (C-4), 76.0, 74.7 (C-5), 66.8 (C-6), 26.8
(C(CH3)2), 26.60 (C(CH3)2), 26.59 (C(CH3)2), 25.1
(C(CH3)2), ꢀ0.3 (Si(CH3)3). Anal. Calcd for
C17H28O6Si: C, 57.28; H, 7.92. Found: C, 57.23; H, 7.96.
Protected nucleoside 36 (245.2 mg, 0.53 mmol) was dis-
solved in satd methanolic ammonia (15 mL). After stir-
ring for 12 h at rt in a sealed flask, the reaction mixture
was evaporated to dryness and coevaporated several
times with absolute EtOH to give an off-white solid
material, which was purified by crystallization from
aqueous EtOH to give 9 (89.9 mg, 57%) as pale yellow
crystals. Repeating the crystallization procedure on the
mother liquor furnished a second crop of crystals
(27.7 mg, 75% combined yield). Rf = 0.2 (40% MeOH
in CH2Cl2, v/v); mp (H2O/EtOH) >220 ꢁC; UV kmax
pH 1, 278 nm, kmax H2O, 270 nm, kmax pH 11, 273 nm;
MALDI-HRMS m/z 320.0841 ([M+Na]+, C12H15N3O6Æ
Na+: Calcd 320.0853); 1H NMR(DMSO- d6): d 7.98
(d, 1H, J = 7.1 Hz, H-6), 7.20 (br s, 1H, ex, NH2), 7.16
(br s, 1H, ex, NH2), 5.87 (d, 1H, J = 8.2 Hz, H-10),
5.29. 3-C-Ethynyl-1,2;5,6-di-O-isopropylidene-a-D-allo-
hexofuranose (39)
To a solution of alcohol 38 (18.72 g, 52.5 mmol) in THF
(100 mL) was added tetrabutylammonium fluoride (1 M

2614
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
solution in THF, 52.5 mL, 52.5 mmol) and reaction mix-
ture stirred for 45 min at rt, whereupon it was evapo-
rated to dryness. The residue was taken up in EtOAc
(250 mL), which was washed with brine (3 · 50 mL),
dried (Na2SO4), evaporated to dryness and purified by
silica gel column chromatography (0–40% EtOAc in
(C-1), 97.5, 95.7, 82.6 (C-2), 78.5, 78.2 (C-4), 70.2 (C-
5), 63.3 (C-6), 26.8 (C(CH3)3), 26.6 (C(CH3)3), 20.9
(CH3CO), 20.8 (CH3CO), ꢀ0.5 (Si(CH3)3). Anal. Calcd
for C20H30O9Si: C, 54.28; H, 6.83. Found: C, 54.21; H,
6.91.
petroleum ether, v/v) to give furanose 39 (11.30 g,
76%) as a white solid material. H NMR(CDCl ) data
5.32. 3,5,6-Tri-O-acetyl-1,2-O-isopropylidene-3-C-ethy-
nyl-a-D-allo-hexofuranose (42)
1
3
are identical with previously published data.35
To a solution of furanose 41 (0.89 g, 2.00 mmol) in THF
(20 mL), was sequentially added glacial AcOH (130 lL,
2.27 mmol) and TBAF (1.0 M solution in THF, 2.0 mL,
2.00 mmol). After stirring for 30 min at rt, the reaction
mixture was evaporated to dryness, and the residue par-
titioned between EtOAc (80 mL) and brine (20 mL). The
phases were separated and the aqueous phase extracted
with EtOAc (2 · 40 mL). The combined organic phase
was dried (MgSO4), evaporated to dryness, and the
resulting residue adsorbed on Kieselguhr and purified
by silica gel column chromatography (30% EtOAc in
petroleum ether, v/v) to afford peracetylated furanose
42 (0.61 g, 82%) as white solid material. Rf = 0.5 (70%
EtOAc in petroleum ether, v/v); MALDI-HRMS m/z
393.1156 ([M+Na]+, C17H22O9ÆNa+: Calcd 393.1147);
1H NMR(CDCl 3): d 5.85 (d, 1H, J = 3.7 Hz, H-1),
5.39–5.46 (m, 1H, H-5), 5.14 (d, 1H, J = 3.7 Hz, H-2),
4.59 (dd, 1H, J = 12.3 Hz, 2.2 Hz, H-6), 4.28 (d, 1H,
J = 8.4 Hz, H-4), 4.18 (dd, 1H, J = 12.3 Hz, 5.5 Hz,
H-6), 2.68 (s, 1H, HC„C), 2.10 (s, 3H, CH3CO), 2.07
(s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 1.53 (s, 3H,
5.30. 1,2-O-Isopropylidene-3-C-[2-(trimethylsilyl)ethyn-
yl]-a-D-allo-hexofuranose (40)
Furanose 38 (6.57 g, 18.4 mmol) was dissolved in 80%
aqueous AcOH (75 mL) and stirred for 24 h at rt, where-
upon the reaction mixture was evaporated to dryness
and coevaporated several times with anhydrous
EtOH–toluene (1:1 v/v). The resulting residue was puri-
fied by crystallization from H2O affording triol 40
(4.80 g, 82%) as a white powder. Rf = 0.2 (70% EtOAc
in petroleum ether, v/v); mp (H2O) 106–111 ꢁC; MAL-
DI-HRMS m/z 339.1223 ([M+Na]+, C14H24O6SiÆNa+:
1
Calcd 339.1234); H NMR(DMSO- d6): d 5.67 (d, 1H,
J = 3.8 Hz, H-1), 5.46 (s, 1H, ex, 3-OH), 4.40–4.47 (m,
3H, 2ex, H-2, 5-OH, 6-OH), 3.71–3.76 (m, 2H, H-4,
H-5), 3.59 (dd, 1H, J = 11.2 Hz, 4.9 Hz, H-6), 3.35
(dd, 1H, J = 11.2 Hz, 5.5 Hz, H-6), 1.43 (s, 3H,
C(CH3)2), 1.27 (s, 3H, C(CH3)2), 0.16 (s, 9H, Si(CH3)3);
13C NMR(DMSO- d6): d 112.1, 105.0, 103.1 (C-1), 91.3,
84.1 (C-2), 79.5 (C-4), 75.8, 71.4 (C-5), 63.4 (C-6), 26.6
(C(CH3)2), 26.5 (C(CH3)2), ꢀ0.2 (Si(CH3)3). Anal.
Calcd for C14H24O6Si: C, 53.14; H, 7.65. Found: C,
52.82; H, 7.56.
C(CH3)2), 1.33 (s, 3H, C(CH3)2); 13C NMR(CDCl ):
3
d 170.7, 169.5, 168.4, 113.6, 104.2 (C-1), 82.7 (C-2),
78.4, 78.3, 77.9 (C-4), 77.1, 70.1 (C-5), 63.4 (C-6), 27.0
(C(CH3)3), 26.7 (C(CH3)3), 21.0 (CH3CO), 20.92
(CH3CO), 20.90 (CH3CO). Anal. Calcd for C17H22O9:
C, 55.13; H, 5.99. Found: C, 55.42; H, 6.06.
5.31. 3,5,6-Tri-O-acetyl-1,2-O-isopropylidene-3-C-
[2-(trimethylsilyl)ethynyl]-a-D-allo-hexofuranose (41)
Furanose 40 (0.40 g, 1.28 mmol) was dried by coevapo-
ration with anhydrous pyridine (15 mL), and dissolved
in anhydrous pyridine (10 mL). Ac2O (1.2 mL,
12.7 mmol) and DMAP (23.4 mg, 0.19 mmol) were
added and the reaction mixture stirred at rt for 3 h,
whereupon crushed ice (3 mL) was added. The reaction
mixture was evaporated to dryness, coevaporated with
toluene (10 mL) and the residue taken up in EtOAc
(20 mL). The organic phase was washed with satd aq
NaHCO3 (2 · 8 mL), and the aqueous phase back-ex-
tracted with EtOAc (10 mL). The combined organic
phase was evaporated to dryness, coevaporated with tol-
uene (2 · 15 mL) and the resulting residue purified by
silica gel column chromatography (30–50% EtOAc in
petroleum ether, v/v) to afford 41 (0.36 g, 64%), as an
amorphous white solid material. Rf = 0.6 (70% EtOAc
in petroleum ether, v/v); MALDI-HRMS m/z 465.1554
([M+Na]+, C20H30O9SiÆNa+: Calcd 465.1551); 1H
5.33. 1,2,3,5,6-Penta-O-acetyl-3-C-ethynyl-a,b-D-allo-
hexofuranose (43)
Method A: Triacetylated furanose 42 (0.58 g, 1.56 mmol)
was dissolved in ice-cold 80% aqueous TFA (5 mL) and
stirred for 2.5 h at 0 ꢁC, whereupon the reaction mixture
was evaporated to dryness and coevaporated with tolu-
ene (10 mL) and anhydrous pyridine (2 · 10 mL). The
crude anomeric diol was dissolved in anhydrous pyri-
dine (10 mL), and Ac2O (1.7 mL, 18.0 mmol) and
DMAP (10.7 mg, 0.09 mmol) were added. After stirring
at rt for 18 h, crushed ice (5 mL) was added and the
reaction mixture was diluted with EtOAc (30 mL) and
washed with satd aq NaHCO3 (2 · 10 mL). The com-
bined aqueous phase was back-extracted with EtOAc
(15 mL) and the combined organic phase evaporated
to dryness and coevaporated with toluene (2 · 30 mL).
The resulting residue purified by silica gel column chro-
matography (20–45% EtOAc in petroleum ether, v/v) to
afford an anomeric mixture (ꢂ1:1.5 by 1H NMR) of gly-
cosyl donor 43 (0.39 g, 61% over two steps) as an amor-
phous white solid material. Method B: Diacetylated
furanose 45 (2.00 g, 6.10 mmol) was dissolved in ice-cold
80% aqueous TFA (15 mL) and stirred at 0 ꢁC for 2 h,
whereupon the reaction mixture was evaporated to dry-
ness, coevaporated with toluene (20 mL) and anhydrous
NMR(CDCl ): d 5.84 (d, 1H, J = 3.7 Hz, H-1), 5.37
3
(ddd, 1H, J = 8.4, 5.3, 2.3 Hz, H-5), 5.11 (d, 1H,
J = 3.7 Hz, H-2), 4.59 (dd, 1H, J = 12.4 Hz, 2.3 Hz, H-
6), 4.26 (d, 1H, J = 8.4 Hz, H-4), 4.19 (dd, 1H,
J = 12.4 Hz, 5.3 Hz, H-6), 2.10 (s, 3H, CH3CO), 2.06
(s, 3H, CH3CO), 2.05 (s, 3H, CH3CO), 1.51 (s, 3H,
C(CH3)2), 1.32 (s, 3H, C(CH3)2), 0.16 (s, 9H, Si(CH3)3);
13C NMR(CDCl ): d 170.6, 169.1, 168.1, 113.2, 104.2
3

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2615
pyridine (2 · 20 mL). The crude anomeric triol was dis-
solved in anhydrous pyridine (30 mL) and Ac2O
(6.0 mL, 63.4 mmol) and DMAP (37.3 mg, 0.30 mmol)
were added. The reaction mixture was stirred for 18 h
at rt, when crushed ice (20 mL) was added, and the reac-
tion mixture diluted with EtOAc (100 mL). The organic
phase was washed with satd aq solution NaHCO3
(2 · 40 mL), and the aqueous phase back extracted with
EtOAc (50 mL). The combined organic phase was evap-
orated to dryness, coevaporated with toluene
(2 · 100 mL), and the resulting residue adsorbed on
Kieselguhr and purified by silica gel column chromatog-
raphy (20–55% EtOAc in petroleum ether, v/v) to afford
5.1 Hz, 2.4 Hz, H-5), 4.55–4.62 (m, 2H, H-6, H-2),
4.18 (dd, 1H, J = 12.3 Hz, 5.1 Hz, H-6), 4.07 (d, 1H,
J = 8.4 Hz, H-4), 2.94 (s, 1H, ex, 3-OH), 2.08 (s, 3H,
CH3CO), 2.05 (s, 3H, CH3CO), 1.58 (s, 3H, C(CH3)2),
1.38 (s, 3H, C(CH3)2), 0.18 (s, 9H, Si(CH3)3);
13C NMR(CDCl ): d 170.6, 169.5, 113.6, 103.9 (C-1),
3
100.3, 94.7, 84.1 (C-2), 79.6 (C-4), 75.8, 70.9 (C-5),
63.1 (C-6), 26.8 (C(CH3)3), 26.7 (C(CH3)3), 20.9
(CH3CO), 20.7 (CH3CO), ꢀ0.5 (Si(CH3)3). Anal. Calcd
for C18H28O8Si: C, 53.98; H, 7.05. Found: C, 54.15; H,
7.05.
5.35. 5,6-Di-O-acetyl-3-C-ethynyl-1,2–O-isopropylidene-
a-D-allo-hexofuranose (45)
1
an anomeric mixture (ꢂ1:3 by H NMR) of glycosyl
donor 43 (2.01 g, 82% over two steps) as an amorphous
white solid material. Physical data for major anomer
in both methods: Rf = 0.5 (70% EtOAc in petroleum
ether, v/v); 1H NMR(CDCl 3): d 6.08 (s, 1H, H-1),
5.74 (s, 1H, H-2), 5.35 (ddd, 1H, J = 9.2, 4.5, 2.6 Hz,
H-5), 4.65 (dd, 1H, J = 12.5 Hz, 2.6 Hz, H-6), 4.48 (d,
1H, J = 9.2 Hz, H-4), 4.15 (dd, 1H, J = 12.5 Hz,
4.5 Hz, H-6), 2.75 (s, 1H, HC„C), 2.07–2.12 (5s,
5 · 3H, 5 · CH3CO; 13C NMR(CDCl 3): d 170.7,
169.6, 169.0, 168.5, 168.3, 98.5, 82.8, 78.7, 78.5, 76.7,
76.3, 71.1, 62.9, 21.1, 21.0, 20.94, 20.89, 20.6. Physical
data for pure fractions of minor anomer in both meth-
To a solution of furanose 44 (2.98 g, 7.44 mmol) in THF
(60 mL) was added glacial AcOH (0.47 mL, 8.21 mmol)
and then TBAF (1 M solution in THF, 7.4 mL,
7.4 mmol), and the reaction mixture was stirred for 2 h
at rt, whereupon it was evaporated to dryness and par-
titioned between EtOAc (100 mL) and brine (20 mL).
The phases were separated and the aqueous phase ex-
tracted with EtOAc (2 · 50 mL). The combined organic
phase was dried (MgSO4), evaporated to dryness, and
the resulting crude yellow oil absorbed on Kieselguhr
and purified by silica gel column chromatography
(40% EtOAc in petroleum ether, v/v) to afford 45
(2.24 g, 92%) as white solid material. Rf = 0.4 (70%
EtOAc in petroleum ether, v/v); MALDI-HRMS m/z
351.1050 ([M+Na]+, C15H20O8ÆNa+: Calcd 351.1050);
1H NMR(CDCl 3): d 5.85 (d, 1H, J = 3.7 Hz, H-1),
5.35 (ddd, 1H, J = 8.4, 5.2, 2.4 Hz, H-5), 4.56–4.61 (m,
2H, H-2, H-6), 4.15 (dd, 1H, J = 12.2 Hz, 5.2 Hz, H-
6), 4.07 (d, 1H, J = 8.4 Hz, H-4), 3.00 (s, 1H, ex, 3-
OH), 2.65 (s, 1H, HC„C), 2.09 (s, 3H, CH3CO), 2.06
(s, 3H, CH3CO), 1.58 (s, 3H, C(CH3)2), 1.38 (s, 3H,
C(CH3)2); 13C NMR(CDCl 3): d 170.8, 169.9, 113.9,
103.9 (C-1), 84.2 (C-2), 79.7, 79.3 (C-4), 77.5, 75.9,
70.7 (C-5), 63.2 (C-6), 26.9 (C(CH3)3), 26.8 (C(CH3)3),
21.0 (CH3CO), 20.9 (CH3CO). Anal. Calcd for
C15H20O8: C, 54.87; H, 6.14. Found: C, 54.86; H, 6.16.
1
ods: Rf = 0.4 (70% EtOAc in petroleum ether, v/v); H
NMR(CDCl ): d 6.47 (d, 1H, J = 4.4 Hz, H-1), 5.76
3
(d, 1H, J = 4.4 Hz, H-2), 5.41 (ddd, 1H, J = 7.9, 5.5,
2.7 Hz, H-5), 4.61 (d, 1H, J = 12.5 Hz, 2.7 Hz, H-6),
4.47 (d, 1H, J = 7.9 Hz, H-4), 4.19 (dd, 1H,
J = 12.5 Hz, 5.5 Hz, H-6), 2.77 (s, 1H, HC„C), 2.07–
2.13 (5s, 5 · 3H, 5 · CH3CO); 13C NMR(CDCl 3): d
170.7, 169.4, 168.8, 168.5, 168.0, 93.6, 80.7, 78.8, 76.4,
76.2, 74.6, 70.2, 62.7, 21.1, 20.94, 20.89, 20.6, 20.3. Phys-
ical data for anomeric mixture: MALDI-HRMS m/z
437.1054 ([M+Na]+, C18H22O11ÆNa+: Calcd 437.1060).
Anal. Calcd for C18H22O11: C, 52.17; H, 5.35. Found:
C, 52.18; H, 5.40.
5.34. 5,6-Di-O-acetyl-1,2-O-isopropylidene-3-C-[2-(tri-
methylsilyl)ethynyl]-a-D-allo-hexofuranose (44)
5.36. 1-[2,3,5,6-Tetra-O-acetyl-3-C-ethynyl-b-D-allo-
hexofuranosyl]uracil (46)
Furanose 40 (3.48 g, 11.0 mmol) was dried by coevapo-
ration with anhydrous pyridine (50 mL), dissolved in
anhydrous pyridine (50 mL) and cooled to 0 ꢁC where-
upon Ac2O (2.6 mL, 27.5 mmol) was added slowly over
10 min. The reaction mixture was allowed to warm up to
rt and was further stirred for 24 h, whereupon crushed
ice (20 mL) was added. The reaction mixture was evap-
orated to dryness, coevaporated with toluene (35 mL),
and the residue taken up in EtOAc (150 mL). The or-
ganic phase was washed with satd aq NaHCO3
(2 · 60 mL), evaporated to dryness, coevaporated with
toluene (2 · 100 mL) and the resulting residue absorbed
on Kieselguhr and purified by silica gel column chroma-
tography (0–50% EtOAc in petroleum ether, v/v) to
afford furanose 44 (3.08 g, 70%) as a white solid material
along with peracetylated furanose 41 (1.10 g, 23%) as a
colorless oil. Rf = 0.6 (70% EtOAc in petroleum ether,
Glycosyl donor 43 (0.96 g, 2.33 mmol) and uracil
(0.33 g, 2.91 mmol) were dried by coevaporation with
anhydrous CH3CN (20 mL), resuspended in anhydrous
CH3CN (25 mL), BSA (1.7 mL, 7.00 mmol) added and
refluxed for 1 h until a homogenous solution was ob-
tained. The solution was cooled to rt, TMSOTf
(0.57 mL, 3.14 mmol) added and the reaction mixture
refluxed for 14 h whereupon it was poured into satd
aq NaHCO3 (25 mL). The phases were separated and
the aqueous phase extracted with EtOAc (3 · 50 mL).
The combined organic phase was evaporated to dryness
and the resulting residue purified by silica gel column
chromatography (0–3% MeOH in CH2Cl2, v/v) to afford
nucleoside 46 (0.50 g, 46%) as a white solid material.
Rf = 0.5 (10% MeOH in CH2Cl2, v/v); UV kmax pH 1,
259 nm, kmax H2O, 259 nm, kmax pH 11, 263 nm; MAL-
DI-HRMS m/z 489.1124 ([M+Na]+, C20H22N2O11ÆNa+:
Calcd 489.1116);1H NMR(CDCl 3): d 8.94 (br s, 1H, ex,
v/v); MALDI-HRMS m/z 423.1456 ([M+Na]+,
1
C18H28O8SiÆNa+: Calcd 423.1446); H NMR(CDCl ):
3
d 5.85 (d, 1H, J = 3.7 Hz, H-1), 5.29 (ddd, 1H, J = 8.4,

2616
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
NH), 7.52 (d, 1H, J = 8.1 Hz, H-6), 6.06 (d, 1H,
J = 3.7 Hz, H-10), 5.83 (d, 1H, J = 8.1 Hz, H-5), 5.58
(d, 1H, J = 3.7 Hz, H-20), 5.52 (ddd, 1H, J = 8.4, 4.8,
2.4 Hz, H-50), 4.65 (dd, 1H, J = 12.3 Hz, 2.4 Hz, H-60),
4.27 (d, 1H, J = 8.4 Hz, H-40), 4.12 (dd, 1H,
J = 12.3 Hz, 4.8 Hz, H-60), 2.89 (s, 1H, HC„C), 2.09–
2.14 (4s, 12H, 4 · CH3CO); 13C NMR(CDCl 3): d
170.7, 169.4, 168.3, 168.0, 162.4, 150.0, 139.2 (C-6),
104.1 (C-5), 88.1 (C-10), 79.7, 79.6, 77.5 (C-20), 77.0,
75.3, 69.8 (C-50), 62.7 (C-60), 20.92 (CH3), 20.86
(CH3), 20.7 (CH3), 20.4 (CH3). Anal. Calcd for
C20H22N2O11: C, 51.50; H, 4.75; N, 6.01. Found: C,
51.22; H, 4.81; N, 5.74.
pH 1, 262 nm, kmax H2O, 262 nm, kmax pH 11, 263 nm;
MALDI-HRMS m/z 321.0700 ([M+Na]+, C12H14N2O7Æ
1
Na+: Calcd 321.0693); H NMR(DMSO- d6): d 11.35
(br s, 1H, ex, NH), 7.83 (d, 1H, J = 8.1 Hz, H-6), 5.84
(d, 1H, ex, J = 6.2 Hz, 20-OH), 5.72–5.75 (m, 2H, 1ex,
H-10, 30-OH), 5.66 (d, 1H, J = 8.1 Hz, H-5), 4.99 (d,
1H, ex, J = 4.8 Hz, 50-OH), 4.59 (t, 1H, ex, J = 5.5 Hz,
60-OH), 4.16 (br t, 1H, H-20), 3.89 (d, 1H, J = 4.4 Hz,
H-40), 3.63–3.79 (m, 2H, H-50, H-60), 3.40–3.56 (m,
1
2H, H-60, HC„C); Selected data H NMR(DMSO-
d6 + one drop D2O): d 5.72 (d, 1H, J = 7.0 Hz, H-10),
4.15 (d, 1H, J = 7.0 Hz, H-20); 13C NMR(DMSO- d6):
d 162.9, 150.8, 140.7 (C-6), 102.0 (C-5), 85.8 (C-10/C-
40), 85.7 (C-10/C-40), 83.0, 78.1 (C-20), 77.8, 72.7, 72.2,
62.4 (C-60). Anal. Calcd for C12H14N2O7Æ11/16 MeOH:
C, 47.68; H, 5.07; N, 8.77. Found: C, 47.30; H, 4.84;
N, 8.64.
5.37. 1-[2,3,5,6-Tetra-O-acetyl-3-C-ethynyl-b-D-allo-
hexofuranosyl]cytosine (47)
Glycosyl donor 43 (0.80 g, 1.94 mmol) and cytosine
(0.27 g, 2.42 mmol) were dried by coevaporation with
anhydrous CH3CN (20 mL), resuspended in CH3CN
(20 mL), BSA (1.4 mL, 5.82 mmol) added and refluxed
for 1 h when a clear homogenous solution was obtained.
After cooling to rt, TMSOTf (0.47 mL, 2.60 mmol) was
added and the reaction mixture refluxed for 40 h. At this
time analytical TLC showed approx 50% conversion
and the reaction mixture was therefore cooled to rt
and a second portion of TMSOTf (0.25 mL, 1.38 mmol)
added. After stirring for further 40 h, the reaction mix-
ture was poured into satd aq NaHCO3 (20 mL), phases
separated and the aqueous phase extracted with EtOAc
(4 · 30 mL). The combined organic phase was evapo-
rated to dryness and the residue purified by silica gel col-
umn chromatography (0–12% MeOH in CH2Cl2, v/v) to
afford nucleoside 47 (0.47 g, 52%) as a white solid mate-
rial. Rf = 0.5 (10% MeOH in CH2Cl2, v/v); UV kmax
pH 1, 277 nm, kmax H2O, 234, 269 nm, kmax pH 11,
272 nm; MALDI-HRMS m/z 488.1256 ([M+Na]+,
C20H23N3O10ÆNa+: Calcd 488.1276); 1H NMR(CDCl 3):
d 7.54 (d, 1H, J = 7.2 Hz, H-6), 6.19 (d, 1H, J = 3.7 Hz,
H-10), 5.87 (d, 1H, J = 7.2 Hz, H-5), 5.69 (d, 1H,
J = 3.7 Hz, H-20), 5.49–5.56 (m, 1H, H-50), 4.67 (dd,
1H, J = 12.3 Hz, 2.2 Hz, H-60), 4.32 (d, 1H,
J = 8.8 Hz, H-40), 4.14 (dd, 1H, J = 12.3 Hz, 4.8 Hz,
H-60), 2.89 (s, 1H, HC„C), 2.04–2.14 (m, 12H,
4 · CH3CO); 13C NMR(CDCl 3): d 170.6, 169.3,
167.85, 167.78, 165.9, 155.4, 140.2 (C-6), 96.4 (C-5),
89.1 (C-10), 80.1, 79.5, 77.4 (C-20), 76.6, 75.4, 69.9 (C-
50), 62.6 (C-60), 20.8 (CH3), 20.7 (CH3), 20.6 (CH3),
20.4 (CH3).
5.39. 1-[3-C-Ethynyl-b-D-allo-hexofuranosyl]cytosine (11)
Protected nucleoside 47 (172 mg, 0.37 mmol) was dis-
solved in satd methanolic ammonia (15 mL) and stirred
in a sealed container for 21 h, whereupon the reaction
mixture was evaporated to dryness and coevaporated
with absolute EtOH (2 · 10 mL) and o-xylene
(3 · 5 mL). The residue was taken up in MeOH
(15 mL) and washed with n-hexane (10 mL). Methanol
was evaporated off and the resulting residue purified
by silica gel column chromatography (0–36% MeOH
in CH2Cl2, v/v) to afford target nucleoside 11 (59 mg,
54%) as a white solid material. Rf = 0.2 (40% MeOH
in CH2Cl2, v/v); UV kmax pH 1, 278 nm, kmax H2O,
269 nm, kmax pH 11, 271 nm; MALDI-HRMS m/z
320.0853
([M+Na]+,
C12H15N3O6ÆNa+:
Calcd
320.0853); 1H NMR(DMSO- d6): d 7.70 (d, 1H,
J = 7.3 Hz, H-6), 7.25 (br s, 1H, ex, NH), 7.21 (br s,
1H, ex, NH), 5.71–5.74 (m, 3H, 1ex, H-5, H-10, 20-
OH), 5.57 (s, 1H, ex, 30-OH), 4.87 (d, 1H, ex,
J = 4.8 Hz, 50-OH), 4.54 (br t, 1H, ex, 60-OH), 4.11 (br
t, 1H, H-20), 3.83 (d, 1H, H-40), 3.76 (m, 1H, H-50),
3.62–3.70 (m, 1H, H-60), 3.41–3.54 (m, 2H, C„CH,
1
H-60); Selected data H NMR(DMSO- d6 + one drop
D2O): d 5.72 (d, 1H, J = 5.9 Hz, H-10), 4.09 (d, 1H,
J = 5.9 Hz, H-20); 13C NMR(DMSO- d6): d 165.5,
155.4, 141.6 (C-6), 94.2 (C-5), 87.5 (C-10), 84.9 (C-40),
83.5, 78.7 (C-20), 77.7, 72.52, 72.49, 62.6 (C-60).
5.40. 3-O-Benzyl-5-O-(tert-butyldimethylsilyl)-6-deoxy-
1,2-O-isopropylidene-a-D-gluco-hexofuranose (49)
5.38. 1-[3-C-Ethynyl-b-D-allo-hexofuranosyl]uracil (10)
To a
solution of 6-deoxyfuranose 4836 (2.97 g,
10.1 mmol) in anhydrous CH2Cl2 (100 mL) was added
TBDMSCl (6.14 g, 40.7 mmol) and imidazole (2.75 g,
40.4 mmol) and the reaction mixture was stirred for
24 h at rt. Solid residues were filtered off and washed
with CH2Cl2. The combined organic phase was washed
with H2O (50 mL) and brine (2 · 50 mL), and evapo-
rated to dryness. The resulting residue was purified by
silica gel column chromatography with (50% Et2O in
petroleum ether, v/v) to afford furanose 49 (3.76 g,
91%) as a clear oil. Rf = 0.8 (50% EtOAc in petroleum
Protected nucleoside 46 (248 mg, 0.53 mmol) was dis-
solved in satd methanolic ammonia (15 mL) and stirred
in a sealed container for 72 h, whereupon the reaction
mixture was evaporated to dryness and taken up in
H2O (15 mL). The aqueous phase was washed with
CH2Cl2 (2 · 20 mL) and ether (2 · 20 mL), evaporated
to dryness and the resulting residue coevaporated with
anhydrous EtOH and purified by silica gel column chro-
matography (0–15% MeOH in CH2Cl2, v/v) to afford
target nucleoside 10 (110 mg, 69%) as a white solid
material. Rf = 0.2 (20% MeOH in CH2Cl2, v/v); UV kmax
ether, v/v); MALDI-HRMS m/z 431.2224 ([M+Na]+,
1
C22H36O5SiÆNa+: Calcd 431.2221); H NMR(CDCl ):
3

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2617
d 7.18–7.29 (m, 5H, Ph), 5.79 (d, 1H, J = 4.0 Hz, H-1),
4.48–4.60 (m, 3H, H-2, CH2Ph), 4.10–4.19 (m, 1H, H-
5), 3.95 (d, 1H, J = 2.8 Hz, H-3), 3.83 (dd, 1H, J = 8.6,
2.8 Hz, H-4), 1.45 (s, 3H, C(CH3)2), 1.21–1.24 (m, 6H,
C(CH3)2, H-6), 0.79 (s, 9H, C(CH3)3), ꢀ0.02 (s, 3H,
(20 min), the mixture was stirred for 20 min, when satd
aq NH4Cl (12 mL) was added. The solution was allowed
to warm up to rt, diluted with H2O (10 mL) phases sep-
arated and the aqueous phase extracted with CH2Cl2
(2 · 20 mL). The combined organic phase was evapo-
rated to dryness and the resulting residue purified by
silica gel column chromatography (50% EtOAc in
petroleum ether, v/v) to afford the furanose 51 (9.17 g,
90%) as a clear oil. Rf = 0.7 (50% EtOAc in petroleum
ether, v/v); MALDI-HRMS m/z 437.2144 ([M+Na]+,
Si(CH3)2), ꢀ0.08 (s, 3H, Si(CH3)2); 13C NMR(CDCl ):
3
d 137.9, 128.3 (Ph), 127.6 (Ph), 127.1 (Ph), 111.5, 104.9
(C-1), 84.8 (C-4), 81.6 (C-3), 81.5, 71.5, 65.2 (C-5),
26.8 (C(CH3)2), 26.2 (C(CH3)2), 25.9 (C(CH3)3), 21.7
(C-6), 17.9, ꢀ3.7 (Si(CH3)2), ꢀ4.7 (Si(CH3)2). Anal.
Calcd for C22H36O5Si: C, 64.67; H, 8.88. Found: C,
64.55; H, 8.89.
1
C20H38O5Si2ÆNa+: Calcd 437.2150); H NMR(DMSO-
d6): d 5.67 (d, 1H, J = 3.7 Hz, H-1), 5.50 (s, 1H, ex, 3-
OH), 4.39 (d, 1H, J = 3.7 Hz, H-2), 4.00–4.08 (m, 1H,
H-5), 3.61 (d, 1H, J = 6.2 Hz, H-4), 1.44 (s, 3H,
C(CH3)2), 1.27 (s, 3H, C(CH3)2), 1.19 (d, 3H,
J = 6.2 Hz, H-6), 0.87 (s, 9H, C(CH3)3, 0.16 (s, 9H,
Si(CH3)3), 0.11 (s, 3H, Si(CH3)2), 0.08 (s, 3H, Si(CH3)2);
13C NMR(DMSO- d6): d 111.9, 105.3, 102.7 (C-1), 91.3,
84.5 (C-2), 83.8 (C-4), 75.4, 67.6 (C-5), 26.53 (C(CH3)2),
26.49 (C(CH3)2), 25.7 (C(CH3)3), 21.0 (C-6), 17.7, 0.4
(Si(CH3)3), ꢀ4.2 (Si(CH3)2), ꢀ4.7 (Si(CH3)2). Anal.
Calcd for C20H38O5Si2: C, 57.93; H, 9.24. Found: C,
57.98; H, 9.34.
5.41. 5-O-(tert-Butyldimethylsilyl)-6-deoxy-1,2-O-iso-
propylidene-a-D-gluco-hexofuranose (50)
Furanose 49 (3.60 g, 8.81 mmol) was dried by coevapo-
ration with toluene (30 mL) and dissolved in EtOAc
(85 mL). To this was added 10% Pd(OH)2/C (0.37 g)
and the reaction mixture was stirred under an H2 atmo-
sphere (balloon) for 19 h at rt. The mixture was then fil-
tered through a Celite pad, which was washed with
EtOAc. The organic phase was evaporated off, and the
resulting residue purified by silica gel column chroma-
tography (20% Et2O in petroleum ether, v/v) to afford
alcohol 50 (2.40 g, 86%) as a clear oil. Rf = 0.7 (50%
EtOAc in petroleum ether, v/v); MALDI-HRMS m/z
341.1764 ([M+Na]+, C15H30O5SiÆNa+: Calcd 341.1755);
1H NMR(DMSO- d6): d 5.76 (d, 1H, J = 3.7 Hz, H-1),
5.11 (d, 1H, ex, J = 4.4 Hz, 3-OH), 4.37 (d, 1H,
J = 3.7 Hz, H-2), 4.01 (m, 1H, H-5), 3.90–3.93 (m, 1H,
H-3), 3.64 (dd, 1H, J = 8.3, 2.9 Hz, H-4), 1.37 (s, 3H,
C(CH3)2), 1.22 (s, 3H, C(CH3)2), 1.15 (d, 3H,
J = 6.2 Hz, H-6), 0.85 (s, 9H, C(CH3)3, 0.05 (s, 6H,
Si(CH3)2); 13C NMR(DMSO- d6): d 110.4, 104.1 (C-1),
84.8 (C-2), 84.2 (C-4), 72.6 (C-3), 64.7 (C-5), 26.6
(C(CH3)2), 26.0 (C(CH3)2), 25.6 (C(CH3)3), 21.4 (C-6),
17.5, ꢀ4.6 (Si(CH3)2), ꢀ5.0 (Si(CH3)2). Anal. Calcd
for C15H30O5Si: C, 56.57; H, 9.49. Found: C, 56.62;
H, 9.53.
5.43. 6-Deoxy-3-C-ethynyl-1,2-O-isopropylidene-a-D-
allo-hexofuranose (52)
To a solution of furanose 51 (9.17 g, 22.1 mmol) in THF
(220 mL) was added TBAF (44 mL, 1 M solution in
THF, 44.0 mmol) and the reaction mixture was stirred
for 3 h at rt, whereupon it was evaporated to dryness.
The resulting residue was purified by silica gel column
chromatography (70% EtOAc in petroleum ether, v/v)
to afford diol 52 (4.97 g, 98%) as a white solid material.
Rf = 0.7 (EtOAc); MALDI-HRMS m/z 251.0891
1
([M+Na]+, C11H16O5ÆNa+: Calcd 251.0890); H NMR
(DMSO-d6): d 5.68 (d, 1H, J = 3.5 Hz, H-1), 5.54 (s,
1H, ex, 3-OH), 4.49 (d, 1H, ex, J = 5.1 Hz, 5–OH),
4.43 (d, 1H, J = 3.5 Hz, H-2), 3.82–3.92 (m, 1H, H-5),
3.54–3.57 (m, 2H, H-4, HC„C), 1.45 (s, 3H,
C(CH3)2), 1.27 (s, 3H, C(CH3)2), 1.14 (d, 3H,
J = 6.2 Hz, H-6); 13C NMR(DMSO- d6): d 111.9, 102.8
(C-1), 84.2 (C-2), 83.2 (C-4), 82.8, 78.2, 75.2, 65.6 (C-
5), 26.6 (C(CH3)2), 26.4 (C(CH3)2), 20.7 (C-6). Anal.
Calcd for C11H16O5: C, 57.88; H, 7.07. Found: C,
57.88; H, 7.06.
5.42. 5-O-(tert-Butyldimethylsilyl)-6-deoxy-1,2-O-iso-
propylidene-3-C-[2-(trimethylsilyl)ethynyl]-a-D-allo-hexo-
furanose (51)
Alcohol 50 (7.83 g, 24.6 mmol) was dried by coevapora-
tion with toluene (50 mL), and dissolved in anhydrous
˚
CH2Cl2 (250 mL). To this, freshly activated 3 A molec-
5.44. 3-C-Ethynyl-1,2-O-isopropylidene-6-O-toluene-
sulfonyl-a-D-allo-hexofuranose (54)
ular sieves powder (21.2 g), glacial AcOH (2.5 mL,
43.7 mmol) and PDC (9.23 g, 24.5 mmol) were added
and the reaction mixture stirred at rt for 22 h. The mix-
ture was evaporated to dryness, coevaporated with tolu-
ene (100 mL) and resuspended in EtOAc. Solid residues
were filtered off using a 2 cm silica pad, which was thor-
oughly washed with additional EtOAc. Evaporation of
the organic phase afforded the crude ketone, which
was dried by coevaporation with toluene and used in
the next step without further purification. Crude ketone
in anhydrous THF (20 mL) was added to a solution of
n-butyllithium (20.0 mL, 2.0 M in hexanes, 40.0 mmol)
and (trimethylsilyl)acetylene (4.5 mL, 0.03 mol) in anhy-
drous THF (40 mL) at ꢀ78 ꢁC. After ended addition
Triol 5328 (3.29 g, 13.4 mmol) was dried by coevapora-
tion with anhydrous pyridine (3 · 50 mL) and dissolved
in anhydrous pyridine (60 mL). To this, a solution of
TsCl (3.87 g, 20.3 mmol) in anhydrous CH2Cl2
(10 mL) was added dropwise at ꢀ50 ꢁC. After ended
addition, the reaction mixture was allowed to warm up
to rt and stirred for 18 h whereupon the reaction mix-
ture was evaporated to dryness. The resulting residue
was purified by silica gel column chromatography (30–
50% EtOAc in petroleum ether, v/v) to afford diol 54
(3.04 g, 57%) as a white solid material, which was used
in the next step without further purification. Rf = 0.6
(EtOAc); MALDI-HRMS m/z 421.0938 ([M+Na]+,

2618
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
C18H22O8SÆNa+: Calcd 421.0928); 1H NMR(DMSO-
d6): 7.77 (d, 2H, J = 8.1 Hz), 7.47 (d, 2H,
to rt, TMSOTf (0.91 mL, 5.06 mmol) was added and
the reaction mixture refluxed for 20 h, whereupon satd
aq NaHCO3 (10 mL) was added, and the mixture evap-
orated to dryness. The residue was partitioned between
CH2Cl2 (25 mL) and brine (25 mL) and after separation
of the phases, the aqueous phase was extracted with
CH2Cl2 (3 · 20 mL). The combined organic phase was
evaporated to dryness and purified by silica gel column
chromatography (0–1% MeOH in CH2Cl2, v/v) to afford
protected nucleoside 56 (0.65 g, 79%) as a white solid
material. Rf = 0.6 (10% MeOH in CH2Cl2, v/v); UV kmax
d
J = 8.1 Hz), 5.77 (s, 1H), 5.65 (d, 1H, J = 3.5 Hz), 5.31
(d, 1H, J = 5.9 Hz), 4.42 (d, 1H, J = 3.5 Hz), 4.16 (d,
1H, J = 8.4 Hz), 3.85–3.97 (m, 2H), 3.75 (d, 1H,
J = 6.6 Hz), 3.58 (s, 1H), 2.42 (s, 3H), 1.42 (s, 3H),
1.27 (s, 3H); 13C NMR(DMSO- d6): d 144.7, 132.1,
130.0, 127.5, 112.2, 102.9, 84.0, 82.1, 79.6, 78.6, 75.1,
72.7, 67.7, 26.5, 26.4, 21.0. EtOAc was identified as a
trace impurity.
5.45. 1,2,3,5-Tetra-O-acetyl-6-deoxy-3-C-ethynyl-a,b-D-
allo-hexofuranose (55)
pH 1, 259 nm, kmax H2O, 259 nm,
pH 11, 263 nm;
max
MALDI-HRMS m/z 431.1065 ([M+Na]+, C18H20N2O9Æ
Na+: Calcd 431.1061); 1H NMR(CDCl 3): d 8.94 (s,
1H, ex, NH), 7.51 (d, 1H, J = 8.1 Hz, H-6), 6.04 (d, 1H,
J = 4.0 Hz, H-10), 5.81 (dd, 1H, J = 8.1, 2.2 Hz, H-5),
5.55 (d, 1H, J = 4.0 Hz, H-20), 5.29–5.38 (m, 1H, H-
50), 4.09 (d, 1H, J = 7.7 Hz, H-40), 2.86 (s, 1H, HC„C),
2.15 (s, 3H, CH3CO), 2.10 (s, 3H, CH3CO), 2.08 (s, 3H,
CH3CO), 1.31 (d, 3H, J = 6.2 Hz, H-60); 13C NMR
(CDCl3): d 169.4, 168.3, 168.0, 162.4, 149.9, 139.1 (C-
6), 103.8 (C-5), 87.6 (C-10), 83.6 (C-40), 79.1, 77.6,
77.2, 75.0, 68.9 (C-50), 21.0 (CH3CO), 20.6 (CH3CO),
20.3 (CH3CO), 17.3 (C-60). Anal. Calcd for
C18H20N2O9Æ1/16 CH2Cl2: C, 52.32; H, 4.89; N, 6.76.
Found: C, 52.24; H, 4.82; N, 6.52.
Diol 52 (1.25 g, 5.48 mmol) was dried by coevaporation
with anhydrous pyridine (20 mL) and dissolved in anhy-
drous pyridine (55 mL). To this Ac2O (2.0 mL,
21.1 mmol) and DMAP (75.6 mg, 0.62 mmol) were
added and the reaction mixture was stirred for 18 h at
rt whereupon analytical TLC showed clean conversion
to a less polar product (Rf = 0.4, 50% EtOAc in petro-
leum ether, v/v). Subsequently, crushed ice (10 mL)
was added and the mixture evaporated to dryness,
coevaporated with toluene (20 mL) and the residue
taken up in EtOAc (25 mL). The organic phase was
washed with satd aq NaHCO3 (2 · 10 mL), evaporated
to dryness and coevaporated with toluene (20 mL) to
leave a residue, which was directly dissolved in 80%
aqueous TFA (55 mL). The reaction mixture was stirred
at rt for 3 h, whereupon analytical TLC showed full con-
version to two polar compounds (both Rf = 0.1, 50%
EtOAc in petroleum ether, v/v). The reaction mixture
was evaporated to dryness, coevaporated with toluene
(3 · 20 mL) and anhydrous pyridine (20 mL) and di-
rectly dissolved in anhydrous pyridine (55 mL). To this
Ac2O (2.0 mL, 21.1 mmol) and DMAP (67.4 mg,
0.55 mmol) were added and the reaction mixture was
stirred at rt for 94 h, whereupon crushed ice (10 mL)
was added and the reaction mixture evaporated to dry-
ness. The residue was taken up in EtOAc (50 mL) and
the organic phase washed with satd aq NaHCO3
(2 · 20 mL). The organic phase was evaporated to dry-
ness and purified by silica gel column chromatography
(50% EtOAc in petroleum ether, v/v) affording an ano-
5.47. 1-[2,3,5-Tri-O-acetyl-6-deoxy-3-C-ethynyl-b-D-allo-
hexofuranosyl]-4-N-benzoylcytosine (57)
Glycosyl donor 55 (0.63 g, 1.77 mmol) and 4-N-benzoyl-
cytosine (0.75 g, 3.48 mmol) were dried by coevapora-
tion with anhydrous CH3CN (2 · 20 mL) and
suspended in anhydrous CH3CN (20 mL). To this
BSA (1.3 mL, 5.26 mmol) was added and the mixture re-
fluxed until it became homogenous (<1 h). After cooling
to rt, TMSOTf (0.80 mL, 4.43 mmol) was added and the
reaction mixture refluxed for 21 h, whereupon satd aq
NaHCO3 (10 mL) was added. The mixture was further
diluted with H2O (20 mL) and EtOAc (20 mL), the
phases separated and the aqueous phase extracted with
CH2Cl2 (3 · 20 mL). The combined organic phase was
evaporated to dryness and the resulting residue purified
by silica gel column chromatography (1% MeOH in
CH2Cl2, v/v) to afford protected nucleoside 57 (0.68 g,
75%) as a white solid material. Rf = 0.7 (10% MeOH
in CH2Cl2, v/v); UV kmax pH 1, 257, 315 nm, kmax
H2O, 261, 301 nm, kmax pH 11, 316 nm; MALDI-
1
meric mixture (1:1 by H NMR) of glycosyl donor 55
(1.41 g, 72%, over three steps) as a white solid material.
Physical data for anomeric mixture: Rf = 0.8 (EtOAc);
MALDI-HRMS m/z 379.0985 ([M+Na]+, C16H20O9Æ
Na+: Calcd 379.1000); 13C NMR(CDCl 3): d 169.5,
169.4, 168.9, 168.8, 168.5, 168.3, 168.1, 168.0, 98.1,
93.3, 86.4, 84.5, 78.6, 78.2, 77.9, 76.6, 76.3, 76.2, 75.8,
74.8, 70.1, 69.4, 21.0, 20.9, 20.8, 20.72, 20.67, 20.4,
20.1, 17.3, 16.5.
HRMS m/z 534.1476 ([M+Na]+, C25H25N3O9ÆNa+:
1
Calcd 534.1483); H NMR(CDCl ): d 8.97 (s, 1H, ex,
3
NH), 7.89–7.94 (m, 3H, H-6, Ph/H-5), 7.47–7.62 (m,
4H, Ph/H-5), 6.21 (d, 1H, J = 3.5 Hz, H-10), 5.68 (d,
1H, J = 3.5 Hz, H-20), 5.34–5.43 (m, 1H, H-50), 4.20
(d, 1H, J = 7.3 Hz, H-40), 2.83 (s, 1H, HC„C), 2.11
(s, 3H, CH3CO), 2.09 (s, 3H, CH3CO), 2.03 (s, 3H,
CH3CO), 1.46 (d, 3H, J = 6.2 Hz, H-60); 13C NMR
(CDCl3): d 169.4, 167.9, 167.8, 162.5, 143.7 (C-6),
133.2 (Ph), 129.0 (Ph), 127.6 (Ph), 97.5 (C-5), 88.5 (C-
10), 84.1 (C-40), 79.4, 77.9 (C-20), 77.0, 75.1, 69.0 (C-
50), 21.0 (CH3CO), 20.6 (CH3CO), 20.4 (CH3CO), 17.3
(C-60). Anal. Calcd for C25H25N3O9Æ5/16 EtOH: C,
58.53; H, 5.15; N, 7.99. Found: C, 58.19; H, 4.85; N,
8.16.
5.46. 1-[2,3,5-Tri-O-acetyl-6-deoxy-3-C-ethynyl-b-D-allo-
hexofuranosyl]uracil (56)
Glycosyl donor 55 (0.72 g, 2.02 mmol) was dried by
coevaporation with anhydrous CH3CN (2 · 20 mL)
and suspended in anhydrous CH3CN (6 mL). To this
was added uracil (0.46 g, 4.10 mmol) and BSA
(1.5 mL, 6.07 mmol) and the solution refluxed until a
homogenous solution was formed (<1 h). After cooling

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
2619
5.48. 1-[6-Deoxy-3-C-ethynyl-b-D-allo-hexofurano-
syl]uracil (12)
after initial minimization of nucleosides using the all-
39
atom AMBERforce field and GB/SA model.40 Non-
bonded interactions were treated without cut-offꢁs.
Throughout conformational searches, the generated
structures were first minimized with the truncated New-
ton conjugate gradient method till a convergence crite-
Protected nucleoside 56 (0.48 g, 1.18 mmol) was dis-
solved in satd methanolic ammonia (12 mL) and stirred
at rt in a sealed container for 71 h whereupon the reac-
tion mixture was evaporated to dryness and coevapo-
rated with anhydrous EtOH (2 · 10 mL). The resulting
residue was adsorbed on silica gel and purified by silica
gel column chromatography (10% MeOH in CH2Cl2,
v/v) to afford target nucleoside 12 (0.26 g, 78%) as a white
solid material. Crystals for use in single crystal X-ray
diffraction studies were obtained by recrystallization
from MeOH. Rf = 0.8 (EtOAc); UVmax pH 1, 261 nm,
kmax H2O, 262 nm, kmax pH 11, 262 nm; MALDI-
HRMS m/z 305.0757 ([M+Na]+, C12H14N2O6ÆNa+:
˚
rion of 0.005 kJ/(A mol) was reached and then refined
using a full matrix Newton Raphson method using the
same convergence criterion. Searches were regarded as
complete when all conformations within 15 kJ/mol of
the global energy minimum were found at least 10 times.
Unique conformations were determined by superimposi-
tion of all heavy (nonhydrogen) atoms and regarded as
duplicate if the interatomic distance in RMS superimpo-
˚
sitions was below 0.25 A.
1
Calcd 305.0744); H NMR(DMSO- d6): d 11.35 (s, 1H,
5.51. Crystallography methods
ex, NH), 7.79 (d, 1H, J = 8.1 Hz, H-6), 5.86 (d, 1H,
ex, J = 6.2 Hz, 20-OH), 5.78 (s, 1H, ex, 30-OH), 5.72
(d, 1H, J = 7.0 Hz, H-10), 5.67 (d, 1H, J = 8.1 Hz, H-
5), 4.80 (d, 1H, ex, J = 4.8 Hz, 50-OH), 4.11 (br t, 1H,
H-20), 3.89–3.94 (m, 1H, H-50), 3.68 (d, 1H,
J = 4.4 Hz, H-40), 3.55 (s, 1H, HC„C), 1.23 (d, 3H,
J = 6.6 Hz, H-60); 13C NMR(DMSO- d6): d 162.9,
150.8, 140.6 (C-6), 102.0 (C-5), 89.0 (C-40), 85.6 (C-10),
83.2, 77.9, 77.8, 71.9, 66.8 (C-50), 19.5 (C-60).
Reflection intensities of 8 and 9 were collected on a
Siemens/Bruker SMART 1K CCD diffractometer
with graphite monochromated Mo Ka radiation,
˚
k = 0.71069 A. Reflection intensities of 12 were collected
on a Bruker–Nonius X8APEX-II CCD diffractometer.
Data collection, integration of frame data and conversion
to intensities were performed using the programs SMART,
SAINT, and SADABS.52,53 Structure solution, refinement
and analysis, and production of crystallographic illustra-
tions were carried out using the programs SIR97,54
SHELXL,55 and X-SEED.56 In no case could the absolute
configuration be established from the X-ray analysis.
5.49. 1-[6-Deoxy-3-C-ethynyl-b-D-allo-hexofurano-
syl]cytosine (13)
Protected nucleoside 57 (0.30 g, 0.59 mmol) was dis-
solved in satd methanolic ammonia (6 mL) and stirred
at rt for 49 h, whereupon the reaction mixture was evap-
orated to dryness and coevaporated with anhydrous
EtOH (2 · 10 mL). The resulting residue was adsorbed
on silica gel and purified by silica gel column chroma-
tography (0–10% MeOH in CH2Cl2, v/v) to afford target
nucleoside 13 (106 mg, 64%) as a white solid material.
Rf = 0.2 (20% MeOH in CH2Cl2, v/v); UV kmax pH 1,
279 nm, kmax H2O, 269 nm, kmax pH 11, 271 nm; MAL-
DI-HRMS m/z 304.0893 ([M+Na]+, C12H15N3O5ÆNa+:
5.52. Crystallographic data of 8
˚
C12H14N2O7, M = 298.25, monoclinic, a = 6.4587(8) A,
˚
˚
b = 6.4659(8) A, c = 14.7700(17) A, b = 97.401(2)ꢁ, V =
3
611.68(13) A , P21 (no. 4), Z = 2, Dx = 1.619 g cmꢀ3
,
˚
F(000) = 312, l = 0.135 mmꢀ1, T = 120 K. 7928 reflec-
tions were measured to hmax = 29.99ꢁ and were merged
(Rint = 0.0325) to 3090 unique reflections (including Fri-
edel equivalents). The refinement using 202 parameters
converged at R1 = 0.0365 (for Fo > 4r(Fo)) and
wR2 = 0.0801 (for all data).
1
Calcd 304.0904); H NMR(DMSO- d6): d 7.66 (d, 1H,
J = 7.3 Hz, H-6), 7.24 (br s, 1H, ex, NH), 7.18 (br s,
1H, ex, NH), 5.72–5.78 (m, 3H, 1ex, H-5, H-10, 20-
OH), 5.63 (s, 1H, ex, 30-OH), 4.74 (d, 1H, ex,
J = 5.1 Hz, 50-OH), 4.05 (br t, 1H, H-20), 3.90–3.95 (m,
1H, H-50), 3.65 (d, 1H, J = 5.1 Hz, H-40), 3.52 (s, 1H,
HC„C), 1.21 (d, 3H, J = 6.2 Hz, H-60). Selected 1H
NMRsignals (DMSO- d6 + one drop D2O): d 5.73 (d,
1H, J = 6.3 Hz, H-10), 4.04 (d, 1H, J = 6.3 Hz, H-20);
13C NMR(DMSO- d6): d 165.5, 155.2, 141.5 (C-6),
94.4 (C-5), 88.4 (C-40), 87.3 (C-10), 83.7, 78.5 (C-20),
77.7, 72.2, 66.7 (C-50), 19.5 (C-60). Anal. Calcd for
C12H15N3O5Æ1/2 H2O: C, 49.65; H, 5.56; N, 14.48.
Found: C, 49.49; H, 5.46; N, 14.08.
5.53. Crystallographic data of 9
˚
C12H15N3O6, M = 297.27, tetragonal, a = 7.8839(14) A,
3
˚
˚
c = 40.106(6) A, V = 2492.8(7) A , P41212 (no. 92), Z =
8, Dx = 1.584 g cmꢀ3, F(000) = 1248, l = 0.129 mmꢀ1
T = 180 K. 6412 reflections were measured to
max = 24.70ꢁ and were merged (Rint = 0.0704) to 2044
,
h
unique reflections (including Friedel equivalents). The
refinement using 208 parameters converged at
R1 = 0.0477 (for Fo > 4r(Fo)) and wR2 = 0.0978 (for all
data).
5.54. Crystallographic data of 12
5.50. Molecular modeling
˚
C12H14N2O6, M = 282.25, monoclinic, a = 8.1725(8) A,
˚
˚
All calculations were performed using the MACROMODEL
V7.2 suite41 of software programs running on 1400 MHz
Athlon PCꢁs under the Linux Mandrake 8.0 operating
system. Conformational searches using the mixed
Monte Carlo49 Low Mode50,51 method were performed
b = 5.5898(6) A, c = 13.5508(14) A, b = 91.749(4), V =
3
618.75(11) A , P21 (no. 4), Z = 2, Dx = 1.515 g cmꢀ3
,
˚
F(000) = 296, l = 0.123 mmꢀ1, T = 180 K. 8924 reflec-
tions were measured to hmax = 29.12ꢁ and were merged
(Rint = 0.0264) to 2777 unique reflections (including

2620
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13 (2005) 2597–2621
7. Shimamoto, Y.; Fujioka, A.; Kazuno, H.; Murakami, Y.;
Ohshimo, H.; Kato, T.; Matsuda, A.; Sasaki, T.; Fuku-
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Friedel equivalents). The refinement using 192 parame-
ters converged at R1 = 0.0302 (for Fo > 4r(Fo)) and
wR2 = 0.0808 (for all data).
5.55. Biological assays
Target nucleosides were evaluated for antiviral activity
against HIV-1 in MT-4 cells and anticancer activity
against human adenocarcinoma breast cancer (MCF-
7) and prostate cancer (PC-3) cell lines as previously
described.43,45
11. Minakawa, N.; Kaga, D.; Kato, Y.; Endo, K.; Tanaka,
M.; Sasaki, T.; Matsuda, A. J. Chem. Soc., Perkin Trans. 1
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Acknowledgements
13. For definitions of pseudorotational phase angle, maximal
puckering amplitude and glycosidic torsion angle see
Saenger, W. Principles of nucleic acid structure; Springer:
Berlin, 1984.
The Danish National Research Foundation and The
Oticon Foundation are thanked for financial support.
We are grateful to the Danish Natural Science Research
Council (SNF) and Carlsbergfondet (Denmark) for pro-
vision of the X-ray equipment and to Dr. Andrew Bond,
Dr. Niels Thorup and Jens K. Bjernemose for data col-
lection and structure solution and refinement of the
crystal structure of 8, 9, and 12. DTU is thanked
for diffractometer access. The technical assistance of
Ms. Laila Ø. Madsen and Birgit E. Reiter is greatly
appreciated.
14. Marquez, V. E.; Ezzitouni, A.; Russ, P.; Siddiqui, M. A.;
Ford, H., Jr.; Feldman, R. J.; Mitsuya, H.; George, C.;
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Supplementary data
1H NMRspectra of 25, 31, and 39 and 13C NMRspec-
tra of 5–7, 11–12, 14, 17, 24, 26, 32–33, 47, 54–55. Sup-
plementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2005.01.029.
21. No attempts were made to identify the configuration at
C4.
Crystallographic data (excluding structure factors) for 8,
9 and 12 have been deposited with the Cambridge Crys-
tallographic Data Centre as supplementary publication
nos. CCDC 261822–261824. Copies of the data can be
obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge, CB2 1EZ, UK, (fax: +44-
(0)1223-336033 or email: deposit@ccdc.cam.uk).
22. Youssefyeh, R. D.; Verheyden, J. P. H.; Moffat, J. G. J.
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27. The relative configuration at C3 could not be determined
at this stage but was proven upon single crystal X-ray
diffraction studies of the 40-C-hydroxymethyl nucleosides
8 and 9.
28. Qureshi, S.; Shaw, G. J. Chem. Soc., Perkin Trans. 1 1985,
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