Synthesis and biological evaluation of nucleobase-modified analogs of the anticancer compounds 3′-C-ethynyluridine (EUrd) and 3′-C- ethynylcytidine (ECyd)

Article abstract of DOI:10.1016/j.bmc.2004.11.054

A series of nucleobase-modified analogs of the anticancer compounds 3′-C-ethynyluridine (EUrd) and 3′-C-ethynylcytidine (ECyd) were designed to overcome the strict substrate specificity of the activating uridine-cytidine kinase. EUrd, ECyd and target nucleosides were obtained using a short convergent synthetic route utilizing diacetone-α-d-glucose as starting material. 5-Iodo-substituted EUrd was the most potent inhibitor among the novel nucleobase-modified analogs in in vitro assays against human adenocarcinoma breast and prostate cancer cells with IC₅₀ values down to 35 nM.

Full text of DOI:10.1016/j.bmc.2004.11.054

Bioorganic & Medicinal Chemistry 13 (2005) 1249–1260
Synthesis and biological evaluation of nucleobase-modified
analogs of the anticancer compounds 30-C-ethynyluridine
(EUrd) and 30-C-ethynylcytidine (ECyd)
Patrick J. Hrdlicka,a Jan S. Jepsen,b Claus Nielsenc and Jesper Wengela,*
aNucleic Acid Center, Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
bDanish Cancer Society, Institute of Cancer Biology, Department of Tumor Endocrinology, DK-2100 Copenhagen, Denmark
cRetrovirus Laboratory, Department of Virology, Statens Serum Institut, DK-2300 Copenhagen, Denmark
Received 3 August 2004; accepted 26 November 2004
Available online 25 December 2004
Abstract—A series of nucleobase-modified analogs of the anticancer compounds 30-C-ethynyluridine (EUrd) and 30-C-ethynylcyt-
idine (ECyd) were designed to overcome the strict substrate specificity of the activating uridine–cytidine kinase. EUrd, ECyd and
target nucleosides were obtained using a short convergent synthetic route utilizing diacetone-a-D-glucose as starting material. 5-
Iodo-substituted EUrd was the most potent inhibitor among the novel nucleobase-modified analogs in in vitro assays against human
adenocarcinoma breast and prostate cancer cells with IC50 values down to 35 nM.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
catalyzed by human RNA polymerases I, II, or III,
which leads to cell apoptosis by a mechanism, which is
not yet fully elucidated.2–4 ECyd is distributed very
selectively into tumor tissue,5,6 accounting for the ab-
sence of severe toxicities.2,7 These properties render
ECyd as a highly promising agent in solid human cancer
therapy and ECyd is, therefore, currently evaluated in
Phase I clinical trials.
30-C-Ethynylcytidine (ECyd, 1) (Fig. 1) displays excel-
lent broad-spectrum antitumor activity in nanomolar
range in vitro as well as in tumor models.1 ECyd is phos-
phorylated by uridine–cytidine kinase (UCK, EC
2.7.1.48) to give the corresponding 50-monophosphate
that upon further phosphorylation by nucleotide kinases
gives the biologically active triphosphate (ECTP). ECTP
competitively inhibits DNA templated RNA synthesis
Structure–activity relationship studies have revealed the
configurational requirements at C20 and C308 and have
clarified the importance of the C30-substituent,8–10
O40-functionality11 and the positioning of the ethynyl
group.8,12 Furthermore, the effect of introduction of
additional branching at C40 and C50 and conformational
restriction of the furanose ring has been studied.13 These
SAR studies strongly indicate that ribo-configuration of
the furanose moiety is essential for UCK-catalyzed acti-
vation and that an ethynyl group placed at the C30(b)-
position is optimal for anticancer activity. Moreover,
UCK has strict substrate specificity, offering limited pos-
sibilities for introduction of sugar modifications for
improvement of activity. Most of the synthesized ana-
logs to date have, therefore, been insufficiently phos-
phorylated to exert a potential pharmacological action.
UCK2, one of at least two subtypes of UCK present
in human cells,14,15 has been proposed to be the more
NH2
N
HO
N
O
O
OH OH
1
Figure 1. Anticancer compound ECyd 1.
Keywords: Antitumor; EUrd; TAS-106; Uridine-cytidine kinase.
*
Corresponding author. Tel.: +45 65502510; fax: +45 66158780;
e-mail: jwe@chem.sdu.dk
0968-0896/$ - see front matter ꢀ 2004Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.11.054

1250
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
important UCK-subtype for activation of ECyd.4
A
was used as starting material. Chemoselective cleavage
of the 5,6-O-isopropylidene group of furanose 2 with
aqueous acetic acid, followed by oxidative cleavage of
the vicinal diol and sodium borohydride reduction of
the resulting aldehyde gave diol 3 in 84% yield over three
steps. Diacetylation of diol 3, followed by isopropylid-
ene cleavage with aqueous trifluoroacetic acid and per-
acetylation of the resulting anomeric diol, gave
glycosyl donor 4 in 74% yield over three steps.
variety of nucleobase-modified ribonucleosides are also
phosphorylated by UCK2, including 3-deazauridine, 4-
thiouridine, 5-halouridines, 5-hydroxyuridine, 6-azaur-
idine and 2-thiocytidine.14 In light of the aforementioned
reasons, it is, therefore, surprising that merely the natu-
ral nucleobases (uracil, thymine, cytosine, adenine, and
guanine), 5-fluorouracil, 5-fluorocytosine, and 4-ami-
no-5-oxopyrido[2,3-d]pyrimidine have been attached to
the pharmacologically relevant 30-C-ethynyl-b-D-ribo-
pentofuranosyl skeleton.1,16 Uracil (EUrd 17), 5-fluoro-
uracil, and 5-fluorocytosine derivatives display signifi-
cant inhibitory effects against leukemia cell lines, albeit
being up to 150 times less active than ECyd.1
Glycosylation of furanose 4 with a series of persilylated
nucleobases (generated in situ from the corresponding
nucleobase and N,O-bis(trimethylsilyl)acetamide, BSA)
and trimethylsilyl triflate (TMSOTf)20 in anhydrous ace-
tonitrile at elevated temperatures (50–60 ꢁC) for 1–3
days, gave b-configured nucleosides 5–12 in good yields
(63–94%, see Table 1 for yields and structures) via anchi-
meric assistance of the O2-acetyl group. In contrast, 5-
azacytidine and cyanuric acid derivatives 13 and 14 were
only obtained in 50% and 30% yield, respectively. Reac-
tion of glycosyl donor 4 with persilylated 2-thiocytosine
or 4-thiouracil under these conditions unexpectedly fur-
nished S-linked ribosides 15 and 16 in 74% and 83%
yield, respectively. A similar glycosylation has only been
observed during synthesis of S-b-D-glucuronides.21,22
Deprotection of nucleosides 5–12 and 14 with saturated
methanolic ammonia proceeded smoothly to afford
target nucleosides 1, 17–23 and 25 in modest yields
(46–83%, see Table 1 for yields and structures), which
primarily reflect loss of compound during column
chromatography or crystallization. Since 5-azacytidine
derivatives are known to be very labile in the presence
of strong nucleophiles,23 a different deprotection proto-
col was chosen for 5-azacytidine derivative 13. Subject-
Herein we describe the synthesis of ECyd 1, EUrd 17
and a series of nucleobase-modified analogs 18–27 here-
of (Scheme 1 and Table 1), which were anticipated to
overcome the crucial activation step catalyzed by
UCK and to be further converted to potentially phar-
macologically active triphosphates.
2. Chemistry
2.1. Synthesis of nucleobase-modified ECyd-analogs
A desirable synthetic route towards EUrd, ECyd, and
nucleobase-modified analogs hereof, would proceed via
a convergent strategy, where a protected 3-C-ethynyl-
D-ribo-pentofuranose as glycosyl donor is reacted with
a series of persilylated nucleobases. Of three synthetic
routes reported to date leading to ECyd,1,17,18 two pro-
ceed via convergent strategies. Both synthetic routes
attract by excellent yields, but require many
chromatographic purification steps1 or employ non-
standard protecting groups, which were selected for
optimized large-scale production of ECyd and EUrd.17
We, therefore, chose a slightly different convergent strat-
egy in which peracetylated furanose 4 was identified as
the key intermediate (Scheme 1). Known furanose 2,19
easily obtained from inexpensive diacetone-a-D-glucose,
´
ing nucleoside 13 to classical Zemplen conditions
(catalytic sodium methoxide in anhydrous MeOH) re-
sulted in full decomposition to very polar products (re-
sults not shown), whereas deprotection with dilute
methanolic ammonia successfully afforded nucleoside
24 in 63% yield. Deprotection of S-ribosides 15 and 16
with methanolic ammonia gave 26 and 27 in 72% and
70% yield, respectively.
O
O
AcO
O
HO
O
O
a,c
d-f
OAc
O
O
OH
O
OAc OAc
OH
O
4
3
2
AcO
B
HO
B
O
O
g
h
OAc OAc
OH OH
5-16
1, 17-27
Scheme 1. Reagents and conditions: (a) 80% aq AcOH, rt; (b) NaIO4, MeOH/H2O (3:1, v/v), 0 ꢁC to rt; (c) NaBH4, MeOH, 0 ꢁC to rt, 84% over
three steps; (d) Ac2O, DMAP, pyridine, rt; (e) 80% aq TFA, 0 ꢁC to rt; (f) Ac2O, DMAP, pyridine, rt, 74% over three steps; (g) nucleobase, BSA,
TMSOTf, CH3CN (yields see Table 1); (h) NH3/MeOH (yields see Table 1).

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
1251
Table 1. Structures of nucleobase-modifications and yields of glycosylations and deacylations
B=
NHR'
Product glycosylation
Yield (%)
Product deacetylation
Yield (%)
5
71
1
74
N
R0 = Bz
R0 = H
N
O
O
N
R = H
R = OH
R = Cl
R = Br
R = I
6
90
64
78
94
80
17
18
19
20
21
77
46
83
68
62
7
R
NH
8
9
10
O
OH
11
12
13
14
15
16
79
63
22
23
24
25
26
27
63
82
63
65
72
70
N
O
O
NH
N
N
N
O
NH2
N
50
N
O
O
N
HN
NH
30
O
O
NH2
N
N
S
74
H
N
O
ꢀ83
N
S
2.2. Structure analysis
the 5-OH group as an exchangeable singlet at 8.71 ppm
in 1H NMR, excluded the possibility of an O-linked
product and indicated the N1-linked structure of nucleo-
side 18. Assignments of 5-halonucleosides 19–21 were
verified by a characteristic downfield signal of H-6 in
1H NMR (ꢀ8.5 ppm) and absence of bathochromic
shifts in their UV spectra in alkaline medium, since
bathochromic shifts are a known characteristic of N3-
nucleosides.26 The regiochemical constitution of nucleo-
The identity of all new compounds was established on
the basis of 1D- and 2D-NMR techniques, UV-measure-
ments, high resolution MALDI-MS, and/or elemental
analysis. ECyd 1 and EUrd 17 were identified by com-
parison with published spectral data.1 Differentiation
between N1-, N3-, N4-, O- or S-linked species, that is
the regiochemical constitution of compounds 18-27
and their intermediates, was ascertained by comparison
of 1H NMR, 13C NMR, and/or UV data of related com-
pounds. Key observations are shown in Table 2 and dis-
cussed below.
1
side 22 was indicated by comparison of H NMR data
of 22 and 3-deazauridine.27 In particular, H-3 was ob-
served as an exchangeable doublet (2.6 Hz coupling to
H-5), characteristic of the dynamic keto–enol tauto-
meric equilibrium of the nucleobase moiety.28 Nucleo-
sides 23 and 24 were identified by comparison of 13C
NMR data with 6-azauridine29 and 5-azacytidine,25
Comparison of UV and 13C NMR data of nucleoside 18
and 5-hydroxyuridine,24,25 along with the appearance of

1252
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
Table 2. Selected NMR and UV data for nucleosides 18–27 and related compounds
Compound
NMR (ppm)a
UV kmax (nm)
Ref.
pH 1
pH 7
pH 11
18
5-Hydroxyuridine
160.4, 149.6, 132.5, 119.9
161.5, 150.8, 133.4, 120.1
—
279
280b
276
278
287
—
279
281
275
278
287
—
305
303c
276
278
278
—
—
24,25
—
—
19
20
—
21
22
—
—
—
7.77, 6.05, 5.91, 5.54d
7.78, 5.98, 5.95, 5.56d
156.7, 149.1, 136.5
158.5, 149.5, 137.3
165.6, 156.6, 153.2
166.6, 157.3, 154.4
150.6, 150.6, 150.1
150.1, 150.1, 149.1
168.6, 163.1, 154.8, 101.6
180.6, 160.8, 142.4, 98.8
—
3-Deazauridine
23
—
—
—
—
—
27
—
256
257e
—
6-Azauridine
24
—
—
—
—
29,40
—
25
5-Azacytidine
25
—
—
—
—
—
—
—
25
1-(b-D-ribo-Pentofuranosyl)cyanuric acid
26
2-Thiocytidine
—
239
—
219/282
—
—
—
31
—
—
S-Methyl-2-thiocytosine
27
4-Thiouridine
241f
224/285
262/298
—
32
—
176.2, 154.0, 144.2, 101.9
190.2, 148.0, 136.0, 112.6
193.7, 149.0, 136.5, 114.2
177.5, 154.3, 143.1, 101.8
263/322
—
—
300
—
33
25
3-(b-D-ribo-Pentofuranosyl)-4-thiouracil
S-Methyl-4-thiouracil
—
270/300
—
300
270/325
34,35
a 13C NMR chemical shift values of nucleobase carbons, unless otherwise mentioned.
b pH 2.
c pH 12.
d 1H NMR chemical shift values of H-6, H-10, H-5 and H-3, respectively.
e 0.2 M NaOH.
f pH 0.
respectively. In accordance with previous observations
3. Biological evaluation
on 1-(b-D-ribo-pentofuranosyl)cyanuric acid,25 13C
NMR signals of C-2 and C-6 of the non-UV active
nucleoside 25 merged due to the symmetrical base moi-
ety, while C-4appeared slightly upfield. Furthermore, a
downfield shift of H-20 (ꢀ0.8 ppm) and upfield shift of
50-OH (ꢀ0.7 ppm), compared to the corresponding
signals of EUrd 17, were observed in the 1H NMR
spectrum of nucleoside 25, in agreement with similar
observations on 1-(b-D-ribo-pentofuranosyl)cyanuric
acid.30 13C NMR signals of the nucleobase carbons
of 26 and 2-thiocytidine31 were very different implying
a different regiochemical constitution of the nucleo-
base moiety of 26. Comparison of the UV spectra of
26 and S-methyl-2-thiocytosine32 allowed identifica-
tion of 26 as the S-linked riboside. 13C NMR signals
of the nucleobase carbons of 27, deviated significantly
from 4-thiouridine33 and 3-(b-D-ribo-pentofuranosyl)-
4-thiouracil,25 while corresponding well with data for
S-methyl-4-thiouracil.34 Furthermore, the UV spectra
of 27 and S-methyl-4-thiouracil35 exhibited similar char-
acteristics supporting further proof for the proposed
assignment.
Compounds 1 and 5–27 were evaluated for antiviral
activity against HIV-1 in MT-4cells as previously de-
scribed.38 All compounds were inactive against HIV-1
at the highest tested concentration of 100 lM. Acylated
nucleosides 5, 6, 9, 10, and 12 and unprotected nucleo-
sides 20, 21, 23, 24 displayed moderate cytotoxicity
(CD50: 0.53–9.7 lM) against MT-4cells, whereas ECyd
and EUrd were strongly cytotoxic with CD50 values of
0.02 lM and 0.03 lM, respectively (Table 3).
Next, novel nucleosides 18–27 were evaluated against
human adenocarcinoma breast cancer (MCF-7) and
prostate cancer (PC-3) cell lines along with the reference
compounds (Table 3). ECyd displayed very potent anti-
cancer activity with IC50 values of 2.2 nM and 0.15 nM
against MCF-7 and PC-3 cells, respectively. In agree-
ment with previous results,1 EUrd was 1.1–11 times less
active than ECyd. 5-Iodouridine derivative 21, the most
potent inhibitor of tumor cell proliferation among the
novel nucleobase-modified nucleosides, displayed IC50
values of 35 nM (MCF-7) and 160 nM (PC-3). Decreas-
ing the size of the 5-substituent by introduction of a bro-
mine or chlorine atom as in nucleosides 19 and 20
resulted in a systematic drop in activity. Interestingly,
the corresponding 5-fluoro-EUrd analog, carrying the
smallest halogen, is known to be only 19–48 fold less ac-
tive than EUrd against L1210 and KB cells.1 Further-
more, the substrate preference of UCK2 is known to
decrease in the order: 5-fluorouridine > 5-bromouri-
dine > 5-iodouridine.14 The very weak anticancer activ-
The furanose ring of ECyd is known to adopt a
South36 conformation in both solid state and solu-
tion.1,11 Substitution of the cytosine moiety with the
different nucleobases, does not perturb the confor-
mation of the furanose ring significantly, as indicated
by the relatively large37 JH-1 ,H-2 values (6.1–8.3 Hz)
0
0
observed in the 1H NMR of target nucleosides 18–
27.

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
1253
Table 3. Cytotoxicity of 30-C-ethynyl-b-D-ribo-pentofuranosyl nucleo-
in reactions were of analytical grade. Anhydrous pyr-
idine was used directly as obtained from commercial
suppliers. Acetonitrile was dried through storage over
sides on MT-4cells and inhibition of tumor cell proliferation in vitro a
Compound
CD50 (lM)
MT-4
IC50 (lM)
MCF-7
IC50 (lM)
PC-3
˚
activated 3 A molecular sieves. Reactions were con-
ducted under an atmosphere of argon when anhydrous
solvents were used. Reactions were monitored by thin-
layer chromatography (TLC) using silica gel coated
plates with fluorescence indicator (SiO2-60, F-254),
which were visualized (1) under UV light, (2) by dipping
in 5% concd sulfuric acid in anhydrous ethanol (v/v) fol-
lowed by heating, or (3) by dipping in a solution of
molybdato-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. Silica gel column chromatography
using moderate pressure (pressure ball) was performed
with Silica gel 60 (particle size 0.040–0.063 mm, Merck).
Evaporation of solvents was carried out under reduced
pressure with temperatures not exceeding 45 ꢁC. After
column chromatography, appropriate fractions were
pooled, evaporated and dried at high vacuum for at least
12 h to give the obtained products in high purity (>95%)
unless stated otherwise. In absence of elemental analysis,
1H NMR and/or 13C NMR ascertained sample purity.
No corrections in yields were made for solvent of crys-
tallization. 1H NMR and 13C NMR spectra were
recorded at 300 MHz 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 by dis-
appearance of peaks on D2O addition. Assignments of
NMR spectra were based on 2D spectra (HETCOR,
COSY) and follow standard carbohydrate/nucleoside
nomenclature. Quaternary carbons were not assigned
in 13C NMR. Traces of solvents in NMR spectra were
identified by reference to published data.39 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 ad-
justed by addition of concd aq HCl or concd aq NaOH.
Elemental analyses were obtained from the Microana-
lytical Department, University of Copenhagen.
5
1.29
0.53
>100
>100
4.1
nd
nd
nd
nd
6
7
nd
nd
nd
nd
8
9
10
nd
nd
nd
nd
0.57
> 100
9.7
11
12
nd
nd
nd
nd
13
14
>100
>100
>100
>100
>100
>100
4.5
nd
nd
nd
nd
15
16
nd
nd
nd
nd
18
19
>25
>25
0.73
0.035
> 25
35
>25
>25
19
20
21
0.46
>100
3.8
0.16
>25
43
22
23
24
25
4.1
4.7
7.2
>100
>100
>30
>25
>25
>25
0.0025
0.0022
>25
>25
>25
0.0017
0.00015
26
27
EUrd 17
ECyd 1
0.03
0.02
nd = Not determined.
a CD50 is the cytotoxic dose of compound required to reduce prolif-
eration of normal uninfected MT-4cells by 50%. IC 50 represents the
concentration at which cell growth was inhibited by 50% as com-
pared to a negative control experiment.
ity of 6-azauridine derivative 23 compared to 5-iodouri-
dine derivative 21, and the lack of activity of 5-hydroxy-
uridine derivative 18 and 3-deazauridine derivative 22, is
also interesting considering that the corresponding
nucleosides without the C30-ethynyl modification are
significantly better substrates for UCK2 than 5-iodouri-
dine.14 These observations lead us to speculate that: (1)
UCK2 may have a different substrate preference for
nucleobase-modified 30-C-ethynyl-D-ribo-pentofurano-
syl nucleosides than for the corresponding nucleobase-
modified ribonucleosides without sugar modifications
and/or (2) nucleotide kinases and RNA polymerases
may have different substrate preferences leading to
either insufficient phosphorylation or to lack of recogni-
tion by RNA polymerase of the corresponding triphos-
phates. However, other factors (lipophilicity, pKa, etc.)
may influence the biological activity of nucleosides 18–
27 studied in our whole cell assays.
4.1. 3-C-Ethynyl-1,2-O-isopropylidene-a-D-ribo-pento-
furanose (3)
Known furanose 219 (11.30 g, 39.8 mmol) was dissolved
in 80% aqueous acetic acid (250 mL). After stirring at rt
for 24h, the solvent was evaporated and the residue co-
evaporated with toluene (2 · 100 mL) and anhydrous
EtOH (100 mL) to furnish the crude triol, which was
directly dissolved in ice-cold MeOH/H2O (300 mL, 3:1
v/v). To this was added sodium periodate (10.20 g,
47.7 mmol) and the reaction mixture warmed to rt and
stirred for 15 h. Solids were filtered off, washed
(MeOH), and the solution concentrated to 1/4volume,
saturated with NaCl (s) and extracted with CH2Cl2
(3 · 100 mL). The combined organic phase was evapo-
rated to dryness to leave the crude hydroxyaldehyde as
a yellow foam (8.5 g), which was directly dissolved in
4. Experimental
General experimental section: All solvents and reagents
were obtained from commercial suppliers and used with-
out further purification unless stated otherwise. All sol-
vents 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. Solvents for use

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P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
ice-cold MeOH (175 mL). Sodium borohydride (4.51 g,
0.12 mol) was added portionwise over 15 min. The reac-
tion mixture was warmed to rt and after stirring for
6.5 h, the reaction mixture was neutralized by addition
of 10% aqueous acetic acid and evaporated to near dry-
ness. The resulting residue was partitioned between
EtOAc (125 mL) and brine (50 mL). The phases were
separated and the aqueous phase extracted with CH2Cl2
(2 · 125 mL) and EtOAc (4 · 125 mL). The combined
organic phase was evaporated to dryness and the result-
ing residue was purified by silica gel column chromato-
graphy (0–7% MeOH in CH2Cl2, v/v) to give diol 3
(7.17 g, 84% over three steps) as a white solid material.
Rf = 0.3 (10% MeOH in CHCl3, v/v); MALDI-HRMS
m/z 237.0736 ([M+Na]+, C10H14O5ÆNa+ calcd
ture: Rf = 0.6 (10% MeOH in CHCl3, v/v); MALDI-
HRMS m/z 365.0825 ([M+Na]+, C15H18O9ÆNa+ calcd
365.0843); 13C NMR (CDCl3) d 170.60, 170.58, 169.2,
169.1, 168.8, 168.6, 168.5, 168.4, 98.6, 93.7, 82.7, 81.5,
78.7, 78.5, 77.9, 76.4, 75.9, 75.6, 75.2, 75.0, 64.4, 63.6,
21.1, 21.0, 20.94, 20.89, 20.87, 20.6, 20.4. Anal. Calcd
for C15H18O9: C, 52.63; H, 5.30. Found: C, 52.62; H,
5.19.
4.3. General procedure for glycosylations to give pro-
tected nucleosides 5–16
Glycosyl donor 4 and nucleobase (quantities of sub-
strates and reagents given in corresponding paragraph)
were dried by co-evaporation with anhydrous CH3CN
(25 mL) and resuspended in anhydrous CH3CN
(10 mL). N,O-Bis(trimethylsilyl)acetamide (BSA) was
added in one portion and the mixture refluxed until a
homogenous solution was obtained (less than 1 h). After
cooling to rt, trimethylsilyl triflate (TMSOTf) was added
in one portion and the reaction mixture stirred under
heating. In some cases (see below) additional TMSOTf
was added. In a typical workup procedure (0.5 g scale),
the reaction mixture was poured into satd aq NaHCO3
(20 mL) and diluted with EtOAc (30 mL). The phases
were separated and the aqueous phase extracted with
EtOAc (3 · 30 mL). The combined organic phase was
evaporated to dryness and the resulting residue purified
by silica gel column chromatography.
1
237.0733); H NMR (DMSO-d6) d 5.94(s, 1H, ex, 3-
OH), 5.74(d, 1H, J = 3.7 Hz, H-1), 4.76 (t, 1H, ex,
J = 5.7 Hz, 5-OH), 4.39 (d, 1H, J = 3.7 Hz, H-2), 3.82
(dd, 1H, J = 8.2 Hz, 2.0 Hz, H-4), 3.49–3.69 (m, 3H,
HC„C, H-5), 1.45 (s, 3H, CH3), 1.28 (s, 3H, CH3);
13C NMR (DMSO-d6) d 111.8, 103.4(C-1), 83.6 (C-2),
82.2, 81.8 (C-4), 77.4, 73.6, 60.9 (C-5), 26.4 (CH3). Anal.
Calcd for C10H14O5Æ1/16 H2O: C, 55.78; H, 6.61. Found:
C, 55.54; H, 6.67.
4.2. 1,2,3,5-Tetra-O-acetyl-3-C-ethynyl-a,b-D-ribo-
pentofuranose (4)
Diol 3 (4.91 g, 22.9 mmol) was dried by co-evaporation
with anhydrous pyridine (75 mL) and dissolved in anhy-
drous pyridine (50 mL). To this, Ac2O (8.7 mL,
91.9 mmol) and DMAP (0.28 g, 2.29 mmol) were added
and the reaction mixture was stirred for 5 h at rt where-
upon analytical TLC showed full conversion of starting
material to one product with high mobility (Rf = 0.8,
10% MeOH in CHCl3, v/v). Crushed ice (20 mL) was
added and the reaction mixture was evaporated to near
dryness and co-evaporated with toluene (30 mL). The
residue was taken up in EtOAc (150 mL), and the organic
phase washed with satd aq NaHCO3 (2 · 50 mL). The
organic phase was evaporated to near dryness and co-
evaporated with toluene (150 mL) to afford a crude
white solid material (6.6 g) which was dissolved in ice-
cold 80% aqueous TFA (50 mL). After stirring the reac-
tion mixture at rt for 6 h analytical TLC showed full
conversion to two more polar compounds (Rf = 0.2
and 0.4, respectively, 10% MeOH in CHCl3, v/v). Sol-
vents were evaporated and the residue coevaporated
with toluene (2 · 50 mL) and anhydrous pyridine
(25 mL). Subsequently, the crude anomeric alcohol
was dissolved in anhydrous pyridine (50 mL) and
Ac2O (8.7 mL, 91.9 mmol) and DMAP (0.28 g,
2.29 mmol) were added. The reaction mixture was stir-
red for 40 h whereupon crushed ice (20 mL) was added.
The reaction mixture was diluted with EtOAc (200 mL),
the phases separated and the organic phase washed with
satd aq NaHCO3 (2 · 75 mL). The organic phase was
evaporated to dryness and the resulting residue co-evap-
orated with toluene (3 · 50 mL) and purified by silica gel
column chromatography (0–3% MeOH in CH2Cl2, v/v)
to give an anomeric mixture (ratio ꢁ 4:5 by 1H NMR) of
glycosyl donor 4 (5.84g, 74% over three steps) as a
transparent yellow oil. Physical data for anomeric mix-
4.3.1. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-4-N-benzoylcytosine (5). After stirring glyco-
syl donor 4 (1.02 g, 2.98 mmol), 4-N-benzoylcytosine
(1.28 g, 5.96 mmol), BSA (3.0 mL, 11.9 mmol) and
TMSOTf (1.6 mL, 8.94mmol) for 24h at 60 ꢁC, the
reaction mixture was worked up. Purification by silica
gel column chromatography (0–4% MeOH in CH2Cl2,
v/v) gave protected nucleoside 5 (1.04g, 71%) as a white
solid material. Rf = 0.4(5% MeOH in CH 2Cl2, v/v); UV
k
max pH 1, 260 nm, 314nm, kmax H2O, 261 nm, 302 nm,
kmax pH 11, 316 nm; MALDI-HRMS m/z 520.1308
([M+Na]+, C24H23N3O9ÆNa+ calcd 520.1327); 1H
NMR (DMSO-d6) d 11.33 (br s, 1H, ex, NH), 8.25 (d,
1H, J = 7.3 Hz, H-6), 7.98–8.02 (m, 2H, Ph), 7.43–7.67
(m, 4H, H-5, Ph), 6.09 (d, 1H, J = 4.8 Hz, H-10), 5.66
(d, 1H, J = 4.8 Hz, H-20), 4.48–4.61 (m, 3H, H-40, H-
50), 4.06 (s, 1H, HC„C), 2.14(s, 3H, CH ), 2.11 (s,
3
3H, CH3), 2.08 (s, 3H, CH3); 13C NMR (DMSO-d6) d
169.9, 168.2, 168.1, 167.3, 163.5, 154.1, 144.7 (C-6),
132.9 (Ph), 132.7 (Ph), 128.41 (Ph), 128.36 (Ph), 96.9
(C-5), 87.3 (C-10), 81.8, 80.8 (C-40), 76.9 (C-20), 76.1,
74.5, 62.9 (C-50), 20.6 (CH3), 20.5 (CH3), 20.2 (CH3).
Anal. Calcd for C24H23N3O9Æ1/16 CH2Cl2: C, 57.48;
H, 4.64; N, 8.36. Found: C, 57.16; H, 4.55; N, 8.23.
4.3.2. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]uracil (6). After stirring peracetylated furanose
4 (0.93 g, 2.72 mmol), uracil (0.61 g, 5.43 mmol), BSA
(2.7 mL, 10.9 mmol) and TMSOTf (1.5 mL, 8.15 mmol)
for 21 h at 60 ꢁC, the reaction mixture was worked up.
Purification by silica gel column chromatography (0–
4% MeOH in CHCl3, v/v) gave protected nucleoside 6
(0.96 g, 90%) as a white solid material. Rf = 0.3 (10%

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
1255
MeOH in CHCl3, v/v); UV kmax pH 1, 259 nm, kmax
H2O, 259 nm, kmax pH 11, 261 nm; MALDI-HRMS m/
(CH3), 20.4(CH 3), 20.2 (CH3). Anal. Calcd for
C17H17ClN2O9: C, 47.62; H, 4.00; N, 6.53. Found: C,
47.70; H, 3.96; N, 6.35.
z
417.0889 ([M +
Na]+, C17H18N2O9ÆNa+ calcd
1
417.0905); H NMR (DMSO-d6) d 11.50 (br s, 1H, ex,
NH), 7.74(d, 1H, J = 8.1 Hz, H-6), 6.01 (d, 1H,
J = 6.0 Hz, H-10), 5.78 (d, 1H, J = 8.1 Hz, H-5), 5.54
(d, 1H, J = 6.0 Hz, H-20), 4.41–4.55 (m, 3H, H-40, H-
50), 4.04 (s, 1H, HC„C), 2.15 (s, 3H, CH3), 2.09 (s,
3H, CH3), 2.06 (s, 3H, CH3); 13C NMR (DMSO-d6) d
169.8, 168.8, 168.3, 162.6, 150.2, 139.6 (C-6), 102.9 (C-
5), 85.0 (C-10), 81.1, 80.1 (C-40), 76.3, 76.1, 74.6, 62.9
(C-50), 20.7 (CH3), 20.5 (CH3), 20.1 (CH3). Anal. Calcd
4.3.5. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-5-bromouracil (9). Peracetylated furanose 4
(0.50 g, 1.46 mmol), 5-bromouracil (0.56 g, 2.92 mmol),
BSA (1.5 mL, 5.84mmol) and TMSOTf (0.79 mL,
4.38 mmol) were stirred at 50 ꢁC for 23 h whereupon
the reaction mixture was worked up. Purification by sil-
ica gel column chromatography (0–3% MeOH in
CH2Cl2, v/v) gave protected nucleoside 9 (0.65 g, 94%)
as a white solid material. Rf = 0.6 (10% MeOH in
CHCl3, v/v); UV kmax pH 1, 277 nm, kmax H2O,
276 nm, kmax pH 11, 276 nm; MALDI-HRMS m/z
for C17H18N2O9Æ1/4CHCl : C, 48.84; H, 4,34; N, 6.60.
3
Found: C, 48.64; H, 4,24; N, 6.54.
4.3.3. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
495.0008
([M+Na]+,
C17H17BrN2O9ÆNa+
calcd
1
furanosyl]-5-hydroxyuracil (7). Glycosyl donor
(0.50 g, 1.46 mmol), 5-hydroxyuracil (0.37 mg,
4
495.0010); H NMR (DMSO-d6) d 12.01 (br s, 1H, ex,
NH), 8.22 (s, 1H, H-6), 6.01 (d, 1H, J = 6.4Hz, H-1 ),
0
0
2.92 mmol), BSA (1.5 mL, 5.84mmol) and TMSOTf
(0.79 mL, 4.38 mmol) were stirred at 50 ꢁC for 44 h,
whereupon the reaction mixture was cooled to rt and a
second portion of TMSOTf (0.40 mL, 2.21 mmol) was
added. Stirring at 50 ꢁC for additional 21 h resulted in
complete consumption of starting material and the reac-
tion mixture was worked up. Purification by silica gel
column chromatography (0–4% MeOH in CH2Cl2,
v/v) gave protected nucleoside 7 (0.38 g, 64%) as a white
solid material. Rf = 0.5 (10% MeOH in CHCl3, v/v);
UV kmax pH 1, 278 nm, kmax H2O, 279 nm, kmax
pH 11, 306 nm; MALDI-HRMS m/z 433.0841
([M+Na]+, C17H18N2O10ÆNa+ calcd 433.0854); 1H
NMR (DMSO-d6) d 11.64(br s, 1H, ex, NH), 9.05 (br
s, 1H, ex, 5-OH), 7.20 (s, 1H, H-6), 6.02 (d, 1H,
J = 6.0 Hz, H-10), 5.48 (d, 1H, J = 6.0 Hz, H-20), 4.41–
4.49 (m, 3H, H-40, H-50), 4.07 (s, 1H, HC„C), 2.15
(s, 3H, CH3), 2.07–2.08 (2s, 2 · 3H, 2 · CH3); 13C
NMR (DMSO-d6) d 169.8, 168.8, 168.4, 160.1, 149.0,
133.3, 117.9 (C-6), 85.0 (C-10), 81.1, 79.6 (C-40), 76.5,
75.8 (C-20), 74.5, 62.8 (C-50), 20.6 (CH3), 20.5 (CH3),
5.63 (d, 1H, J = 6.4Hz, H-2 ), 4.46–4.60 (m, 3H, H-40,
H-50), 4.08 (s, 1H, HC„C), 2.14(s, 3H, CH ), 2.08–
3
2.10 (2s, 2 · 3H, 2 · CH3); 13C NMR (DMSO-d6) d
169.7, 168.9, 168.4, 158.7, 149.6, 139.2 (C-6), 97.0, 84.8
(C-10), 81.2, 80.5 (C-40), 76.3, 76.1, 74.6, 63.0 (C-50),
20.8 (CH3), 20.5 (CH3), 20.2 (CH3). Anal. Calcd for
C17H17BrN2O9: C, 43.15; H, 3.62; N, 5.92. Found: C,
43.13; H, 3.52; N, 5.82.
4.3.6. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-5-iodouracil (10). Glycosyl donor 4 (0.50 g,
1.46 mmol), 5-iodouracil (0.70 g, 2.92 mmol), BSA
(1.5 mL, 5.84mmol) and TMSOTf (0.79 mL,
4.38 mmol) were stirred at 50 ꢁC for 29 h whereupon
the reaction mixture was worked up. Purification by sil-
ica gel column chromatography (0–4% MeOH in
CH2Cl2, v/v) gave protected nucleoside 10 (0.61 g,
80%) as a white solid material. Rf = 0.8 (10% MeOH
in CHCl3, v/v); UV kmax pH 1, 286 nm, kmax H2O,
286 nm, kmax pH 11, 279 nm; MALDI-HRMS m/z
542.9890
([M+Na]+,
C17H17IN2O9ÆNa+
calcd
1
20.1 (CH3). Anal. Calcd for C17H18N2O10Æ1/4CH Cl2:
542.9871); H NMR (DMSO-d6) d 11.87 (br s, 1H, ex,
NH), 8.19 (s, 1H, H-6), 6.00 (d, 1H, J = 6.2 Hz, H-10),
5.59 (d, 1H, J = 6.2 Hz, H-20), 4.45–4.58 (m, 3H, H-40,
2
C, 48.01; H, 4.32; N, 6.49. Found: C, 48.06; H, 4.24;
N, 6.44.
H-50), 4.10 (s, 1H, HC„C), 2.14(s, 3H, CH ), 2.09–
3
4.3.4. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-5-chlorouracil (8). Peracetylated furanose 4
(0.50 g, 1.46 mmol), 5-chlorouracil (0.43 g, 2.92 mmol),
BSA (1.5 mL, 5.84mmol) and TMSOTf (0.79 mL,
4.38 mmol) were stirred at 50 ꢁC for 29 h, whereupon
the reaction mixture was worked up. Purification by sil-
ica gel column chromatography (0–3% MeOH in
CH2Cl2, v/v) gave protected nucleoside 8 (0.49 g, 78%)
as a white solid material. Rf = 0.6 (10% MeOH in
CHCl3, v/v); UV kmax pH 1, 274nm, kmax H2O,
275 nm, kmax pH 11, 274nm; MALDI-HRMS m/z
2.10 (2s, 2 · 3H, 2 · CH3); 13C NMR (DMSO-d6) d
169.7, 168.9, 168.4, 160.1, 149.9, 143.8 (C-6), 84.7 (C-
10), 81.1, 80.4(C-4 ), 76.3, 76.1, 74.6, 71.0, 62.9 (C-50),
20.7 (CH3), 20.5 (CH3), 20.1 (CH3).
0
4.3.7. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-3-deazauracil (11). Peracetylated furanose 4
(0.50 g, 1.46 mmol), 3-deazauracil (0.32 g, 2.92 mmol),
BSA (1.5 mL, 5.84mmol) and TMSOTf (0.79 mL,
4.38 mmol) were stirred at 50 ꢁC for 24h, whereupon
the reaction mixture was cooled to rt and a second por-
tion of TMSOTf (0.30 mL, 1.65 mmol) added. After
stirring at 50 ꢁC for additional 16 h a third portion of
TMSOTf (0.10 mL, 0.56 mmol) was added. After stir-
ring for additional 26 h at 50 ꢁC, the reaction mixture
was worked up. Purification by silica gel column chro-
matography (0–4% MeOH in CH2Cl2, v/v) gave pro-
tected nucleoside 11 (0.51 g, 79%) as a white solid
material. Rf = 0.5 (10% MeOH in CH2Cl2, v/v); UV kmax
451.0522
([M+Na]+,
C17H17ClN2O9ÆNa+
calcd
1
451.0515); H NMR (DMSO-d6) d 12.04(br s, 1H, ex,
0
NH), 8.17 (s, 1H, H-6), 6.010 (d, 1H, J = 6.4Hz, H-1 ),
5.63 (d, 1H, J = 6.4Hz, H-2 ), 4.57 (t, 1H, J = 4.8 Hz,
H-40), 4.48 (d, 2H, J = 4.8 Hz, H-50), 4.08 (s, 1H,
HC„C), 2.14(s, 3H, CH ), 2.09 (s, 3H, CH3), 2.07 (s,
3
3H, CH3); 13C NMR (DMSO-d6) d 169.7, 168.9,
168.4, 158.6, 149.4, 136.9 (C-6), 108.4, 84.8 (C-10),
81.2, 80.5 (C-40), 76.3, 76.1, 74.6, 63.0 (C-50), 20.8
pH 1, 278 nm, kmax H2O, 282 nm,
pH 11, 256 nm;
max

1256
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
MALDI-HRMS m/z 416.0965 ([M+Na]+, C18H19NO9Æ
2.13 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.07 (s, 3H,
CH3); (DMSO-d6) d 169.9, 168.5, 168.2, 165.4, 155.8
(C-6), 152.3, 86.1 (C-10), 81.4, 80.5 (C-40), 76.3
(C-20), 74.7, 63.0 (C-50), 20.7 (CH3), 20.5 (CH3), 20.2
(CH3).
1
Na+ calcd 416.0952); H NMR (DMSO-d6) d 10.91 (br
s, 1H, ex, OH), 7.58 (d, 1H, J = 7.9 Hz, H-6), 6.26 (d,
1H, J = 5.3 Hz, H-10), 6.02 (dd, 1H, J = 7.9 Hz,
2.6 Hz, H-5), 5.55 (d, 1H, ex, J = 2.6 Hz, H-3), 5.51 (d,
1H, J = 5.3 Hz, H-20), 4.41–4.50 (m, 3H, H-40, H-50),
4.03 (s, 1H, HC„C), 2.14(s, 3H, CH ), 2.06–2.07 (2s,
4.3.10. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]cyanuric acid (14). Glycosyl donor 4 (0.40 g,
1.27 mmol), cyanuric acid (0.30 g, 2.34mmol), BSA
(1.7 mL, 7.04mmol) and TMSOTf (0.63 mL,
3.51 mmol) were stirred at 55 ꢁC for 21 h, whereupon
analytical TLC showed approx. 70% conversion of start-
ing material into several non-UV active compounds.
The reaction mixture was cooled to rt and a second por-
tion of TMSOTf (0.20 mL, 1.11 mmol) added. After
stirring for additional 24h the reaction mixture was
worked up. Purification by silica gel column chromato-
graphy (0–2% MeOH in CHCl3, v/v) afforded nucleoside
14 (145 mg, 30%) as a yellow solid material, which was
used in the next step without further purification.
Rf = 0.5 (10% MeOH in CH2Cl2, v/v); MALDI-HRMS
m/z 434.0785 ([M+Na]+, C16H17N3O10ÆNa+ calcd
434.0806); 1H NMR (DMSO-d6) d 6.28 (d, 1H,
J = 7.5 Hz), 6.07 (d, 1H, J = 7.5 Hz), 4.49–4.55 (m,
2H), 4.21 (dd, 1H, J = 12.1 Hz, J = 8.8 Hz), 3.93 (s,
1H, HC„C), 2.14(s, 3H, CH 3), 2.07 (s, 3H, CH3),
2.02 (s, 3H, CH3); 13C NMR (DMSO-d6) d 169.9,
169.1, 168.4, 148.4, 147.1, 83.7, 80.4, 80.3, 76.6, 75.2,
73.0, 63.6, 20.9, 20.5, 20.2. Acetamide was identified as
a trace impurity.
3
2 · 3H, 2 · CH3); 13C NMR (DMSO-d6) d 169.9,
168.3, 168.2, 166.8, 162.4, 133.9 (C-6), 101.4 (C-5),
97.6 (C-3), 84.7 (C-10), 81.1, 79.9 (C-40), 76.9, 76.6,
74.5, 62.8 (C-50), 20.6 (CH3), 20.4(CH ), 20.1 (CH3).
3
Anal. Calcd for C18H19NO9Æ1/16 CH2Cl2: C, 54.42; H,
4.84; N, 3.51. Found: C, 54.25; H, 4.82; N, 3.52.
4.3.8. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-6-azauracil (12). Peracetylated furanose 4
(0.50 g, 1.46 mmol), 6-azauracil (0.33 g, 2.92 mmol),
BSA (1.5 mL, 5.84mmol) and TMSOTf (0.79 mL,
4.38 mmol) were stirred at 50 ꢁC for 48 h. The reaction
mixture was cooled to rt and a second portion TMSOTf
(0.40 mL, 2.21 mmol) added. After heating at 50 ꢁC for
additional 19 h the reaction mixture was worked up.
Purification by silica gel column chromatography (0–
2% MeOH in CH2Cl2, v/v) afforded protected nucleo-
side 12 (0.37 g, 63%) as a slightly yellow solid material.
Rf = 0.5 (5% MeOH in CH2Cl2, v/v); UV kmax pH 1,
258 nm, kmax H2O, 257 nm, kmax pH 11, 255 nm; MAL-
DI-HRMS m/z 418.0865 ([M+Na]+, C16H17N3O9ÆNa+
1
calcd 418.0857); H NMR (DMSO-d6) d 12.40 (br s,
1H, ex, NH), 7.66 (s, 1H, H-5), 6.08 (d, 1H,
J = 6.3 Hz, H-10), 5.88 (d, 1H, J = 6.3 Hz, H-20), 4.45–
4.52 (m, 2H, H-40, H-5A 0), 4.17 (dd, 1H, J = 12.5 Hz,
7.3 Hz, H-50B), 3.91 (s, 1H, HC„C), 2.15 (s, 3H, CH3),
2.05–2.07 (2s, 2 · 3H, 2 · CH3); 13C NMR (DMSO-d6)
d 169.9, 168.7, 168.3, 156.1, 148.1, 137.1 (C-5), 85.9
(C-10), 80.4, 80.0, 76.4, 74.7, 74.4, 62.8 (C-50), 20.7,
20.5, 20.2. Anal. Calcd for C16H17N3O9: C, 48.61; H,
4.33; N, 10.63; Found: C, 48.41; H, 4.31; N, 10.31.
4.3.11. S-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-2-thiocytosine (15). Glycosyl donor 4 (0.40 g,
1.17 mmol), 2-thiocytosine (0.30 g, 2.34mmol), BSA
(1.2 mL, 4.67 mmol) and TMSOTf (0.63 mL,
3.51 mmol) were stirred at 50 ꢁC for 40 h, whereupon
the reaction mixture was worked up. Purification by sil-
ica gel column chromatography (0–2% MeOH in
CH2Cl2, v/v) gave protected nucleoside 15 (0.35 g,
74%) as a white solid material. Rf = 0.5 (10% MeOH
in CHCl3, v/v); UV kmax pH 1, 237 nm, kmax H2O,
281 nm, kmax pH 11, 281 nm; MALDI-HRMS m/z
4.3.9. 1-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-5-azacytosine (13). Glycosyl donor 4 (0.41 g,
1.20 mmol), 5-azacytosine (0.27 g, 2.41 mmol), BSA
(1.2 mL, 4.81 mmol) and TMSOTf (0.65 mL,
3.61 mmol) were stirred at 50 ꢁC for 24h. The reaction
mixture was cooled to rt and a second portion of
TMSOTf (0.20 mL, 1.11 mmol) added. The reaction
mixture was stirred for additional 6 h at 50 ꢁC where-
upon it was worked up. To prevent decomposition of
the nucleobase moiety, the organic phase was washed
quickly with ice-cold satd aq NaHCO3 (25 mL). The
phases were separated and the aqueous phase extracted
with EtOAc (3 · 25 mL). The combined organic phase
was evaporated to dryness and the resulting residue
was purified by silica gel column chromatography (0–
3% MeOH in EtOAc, v/v) to afford nucleoside 13
(0.24g, 50%) as a white solid material. Rf = 0.4(10%
MeOH in EtOAc, v/v); UV kmax pH 1, 255 nm, kmax
H2O, 243 nm, kmax pH 11, 251 nm; MALDI-HRMS
m/z 417.1006 ([M+Na]+, C16H18N4O8ÆNa+ calcd
432.0839
([M+Na]+,
C17H19N3O7SÆNa+
calcd
432.0836); 1H NMR (DMSO-d6) d 7.90 (d, 1H,
J = 5.9 Hz, H-6), 7.07 (br s, 2H, 2 ex, NH2), 6.22 (d,
1H, J = 3.3 Hz, H-10), 6.19 (d, 1H, J = 5.9 Hz, H-5),
5.68 (d, 1H, J = 3.3 Hz, H-20), 4.32–4.44 (m, 2H, H-40,
H-50A), 4.16–4.24 (dd, 1H, J = 11.4Hz, 6.6 Hz, H-5 0B),
4.09 (s, 1H, HC„C), 2.12 (s, 3H, CH3), 2.11 (s, 3H,
CH3), 2.05 (s, 3H, CH3); 13C NMR (DMSO-d6) d
169.9, 168.5, 168.2, 166.8, 163.2, 154.9 (C-6), 102.0 (C-
5), 85.3 (C-10), 81.9, 80.9, 78.3 (C-20), 75.9, 75.2, 63.3
(C-50), 20.5 (CH3), 20.4(CH ), 20.2 (CH3). Anal. Calcd
3
for C17H19N3O7S: C, 49.87; H, 4.68; N, 10.26; S, 7.83.
Found: C, 49.69; H, 4.55; N, 10.11; S, 7.83.
4.3.12. S-[2,3,5-Tri-O-acetyl-3-C-ethynyl-b-D-ribo-pento-
furanosyl]-4-thiouracil (16). Peracetylated furanose 4
(0.50 g, 1.46 mmol), 4-thiouracil (0.37 g, 2.85 mmol),
BSA (1.5 mL, 5.84mmol) and TMSOTf (0.79 mL,
4.38 mmol) were stirred at 50 ꢁC for 28 h whereupon
the reaction mixture was worked up. Purification by sil-
ica gel column chromatography (0–4% MeOH in
1
417.1017); H NMR (DMSO-d6) d 8.43 (s, 1H, H-6),
7.75 (s, 1H, ex, NH2), 7.73 (s, 1H, ex, NH2), 5.91
(d, 1H, J = 5.7 Hz, H-10), 5.77 (d, 1H, J = 5.7 Hz, H-
20), 4.44–4.55 (m, 3H, H-40, H-50), 4.06 (s, 1H, HC„C),

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
1257
CH2Cl2, v/v) afforded pure nucleoside 16 (0.17 g) as a
yellow solid material. Furthermore, 16 (0.33 g, ꢁ83%
combined yield) contaminated with traces of 4-thioura-
cil was isolated. The two fractions were combined and
used in the next step without further purification. Phys-
ical data for pure fraction: Rf = 0.4(5% MeOH in
CH2Cl2, v/v); UV kmax pH 1, 261 nm, 302 nm, kmax
H2O, 258 nm, 297 nm, kmax pH 11, 301 nm; MALDI-
HRMS m/z 433.0662 ([M + Na]+, C17H18N2O8SÆNa+
4.4.3. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-5-hydr-
oxyuracil (18). Nucleoside 7 (0.30 g, 0.74mmol) was
stirred for 50 h. The residue resulting after workup
was purified by silica gel column chromatography (0–
15% MeOH in CH2Cl2, v/v) to afford nucleoside 18
(98 mg, 46%). Rf = 0.6 (40% MeOH in CH2Cl2, v/v);
UV kmax pH 1, 279 nm, kmax H2O, 279 nm, kmax
pH 11, 305 nm; MALDI-HRMS m/z 307.0534
([M+Na]+, C11H12N2O7ÆNa+ calcd 307.0537); 1H
NMR (DMSO-d6) d 11.47 (s, 1H, ex, NH), 8.71, (s,
1H, ex, 5-OH), 7.52 (s, 1H, H-6), 5.85 (s, 1H, ex, 30-
OH), 5.83 (d, 1H, J = 7.3 Hz, H-10), 5.79 (d, 1H, ex,
J = 6.6 Hz, 20-OH), 5.12 (t, 1H, ex, J = 4.2 Hz, 50-OH),
4.15 (br t, 1H, H-20), 3.85 (br t, 1H, H-40), 3.60–3.75
(m, 2H, H-50), 3.54(s, 1H, HC „C); 13C NMR
(DMSO-d6) d 160.4, 149.6, 132.5, 119.9 (C-6), 86.2 (C-
40), 85.8 (C-10), 82.9, 77.3 (C-20), 76.9, 72.6, 61.3 (C-
50). Anal. Calcd for C11H12N2O7Æ3/16 MeOH: C,
46.30; H, 4.43; N, 9.65. Found: C, 45.90; H, 4.37; N,
9.76.
1
calcd 433.0676); H NMR (DMSO-d6) d 11.73 (br s,
1H, ex), 7.75 (d, 1H, J = 6.6 Hz, H-6), 6.35 (d, 1H,
J = 6.6 Hz, H-5), 6.27 (d, 1H, J = 2.9 Hz, H-10), 5.67
(d, 1H, J = 2.9 Hz, H-20), 4.38–4.44 (m, 2H, H-40, H-
50A), 4.18–4.26 (dd, 1H, J = 12.5 Hz, 7.7 Hz, H-50B),
4.13 (s, 1H, HC„C), 2.13 (s, 3H, CH3), 2.12 (s, 3H,
CH3), 2.06 (s, 3H, CH3); 13C NMR (DMSO-d6) d
173.6, 169.9, 168.7, 168.2, 153.8, 144.9 (C-6), 101.7 (C-
5), 83.1 (C-10), 82.1, 81.2 (C-40), 78.3 (C-20), 75.7, 75.0,
63.1 (C-50), 20.5 (CH3), 20.4(CH ), 20.2 (CH3). Anal.
3
Calcd for C17H18N2O8S: C, 49.75; H, 4.42; N, 6.83; S,
7.81. Found: C, 49.36; H, 4.31; N, 6.78; S, 7.91.
4.4.4. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-5-chloro-
uracil (19). Nucleoside 8 (0.31 g, 0.72 mmol) was stirred
for 48 h. The residue resulting after workup was purified
by crystallization (MeOH and minimal H2O), which
after washing with cold MeOH afforded unprotected
nucleoside 19 (146 mg) as white needles. Repeated crys-
tallization of the mother liquor afforded additional 19
(combined yield 180 mg, 83%). Rf = 0.3 (20% MeOH
in CH2Cl2, v/v); mp (MeOH/H2O) 133–136 ꢁC; UV kmax
pH 1, 276 nm, kmax H2O, 275 nm, kmax pH 11, 276 nm;
4.4. General procedure for deacylations to give nucleo-
sides 1, 17–23, 25–27
The protected nucleoside (quantity of substrate given in
the corresponding paragraph) was dissolved in saturated
methanolic ammonia (20 mL for 0.3 g of protected
nucleoside) and stirred in a sealed container for 24–
48 h (exact time indicated in corresponding paragraph)
at rt. The reaction mixture was evaporated to dryness
and co-evaporated several times with absolute EtOH
to afford a residue, which was purified by either washing
with an appropriate solvent, silica gel column chroma-
tography or crystallization.
MALDI-HRMS
m/z
325.0213
([M+Na]+,
C11H11ClN2O6ÆNa+ calcd 325.0198); 1H NMR
(DMSO-d6) d 11.92 (br s, 1H, ex, NH), 8.44 (s, 1H, H-
6), 5.96 (s, 1H, ex, 30-OH), 5.81–5.86 (m, 2H, 1 ex, 20-
OH, H-10), 5.31 (t, 1H, ex, J = 3.9 Hz, 50-OH), 4.21
(br t, 1H, H-20), 3.93 (t, 1H, J = 2.9 Hz, H-40), 3.72–
3.80 (m, 1H, H-50A), 3.63–3.69 (m, 1H, H-50B), 3.57 (s,
1H, HC„C); Selected data 1H NMR (DMSO-d6 + 1
drop D2O) 5.82 (d, 1H, J = 7.3 Hz, H-10), 4.20 (d, 1H,
J = 7.3 Hz, H-20); 13C NMR (DMSO-d6) d 158.8,
149.9, 138.0 (C-6), 107.6, 86.8 (C-40), 86.3 (C-10), 82.5,
78.0 (C-20), 77.1, 72.7, 61.2 (C-50). Anal. Calcd for
C11H11ClN2O6Æ7/8 MeOH: C, 43.27; H, 4.13; N, 8.54.
Found: C, 42.87; H, 4.33; N, 8.31.
4.4.1. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]cytosine
(ECyd, 1). Nucleoside 5 (0.51 g, 1.03 mmol) was stirred
46 h. The residue resulting after workup was washed
with ice-cold EtOH to afford the known nucleoside 1
(0.21 g, 74%) as a white solid material. Rf = 0.1 (20%
MeOH in CH2Cl2, v/v); Selected data 1H NMR
(DMSO-d6 + 1 drop D2O) d 5.83 (d, 1H, J = 6.6 Hz,
H-10), 4.11 (d, 1H, J = 6.6 Hz, H-20). 1H NMR
(DMSO-d6), 13C NMR (DMSO-d6) and UV data were
identical to previously published data.1,18
4.4.5. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-5-bromo-
uracil (20). Nucleoside 9 (0.34g, 0.72 mmol) was stirred
for 48 h. The residue resulting after workup was purified
by crystallization (MeOH), which after washing with
cold CH2Cl2 afforded the unprotected nucleoside 20
(150 mg) as white needles. Repeated crystallization of
the mother liquor afforded additional 20 (combined
4.4.2.
1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]uracil
(EUrd, 17). Nucleoside 6 (0.66 g, 1.68 mmol) was stirred
46 h. The residue resulting after workup was adsorbed
on Kieselguhr and purified by silica gel column chroma-
tography (0–40% MeOH in CH2Cl2, v/v) to afford the
known nucleoside 17 (0.35 g, 77%) as a white solid mate-
rial. Rf = 0.3 (20% MeOH in CH2Cl2, v/v); UV kmax
pH 1, 261 nm, kmax H2O, 261 nm, kmax pH 11, 261 nm;
MALDI-HRMS m/z 291.0597 ([M+Na]+, C11H12N2O6Æ
yield 171 mg, 68%). Rf = 0.4(20% MeOH in CH Cl2,
2
v/v); mp (MeOH) 125–128 ꢁC; UV kmax pH 1, 278 nm,
kmax H2O, 278 nm, kmax pH 11, 278 nm; MALDI-
HRMS m/z 368.9708 ([M+Na]+, C11H11BrN2O6ÆNa+
Na+ calcd 291.0587); Selected data H NMR (DMSO-
1
1
d6 + 1 drop D2O) d 5.82 (d, 1H, J = 7.3 Hz, H-10), 4.17
(d, 1H, J = 7.3 Hz, H-20); 13C NMR (DMSO-d6) d
162.9, 150.8, 140.8 (C-6), 102.1 (C-5), 86.5 (C-40), 85.9
(C-10), 82.6, 77.7 (C-20), 77.0, 72.6, 61.3 (C-60). 1H
NMR (DMSO-d6) data were identical to previously
published data.1
calcd 368.9693); H NMR (DMSO-d6) d 11.87 (br s,
1H, ex, NH), 8.52 (s, 1H, H-6), 5.96 (s, 1H, ex, 30-
OH), 5.81–5.86 (m, 2H, 1 ex, 20-OH, H-10), 5.31 (t,
1H, ex, J = 4.0 Hz, 50-OH), 4.21 (br t, 1H, H-20), 3.93
(t, 1H, J = 2.9 Hz, H-40), 3.72–3.80 (m, 1H, H-50A),
3.62–3.70 (m, 1H, H-50B), 3.56 (s, 1H, HC„C); Selected

1258
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
1
data H NMR (DMSO-d6 + 1 drop D2O) 5.82 (d, 1H,
J = 7.2 Hz, H-10), 4.20 (d, 1H, J = 7.2 Hz, H-20); 13C
NMR (DMSO-d6) d 158.7, 150.2, 140.5 (C-6), 96.2,
86.8 (C-40), 86.2 (C-10), 82.5, 78.0 (C-20), 77.1, 72.8,
61.1 (C-50). Anal. Calcd for C11H11BrN2O6Æ7/16 MeOH:
C, 38.09; H, 3.44; N, 7.77. Found: C, 38.18; H, 3.69; N,
7.37.
3.3 Hz, H-40), 3.59–3.67 (m, 1H, H-5A0 ), 3.51 (s, 1H,
HC„C), 3.41–3.49 (m, 1H, H-50B); 13C NMR (DMSO-
d6) d 156.7, 149.1, 136.5 (C-5), 87.3 (C-10), 87.2 (C-40),
0
82.8, 76.6, 76.0 (C-20), 71.9, 62.4(C-5 ). Anal. Calcd
for C10H11N3O6Æ1/4H O: C, 43.88; H, 4.23; N, 15.35.
2
Found: C, 44.06; H, 4.14; N, 15.01.
4.4.9. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-5-azacy-
tosine (24). To an ice-cold solution of protected nucleo-
side 13 (174.4 mg, 0.44 mmol) in anhydrous MeOH
(15 mL) was added saturated methanolic ammonia
(1.00 mL). The reaction mixture was warmed to rt
and stirred for 27 h, whereupon it was evaporated to
dryness and coevaporated with anhydrous EtOH
(2 · 10 mL). The resulting residue was purified by silica
gel column chromatography (0–20% MeOH in
CH2Cl2, v/v) to afford nucleoside 24 (75 mg, 63%) as
a white solid material. Rf = 0.3 (40% MeOH in CHCl3,
v/v); UV kmax pH 1, 257 nm, kmax H2O, 242 nm, kmax
pH 11, 249 nm; MALDI-HRMS m/z 291.0692
([M+Na]+, C10 H12N4O5ÆNa+ calcd 291.0700); 1H
NMR (DMSO-d6) d 8.54(s, 1H, H-6), 7.63 (br s,
1H, ex, NH2), 7.61 (br s, 1H, ex, NH2), 5.93 (s, 1H,
ex, 30-OH), 5.86 (d, 1H, ex, J = 6.6 Hz, 20-OH), 5.74
(d, 1H, J = 6.6 Hz, H-10), 5.10 (t, 1H, ex, J = 4.8 Hz,
50-OH), 4.29 (t, 1H, J = 6.6 Hz, H-20), 3.92 (br t, 1H,
H-40), 3.65–3.73 (m, 2H, H-50), 3.57 (s, 1H, HC„C);
13C NMR (DMSO-d6) d 165.6, 156.6 (C-6), 153.2,
87.3 (C-10), 86.7 (C-40), 82.6, 78.1 (C-20), 77.4, 72.5,
61.3 (C-50).
4.4.6.
1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-5-iodo-
uracil (21). Nucleoside 10 (0.33 g, 0.64mmol) was stirred
for 21 h. The residue resulting after workup was purified
by silica gel column chromatography (0–100% EtOAc in
petroleum ether, v/v, then 0–3% MeOH in EtOAc, v/v)
to afford nucleoside 21 (156 mg, 62%). Rf = 0.4(20%
MeOH in CH2Cl2, v/v); UV kmax pH 1, 287 nm, kmax
H2O, 287 nm, kmax pH 11, 278 nm; MALDI-HRMS m/
z
416.9574 ([M+Na]+, C11H11IN2O6ÆNa+ calcd
1
416.9554); H NMR (DMSO-d6) d 11.72 (br s, 1H, ex,
NH), 8.53 (s, 1H, H-6), 5.93 (s, 1H, ex, 30-OH), 5.84
(d, 1H, ex, J = 6.6 Hz, 20-OH), 5.80 (d, 1H, J = 7.0 Hz,
H-10), 5.29 (t, 1H, ex, J = 3.8 Hz, 50-OH), 4.20 (br t,
1H, H-20), 3.92 (t, 1H, J = 2.9 Hz, H-40), 3.71–3.79 (m,
1H, H-50A), 3.62–3.69 (m, 1H, H-50B), 3.56 (s, 1H,
HC„C); 13C NMR (DMSO-d6) d 160.3, 150.5, 145.3
(C-6), 86.7 (C-40), 86.0 (C-10), 82.5, 78.0 (C-20), 77.0,
72.7, 69.9, 61.1 (C-50). Anal. Calcd for C11H11IN2O6Æ
1/2 MeOH: C, 33.68; H, 3.19; N, 6.83. Found: C,
34.08; H, 3.35; N, 6.54.
4.4.7. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-3-deaza-
uracil (22). Nucleoside 11 (0.29 g, 0.73 mmol) was stirred
for 23 h. The residue resulting after workup was purified
by silica gel column chromatography (0–12% MeOH in
CHCl3, v/v) to afford nucleoside 22 (123 mg, 63%) as a
4.4.10. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]cyanuric
acid (25). Protected nucleoside 14 (115 mg, 0.28 mol)
was stirred for 40 h. The residue resulting after workup
was purified using silica gel column chromatography (0–
30% MeOH in CHCl3, v/v) to afford nucleoside 25
(51 mg, 65%) as a white solid material. Rf = 0.2 (20%
MeOH in CHCl3, v/v); MALDI-HRMS m/z 308.0481
([M+Na]+, C10H11N3O7ÆNa+ calcd 308.0489); 1H
NMR (DMSO-d6) d 5.91 (d, 1H, J = 8.3 Hz, H-10),
5.83 (s, 1H, ex, 30-OH), 5.74(d, 1H, ex, J = 6.6 Hz, 20-
OH), 4.94 (dd, 1H, J = 8.3 Hz, 6.6 Hz, H-20), 4.44 (t,
1H, ex, J = 5.7 Hz, 50-OH), 3.85 (t, 1H, J = 4.9 Hz, H-
40), 3.59–3.65 (m, 2H, H-50), 3.49 (s, 1H, HC„C); 13C
NMR (DMSO-d6) d 150.6, 150.2, 87.1, 85.7, 83.1,
76.3, 73.4, 72.3, 62.2.
white solid material. Rf = 0.4(20% MeOH in CH Cl2,
2
v/v); UV kmax pH 1, 235 nm, 266 nm, kmax H2O,
279 nm, kmax pH 11, 256 nm; MALDI-HRMS m/z
290.0626 ([M+Na]+, C12H13NO6ÆNa+ calcd 290.0635);
1H NMR (DMSO-d6) d 10.66 (br s, 1H, ex, OH), 7.77
(d, 1H, J = 7.7 Hz, H-6), 6.05 (d, 1H, J = 6.6 Hz, H-
10), 5.91 (dd, 1H, J = 7.7 Hz, 2.6 Hz, H-5), 5.80 (br s,
1H, ex, 3-OH), 5.74(br d, 1H, ex, J = 6.2 Hz, 20-OH)
5.54(d, 1H, ex, J = 2.6 Hz, H-3), 5.05 (br t, 1H, ex, 50-
OH), 4.14 (br t, 1H, H-20), 3.88 (t, 1H, J = 3.7 Hz, H-
40), 3.63–3.74(m, 2H, H-5 ), 3.51 (s, 1H, HC„C); 13C
NMR (DMSO-d6) d 166.5, 163.2, 135.4(C-6), 100.4
(C-5), 97.8 (C-3), 86.2 (C-10/C-40), 86.0 (C-10/C-40),
83.0, 78.9 (C-20), 77.0, 72.6, 61.5 (C-50).
0
4.4.11. S-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-2-thio-
cytosine (26). Protected nucleoside 15 (0.26 g,
0.63 mmol) was stirred for 40 h. The residue resulting
after workup was crystallized (EtOH and minimal
H2O), which after washing with cold EtOH afforded
nucleoside 26 (108 mg) as a white solid material. Crys-
tallization from the mother liquor afforded additional
nucleoside 26 (combined yield 128 mg, 72%). Rf = 0.4
(20% MeOH in CH2Cl2, v/v); mp (EtOH/H2O) 176–
180 ꢁC; UV kmax pH 1, 239 nm, kmax H2O, 219 nm,
4.4.8. 1-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-6-azaura-
cil (23). Nucleoside 12 (0.27 g, 0.69 mmol) was stirred
for 24h. The residue resulting after workup was ad-
sorbed on silica gel and purified by silica gel column
chromatography (0–30% MeOH in CH2Cl2, v/v) to af-
ford nucleoside 23 (152 mg, 82%) as a pale yellow solid
material. Rf = 0.6 (40% MeOH in CH2Cl2, v/v); UV kmax
pH 1, 261 nm, kmax H2O, 261 nm, kmax pH 11, 256 nm;
MALDI-HRMS m/z 292.0538 ([M+Na]+, C10H11N3O6Æ
282 nm, kmax pH 11, 282 nm; MALDI-HRMS m/z
1
+
Na+ calcd 292.0540); H NMR (DMSO-d6) d 12.29 (br
306.0524([M+Na]
,
C11H13N3O4SÆNa+
calcd
s, 1H, ex, NH), 7.61 (s, 1H, H-5), 5.91 (br s, 1H,
ex, 30-OH), 5.84(d, 1H, J = 7.3 Hz, H-10), 5.76–5.81
(m, 1H, ex, 20-OH), 4.54 (br t, 1H, ex, J = 7.3 Hz, 50-
OH), 4.45 (m, 1H, H-20), 3.84(dd, 1H, J = 7.3 Hz,
306.0519); 1H NMR (DMSO-d6) d 7.89 (d, 1H,
J = 5.9 Hz, H-6), 6.97 (br s, 2H, 2 ex, NH2), 6.15 (d,
1H, J = 5.9 Hz, H-5), 5.87 (d, 1H, J = 6.6 Hz, H-10),
5.84(d, 1H, ex, J = 6.6 Hz, 20-OH), 5.67 (br s, 1H, ex,

P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
1259
3-OH), 4.73 (br t, 1H, ex, J = 5.3 Hz, 50-OH), 4.03 (t,
1H, J = 6.6 Hz, H-20), 3.77 (dd, 1H, J = 6.2 Hz,
2.8 Hz, H-40), 3.49–3.64 (m, 3H, H-50, HC„C); 13C
NMR (DMSO-d6) d 168.6, 163.1, 154.8 (C-6), 101.6
(C-5), 86.7 (C-40), 85.7 (C-10), 83.1, 79.9 (C-20), 77.0,
72.6, 61.9 (C-50). Anal. Calcd for C11H13N3O4S: C,
46.63; H, 4.63; N, 14.83; S, 11.32. Found: C, 46.52; H,
4.61; N, 14.72; S, 11.22.
three additional days the experimental medium was re-
newed. Five days after initial addition of experimental
medium, the cell cultures were washed once in PBS buf-
fer (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4Æ2H2O,
0.24g/L KH 2PO4, pH 7.4) and then cells were fixed and
stained for 10 min with a solution of 0,5% (w/v) crystal
violet (Sigma–Aldrich) and 25% (v/v) methanol. Un-
bound crystal violet was removed by gentle wash in
tap water. After drying, the crystal violet stained cells
were re-dissolved in a solution 0.1 M sodium citrate/
50% (v/v) ethanol solution. The optical density mea-
sured at 570 nm was a relative measure for the cell num-
ber. Growth inhibition of cancer cells was expressed as
percent of control, that is untreated cells, and data
points were mean of triplicate samples. This assay was
applied for practical reasons and yields the same results
as directly counting the number of cells.42
4.4.12. S-[3-C-Ethynyl-b-D-ribo-pentofuranosyl]-4-thio-
uracil (27). Protected nucleoside 16 (0.40 g, 0.98 mmol
with traces of 4-thiouracil), was stirred for 41 h. The res-
idue resulting after workup was adsorbed on silica gel
and purified by silica gel column chromatography (0–
12% EtOH in CHCl3, v/v) to furnish nucleoside 27
(195 mg, 70%). Rf = 0.2 (20% EtOH in CH2Cl2, v/v);
UV kmax pH 1, 263 nm, 322 nm, kmax H2O, 262 nm,
298 nm, kmax pH 11, 300 nm; MALDI-HRMS m/z
307.0356
([M+Na]+,
C11H12N2O5SÆNa+
calcd
307.0359); 1H NMR (DMSO-d6) d 11.57 (br s, 1H,
ex), 7.68 (d, 1H, J = 6.6 Hz, H-6), 6.28 (d, 1H,
J = 6.6 Hz, H-5), 5.89–5.94(m, 2H, 1 ex, H-1 0, 20-
OH), 5.82 (br s, 1H, ex, 3-OH), 4.79 (t, 1H, ex,
J = 5.3 Hz, 50-OH), 4.12 (br t, 1H, H-20), 3.85 (dd, 1H,
J = 5.5 Hz, 2.9 Hz, H-40), 3.40–3.64 (m, 3H, H-50,
Acknowledgements
The Danish National Research Foundation is thanked
for financial support. The technical assistance of Birgit
E. Reiter is greatly appreciated.
1
HC„C); Selected data H NMR (DMSO-d6 + 1 drop
D2O) d 5.90 (d, 1H, J = 6.1 Hz, H-10), 4.12 (d, 1H,
J = 6.1 Hz, H-20); 13C NMR (DMSO-d6) d 176.2,
154.0, 144.2 (C-6), 101.9 (C-5), 87.3 (C-40), 84.2 (C-10),
82.8, 80.0 (C-20), 77.3, 72.3, 61.7 (C-50). Traces of EtOH
were observed.
Supplementary data
1H NMR spectra of 27 and 13C NMR spectra of 1, 10,
13–14, 17–18, and 24–25. Supplementary data associ-
ated with this article can be found, in the online version,
at doi:10.1016/j.bmc.2004.11.054.
5. Anticancer assay
References and notes
Human adenocarcinoma breast cancer cell line MCF-7
was originally obtained from the Human Cell Culture
Bank, Mason Research Institute (Rockville, MD).
MCF-7 cells were routinely propagated in growth med-
ium composed of Dulbecco0s Modified Eagle Medium:
Ham0s Nutrient Mixture F-12, 1:1 mix (Invitrogen,
Denmark, catalog number 21041–025), supplemented
with 2.5 mM Glutamax (Invitrogen, Denmark), 6 ng/
mL insulin (Novo Nordic, Denmark) and 1% fetal calf
serum (FCS).41 Human adenocarcinoma prostate cancer
cell line PC-3 was purchased from ATCC-LGC Promo-
chem (United Kingdom, ATCC catalog number CRL-
1435) and grown in ATCC complete growth medium,
1. Hattori, H.; Tanaka, M.; Fukushima, M.; Sasaki, T.;
Matsuda, A. J. Med. Chem. 1996, 39, 5005–5011.
2. Takatori, S.; Kanda, H.; Takenaka, K.; Wataya, Y.;
Matsuda, A.; Fukushima, M.; Shimamoto, Y.; Tanaka,
M.; Sasaki, T. Cancer Chemother. Pharmacol. 1999, 44,
97–104.
3. Matsuda, A.; Fukushima, M.; Wataya, Y.; Sasaki, T.
Nucleosides Nucleotides 1999, 18, 811–814.
4. Shimamoto, Y.; Koizumi, K.; Okabe, H.; Kazuno, H.;
Murakami, Y.; Nakagawa, F.; Matsuda, A.; Sasaki, T.;
Fukushima, M. Jpn. J. Cancer Res. 2002, 93, 825–833.
5. Tabata, S.; Tanaka, M.; Matsuda, A.; Fukushima, M.;
Sasaki, T. Oncol. Rep. 1996, 3, 1029–1034.
6. Azuma, A.; Matsuda, A.; Sasaki, T.; Fukushima, M.
Nucleosides Nucleotides Nucleic Acids 2001, 20, 609–619.
7. Shimamoto, Y.; Fujioka, A.; Kazuno, H.; Murakami, Y.;
Ohshimo, H.; Kato, T.; Matsuda, A.; Sasaki, T.; Fuku-
shima, M. Jpn. J. Cancer. Res. 2001, 92, 343–351.
8. Hattori, H.; Nozawa, E.; Iino, T.; Yoshimura, Y.; Shuto,
S.; Shimamoto, Y.; Nomura, M.; Fukushima, M.;
Tanaka, M.; Sasaki, T.; Matsuda, A. J. Med. Chem.
1998, 41, 2892–2902.
0
ATCC catalog number 30–2004(Ham s F12K medium
with 2 mM L-glutamine, 1.5 g/L sodium bicarbonate,
also known as Kaighn0s modification of Ham0s F-12
medium) supplemented with 10% FCS. MCF-7 cells
and PC-3 cells were propagated at 37 ꢁC in a humidified
5% CO2 incubator. In all experiments 250 U/mL penicil-
lin and 31.25 lg/mL streptomycin sulfate were added to
prevent bacterial infections.
9. Dauvergne, J.; Burger, A.; Biellmann, J.-F. Nucleosides
Nucleotides Nucleic Acids 2001, 20, 1775–1781.
10. Jung, F.; Burger, A.; Biellmann, J.-F. Org. Lett. 2003, 5,
383–385.
11. Minakawa, N.; Kaga, D.; Kato, Y.; Endo, K.; Tanaka,
M.; Sasaki, T.; Matsuda, A. J. Chem. Soc., Perkin Trans. 1
2002, 19, 2182–2189.
Growth experiments were performed in 24multi well
dishes (Nunc, Roskilde, Denmark), which were seeded
with 1.5 · 104 MCF-7 cells per well or 2.6 · 104 PC-3
cell per well. Two days after seeding, the medium was
changed to experimental medium containing the tested
compounds, which were dissolved in sterile water. After

1260
P. J. Hrdlicka et al. / Bioorg. Med. Chem. 13(2005) 1249–1260
12. Nomura, M.; Shuto, S.; Tanaka, M.; Sasaki, T.; Mori, S.;
Shigeta, S.; Matsuda, A. J. Med. Chem. 1999, 42, 2901–
2908.
13. Hrdlicka, P. J.; Andersen, N. K.; Jepsen J. S.; Hansen, F.
G.; Haselmann, K. F.; Nielsen, C.; Wengel, J., submitted.
14. Van Rompay, R.; Norda, A.; Linden, K.; Johansson, M.;
Karlsson, A. Mol. Pharmacol. 2001, 59, 1181–1186.
15. Koizumi, K.; Shimamoto, Y.; Azuma, A.; Wataya, Y.;
Matsuda, A.; Sasaki, T.; Fukushima, M. Int. J. Mol. Med.
2001, 8, 273–278.
16. Girardet, J.-L.; Gunic, E.; Esler, C.; Cieslak, D.; Pietrz-
kowski, Z.; Wang, G. J. Med. Chem. 2000, 43, 3704–
3713.
17. Nomura, M.; Sato, T.; Washinosu, M.; Tanaka, M.; Asao,
T.; Shuto, S.; Matsuda, A. Tetrahedron 2002, 58, 1279–
1288.
27. Cook, P. D.; Day, R. T.; Robins, R. K. J. Heterocycl.
Chem. 1977, 14, 1295–1298.
28. Currie, B. L.; Robins, R. K.; Robins, M. J. J. Heterocycl.
Chem. 1970, 7, 323–329.
29. Harry-Oꢀkuru, R. E.; Smith, J. M.; Wolfe, M. S. J. Org.
Chem. 1997, 62, 1754–1759.
30. Dugas, H.; Blackburn, B. J.; Robins, R. K.; Deslauriers,
R.; Smith, I. C. P. J. Am. Chem. Soc. 1971, 93, 3468–3470.
31. Jones, A. J.; Grant, D. M.; Winkley, M. W.; Robins, R. K.
J. Am. Chem. Soc. 1970, 92, 4079–4087.
32. Boarland, M. P. V.; McOmie, J. F. W. J. Chem. Soc. 1952,
3722–3728.
33. Miah, A.; Reese, C. B.; Song, Q. Nucleosides Nucleotides
1997, 16, 53–65.
34. Still, I. W. J.; Plavac, N.; McKinnon, D. M.; Chauhan, M.
S. Can. J. Chem. 1978, 56, 725–729.
18. Ludwig, P. S.; Schwendener, R. A.; Schott, H. Synthesis
2002, 16, 2387–2392.
35. Psoda, A.; Kazimierczuk, Z.; Shugar, D. J. Am. Chem.
Soc. 1974, 96, 6832–6838.
19. Kakinuma, K.; Iihama, Y.; Takagi, I.; Ozawa, K.;
Yamauchi, N.; Imamura, N.; Esumi, Y.; Uramoto, M.
Tetrahedron 1992, 48, 3763–3774.
36. For definitions of nucleosides conformations see: Altona,
C.; Sundaranlingam, M. J. Am. Chem. Soc. 1972, 94,
8205–8212.
20. Vorbruggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber.
37. Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973,
95, 2333–2344.
¨
1981, 114, 1234–1255.
21. Wagner, G.; Mahrwald, R. Pharmazie 1980, 35, 663–668.
22. Wagner, G.; Mahrwald, R. Pharmazie 1981, 36, 12–15.
23. Ben-Hattar, J.; Jiricny, J. J. Org. Chem. 1986, 51, 3211–
3213.
38. Danel, K.; Pedersen, E. B.; Nielsen, C. J. Med. Chem.
1998, 41, 191–198.
39. Gottlieb, H. E.; Kotlyar, V.; Nudelman, A. J. Org. Chem.
1997, 62, 7512–7515.
24. Ozinskas, A. J.; Bobst, A. M. Helv. Chim. Acta 1979, 62,
1677–1681.
40. Restivo, A. R.; Dondzila, F. A. J. Org. Chem. 1962, 27,
2281–2282.
25. Jones, A. J.; Grant, D. M.; Winkley, M. W.; Robins, R. K.
J. Phys. Chem. 1970, 74, 2684–2689.
41. Lykkesfeldt, A. E.; Larsen, S. S.; Briand, P. Int. J. Cancer
1995, 61, 529–534.
26. Shapiro, R.; Kang, S. Biochim. Biophys. Acta 1971, 232,
1–4.
42. Lundholt, B. K.; Briand, P.; Lykkesfeldt, A. E. Breast
Cancer Res. Treat. 2001, 67, 199–214.