Stereocontrolled synthesis of 4′-C-cyano and 4′-C-cyano- 4′-deoxy pyrimidine pyranonucleosides as potential chemotherapeutic agents

Article abstract of DOI:10.1016/j.carres.2012.10.012

A new series of 4′-C-cyano and 4′-C-cyano-4′-deoxy pyrimidine pyranonucleosides has been designed and synthesized. Commercially available 1,2,3,4,6-penta-O-acetyl-d-mannopyranose (1) was condensed with silylated 5-fluorouracil, uracil, and thymine, respec

Full text of DOI:10.1016/j.carres.2012.10.012

Carbohydrate Research 364 (2012) 8–14
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Stereocontrolled synthesis of 40-C-cyano and 40-C-cyano-40-deoxy pyrimidine
pyranonucleosides as potential chemotherapeutic agents
Christos Kiritsis a, Stella Manta a, Vanessa Parmenopoulou a, Athina Dimopoulou a, Nikolaos Kollatos a,
Ioannis Papasotiriou b, Jan Balzarini c, Dimitri Komiotis a,
⇑
a Department of Biochemistry and Biotechnology, Laboratory of Bio-Organic Chemistry, University of Thessaly, 26 Ploutonos Str., 41221 Larissa, Greece
b Research Genetic Cancer Center (R.G.C.C. Ltd.), Megalou Alexandrou 155 str., GR-53070 Filotas, Greece
c Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
A new series of 40-C-cyano and 40-C-cyano-40-deoxy pyrimidine pyranonucleosides has been designed and
Article history:
Received 17 September 2012
Received in revised form 15 October 2012
Accepted 16 October 2012
Available online 24 October 2012
synthesized. Commercially available 1,2,3,4,6-penta-O-acetyl-
silylated 5-fluorouracil, uracil, and thymine, respectively to afford after deacetylation 1-(
D
-mannopyranose (1) was condensed with
-mannopyr-
a
-D
anosyl)nucleosides (2a–c). Subjecting 2a–c to the sequence of specific acetalation, selective protection
of the primary hydroxyl group and oxidation, the 40-ketonucleosides 6a–c and 7c were obtained. Reaction
of compounds 6a,b, and 7c with sodium cyanide and subsequent deprotection gave the target 1-(40-C-
Keywords:
cyano-
a-D
-mannopyranosyl)nucleosides 12a–c. Deoxygenation at the 40-position of cyanohydrins 8a,b,
C-Cyano pyranonucleosides
Stereocontrolled synthesis
Cytotoxicity
and 11c followed by deprotection led to the desired 1-(40-C-cyano-40-deoxy-
a-D-talopyranosyl)nucleo-
sides (15a–c). The newly synthesized compounds were evaluated for their potential antiviral and cyto-
static activities in cell culture.
5-Fluorouracil
Ó 2012 Elsevier Ltd. All rights reserved.
Nucleoside chemistry represents an important research area for
drug discovery, as many nucleoside analogues are prominent drugs
and have been widely applied to cancer and viral chemotherapy.1–5
Extensive modifications have been performed on both the hetero-
cyclic base and the sugar moiety in the search for effective and
selective derivatives with a therapeutic profile.
Considering that glycosidic bond cleavage is a frequently
encountered degradative pathway of nucleosides,6 special atten-
tion has been drawn to six-membered nucleosides, which showed
resistance to hydrolysis7 and exhibited antiviral,8,9 antibiotic,10
antioxidant,11,12 and antitumor3 properties. The introduction of
fluoro, keto,13–18 exomethylene,19–21 ethynyl,22 azido, and amino23
groups to pyranonucleosides has led to novel scaffolds endowed
with interesting biological activities. Experimental data also sug-
gested, that human poly(A)-specific ribonuclease24 and glycogen
phosphorylase25–28 are among the molecular targets of several of
these compounds, while it was revealed that 5-fluorouracil nucle-
oside analogues likely represent novel prodrugs of 5-fluorouracil
targeting thymidylate synthase.29
change in biological activity and stability.30,31 A number of cyano
ribofuranoses have demonstrated substantial antiviral32–34 and
anticancer properties,31,35 while their promising therapeutic poten-
tial has been partly attributed to the small size and high electroneg-
ativity of the cyano group.
Taking into consideration our recent findings on 30-C-cyano and
30-deoxy-C-cyano pyranonucleosides as cytotoxic agents36 and
based on the fact that various 40-C substituted nucleosides,37,38 such
as 40-cyano-thymidine,31 exhibitvery potentanti-HIV and/or antitu-
mor activity,39 it was envisaged that the transposition of nitrile from
C-30 to C-40 could also result in nucleosides of significant biological
interest. Thus, we report herein the stereocontrolled synthesis of a
novel series of branched-chain C-cyano pyrimidine pyranonucleo-
sides, 40-C-cyano- -mannopyranonucleosides (12a–c), and 40-C-
cyano-40-deoxy-
-D-talopyranonucleosides (15a–c), containing
a
-D
a
5-fluorouracil, uracil, and thymine as base moieties. The synthesis
and biological assessment of these compounds are presented herein.
Retrosynthetic analysis suggested that the 40-C-cyano nucleo-
sides could be obtained from the corresponding 40-keto nucleo-
sides by a nucleophilic addition reaction. The synthesis of the
keto pyranonucleosides 6a–c and 7c, as key intermediates is out-
lined in Scheme 1.
In the field of modified nucleosides, considerable interest has
been drawn in the synthesis of cyano-substituted nucleosides and
their analogues, with the cyano group incorporated either on the
base moiety or on the sugar part frequently leading to a drastic
The a
-pyranonucleosides of 5-fluorouracil 2a,40 uracil 2b,20 and
thymine 2c (Scheme 1), were readily prepared by condensing per-
acetylated mannose (1) with the proper silylated base followed by
subsequent deacetylation. The 1H NMR spectra of 2a–c showed a
⇑
Corresponding author. Tel.: +30 2410 565285; fax: +30 2410 565290.
E-mail address: dkom@bio.uth.gr (D. Komiotis).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.10.012

C. Kiritsis et al. / Carbohydrate Research 364 (2012) 8–14
9
OAc
HO
OH
OAc
O
X
AcO
AcO
O
i
N
OH
O
OAc
O
N
H
OH
1
2a-c
a: X = F (5-FU)
b: X = H (U)
ii
c: X = CH3 (Thy)
RO
RO
R3O
X
X
X
O
O
O
O
O
O
v
iv
N
N
R1
N
O
O
O
O
O
O
O
N
H
N
H
N
H
O
O
O
R2
OH
6a-c: R = Tr
3a-c: R = H
4a-c: R = Tr
8a,b: R1 = CN, R2 = OH, R3 = Tr
9c: R1 = CN, R2 = OH, R3 = Tr
10c: R1 = OH, R2 = CN, R3 = Tr
7c: R = TBDMS
iii
11c: R1 = CN, R2 = OH, R3 = TBDMS
5c: R = TBDMS
vii
vi
HO
HO
RO
OH
OH
X
X
X
O
vi
O
O
O
N
N
NC
N
NC
OH
O
NC
OH
O
O
O
O
N
H
N
N
H
O
O
H
OH
15a-c
12a-c
13a-c: R = Tr
14c: R = TBDMS
Scheme 1. Reagents and conditions: (i) silylated base, CH3CN, Me3SiOSO2CF3; methanolic ammonia; (ii) acetone, 2,2-dimethoxypropane, p-toluenesulfonic acid, water; (iii)
TrCl or TBDMS, pyridine, DMAP; (iv) PDC, Ac2O, dry CH2Cl2; (v) (a) (R = Tr) H2O, Et2O, NaHCO3, NaCN, (b) (R = TBDMS) H2O, Et2O, NaCN; (vi) TFA 90% in MeOH; (vii) phenyl
chlorothionoformate, Et3N, DMAP, CH3CN, 0 °C; Bu3SnH, AIBN, toluene, 100 °C.
large coupling between protons H-10 and H-20, indicating an axial
orientation of both protons and an equatorially oriented base ring.
Subjecting 2a–c to the sequence of specific acetalation, selective
tritylation of the primary hydroxyl group and oxidation,20 the
desired 40-ketonucleosides 6 were obtained. The 1H NMR spectra
of 6a–c revealed the presence of H-30 at d 4.74–4.65 as a doublet,
due to coupling with H-20, confirming thus the absence of proton
at C-40.
Treatment of ketonucleosides 6a,b with sodium cyanide (NaCN)
and sodium bicarbonate (NaHCO3) in a two-phase ethyl ether
(Et2O)/water (H2O) system,32 favoured the equatorial approach of
CNꢀ affording the cyanohydrins 8a,b as the only stable products.
Their stability may be related to the trans diaxially oriented two
oxygen atoms attached to C-30 and C-40. The structure of these
compounds was assigned on the basis of NOE experiments. Thus,
the mutual NOE enhancements observed between the free OH
group of cyanohydrin 8a with both H-30 and H-50 show that these
protons are on the same side of the plane (Fig. 1). Interestingly, the
same reaction of CNꢀ on thymine nucleoside 6c resulted in an
inseparable ca. 1:1 mixture of two 40-epimers 9c and 10c, while
changes in solvent, pH, or temperature had no significant effect
on the epimers’ ratio. In an effort to improve the stereoselectivity,
we also examined the influence of the 60-protective group on the
steric course of the reaction. Thus, treatment of thymine analogue
3c with t-butyldimethylsilyl chloride (TBDMSCl)19 followed by oxi-
dation, furnished 40-keto analogue 7c, which upon reaction with
NaCN in aqueous ether35 gave the single manno cyanohydrin
11c. It seems that the presence of the TBDMS group on C-60 facili-
tated the equatorial orientation of the C-40 cyano group, possibly
due to steric effects. NOE data confirmed this stereochemical trend
since the free OH group of 11c exhibited positive NOEs with the
nearby protons H-30 and H-50, suggesting their close proximity.
Finally, full deprotection of 8a,b, and 11c, using trifluoroacetic acid
(TFA) gave the desired nucleosides 12a–c.
With the stereochemistry of the newly synthesized cyanohy-
drins secure, we turned our attention to the synthesis of their 40-
deoxy derivatives 15a–c (Scheme 1). Phenoxythiocarbonylation
of 8a,b, and 11c under commonly used conditions, phenyl chlo-
rothionoformate, 4-(dimethylamino)pyridine (DMAP), and trieth-
ylamine (Et3N) in CH3CN,35 followed by reaction with Bu3SnH in
the presence of 2,20-azobis(isobutyronitrile) (AIBN), gave stereo-
specifically, the 40-deoxy derivatives 13a,b, and 14c. Interestingly,
radical deoxygenation of the epimers 9c and 10c also proceeded
stereospecifically to furnish thymine derivative 13c, as the only
product. The stereochemistry of C-40 of the deoxygenated com-
pounds was determined by NOE measurements. Irradiation of
H-40 of compound 13b, chosen as model molecule, produced strong
enhancements for the H-30 (12%) and H-50 (12%); conversely, when
H-30 and H-50 was irradiated, a positive NOE effect was observed
for H-40 (10% and 13%, respectively) (Fig. 1). These data suggest
that all of these protons are in the same face of the pyranose ring,
concluding that the hydrogen atom enters from the less hindered,
a
-face of the planar radical intermediate, opposite to the bulky
20,30-O-isopropylidene group. Finally, compounds 13a,b, and 13c

10
C. Kiritsis et al. / Carbohydrate Research 364 (2012) 8–14
20%
15%
18%
H
H
H
H
TrO
TrO
15%
F
O
O
12%
O
O
H
NC
H
N
N
O
NC
O
O
O
N
H
N
H
O
O
H
H
13%
3%
4%
12%
10%
H
O
3%
H
2%
13b
Figure 1. Selected NOEs observed for compounds 8a and 13b.
8a
or 14c were converted to the fully unprotected derivatives 15a–c,
respectively, by treatment with TFA 90% in MeOH.
In conclusion, we present herein a stereocontrolled synthesis of
a new series of 40-C-cyano and 40-C-cyano-40-deoxy pyrimidine
pyranonucleosides, which involves simple protection–deprotec-
tion sequences on suitable intermediates. These results allow
access to 40-C branched chain nucleoside analogues, in a highly
predictable and efficient manner. The target nucleosides were eval-
uated for their antiviral and cytostatic activity. Although the syn-
The target nucleosides 12a–c and 15a–c were tested for their
inhibitory effects on the proliferation of murine leukemia cells
(L1210), human T-lymphocyte cells (CEM) and human cervix carci-
noma cells (HeLa). The uracil (12b, 15b) and thymine (12c, 15c)
40-CN(40-deoxy) derivatives hardly inhibit tumor cell proliferation.
Their IC50s were invariably around or above 200
lM. Instead, the
thesized compounds were devoid of a significant antiviral
5-fluorouracil 40-CN(40-deoxy) nucleoside derivatives 12a and
15a were endowed with a much more pronounced antiprolifera-
tive effect. In fact, they inhibited L1210 and HeLa tumor cell prolif-
potential, the 5-fluorouracil 40-CN(40-deoxy) nucleoside derivatives
12a and 15a showed a similar cytostatic activity spectrum as the
free base 5-fluorouracil.
eration at IC50s that ranged between 2.6 and 4.1 lM. They were
somewhat less inhibitory against CEM cell proliferation. The cyto-
static profile of the new compounds 12a and 15a closely parallels
that of 5-fluorouracil. When evaluated against thymidine kinase-
deficient L1210 and HeLa tumor cell lines, the compounds fully
kept their cytostatic activity (Table 1). These findings strongly sug-
gest that the cytostatic activity of the 5-fluorouracil derivatives
does not require TK-dependent phosphorylation (like 5-fluoro-20-
deoxyuridine) and thus most likely function as prodrugs of 5-fluo-
rouracil that need to be converted first to the free 5-fluorouracil
base prior to further metabolic activation. In this respect both 40-
C-cyano- and 40-C-cyano-40-deoxy pyrimidine pyranonucleoside
analogues behave similarly as the earlier described 30-C-cyano-
and 30-C-cyano-30-deoxy pyrimidine pyranonucleoside analogues
of 5-fluorouracil.36 None of the compounds showed antiviral activ-
ity in cell culture. It is currently unclear how 5-fluorouracil is re-
leased from the pyranonucleoside derivatives and what the
kinetics of 5-FU release in the intact cell cultures or in an in vivo
environment are. Although it can be argued that administration
of 5-FU itself might be the most efficient modality for susceptible
cancer drug treatment, it cannot be excluded that 5-FU derivatives
of the 30- or 40-C-cyano(deoxy)pyrimidine pyranonucleosides
might have eventual advantages in efficacy or therapeutic window
by potentially altered delivery characteristics of these compounds
versus the free 5-FU parent drug. In vivo studies in animal models
may further address this issue.
1. Experimental
1.1. General procedure
Melting points were recorded in a Mel-Temp apparatus and are
uncorrected. Thin layer chromatography (TLC) was performed on
Merck precoated 60F254 plates. Reactions were monitored by TLC
on silica gel, with detection by UV light (254 nm) or by charring
with sulfuric acid. Flash chromatography was performed using sil-
ica gel (240–400 mesh, Merck). 1H NMR spectra were recorded at
300 MHz on a Bruker AVANCEIII 300 spectrometer and 13C NMR
spectra at 75.5 MHz on the same spectrometer, using chloro-
form-d (CDCl3), methanol-d4 (CD3OD). Chemical shifts are reported
in parts per million (d) downfield from tetramethylsilane (TMS) as
internal standard.
UV–Vis spectra were recorded on a PG T70 UV–VIS spectrome-
ter and mass spectra were obtained on a Thermo Quest Finnigan
AQA Mass Spectrometer (electrospray ionization). Optical rotations
were measured using an Autopol I polarimeter. Infrared spectra
were obtained with a Thermo Scientific Nicolet IR100 FT-IR spec-
trometer. All reactions were carried out in dry solvents. Dichloro-
methane (CH2Cl2) was distilled from phosphorus pentoxide and
stored over 4 Å molecular sieves. Acetonitrile and toluene were
distilled from calcium hydride and stored over 3 Å molecular
sieves. Pyridine was stored over potassium hydroxide pellets. All
Table 1
Cytostatic activity of 12a–c and 15a–c against a panel of tumor cell lines
a
Compound
IC50
(lM)
L1210
CEM
57
>500
>500
18
221
332 64
18
0.022 0.006
HeLa
3.2 0.0
494
433 94
2.6 0.9
195 21
L1210/TKꢀ
HeLa TKꢀ
12a
12b
12c
15a
15b
15c
5-Fluorouracil
5-Fluoro-dUrd
3.0 0.9
>500
>500
4.1 0.6
210 16
262 12
0.38 0.17
0.0011 0.0002
7
2.6 0.7
>500
>500
0.81 0.10
>500
P500
8
6
8
4.6 1.5
0.49 0.02
227
6
205
8
188
4
266 17
0.32 0.31
3.0 0.1
214 18
0.23 0.01
1.4 0.4
5
0.54 0.12
0.050 0.011
a
50% inhibitory concentration or compound concentration required to inhibit tumoral proliferation by 50%.

C. Kiritsis et al. / Carbohydrate Research 364 (2012) 8–14
11
reactions sensitive to oxygen or moisture were carried out under
nitrogen atmosphere using oven-dried glassware.
compound 8a as a colorless oil: ½a D22
ꢁ
ꢀ18 (c 1.3, CHCl3); UV
(CHCl3): kmax 262 nm (e
9356); 1H NMR (CDCl3, 300 MHz): d 8.96
(br s, 1H, NH), 7.43–7.28 (m, 16H, 3C6H5, H-6), 5.38 (d, 1H,
1.2. Synthesis of 1-(40-C-cyano-
fluorouracil (12a)
a-D
-mannopyranosyl)5-
J1 ,2 = 2.7 Hz, H-10), 4.83 (s, 1H, 40-OH), 4.77 (dd, 1H, J2 ,3 = 6.8 Hz,
0
0
0
0
H-20), 4.60 (d, 1H, H-30), 3.89 (t, 1H, J5 ,6a = J5 ,6b = 6.5 Hz, H-50),
3.78–3.67 (m, 2H, H-6a0,b0), 1.67, 1.38 (2s, 6H, 2CH3). Anal. Calcd
for C33H30FN3O7: C, 66.10; H, 5.04; N, 7.01. Found: C, 66.29; H,
5.11; N, 7.23. ESIMS m/z 600.58 [M+H+].
0
0
0
0
1.2.1. 1-(20,30-O-Isopropylidene-
a-D-mannopyranosyl)5-
fluorouracil (3a)
To a stirred suspension of 2a40 (2.50 g, 8.6 mmol) in anhydrous
acetone (17.0 mL) and 2,2-dimethoxypropane (17.0 mL) was added
p-toluenesulfonic acid monohydrate (0.34 g, 1.8 mmol). After
50 min, water (34.0 mL) was added and the reaction mixture was
stirred for 16 h. The resulting solution was neutralized with satu-
rated sodium bicarbonate so that pH did not exceed 7, concen-
trated and the residue was purified by flash chromatography (9:1
CH2Cl2–MeOH, Rf 0.37) to give 2.22 g (78%) of compound 3a as a
1.2.5. 1-(40-C-Cyano-
a-D-mannopyranosyl)5-fluorouracil (12a)
Compound 8a (0.59 g, 1.0 mmol) was dissolved in 4.5 mL of 90%
TFA in MeOH. The solution was stirred for 10 min at room temper-
ature and then concentrated under reduced pressure, in order to
remove traces of TFA. The residue was purified by flash chromatog-
raphy (9:1 CH2Cl2–MeOH, Rf 0.12) to give 0.24 g (77%) of compound
12a as a white foam. ½a D22
ꢁ
+6 (c 0.2, MeOH); UV (MeOH): kmax
263 nm (e
5380); IR (Nujol, cmꢀ1): 2235 (CN); 1H NMR (CD3OD,
white solid, mp 200–202 °C: ½a D22
ꢁ
+2 (c 0.5, MeOH); UV (MeOH):
300 MHz): d 7.74 (d, 1H, J5,6 = 6.5 Hz, H-6), 5.93 (d, 1H, J1 ,2 = 9.5 Hz,
0
0
11,076); 1H NMR (CDCl3, 300 MHz): d 7.47 (d, 1H,
H-10), 4.30 (dd, 1H, J5 ,6a = 9.3 Hz, J6a ,6b = 12.8 Hz, H-6a0), 4.13–3.91
0
0
0
0
kmax 262 nm (
e
J5,6 = 5.9 Hz, H-6), 5.70 (d, 1H, J1 ,2 = 7.8 Hz, H-10), 4.51–4.41 (m,
(m, 3H, H-20, H-30, H-50), 3.76 (dd, 1H, J5 ,6b = 2.6 Hz, H-6b0); 13C
NMR (CD3OD, 75.5 MHz): d 158.6, 149.6, 141.7, 128.1, 118.5, 82.9,
73.1, 71.9, 68.4, 65.6, 59.1. Anal. Calcd for C11H12FN3O7: C, 41.65;
H, 3.81; N, 13.25. Found: C, 41.87; H, 3.76; N, 13.32. ESIMS m/z
318.22 [M+H+].
0
0
0
0
2H, H-20, H-30), 4.15 (t, 1H, J3 ,4 = J4 ,5 = 7.1 Hz, H-40), 3.89–3.75
(m, 3H, H-50, H-6a0, H-6b0), 1.53, 1.39 (2s, 6H, 2CH3). Anal. Calcd
for C13H17FN2O7: C, 46.99; H, 5.16; N, 8.43. Found: C, 46.90; H,
5.29; N, 8.66. ESIMS m/z 333.29 [M+H+].
00
0
0
0
1.2.2. 1-(20,30-O-Isopropylidene-60-O-trityl-
a
-D
-
1.3. Synthesis of 1-(40-C-cyano-
a-D-mannopyranosyl)uracil
mannopyranosyl)5-fluorouracil (4a)
(12b)
To a solution of 3a (2.22 g, 6.7 mmol) in pyridine (34.0 mL) were
added triphenylmethyl chloride (2.43 g, 8.7 mmol) and a catalytic
amount of 4-dimethylaminopyridine. The reaction mixture was
stirred overnight at room temperature. After being quenched with
MeOH and concentrated, the residue was purified by flash chroma-
tography (1:1 hexane–EtOAc, Rf 0.37) to give 2.69 g (70%) of com-
1.3.1. 1-(40-C-Cyano-20,30-O-isopropylidene-60-O-trityl-
a-D-
mannopyranosyl)uracil (8b)
Uracil derivative 8b was synthesized from 1-(20,30-O-isopropyl-
idene-60-O-trityl- -lyxo-hexopyranosyl-4-ulose)uracil (6b)20 by
a-D
pound 4a as a colorless oil: ½a D22
ꢁ
+10 (c 0.8, CHCl3); UV (CHCl3):
the similar procedure as described for 8a. It was purified by flash
kmax 265 nm (e
1048); 1H NMR (CDCl3, 300 MHz): d 8.42 (br s,
chromatography (9.6:0.4 CH2Cl2–MeOH, Rf 0.22) to give 1.12 g
1H, NH), 7.43–7.21 (m, 16H, 3C6H5, H-6), 5.77 (d, 1H, J1 ,2 = 5.4 Hz,
(60%) of compound 8b as a colorless oil: ½a D22
ꢀ9 (c 0.5, CHCl3);
ꢁ
0
0
H-10), 4.41–4.35 (m, 2H, H-20, H-30), 4.22 (t, 1H, J3 ,4 = J4 ,5 = 7.0 Hz,
H-40), 3.87–3.79 (m, 1H, H-50), 3.58–3.53 (m, 2H, H-6a0, H-6b0),
1.50, 1.37 (2s, 6H, 2CH3). Anal. Calcd for C32H31FN2O7: C, 66.89;
H, 5.44; N, 4.88. Found: C, 66.98; H, 5.27; N, 4.81. ESIMS m/z
575.59 [M+H+].
UV (CHCl3): kmax 261 nm (e
6756); 1H NMR (CDCl3, 300 MHz): d
0
0
0
0
9.01 (br s, 1H, NH), 7.43–7.28 (m, 15H, 3C6H5), 7.19 (d, 1H,
0
0
J5,6 = 8.1 Hz, H-6), 5.79 (d, 1H, H-5), 5.39 (d, 1H, J1 ,2 = 2.0 Hz, H-
10), 4.87 (s, 1H, 40-OH), 4.82 (dd, 1H, J2 ,3 = 6.8 Hz, H-20), 4.64 (d,
0
0
1H, H-30), 3.96 (t, 1H, J5 ,6a = J5 ,6b = 6.5 Hz, H-50), 3.70 (d, 2H, H-
6a0,b0), 1.68, 1.38 (2s, 6H, 2CH3). Anal. Calcd for C33H31N3O7: C,
68.15; H, 5.37; N, 7.22. Found: C, 68.23; H, 5.19; N, 7.41. ESIMS
m/z 582.64 [M+H+].
0
0
0
0
1.2.3. 1-(20,30-O-Isopropylidene-60-O-trityl-
a-D-lyxo-
hexopyranosyl-4-ulose)5-fluorouracil (6a)
A mixture of 4a (2.69 g, 4.7 mmol), PDC (2.10 g, 5.6 mmol), and
Ac2O (1.3 mL, 13.9 mmol) was stirred in dry CH2Cl2 (47.0 mL) for
5 h, under nitrogen at room temperature. Purification by flash
chromatography (1:1 hexane–EtOAc, Rf 0.51) yielded 1.82 g (68%)
1.3.2. 1-(40-C-Cyano-
a-D-mannopyranosyl)uracil (12b)
Uracil derivative 12b was synthesized from 8b by the similar
procedure as described for 12a. It was purified by flash chromatog-
raphy (9:1 CH2Cl2–MeOH, Rf 0.10) to give 0.20 g (70%) of com-
of compound 6a as a white foam: ½a D22
ꢀ4 (c 0.2, CHCl3); UV
ꢁ
(CHCl3): kmax 265 nm (
e
10,487); 1H NMR (CDCl3, 300 MHz): d
pound 12b as a white foam: ½a D22
ꢁ
+74 (c 0.9, MeOH); UV (MeOH):
8.47 (br s, 1H, NH), 7.47–7.24 (m, 16H, 3C6H5, H-6), 5.73 (d, 1H,
kmax 257 nm (e
7149); IR (Nujol, cmꢀ1): 2240 (CN); 1H NMR
J1 ,2 = 4.7 Hz, H-10), 4.92 (dd, 1H, J2 ,3 = 7.5 Hz, H-20), 4.74 (d, 1H,
(CD3OD, 300 MHz): d 7.57 (d, 1H, J5,6 = 8.1 Hz, H-6), 5.94 (d, 1H,
0
0
0
0
H-30), 4.24 (dd, 1H, J5 ,6a = 2.7 Hz, J5 ,6b = 5.3 Hz, H-50), 3.77 (dd,
J1 ,2 = 9.6 Hz, H-10), 5.65 (d, 1H, H-5), 4.31 (dd, 1H, J5 ,6a = 9.2 Hz,
0
0
0
0
0
0
0
0
1H, J6a ,6b = 10.6 Hz, H-6a0), 3.49 (dd, 1H, H-6b0), 1.57, 1.43 (2s,
6H, 2CH3). Anal. Calcd for C32H29FN2O7: C, 67.12; H, 5.11; N,
4.89. Found: C, 66.99; H, 5.22; N, 4.84. ESIMS m/z 573.60 [M+H+].
J6a ,6b = 12.8 Hz, H-6a0), 4.04–3.94 (m, 3H, H-20, H-30, H-50), 3.76
0
0
0
0
(dd, 1H, J5 ,6b = 2.7 Hz, H-6b0); 13C NMR (CD3OD, 75.5 MHz): d
162.7, 150.7, 140.2, 119.1, 103.1, 84.5, 73.6, 70.5, 67.8, 66.2, 59.2.
Anal. Calcd for C11H13N3O7: C, 44.15; H, 4.38; N, 14.04. Found: C,
44.49; H, 4.56; N, 13.96. ESIMS m/z 300.22 [M+H+].
0
0
1.2.4. 1-(40-C-Cyano-20,30-O-isopropylidene-60-O-trityl-
a-D-
mannopyranosyl)5-fluorouracil (8a)
A
mixture of 6a (1.82 g, 3.2 mmol), H2O (25.0 mL), Et2O
1.4. Synthesis of 1-(40-C-cyano-
a-D-mannopyranosyl)thymine
(50.0 mL), NaHCO3 (0.54 g, 6.4 mmol) and NaCN (0.16 g, 3.2 mmol)
was stirred vigorously at room temperature for 72 h. The organic
phase was separated, and the aqueous phase was washed with
Et2O. The combined ether phases were dried over Na2SO4, filtered,
and evaporated to dryness. The residue was purified by flash chro-
matography (9.6:0.4 CH2Cl2–MeOH, Rf 0.49) to give 1.18 g (62%) of
(12c)
1.4.1. 1-(a-D-Mannopyranosyl)thymine (2c)
A mixture of thymine (2.66 g, 21.1 mmol), hexamethyldisilaz-
ane (HMDS) (5.5 mL, 26.2 mmol) and saccharine (0.18 g,

12
C. Kiritsis et al. / Carbohydrate Research 364 (2012) 8–14
1.0 mmol) in dry CH3CN (69.0 mL) was refluxed for 30 min. To
this were added 1,2,3,4,6-penta-O-acetyl- -mannopyranose (1)
Si: C, 54.28; H, 7.74; N, 6.33. Found: C, 54.07; H, 7.72; N, 6.22.
D
ESIMS m/z 443.60 [M+H+].
(5.89 g, 15.1 mmol) and Me3SiOSO2CF3 (2.5 mL, 21.1 mmol). The
reaction mixture was refluxed at 110 °C for 1 h, cooled, neutral-
ized with aqueous sodium bicarbonate, and extracted with
CH2Cl2. The organic extract was dried over anhydrous sodium sul-
fate, filtered, and evaporated to dryness. The residue was then
treated with ammonia/MeOH (saturated at 0 °C, 840.0 mL). The
solution was stirred overnight at room temperature and then
was concentrated under reduced pressure. The residue was puri-
fied by flash chromatography (8:2 EtOAc–MeOH, Rf 0.14) to give
1.4.5. 1-(20,30-O-Isopropylidene-60-O-trityl-
a-D-lyxo-
hexopyranosyl-4-ulose)thymine (6c)
Thymine derivative 6c was synthesized from 4c by the similar
procedure as described for 6a. It was purified by flash chromatog-
raphy (1:1 hexane–EtOAc, Rf 0.34) to give 1.39 g (67%) of com-
pound 6c as a white foam: ½a D22
ꢀ19 (c 0.9, CHCl3); UV (CHCl3):
ꢁ
kmax 265 nm (e
9562); 1H NMR (CDCl3, 300 MHz): d 8.44 (br s,
1H, NH), 7.44–7.16 (m, 15H, 3C6H5), 7.04 (s, 1H, H-6), 5.58 (d,
3.25 g (75%) of compound 2c as a colorless oil: ½a D22
ꢁ
+26 (c 0.7,
1H, J1 ,2 = 3.5 Hz, H-10), 4.99 (dd, 1H, J2 ,3 = 7.5 Hz, H-20), 4.72 (d,
0
0
0
0
11,504); 1H NMR (CD3OD,
1H, H-30), 4.16 (dd, 1H, J5 ,6a = 2.5 Hz, J5 ,6b = 5.5 Hz, H-50), 3.71
0
0
0
0
MeOH); UV (MeOH): kmax 262 nm (
e
300 MHz): d 7.64 (s, 1H, H-6), 6.00 (d, 1H, J1 ,2 = 9.1 Hz, H-10),
(dd, 1H, J6a ,6b = 10.5 Hz, H-6a0), 3.43 (dd, 1H, H-6b0), 1.91 (1s, 3H,
5-CH3), 1.55, 1.39 (2s, 6H, 2CH3). Anal. Calcd for C33H32N2O7: C,
69.70; H, 5.67; N, 4.93. Found: C, 69.22; H, 5.34; N, 5.06. ESIMS
m/z 569.64 [M+H+].
0
0
0
0
4.15 (dd, 1H, J5 ,6a = 8.5 Hz, J6a ,6b = 11.9 Hz, H-6a0), 4.08–3.97
0
0
0
0
(m, 3H, H-20, H-30, H-50), 3.83 (d, 1H, J3 ,4 = 1.9 Hz, H-40), 3.68
0
0
(dd, 1H, J5 ,6b = 4.2 Hz, H-6b0), 1.93 (1s, 3H, 5-CH3). Anal. Calcd
for C11H16N2O7: C, 45.83; H, 5.59; N, 9.72. Found: C, 45.71; H,
5.33; N, 10.01. ESIMS m/z 289.27 [M+H+].
0
0
1.4.6. 1-(60-O-t-Butyldimethylsilyl-20,30-O-isopropylidene-
a-D-
lyxo-hexopyranosyl-4-ulose)thymine (7c)
1.4.2. 1-(20,30-O-Isopropylidene-
(3c)
a-
D
-mannopyranosyl)thymine
Thymine derivative 7c was synthesized from 5c by the similar
procedure as described for 6a. It was purified by flash chromatog-
raphy (1:1 hexane–EtOAc, Rf 0.38) to give 1.04 g (66%) of com-
Thymine derivative 3c was synthesized from 2c by the similar
procedure as described for 3a. It was purified by flash chromatog-
raphy (9:1 CH2Cl2–MeOH, Rf 0.37) to give 3.04 g (82%) of com-
pound 7c as a colorless oil: ½a D22
ꢁ
+4 (c 0.6, CHCl3); UV (CHCl3):
kmax 263 nm (e
3533); 1H NMR (CDCl3, 300 MHz): d 8.38 (br s,
pound 3c as a white solid, mp 150–152 °C: ½a D22
ꢁ
+16 (c 0.2,
1H, NH), 7.13 (s, 1H, H-6), 5.71 (d, 1H, J1 ,2 = 4.2 Hz, H-10), 4.91
0
0
0
0
MeOH); UV (MeOH): kmax 263 nm (
e
7308); 1H NMR (CDCl3,
(dd, 1H, J2 ,3 = 7.6 Hz, H-20), 4.72 (d, 1H, H-30), 4.24–4.22 (m, 1H,
300 MHz): d 7.18 (s, 1H, H-6), 5.64 (d, 1H, J1 ,2 = 7.3 Hz, H-10),
H-50), 4.10 (dd, 1H, J5 ,6a = 5.2 Hz, J6a ,6b = 11.3 Hz, H-6a0), 3.99
0
0
0
0
0
0
4.46–4.37 (m, 2H, H-20, H-30), 4.02 (dd, 1H, J3 ,4 = 8.8 Hz, J4 ,5
=
(dd, 1H, J5 ,6b = 2.6 Hz, H-6b0), 1.89 (1s, 3H, 5-CH3), 1.55, 1.32 (2s,
6H, 2CH3), 0.89 (s, 9H, t-Bu), 0.07 (s, 6H, 2Si-CH3). Anal. Calcd for
0
0
0
0
0
0
6.3 Hz, H-40), 3.80–3.65 (m, 3H, H-50, H-6a0, H-6b0), 1.86 (1s, 3H,
5-CH3), 1.44, 1.31 (2s, 6H, 2CH3). Anal. Calcd for C14H20N2O7: C,
51.22; H, 6.14; N, 8.53. Found: C, 51.38; H, 6.24; N, 8.67. ESIMS
m/z 329.33 [M+H+].
C
20H32N2O7Si: C, 54.52; H, 7.32; N, 6.36. Found: C, 54.35; H, 7.54;
N, 6.21. ESIMS m/z 441.55 [M+H+].
1.4.7. 1-(60-O-t-Butyldimethylsilyl-40-C-cyano-20,30-O-
isopropylidene-a-D-mannopyranosyl)thymine (11c)
1.4.3. 1-(20,30-O-Isopropylidene-60-O-trityl-
a
-D
-
mannopyranosyl)thymine (4c)
Sodium cyanide (0.10 g, 2.1 mmol) was added to a solution of
7c (2.36 g, 1.0 mmol) in a mixture of Et2O–H2O (2:1 v/v, 24.0 mL).
The mixture was vigorously stirred for 36 h at room temperature.
AcOEt was added to the mixture and the whole was extracted
with H2O. The residue was purified by flash chromatography
(9.6:0.4 CH2Cl2–MeOH, Rf 0.48) to give 0.67 g (61%) of compound
Thymine derivative 4c was synthesized from 3c by the similar
procedure as described for 4a. It was purified by flash chromatog-
raphy (1:1 hexane–EtOAc, Rf 0.21) to give 2.09 g (79%) of com-
pound 4c as a white foam: ½a D22
ꢁ
+18 (c 0.9, CHCl3); UV (CHCl3):
kmax 265 nm (e
9562); 1H NMR (CDCl3, 300 MHz): d 8.37 (br s,
1H, NH), 8.00 (s, 1H, H-6), 7.43–7.23 (m, 15H, 3C6H5), 5.71 (d,
11c as a colorless oil: ½a D22
ꢀ40 (c 0.6, CHCl3); UV (CHCl3): kmax
ꢁ
1H, J1 ,2 = 6.9 Hz, H-10), 4.51–4.37 (m, 2H, H-20, H-30), 4.11 (t, 1H,
265 nm (e
11,261); 1H NMR (CDCl3, 300 MHz): d 8.81 (s, 1H,
0
0
J3 ,4 = J4 ,5 = 8.5 Hz, H-40), 3.86–3.80 (m, 1H, H-50), 3.48–3.36 (m,
2H-6a0, H-6b0), 1.93 (1s, 3H, 5-CH3), 1.46, 1.33 (2s, 6H, 2CH3). Anal.
Calcd for C33H34N2O7: C, 69.46; H, 6.01; N, 4.91. Found: C, 69.24; H,
6.31; N, 5.11. ESIMS m/z 571.61 [M+H+].
NH), 7.06 (s, 1H, H-6), 5.40 (d, 1H, J1 ,2 = 1.5 Hz, H-10), 5.26 (br
0
0
0
0
0
0
s, 1H, 40-OH), 4.84 (dd, 1H, J2 ,3 = 6.9 Hz, H-20), 4.69 (d, 1H, H-
0
0
30), 4.12 (t, 1H, J5 ,6a = J5 ,6b = 6.5 Hz, H-50,), 4.02 (m, 2H, H-6a0,b0),
1.95 (1s, 3H, 5-CH3), 1.72, 1.39 (2s, 6H, 2CH3), 0.89 (s, 9H, t-Bu),
0.15, 0.11 (2s, 6H, 2Si-CH3). Anal. Calcd for C21H33N3O7Si: C,
53.94; H, 7.11; N, 8.99. Found: C, 54.11; H, 7.31; N, 8.87. ESIMS
m/z 468.60 [M+H+].
0
0
0
0
1.4.4. 1-(60-O-t-Butyldimethylsilyl-20,30-O-isopropylidene-
a-D-
mannopyranosyl)thymine (5c)
To a stirred solution of 3c (1.52 g, 4.6 mmol) in pyridine
(23.0 mL) were added successively TBDMSCl (0.92 g, 6.1 mmol)
and a catalytic amount of 4-dimethylaminopyridine. The reaction
mixture was stirred for 30 min at 0 °C under nitrogen and then
at room temperature for 5 h. After, the reaction mixture was
quenched with MeOH and evaporated under reduced pressure.
The resulting residue was purified by flash chromatography (1:1
hexane–EtOAc, Rf 0.21) to give 1.58 g (77%) of compound 5c as a
1.4.8. 1-(40-C-Cyano-
a-D-mannopyranosyl)thymine (12c)
Thymine derivative 12c was synthesized from 11c by the simi-
lar procedure as described for 12a. It was purified by flash chroma-
tography (1:1 CH2Cl2–MeOH, Rf 0.10) to give 0.16 g (72%) of
compound 12c as a white foam: ½a D22
ꢁ
+30 (c 0.7, MeOH); UV
(MeOH): kmax 267 nm (e
2810); IR (Nujol, cmꢀ1): 2245 (CN); 1H
NMR (CD3OD, 300 MHz):
d 7.49 (s, 1H, H-6), 6.03 (d, 1H,
colorless oil: ½a D22
ꢁ
ꢀ55 (c 1.2, CHCl3); UV (CHCl3): kmax 266 nm (
e
J1 ,2 = 9.5 Hz, H-10), 4.41 (dd, 1H, J5 ,6a = 9.3 Hz, J6a ,6b = 12.8 Hz, H-
0
0
0
0
0
0
9575); 1H NMR (CDCl3, 300 MHz): d 8.76 (br s, 1H, NH), 7.13 (s,
6a0), 4.14–4.03 (m, 3H, H-20, H-30, H-50), 3.85 (dd, 1H, J5 ,6b = 2.8 Hz,
H-6a0,b0), 1.89 (1s, 3H, 5-CH3); 13C NMR (CD3OD, 75.5 MHz): d
163.7, 150.1, 138.5, 118.8, 110.4, 83.6, 72.5, 71.9, 68.2, 66.4, 59.7,
13.4. Anal. Calcd for C12H15N3O7: C, 46.01; H, 4.83; N, 13.41. Found:
C, 46.23; H, 4.77; N, 13.69. ESIMS m/z 314.28 [M+H+].
0
0
1H, H-6), 5.65 (d, 1H, J1 ,2 = 5.7 Hz, H-10), 4.52–4.44 (m, 2H, H-20,
0
0
H-30), 4.14 (t, 1H, J3 ,4 = J4 ,5 = 8.2 Hz, H-40), 3.92–3.75 (m, 3H, H-
50, H-6a0, H-6b0), 1.94 (1s, 3H, 5-CH3), 1.53, 1.37 (2s, 6H, 2CH3),
0.89 (s, 9H, t-Bu), 0.07 (s, 6H, 2Si-CH3). Anal. Calcd for C20H34N2O7-
0
0
0
0

C. Kiritsis et al. / Carbohydrate Research 364 (2012) 8–14
13
1.5. Synthesis of 1-(40-C-cyano-40-C-deoxy)5-fluorouracil (15a)
1.5.1. 1-(40-C-Cyano-40-C-deoxy-20,30-O-isopropylidene-60-O-
J6a ,6b = 12.4 Hz, H-6a0), 4.23–4.18 (m, 2H, H-30, H-50), 3.73–3.63
0
0
(m, 2H, H-20, H-6b0), 3.45 (dd, 1H, J3 ,4 = 6.4 Hz, J4 ,5 = 2.6 Hz, H-
40); 13C NMR (CD3OD, 75.5 MHz): d 162.1, 149.5, 141.2, 117.1,
104.6, 82.8, 74.6, 72.3, 65.2, 64.6, 30.1. Calcd for C11H13N3O6: C,
46.65; H, 4.63; N, 14.84. Found: C, 46.35; H, 4.54; N, 14.96. ESIMS
m/z 284.23 [M+H+].
0
0
0
0
trityl-a-D-mannopyranosyl)5-fluorouracil (13a)
Phenyl chlorothionoformate (0.2 mL, 1.5 mmol) was added to a
solution of 8a (0.59 g, 1.0 mmol), DMAP (0.05 g, 0.4 mmol), and
Et3N (0.2 mL, 1.5 mmol) in CH3CN (10 mL) under nitrogen at 0 °C.
The mixture was stirred for 1 h and then diluted with AcOEt. The
whole was washed with H2O and the separated organic phase
was dried over anhydrous sodium sulfate, filtered, and evaporated
to dryness. The residue was coevaporated two times with toluene
and then was dissolved in toluene (10 mL). Bu3SnH (0.4 mL,
1.6 mmol) was added to the above solution containing AIBN
(0.03 g, 0.2 mmol) at 100 °C under nitrogen. After being heated
for 45 min, the solvent was removed under reduced pressure.
The residue was purified by flash chromatography (9.8:0.2
CH2Cl2–MeOH, Rf 0.43) to give 0.44 g (77%) of compound 13a as
1.7. Synthesis of 1-(40-C-cyano-40-C-deoxy)thymine (15c)
1.7.1. 1-(40-C-Cyano-40-C-deoxy-20,30-O-isopropylidene-60-O-
trityl-a-D-mannopyranosyl)thymine (13c)
A mixture of the 6c (1.39 g, 2.4 mmol), water (20.0 mL), ethyl
ether (40.0 mL), sodium bicarbonate (0.40 g, 4.8 mmol), and sodium
cyanide (0.12 g, 2.4 mmol) was stirred vigorously at room tempera-
ture for 24 h. The organic phase was separated, and the aqueous
phase was washed with Et2O. The combined ethereal phases were
dried over anhydrous sodium sulfate, filtered, and evaporated to
dryness. The residue, a mixture of two epimeric cyanohydrins 9c
and 10c, was dissolved in acetonitrile (17.0 mL). To this solution
were added DMAP (0.13 mg, 1.1 mmol), Et3N (0.5 mL, 3.7 mmol),
and phenyl chlorothionoformate (0.5 mL, 3.7 mmol). The mixture
was stirred at room temperature for 1 h, and the solvent was evap-
orated under reduced pressure. The residue was coevaporated two
times with toluene and then was dissolved in toluene (17.0 mL).
Bu3SnH (1.1 mL, 3.8 mmol) was added to the above solution con-
taining AIBN (0.07 g, 0.4 mmol) at 100 °C under nitrogen. After being
heated for 45 min, the solvent was removed under reduced pressure.
The residue was purified by flash chromatography (9.8:0.2 CH2Cl2–
MeOH, Rf 0.38) to give 0.71 g (50%) of compound 13c as a colorless
a colorless oil: ½a D22
ꢀ48 (c 0.7, CHCl3); UV (CHCl3): kmax 266 nm
ꢁ
(e
9518); 1H NMR (CDCl3, 300 MHz): d 8.62 (br s, 1H, NH), 7.44–
7.28 (m, 16H, 3C6H5, H-6), 5.50 (d, 1H, J1 ,2 = 3.8 Hz, H-10), 4.67
0
0
(t, 1H, J2 ,3 = J3 ,4 = 6.5 Hz, H-30), 4.55 (dd, 1H, H-20), 3.99 (dt, 1H,
0
0
0
0
J4 ,5 = 3.0 Hz, J5 ,6a = J5 ,6b = 6.9 Hz, H-50), 3.60 (dd, 1H, J6a ,6b
=
0
0
0
0
0
0
0
0
10.3 Hz, H-6a0), 3.51 (dd, 1H, H-40), 3.38 (dd, 1H, H-6b0), 1.66, 1.39
(2s, 6H, 2CH3). Anal. Calcd for C33H30FN3O6: C, 67.91; H, 5.18; N,
7.20. Found: C, 68.08; H, 5.12; N, 7.26. ESIMS m/z 584.62 [M+H+].
1.5.2. 1-(40-C-Cyano-40-C-deoxy)5-fluorouracil (15a)
5-Fluorouracil derivative 15a was synthesized from 13a by the
similar procedure as described for 12a. It was purified by flash
chromatography (9:1 CH2Cl2–MeOH, Rf 0.15) to give 0.16 g (71%)
oil: ½a 2D2
ꢁ
ꢀ4 (c 0.5, CHCl3); UV (CHCl3): kmax 265 nm (
e
7429); 1H
of compound 15a as a white foam: ½a D22
ꢁ
+9 (c 0.9, MeOH); UV
NMR (CDCl3, 300 MHz): d 8.54 (br s, 1H, NH), 7.43–7.24 (m, 15H,
(MeOH): kmax 262 nm (
e
2649); IR (Nujol, cmꢀ1): 2245 (CN); 1H
3C6H5), 7.03 (s, 1H, H-6), 5.46 (d, 1H, J1 ,2 = 2.5 Hz, H-10), 4.74 (t,
0
0
NMR (CD3OD, 300 MHz): d 7.90 (d, 1H, J5,6 = 6.7 Hz, H-6), 6.01 (d,
1H, J2 ,3 = J3 ,4 = 6.8 Hz, H-30), 4.65 (dd, 1H, H-20), 4.05 (dt, 1H,
0
0
0
0
1H, J1 ,2 = 9.4 Hz, H-10), 4.41–4.26 (m, 3H, H30, H50, H-6a0), 3.79
J4 ,5 = 2.7 Hz,
J5 ,6a = J5 ,6b = 6.2 Hz, H-50),
3.56
(dd, 1H,
0
0
0
0
0
0
0
0
(dd, 1H, J5 ,6 b = 1.5 Hz, J6a ,6 b = 11.7 Hz, H-6b0), 3.70 (dd, 1H,
J6a ,6b = 10.0 Hz, H-6a0), 3.48 (dd, 1H, H-40), 3.31 (dd, 1H, H-6b0),
0
0
0
0
0
0
J2 ,3 = 2.8 Hz, H-20), 3.53 (dd, 1H, J3 ,4 = 6.3 Hz, J4 ,5 = 2.6 Hz, H-40);
13C NMR (CD3OD, 75.5 MHz): d 157.9, 149.3, 140.8, 128.7, 116.8,
83.1, 74.2, 72.1, 67.3, 65.6, 29.1. Anal. Calcd for C11H12FN3O6: C,
43.86; H, 4.02; N, 13.95. Found: C, 43.70; H, 4.32; N, 14.22. ESIMS
m/z 302.21 [M+H+].
1.93 (1s, 3H, 5-CH3), 1.64, 1.36 (2s, 6H, 2CH3). Anal. Calcd for
0
0
0
0
0
0
C
34H33N3O6: C, 70.45; H, 5.74; N, 7.25. Found: C, 70.71; H, 5.62; N,
7.33. ESIMS m/z 580.66 [M+H+].
1.7.2. 1-(60-O-t-Butyldimethylsilyl-40-C-cyano-40-C-deoxy-20,30-O-
isopropylidene-a-D-mannopyranosyl)thymine (14c)
1.6. Synthesis of 1-(40-C-cyano-40-C-deoxy)uracil (15b)
Thymine derivative 14c was synthesized from 11c by the simi-
lar procedure as described for 13a. It was purified by flash chroma-
tography (9.8:0.2 CH2Cl2–MeOH, Rf 0.37) to give 0.23 g (70%) of
1.6.1. 1-(40-C-Cyano-40-C-deoxy-20,30-O-isopropylidene-60-O-
trityl-
a
-D
-mannopyranosyl)uracil (13b)
compound 14c as a colorless oil: ½a D22
ꢀ36 (c 1.0, CHCl3); UV
ꢁ
Uracil derivative 13b was synthesized from 8b by the similar pro-
cedure as described for 13a. It was purified by flash chromatography
(9.8:0.2 CH2Cl2–MeOH, Rf , Rf 0.25) to give 0.39 g (72%) of compound
(CHCl3): kmax 263 nm (e
6376); 1H NMR (CDCl3, 300 MHz): d 8.77
(br s, 1H, NH), 7.08 (s, 1H, H-6), 5.52 (d, 1H, J1 ,2 = 2.6 Hz, H-10),
0
0
4.79 (t, 1H, J2 ,3 = J3 ,4 = 6.7 Hz, H-30), 4.68 (dd, 1H, H-20), 4.14 (m,
0
0
0
0
13b as a colorless oil: ½a D22
ꢁ
+20 (c 1.4, CHCl3); UV (CHCl3): kmax
1H, H-50), 3.88 (dd, 1H, J6a ,6b = 10.2 Hz, J5 ,6a = 5.9 Hz, H-6a0), 3.49
0
0
0
0
258 nm (e
5454); 1H NMR (CDCl3, 300 MHz): d 8.23 (br s, 1H, NH),
(dd, 1H, H-40), 3.39 (dd, 1H, J5 ,6b = 7.9 Hz, H-6b0), 1.95 (1s, 3H, 5-
CH3), 1.70, 1.38 (2s, 6H, 2CH3), 0.86 (s, 9H, t-Bu), 0.08, 0.06 (2s,
6H, 2Si-CH3). Anal. Calcd for C21H33N3O6Si: C, 55.85; H, 7.37; N,
9.30. Found: C, 55.97; H, 7.49; N, 9.37. ESIMS m/z 452.61 [M+H+].
0
0
7.46–7.28 (m, 15H, 3C6H15), 7.22 (d, 1H, J5,6 = 8.1 Hz, H-6), 5.79 (d,
1H, H-5), 5.51 (d, 1H, J1 ,2 = 2.8 Hz, H-10), 4.74 (t, 1H,
0
0
J2 ,3 = J3 ,4 = 6.7 Hz, H-30), 4.66 (dd, 1H, H-20), 4.06 (dt, 1H,
0
0
0
0
J4 ,5 = 2.3 Hz, J5 ,6a = J5 ,6b = 6.5 Hz, H-50), 3.60 (dd, 1H, J6a ,6b
=
0
0
0
0
0
0
0
0
10.0 Hz, H-6a0), 3.49 (dd, 1H, H-40), 3.36 (dd, 1H, H-6b0), 1.66, 1.39
(2s, 6H, 2CH3). Anal. Calcd for C33H31N3O6: C, 70.07; H, 5.52; N,
7.43. Found: C, 69.93; H, 5.66; N, 7.38. ESIMS m/z 566.60 [M+H+].
1.7.3. 1-(40-C-Cyano-40-C-deoxy)thymine (15c)
Thymine derivative 15c was synthesized from 13c or 14c by the
similar procedure as described for 15a. It was purified by flash
chromatography (9:1 CH2Cl2–MeOH, Rf 0.14) to give 15c (0.10 g,
69% from 13c or 0.11 g, 73% from 14c) of compound as a white
1.6.2. 1-(40-C-Cyano-40-C-deoxy)uracil (15b)
Uracil derivative 15b was synthesized from 13b by the similar
procedure as described for 12a. It was purified by flash chromatog-
raphy (9:1 CH2Cl2–MeOH, Rf 0.13) to give 0.14 g (72%) of com-
foam: ½a 2D2
ꢁ
+4 (c 0.4, CHCl3); UV (CHCl3): kmax 266 nm (e 2447);
IR (Nujol, cmꢀ1): 2235 (CN); 1H NMR (CD3OD, 300 MHz): d 7.52
(s, 1H, H-6), 6.02 (d, 1H, J1 ,2 = 9.7 Hz, H-10), 4.43–4.26 (m, 3H, H-
0
0
pound 15b as a white foam: ½a D22
ꢁ
+23 (c 0.5, CHCl3); UV (CHCl3):
30, H-50, H-6a0), 3.73–3.63 (m, 2H, H-20, H-6b0), 3.55 (dd, 1H,
0
0
0
0
kmax 256 nm (
e
3062); IR (Nujol, cmꢀ1): 2250 (CN); 1H NMR
J3 ,4 = 6.3 Hz, J4 ,5 = 2.4 Hz, H-40), 1.89 (1s, 3H, 5-CH3); 13C NMR
(CD3OD, 75.5 MHz): d 163.2, 151.5, 140.8, 116.9, 111.2, 83.2,
74.1, 72.4, 64.8, 64.3, 29.8, 15.4. Anal. Calcd for C12H15N3O6: C,
(CD3OD, 300 MHz): d 7.58 (d, 1H, J5,6 = 8.1 Hz, H-6), 5.93 (d, 1H,
J1 ,2 = 9.5 Hz, H-10), 5.64 (d, 1H, H-5), 4.30 (dd, 1H, J5 ,6a = 9.2 Hz,
0
0
0
0

14
C. Kiritsis et al. / Carbohydrate Research 364 (2012) 8–14
7. Barral, K.; Halfon, P.; Pèpe, G.; Camplo, M. Tetrahedron Lett. 2002, 43, 81–84.
8. Verheggen, I.; Van Aerschot, A.; Toppet, S.; Snoeck, R.; Janssen, G.; Balzarini, J.;
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48.48; H, 5.09; N, 14.14. Found: C, 48.24; H, 5.21; N, 14.27. ESIMS
m/z 298.24 [M+H+].
1.8. Cytostatic activity assays
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Murine leukemia L1210 and thymidine kinase (TK)-deficient
L1210 (L1210/TKꢀ), human T-lymphocyte CEM and human cervix
carcinoma (HeLa) and HeLa/TKꢀ cells were suspended at
300,000–500,000 cells/mL of culture medium, and 100
lL of a cell
suspension was added to 100 L of an appropriate dilution of the
l
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tion at 37 °C for two (L1210, FM3A) or three (CEM, HeLa) days, the
cell number was determined using a Coulter counter. The IC50 was
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proliferation by 50%.
ˇ
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1.9. Antiviral activity assays
The antiviral assays [except anti-human immunodeficiency
virus (HIV) assays] were based on inhibition of virus-induced cyto-
pathicity in HEL [herpes simplex virus type 1 (HSV-1), HSV-2 (G),
vaccinia virus and vesicular stomatitis virus], Vero (parainflu-
enza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus),
or HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respira-
tory syncytial virus) cell cultures. Confluent cell cultures in micro-
titer 96-well plates were inoculated with 100 cell culture
inhibitory dose-50 (CCID50) of virus (1 CCID50 being the virus dose
to infect 50% of the cell cultures) in the presence of varying concen-
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trations (200, 40, 8, . . . lM) of the test compounds. Viral cytopath-
icity was recorded as soon as it reached completion in the control
virus-infected cell cultures that were not treated with the test
compounds. The methodology of the anti-HIV assays was as fol-
lows: human CEM (ꢂ3 ꢃ 105 cells/cm3) cells were infected with
100 CCID50 of HIV-1(IIIB) or HIV-2(ROD)/mL and seeded in 200 lL
wells of a microtiter plate containing appropriate dilutions of the
test compounds. After 4 days of incubation at 37 °C, HIV-induced
CEM giant cell formation was examined microscopically.
Acknowledgements
29. Tzioumaki, N.; Manta, S.; Tsoukala, E.; Vande Voorde, J.; Liekens, S.; Komiotis,
D.; Balzarini, J. Eur. J. Med. Chem. 2011, 46, 993–1005.
This work was supported in part by the Postgraduate Pro-
grammes ‘Biotechnology-Quality assessment in Nutrition and the
Environment’, ‘Application of Molecular Biology-Molecular Genet-
ics-Molecular Markers’, Department of Biochemistry and Biotech-
nology, University of Thessaly, and the KU Leuven (GOA 10/14).
We thank Lizette van Berckelaer, Leen Ingels, Leentje Persoons and
Frieda De Meyer for excellent technical assistance in the biological
assays.
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