Exomethylene pyranonucleosides: Efficient synthesis and biological evaluation of 1-(2,3,4-trideoxy-2-methylene-β-d-glycero-hex-3-enopyranosyl)thymine

Article abstract of DOI:10.1016/j.bmc.2007.05.055

A new series of unsaturated pyranonucleosides with an exocyclic methylene group and thymine as heterocyclic base have been designed and synthesized. d-Galactose (1) was readily transformed in three steps into the corresponding 1-(β-d-galactopyranosyl)thym

Full text of DOI:10.1016/j.bmc.2007.05.055

Bioorganic & Medicinal Chemistry 15 (2007) 5448–5456
Exomethylene pyranonucleosides: Efficient synthesis and
biological evaluation of 1-(2,3,4-trideoxy-2-methylene-b-D-
glycero-hex-3-enopyranosyl)thymine
a
a
b
George Agelis, Niki Tzioumaki, Tanja Botic, Avrelija Cencic and Dimitri Komiotisa,
ˇb,c
*
´
aDepartment of Biochemistry and Biotechnology, Laboratory of Organic Chemistry, University of Thessaly,
26 Ploutonos Street, 41221 Larissa, Greece
bDepartment of Microbiology, Biochemistry and Biotechnology, Faculty of Agriculture, University of Maribor,
Vrbanska c.30, 2000 Maribor, Slovenia
cDepartment of Biochemistry, Medical Faculty, University of Maribor, Slovenia
Received 17 October 2006; revised 22 May 2007; accepted 23 May 2007
Available online 26 May 2007
Abstract—A new series of unsaturated pyranonucleosides with an exocyclic methylene group and thymine as heterocyclic base have
been designed and synthesized. D-Galactose (1) was readily transformed in three steps into the corresponding 1-(b-D-galactopyrano-
syl)thymine (2). Selective protection of the primary hydroxyl group of 2 with a t-butyldimethylsilyl (TBDMS) group, followed by
specific acetalation, and oxidation gave 1-(6-O-t-butyldimethylsilyl-3,4-O-isopropylidene-b-D-lyxo-hexopyranosyl-2-ulose)thymine
(5). Wittig reaction of the ketonucleoside 5, deprotection and tritylation of the 60-hydroxyl group gave 1-(2-deoxy-2-methylene-
6-O-trityl-b-D-lyxo-hexopyranosyl)thymine (9). Exomethylene pyranonucleoside 9 was converted to the olefinic derivative 10, which
after detritylation afforded the title compound 1-(2,3,4-trideoxy-2-methylene-b-D-glycero-hex-3-enopyranosyl)thymine (11). These
novel synthesized compounds were evaluated for antiviral activity against rotaviral infection and cytotoxicity in colon cancer. As
compared to AZT, compounds 1-(2-deoxy-2-methylene-b-D-lyxo-hexopyranosyl)thymine (7) and 1-(b-D-lyxo-hexopyranosyl-2-
ulose)thymine (8) showed to be more efficient, in rotavirus infections and in treatment of colon cancer.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Nucleoside analogues display a wide range of biological
activities as anti-tumor, antiviral, and chemotherapeutic
agents.2–5 Consequently, a large number of modifica-
tions have been made to both the base and sugar moie-
ties of natural nucleosides, accordingly, with the
objective of increasing the therapeutic index of estab-
lished antiviral agents.6 Over the past two decades,
nucleoside chemistry has evolved to facilitate efficient
routes to effective agents for the treatment of cancer
and antiviral agents against several types of viruses
including rotavirus.7–11
Rotaviruses are the major viral cause of infantile gastro-
enteritis and first contact with host represents the gas-
trointestinal tract. In many cases they enter via
contaminated food or water. Rotaviruses represent
80% of recognized viral etiologies and 140 million cases
of diarrhea per year.1 They strike young children with
similar frequency throughout the world, but the mortal-
ity rate is high especially in developing countries where
870,000 deaths per year have been reported (WHO,
1997). Therefore, it is important to find an effective
anti-rotaviral therapy. As to our knowledge there is no
efficient therapy or golden standard among nucleoside
analogues against rotavirus infection.
Lately, nucleosides with a six-membered carbohydrate
moiety have been evaluated for their potential antivi-
ral12–15 and antibiotic16 properties and as building
blocks in nucleic acid synthesis.17,18 Among them, the
unsaturated ketonucleosides are well established for
their antineoplastic activity and immunosuppressive ef-
fects.19–21 It appeared that the presence of a carbon–car-
bon double bound in a,b disposition to the keto group
of the sugar moiety enhances activity and the presence
Keywords: Exomethylene nucleosides; Ketonucleosides; Unsaturated
nucleosides; Olefination; Antiviral, anti-tumor activity.
*
Corresponding author. Tel.: +30 2410 565285; fax: +30 2410
565299; e-mail: dkom@bio.uth.gr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.05.055

G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
5449
of a primary hydroxyl group seemed not to be a prere-
quisite for biological activity.19,22,23 Those nucleosides
not only exhibit growth inhibitory activity against a
variety of tumor cells23,24 in vitro and L1210 leuke-
mia25,26 in vivo, but they also may constitute important
synthetic intermediates in the nucleoside field owing to
their chemical reactivity in various media.27,28
urated exomethylene pyranonucleosides shown in
Schemes 1 and 2.
2. Results and discussion
2.1. Synthesis
We have recently reported10,11 the synthesis of a new
generation of unsaturated ketopyranosyl nucleoside
derivatives and a number of them proved to be more
efficient in rotavirus and anti-tumor growth inhibition
as compared to AZT and to 5FU, respectively. Our find-
ings indicated that some of the new ketonucleoside ana-
logues interfere with the replication of rotavirus in cell
model of Caco-2 cells and by that limit the destruction
of cells originated from human colon.
Commercially available D-galactose (1) was readily
transformed in three steps into the corresponding 1-(b-
D-galactopyranosyl)thymine39 (2) in which the primary
hydroxyl group was selectively protected with TBDMS
group to yield 3. Specific acetalation of 3 using 2,2-
dimethoxypropane40 in acetone led to the 30,40-O-iso-
propylidene derivative 4. Oxidation of the free hydroxyl
with the pyridinium dichromate (PDC)/acetic anhydride
(Ac2O) method41 gave the desired 20-ketonucleoside 5
1
isolated as a white solid. The H NMR spectrum of 5
showed the H-10 signal as sharp singlet at 6.18 ppm
Furthermore, modified nucleosides containing an exocy-
clic methylene or fluoromethylene group in position 20,
30 or 40 exhibited potent anti-tumor and antiviral activ-
ities. Their satisfactory antiviral and anti-cancer proper-
ties were attributed, at least in part, to the ability of
these nucleosides to irreversibly inactivate ribonucleo-
tide reductase after phosphorylation via specific nucleo-
side kinases.29–38 Based on these findings we designed,
synthesized, and pharmacologically evaluated the rota-
virus and anti-tumor growth inhibition of a new series
of unusual nucleosides, i.e., the exomethylene and unsat-
0
0
and J3 ;4 ¼ 5:4 as a doublet, confirming the absence of
a proton at C-20.
Wittig reaction of the keto intermediate 5 with sodium
hydride (NaH) and methyl triphenylphosphonium bro-
mide (Ph3PCH3Br) at 0 ꢁC, in the presence of t-amyl
alcohol in tetrahydrofuran (THF), resulted in 6 in excel-
1
lent yield (92%).36 The H NMR spectrum of 6 showed
the presence of two vinylic protons at 5.62 and 5.49 ppm
each as a broad singlet, corresponding to the 20-exom-
OTBDMS
OH
OH
O
HO
HO
O
HO
O
Thy
Thy
Ref. 39
a
OH
OH
OH
OH
HO
HO
HO
2
1
3
b
OTBDMS
OTBDMS
OTBDMS
H3C
H3C
H3C
H3C
H3C
O
O
O
O
O
O
Thy
Thy
Thy
d
c
O
O
O
H3C
CH2
O
HO
5
6
4
e
e
OH
O
OH
O
O
HO
HO
H C
Thy
Thy
3
NH
Thy =
OH
OH
NH
O
CH2
O
7
8
Scheme 1. Reagents and conditions: (a) TBDMSCl, pyridine, 0–25 ꢁC, 5 h; (b) acetone, 2,2-dimethoxypropane, p-toluenesulfonic acid, 25 ꢁC, 3 h; (c)
PDC, Ac2O, dry CH2Cl2, 25 ꢁC, under nitrogen, 4 h; (d) Ph3PCH3Br, NaH, t-amyl alcohol in THF, 0 ꢁC, 2 h, 25 ꢁC, 30 min, under nitrogen; (e) TFA
90% in MeOH, 25 ꢁC, 10 min.

5450
G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
OTr
OH
O
OH
O
OTr
HO
HO
O
O
Thy
Thy
Thy
Thy
g
f
h
OH
OH
CH2
CH2
CH2
CH2
10
11
9
7
i
OTr
O
OTr
O
O
I3C
H C
Thy
Thy
3
NH
Thy =
NH
O
I
CH3
CH3
12
13
Scheme 2. Reagents and conditions: (f) TrCl, pyridine, 25 ꢁC, 4 h; (g) dry Tol/DMF (4:1), iodine–imidazole–Ph3P, 100 ꢁC, 1 h; (h) formic acid/diethyl
ether, 1:2, 25 ꢁC, 7 min; (i) dry Tol/DMF (4:1), CHI3–imidazole–Ph3P, 100 ꢁC, 1 h.
ethylene group. Compounds 5 and 6 were converted to
the fully unprotected derivatives 8 and 7, respectively,
by treatment with trifluoroacetic acid (TFA) 90% in
methanol (MeOH), for 10 min (Scheme 1).
2.2. Biological activity
The biological activity of compounds was evaluated in
intestinal epithelial cell cultures of the human colon ade-
nocarcinoma cell line Caco-2, non-tumorigenic human
fetal cell line H4, and embryonic monkey kidney cells
MA 104. Antiviral activity of synthesized compounds
was evaluated against rotavirus infection of Caco-2
and MA 104 cells and potential anti-tumor activity
was evaluated by cytotoxicity and anti-proliferation test,
using tumorous Caco-2 and H4, non-tumorigenic cell
counterpart, respectively.
The olefination of the 3,4-vicinal diol proved to be a cru-
cial step for our synthetic procedure and several possible
synthetic approaches to the olefinic nucleoside 10 were
investigated. Selective protection of the 60-hydroxyl
group of 1-(2-deoxy-2-methylene-b-D-galactopyranos-
yl)thymine (7) by a trityl group (Tr) afforded 9 in high
yield (85%). The Corey–Winter42,43 reaction by treating
the diol 9 with thiocarbonyldiimidazole followed by
reductive olefination with trimethylphosphite, the Bar-
ton44 deoxygenation via xanthate ester upon treatment
with tributyltin hydride, as the Hanessian45 method with
N,N-dimethylformamide dimethylacetal and methyl io-
dide, afforded an untreatable mixture of products. In
contrast, the Garegg–Samuelsson method,46–49 with io-
dine-triphenylphosphine (Ph3P)-imidazole,49 led to the
direct conversion of diol 9 to the desired olefinic com-
pound 10 in 56% yield. Noteworthy, when the same
reaction was attempted with iodoform (CHI3)–Ph3P–
imidazole50–52 in toluene/dimethylformamide, 4:1, at
100 ꢁC, 10 was obtained in only 26% yield along with
the tentatively proposed byproduct 12, as it has been
characterized by NMR, FAB-MS, and chemical analy-
sis. Compound 12 was possibly formed53 via the inter-
mediate 13 and its 1H NMR spectrum showed the
disappearance of the two exomethylene proton signals,
the presence of one vinylic proton signal due to the H-
2.2.1. Antiviral activity. To examine the potential antivi-
ral properties of the compounds, two different ap-
proaches were followed to test the possible inhibitory
activity of the compounds on rotavirus infection of
Caco-2 and MA 104 cells, as described in experimental
part. Only compounds 7 and 8 showed a significant pro-
tection against rotavirus on Caco-2 cells by neutraliza-
tion of virus before its attachment on cell surface at
very low dose of 2.46 and 2.44 lM, respectively (Table
1) in comparison to golden standard used (AZT,
74.62 lM). The same antiviral activity was observed also
on MA 104 cells by compounds 7 and 8. None of the
tested compounds was able to elicit antiviral activity
by inhibition of virus infectivity in Caco-2 cells (Table
1). On the contrary, the inhibition of virus replication
following virus attachment was different on MA 104
cells as the one observed for Caco-2 cells (Table 2 vs Ta-
ble 1). Compounds 7, 8, and 10 achieve antiviral activity
against rotavirus at the concentrations of 7.03, 6.98, and
4.06 lM. In comparison to standard (AZT), concentra-
tion used for compounds was three to four times lower,
except for the compound 11, where the IC50 was
achieved at the concentration of 79.92 lM (Table 2).
From the results obtained in this study and results of tis-
sue selectivity index (TSI values) it can be concluded
that the antiviral effect of the compounds depends on
the mechanisms of establishment of antiviral state in in-
fected cells and is cell line specific.
30 at 7.10 ppm as a doublet ðJ3 ;4 ¼ 1:3 HzÞ, and a three
protons singlet due to the methyl group in 20-position at
1.90 ppm. Finally, detritylation of 10 was performed
with formic acid/diethyl ether,54 1:2, for 7 min, to afford
the corresponding title nucleoside derivative 1-(2,3,4-tri-
deoxy-2-methylene-b-D-glycero-hex-3-enopyranosyl)thymine
(11) (Scheme 2).
0
0
Compounds 7, 8, 10, and 11 were evaluated for antiviral
and cytotoxic activity.

G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
5451
Table 1. Antiviral activity of compounds 7, 8, 10, 11 and AZT against rotavirus RF strain on Caco-2 cells (IC50
)
Compound
Treatment Aa
Treatment Bb
c
IC50
CC50/IC50
IC50
CC50/IC50
mg/mL
lM
mg/mL
lM
7
0.0007
0.0007
n.e.
2.46
2.44
n.e.
28.61
28.65
n.e.
n.e.
n.e.
n.e.
n.e.
0.006
n.e.
n.e.
n.e.
n.e.
22.39
n.e.
n.e.
n.e.
n.e.
2.5
8
10
11
AZT
n.e.
0.0200
n.e.
74.62
n.e.
0.75d
n.e., no effect observed.
a Neutralization of the virus in the solution before its attachment.
b Inhibition of infectivity following virus attachment.
c CC50/IC50 values were calculated using CC50 values in Table 3.
d CC50 for AZT on Caco-2 cells = 56.1 lM.
Table 2. Antiviral activity of compounds 7, 8, 10, 11 and AZT against rotavirus RF strain on MA 104 cells (IC50
)
Compound
Treatment Aa
Treatment Bb
c
IC50
CC50/IC50
IC50
CC50/IC50
mg/mL
lM
mg/mL
lM
7
0.0007
0.0007
n.e.
2.46
2.44
n.e.
85.80
85.90
n.e.
0.002
0.002
0.002
0.020
0.006
7.03
6.98
30.03
30.03
60.00
5.99
8
10
11
AZT
4.06
79.92
22.39
n.e.
0.020
n.e.
74.62
n.e.
3.01d
10.03
n.e., no effect observed.
a Neutralization of the virus in the solution before its attachment.
b Inhibition of infectivity following virus attachment.
c CC50/IC50 values were calculated using CC50 values in Table 3.
d CC50 for AZT on MA 104 cells = 224.5 lM.
2.2.2. Cytotoxic and growth inhibition. All tested com-
pounds have potential anti-tumor activity but at dif-
ferent doses. Cytotoxic effect (IC50) measured in H4
and Caco-2 cells for compounds 7 and 8 showed that
tested compounds are four times more cytotoxic and
2.5 times more selective (see TSI values) against colon
carcinoma cells than standard 5FU compound (Table
3). CC50 for both compound were 69.9 and 70.4 lM,
respectively, in comparison to 5FU where concentra-
tion of 384.4 lM was needed to obtain same anti-tu-
Additionally, cell growth inhibition assay was performed
in Caco-2 cells. Compounds 8 and 10 diminished the
growth of Caco-2 cells as determined by cell colony num-
bers after 10 days of incubation at very low concentration
of 0.70 and 0.40 lM, respectively, in comparison to 5FU,
whereas IC50 for compound 7 was achieved at higher con-
centration of 21.1 lM. From the results obtained it can be
stated that compound 8 was the most efficient in achieving
anti-tumor activity against Caco-2 cells if results of cyto-
toxic activity on H4 are as well taken into the account.
mor
activity
against
Caco-2
cells.
Higher
concentration of compounds 10 and 11 resulted in
intensive destruction of H4 cells, whereas in the case
of Caco-2 cells, cytotoxic activity was observed not
before concentration of 1015.1 and 1998.0 lM
(Table 3).
3. Conclusions
In conclusion, compounds 7 and 8 are potential anti-tu-
mor and anti-rotavirus agents, as first, they exhibit high
Table 3. Cytotoxic effect (CC50, lM) of compounds 7, 8, 10, 11 and 5FU on H4, Caco-2, and MA 104 cells and growth inhibition (IC50, lM) on
Caco-2 cells
Compound
Cytotoxic effect (CC50, lM)
TSIa
Growth inhibition (IC50, lM)
Caco-2
H4
Caco-2
MA 104
Caco-2
MA 104
7
1758.9
1746.8
40.6
70.4
69.9
211.1
209.6
243.6
479.5
230.6
24.9
25
8.3
8.3
21.1
0.70
0.40
79.92
1.50
8
10
11
5FU
1015.1
1998.0
384.4
0.04
0.0014
10
0.160
0.0058
16.67
2.8
3843.8
a Tumor selectivity index (CC50 on H4 cells/CC50 on the specific host cells).

5452
G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
0
0
0
0
anti-tumor activity that is selective to tumor cells where
much lower concentration is needed to exhibit the cyto-
toxic and anti-proliferative effect as compared to normal
non-cancerogenic cells, and second, as they have anti-
rotavirus activity at much lower concentrations than
the observed cytotoxicity effect. The compound 7 is even
more interesting, as it is chemically more stable than
corresponding keto compound 8. In contrast to the
ketonucleosides,55,56 the introduction of an additional
unsaturation in the exomethylene nucleosides, com-
pounds 10 and 11, did not enhance growth inhibition
of Caco-2 cells, suggesting that the keto and the exom-
ethylene derivatives are acting via different mechanisms.
Compounds 7 and 8 showed to be more efficient than
currently used nucleoside analogue AZT, in rotavirus
infections and furthermore in treatment of colon cancer
as used 5FU.
40, J3 ;4 ¼ 3:2 Hz, J4 ;5 ¼ 0:9 Hz), 3.88-3.78 (m, 3H, H-
20
and
H-6a0,6b0)
3.71
3.63
(dt,
(dd,
1H,
1H,
H-50,
H-30,
0
0
0
J5 ;6a0 ¼ J5 ;6b ¼ 6:2 Hz),
0
0
J2 ;3 ¼ 9:4 Hz), 1.94 (s, 3H, 5-CH3).
Found: C, 50.42; H, 7.74; N, 6.83. Calcd for
C17H30N2O7Si: C, 50.73; H, 7.51; N, 6.96.
ESI-MS (m/z): Found 403.32 (M+H+).
4.1.2. 1-(6-O-t-Butyldimethylsilyl-3,4-O-isopropylidene-
b-D-galactopyranosyl)thymine (4). To a stirred suspen-
sion of 3 (6.38 g, 15.85 mmol) in anhydrous acetone
(280 mL) and 2,2-dimethoxypropane (15.74 mL) was
added p-toluenesulfonic acid monohydrate (0.58 g,
3.05 mmol). After 3 h the resulting solution was neu-
tralized with triethylamine so pH did not exceed 7.
The solution was concentrated and the residue was
purified by flash chromatography (ethyl acetate/hex-
4. Experimental
ane, 6:4), to give 4 (5.26 g, 75%, Rf = 0.35) as a white
22
D
foam. ½aꢀ þ 32:2 (c 0.1, CHCl3); kmax 260 nm; (e
4.1. General procedure
4332).
Thin-layer chromatography (TLC) was performed on
Merck precoated 60F254 plates. Reactions were moni-
tored by TLC on silica gel, with detection by UV light
(254 nm) or by charring with sulfuric acid. Flash chro-
matography was performed using Silica gel (240–400
mesh, Merck).
1H NMR (CD3OD): d 7.43 (s, 1H, H-6), 5.48 (d, 1H, H-
10, J1 ;2 ¼ 9:4 Hz), 4.33 (dd, 1H, H-40, J3 ;4 ¼ 5:5 Hz,
0
0
0
0
J4 ;5 ¼ 2:2 Hz), 4.20 (dd, 1H, H-30, J2 ;3 ¼ 7:1 Hz),
0
0
0
0
4.10 (dt, 1H, H-50, J5 ;6a0 ¼ J5 ;6b ¼ 6:7 Hz), 3.91–3.82
(m, 2H, H-6a0,6b0), 3.78 (dd, 1H, H-20), 1.90 (s, 3H, 5-
CH3), 1.56 and 1.36 (2s, 6H, 2· CH3).
0
0
0
1H NMR spectra were recorded at room temperature
with a Brucker 400 MHz spectrometer using chloro-
form-d (CDCl3) and methanol-d4 (CD3OD). Chemical
shifts are reported in parts per million (d) downfield
from tetramethylsilane (TMS) as internal standard.
Mass spectra were obtained with a Micromass Platform
LC (ESI-MS). Optical rotations were measured using a
Schmidt and Haensch polarimeter. All reactions were
carried out in dry solvents. Dichloromethane (CH2Cl2)
was distilled from phosphorus pentoxide and stored
over 4E molecular sieves. Acetonitrile, toluene (Tol),
and dimethylformamide (DMF) were distilled from cal-
cium hydride and stored over 3E molecular sieves. THF
was freshly distilled under nitrogen from sodium/benzo-
phenone before use and pyridine stored over pellets of
potassium hydroxide.
Found: C, 54.46; H, 7.48; N, 6.51. Calcd for
C20H34N2O7Si: C, 54.28; H, 7.74; N, 6.33.
ESI-MS (m/z): Found 443.42 (M+H+).
4.1.3. 1-(6-O-t-Butyldimethylsilyl-3,4-O-isopropylidene-
b-D-lyxo-hexopyranosyl-2-ulose)thymine (5). A mixture
of 4 (5.26 g, 11.88 mmol; dried by co-evaporation with
Tol), PDC (5.36 g, 14.25 mmol), and Ac2O (3.36 mL,
35.64 mmol) was stirred in dry CH2Cl2 (42 mL) for
4 h, under nitrogen at room temperature. Ethyl acetate
(15 mL) was added and the resulting slurry was trans-
ferred on top of a silica gel column packed in ethyl ace-
tate. The solution was filtered through the column and
washed with ethyl acetate (200 mL) until the product
was eluted completely. The solvent was evaporated
and the residue was rendered free of Ac2O and pyridine
by co-evaporation with Tol (3·). Purification by flash
chromatography (ethyl acetate/hexane, 5:5) yielded pure
4.1.1.
1-(6-O-t-Butyldimethylsilyl-b-D-galactopyranos-
yl)thymine (3). To a stirred solution of 2 (4.97 g,
17.24 mmol) in pyridine (86 mL) were added succes-
sively TBDMSCl, (3.40 g, 22.56 mmol) and a catalytic
amount of 4-dimethylaminopyridine. The reaction mix-
ture 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 was evaporated
under reduced pressure. The resulting residue was puri-
fied by flash chromatography (CH2Cl2/MeOH, 9:1) to
5 (4.39 g, 84%, Rf = 0.4) as a white solid, mp 144–
22
D
146 ꢁC; ½aꢀ ꢁ 26:3 (c 0.1, CHCl3); kmax 260 nm; (e
8304).
1H NMR (CDCl3): d 8.46 (br s, 1H, NH), 7.26 (s, 1H,
H-6), 6.18 (s, 1H, H-10), 4.71 (dd, 1H, H-40,
J3 ;4 ¼ 5:4 Hz, J4 ;5 ¼ 1:4 Hz), 4.65 (d, 1H, H-30),
4.32-4.29 (m, 1H, H-50) 3.94-3.86 (m, 2H, H-6a0,6b0),
1.92 (s, 3H, 5-CH3), 1.48 and 1.41 (2s, 6H, 2· CH3).
0
0
0
0
give 3 (6.38 g, 92%, Rf = 0.25) as a white solid. Mp
22
D
140–142 ꢁC; ½aꢀ þ 25:5 (c 0.1, MeOH); kmax 260 nm;
(e 3911).
Found: C, 54.68; H, 7.21; N, 6.52. Calcd for
C20H32N2O7Si: C, 54.52; H, 7.32; N, 6.36.
1H NMR (CD3OD): d 8.53 (br s, 1H, NH), 7.65 (s, 1H,
H-6), 5.50 (d, 1H, H-10, J1 ;2 ¼ 9:2 Hz), 3.95 (dd, 1H, H-
ESI-MS (m/z): Found 441.43 (M+H+).
0
0

G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
5453
4.1.4. 1-(6-O-t-Butyldimethylsilyl-2-deoxy-3,4-O-isopro-
pylidene-2-methylene-b-D-lyxo-hexopyranosyl)thymine (6).
To a stirred suspension of methyl triphenyl-phosphonium
bromide (8.89 g, 24.90 mmol) and t-amyl alcohol
(2.96 mL, 27.13 mmol) in dry THF (73 mL) was added
NaH (0.65 g, 60% in oil, 27.13 mmol) at 0 ꢁC under
nitrogen and the reaction mixture was stirred for 2 h at
ambient temperature under nitrogen. To this yellow
phosphorus ylide was added a solution of 5 (4.39 g,
9.96 mmol) in dry THF (16 mL) dropwise, at 0 ꢁC under
nitrogen. After the mixture was stirred for 30 min at
ambient temperature under nitrogen, the reaction
mixture was quenched with saturated sodium bicarbon-
ate (NaHCO3) solution and extracted with ethyl acetate.
The organic layer was washed with water, dried with
anhydrous magnesium sulfate (MgSO4), and evaporated.
The residue was purified by flash chromatography (ethyl
4.27 (m, 1H, H-50) 3.83–3.78 (m, 2H, H-6a0,6b0), 1.87 (s,
3H, 5-CH3).
Found: C, 46.27; H, 4.70; N, 9.63. Calcd for
C11H14N2O7: C, 46.16; H, 4.93; N, 9.79.
ESI-MS (m/z): Found 287.21 (M+H+).
4.1.7. 1-(2-Deoxy-2-methylene-6-O-trityl-b-D-lyxo-hexo-
pyranosyl)thymine (9). To a solution of 7 (2.19 g,
7.70 mmol) in pyridine (80 mL) were added TrCl
(2.58 g, 9.24 mmol) and a catalytic amount of 4-dimeth-
ylaminopyridine. The reaction mixture was stirred for
4 h at room temperature. After being quenched with
MeOH and concentrated, the resulting residue was puri-
fied by flash chromatography (ethyl acetate/hexane, 7:3)
to give 9 (3.45 g, 85%, Rf = 0.6 in ethyl acetate).
22
D
acetate/hexane, 3:7) to give 6 (4.02 g, 92%, Rf = 0.25) as
½aꢀ ꢁ 15:7 (c 0.1, CHCl3); kmax 260 nm; (e 7712).
22
D
a viscous oil. ½aꢀ ꢁ 6:32 (c 0.1, CHCl3); kmax 260 nm;
(e 6016).
1H NMR (CDCl3): d 7.43–7.20 (m, 16H, 3C6H5 and
H-6), 6.18 (s, 1H, H-10), 5.52 and 4.89 (br s, 2H,
methylene), 4.78 (br s, 1H, H-30), 4.11 (m, 1H, H-40),
3.85–3.83 (m, 1H, H-50), 3.46–3.39 (m, 2H, H-6a0,6b0),
1.93 (s, 3H, 5-CH3).
1H NMR (CDCl3): d 8.91 (br s, 1H, NH), 7.28 (s, 1H,
H-6), 6.53 (s, 1H, H-10), 5.62 and 5.49 (br s, 2H, meth-
ylene), 4.85 (d, 1H, H-30, J3 ;4 ¼ 7:4 Hz), 4.38 (dd, 1H,
0
0
H-40, J4 ;5 ¼ 1:8 Hz), 3.85-3.74 (m, 2H, H-6a0,6b0),
3.57-3.53 (m, 1H, H-50), 1.90 (s, 3H, 5-CH3), 1.56 and
1.41 (2s, 6H, 2· CH3).
0
0
Found: C, 70.58; H, 5.43; N, 5.47. Calcd for
C31H30N2O6: C, 70.71; H, 5.74; N, 5.32.
Found: C, 57.38; H, 7.65; N, 6.24. Calcd for
C21H34N2O6Si: C, 57.51; H, 7.81; N, 6.39.
ESI-MS (m/z): Found 527.31 (M+H+).
4.1.8. 1-(2,3,4-Trideoxy-2-methylene-6-O-trityl-b-D-gly-
cero-hex-3-enopyranosyl)thymine (10)
ESI-MS (m/z): Found 439.31 (M+H+).
4.1.8.1. Method A. Imidazole (0.89 g, 13.10 mmol),
Ph3P (6.87 g, 26.20 mmol), and iodine (3.33 g,
13.10 mmol) were added to the suspension of 9 (3.45 g,
6.55 mmol) in 50 mL of dry Tol/DMF (4:1). The result-
ing reaction mixture was heated (100 ꢁC, oil bath) under
nitrogen for 1 h where upon the color changed from a
yellow–white to a red–brown. The reaction mixture
was concentrated in vacuum, the residue diluted with
ethyl acetate, washed with aqueous NaHCO3, sodium
thiosulfate, and water. The organic phase was dried with
MgSO4, the solvent was removed in vacuum, and the
residue was purified by flash chromatography (ethyl ace-
tate/hexane, 4:6). The olefinic nucleoside derivative 10
4.1.5.
1-(2-Deoxy-2-methylene-b-D-lyxo-hexopyranos-
yl)thymine (7). Compound 6 (3.97 g, 9.05 mmol) was dis-
solved in 40 mL of 90% TFA in MeOH. The solution
was stirred for 10 min at room temperature and then
was concentrated at 40 ꢁC under reduced pressure, in or-
der to remove traces of TFA. The residue was purified
by flash chromatography (ethyl acetate/MeOH, 9:1) to
give 7 (2.19 g, 85%, Rf = 0.3) as a white foam.
22
D
½aꢀ ꢁ 12:4 (c 0.1, MeOH); kmax 260 nm; (e 5893).
1H NMR (CD3OD): d 7.68 (s, 1H, H-6), 6.22 (s, 1H, H-
10), 5.46 and 4.83 (br s, 2H, methylene), 4.37 (br s, 1H,
H-30), 3.93 (m, 1H, H-40), 3.88–3.84 (m, 1H, H-50), 3.79–
3.70 (m, 2H, H-6a0,6b0), 1.89 (s, 3H, 5-CH3).
(1.81 g, 56%, Rf = 0.75 in ethyl acetate/hexane, 7:3)
22
D
was obtained as colorless syrup. ½aꢀ ꢁ 21:1 (c 0.1,
CHCl3); kmax 260 nm; (e 5282).
1H NMR (CDCl3): d 8.31 (br s, 1H, NH), 7.41–7.18 (m,
Found: C, 50.57; H, 5.45; N, 9.71. Calcd for
C12H16N2O6: C, 50.70; H, 5.67; N, 9.85.
16H, 3C6H5 and H-6), 6.41 (s, 1H, H-10), 6.31 (dd, 1H,
H-40, J3 ;4 ¼ 10:0 Hz, J4 ;5 ¼ 1:8 Hz), 5.91 (d, 1H, H-
30), 5.04 and 4.68 (br s, 2H, methylene), 4.63 (m, 1H,
H-50), 3.28–3.03 (m, 2H, H-6a0,6b0), 1.85 (s, 3H, 5-CH3).
0
0
0
0
ESI-MS (m/z): Found 285.28 (M+H+).
4.1.6. 1-(b-D-Lyxo-hexopyranosyl-2-ulose)thymine (8).
Compound 8 was prepared from 5 (50 mg, 0.11 mmol)
as for 7. Purification by flash chromatography (ethyl
Found: C, 75.39; H, 5.58; N, 5.82. Calcd for
C31H28N2O4: C, 75.59; H, 5.73; N, 5.69.
acetate/MeOH, 9:1) yielded 8 (25 mg, 79%, Rf = 0.2) as
22
D
ESI-MS (m/z): Found 493.23 (M+H+).
a white foam. ½aꢀ ¼ þ26:0 (c 0.1, MeOH); kmax 260
nm; (e 6041).
4.1.8.2. Method B. Imidazole (26 mg, 0.38 mmol),
Ph3P (200 mg, 0.76 mmol), and CHI3 (150 mg,
0.38 mmol) were added to the suspension of 9 (100 mg,
1H NMR (CD3OD): d 7.33 (s, 1H, H-6), 6.31 (s, 1H, H-
10), 4.69 (d, 1H, H-40, J3 ;4 ¼ 3:5 Hz), 4.37 (d, 1H, H-30),
0
0

5454
G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
0.19 mmol) in 2 mL of dry Tol/DMF (4:1). The resulting
reaction mixture was heated (100 ꢁC, oil bath) under
nitrogen for 1 h, was concentrated in vacuum, and the
residue diluted with ethyl acetate, washed with aqueous
NaHCO3, sodium thiosulfate, and water. The organic
phase was dried with MgSO4, the solvent was removed
in vacuum, and after purification by flash chromatogra-
phy (ethyl acetate/hexane, 4:6) were isolated the desired
product 10 (24 mg, 26%) and byproduct 12 (35 mg, 21%,
Rf = 0.55 in ethyl acetate/hexane, 7:3).
concentration of DMSO was below 0.1% of cell culture
medium. All solutions were protected against light.
AZT (Retrovirꢂ) GlaxoSmithKline, USA, a drug used
for anti-retroviral therapy (ART) was used as a stan-
dard compound in antiviral experiments and 5FU as a
standard compound in anti-tumor experiments. Solu-
tions were prepared in the same way as those of unsatu-
rated exomethylene pyranonucleosides.
4.2.3. Virus propagation. The rotavirus RF strain was
propagated in Caco-2 cells in the presence of trypsin
(1 lg per mL of DMEM) as described previously.58
Supernatant containing the virus was collected from
the flasks when cytopathic effect (CPE) was observed
(24–48 h at 37 ꢁC) by microscopy and clarified by centri-
fugation. Virus was stored at ꢁ70 ꢁC until used.
Data for compound 12: 1H NMR (CDCl3): d 8.71 (br s,
1H, NH), 7.69–7.21 (m, 16H, 3C6H5 and H-6), 7.10 (d,
1H, H-30, J3 ;4 ¼ 1:3 Hz), 6.69 (s, 1H, H-10), 4.40-4.38
(m, 1H, H-50), 3.62–3.53 (m, 2H, H-6a0,6b0), 2.85 (t,
1H, H-40), 1.90 (s, 3H, 20-CH3), 1.84 (s, 3H, 5-CH3).
0
0
Found: C, 43.47; H, 3.18; N, 3.28. Calcd for
C32H29I3N2O4: C, 43.36; H, 3.30; N, 3.16.
4.2.4. Antiviral assay. The potential antiviral activity of
the newly synthesized compounds was tested against
rotavirus by investigating:
ESI-MS (m/z): Found 887.10 (M+H+).
4.1.9. 1-(2,3,4-Trideoxy-2-methylene-b-D-glycero-hex-3-
enopyranosyl)thymine (11). Compound 10 (1.81 g,
3.67 mmol) was dissolved in a mixture of formic acid/
diethyl ether (93 mL, 1:2). The mixture was stirred for
7 min at room temperature, diluted with toluene, and
co-distilled several times with the same solvent to avoid
ester formation.57 The concentrated residue was purified
4.2.4.1. The inhibition of infectivity following virus
attachment. Washed monolayer of Caco-2 and MA 104
cells was first incubated with rotavirus for 1 h at 37 ꢁC in
the atmosphere of 5% CO2 (time for virus to attach to
cell membrane receptors). After incubation the remain-
ing virus was washed off with DMEM without supple-
ments and monolayer was treated immediately with
the tested compounds or AZT added in 3-fold serial
dilutions (initial concentration of 0.5 mg/mL). After
72 h of incubation for rotavirus, the plates were stained
with Crystal Violet in ethanol, rinsed with water, and
destained with 10% (v/v) acetic acid. The A590 was mea-
sured, and the results were expressed, for each dilution,
by the mean ratios (%, SD) of absorbances in virus-in-
fected wells (n = 6) to those in control (only virus-in-
fected) wells (n = 6). The minimal inhibitory
concentration (IC50) of the tested compounds was ob-
tained from the concentration–effect curve.
by flash chromatography (ethyl acetate/hexane, 4:6) to
22
D
afford pure 11 (0.75 g, 82%, Rf = 0.25). ½aꢀ ꢁ 18:4 (c
0.1, CHCl3); kmax 260 nm; (e 4421).
1H NMR (CDCl3): d 8.63 (br s, 1H, NH), 7.27 (s, 1H,
H-6), 6.55 (s, 1H, H-10), 6.49 (dd, 1H, H-40,
0
0
0
0
J3 ;4 ¼ 10:1 Hz, J4 ;5 ¼ 2:1 Hz), 5.80 (d, 1H, H-3), 5.20
and 4.82 (br s, 2H, methylene), 4.52 (m, 1H, H-50),
3.40-3.30 (m, 2H, H-6a0,6b0), 1.91 (s, 3H, 5-CH3).
Found: C, 57.74; H, 5.46; N, 11.28. Calcd for
C12H14N2O4: C, 57.59; H, 5.64; N, 11.19.
4.2.4.2. The neutralization of the virus in solution
before attachment. Threefold dilutions of each of the
tested compounds or AZT (initial concentration of
0.5 mg/mL) were first co-incubated with rotavirus in
DMEM supplemented with trypsin for 12 h prior to
the infection of cell monolayer at 37 ꢁC and 5%
CO2. Residual viral infectivity was measured after
72 h post-infection. Rotavirus alone was treated in
the same way as the control. After 72 h of incubation,
the plates were stained with Crystal Violet in ethanol,
rinsed with water, and destained with 10% (v/v) acetic
acid. The A590 was measured, and the results were ex-
pressed, for each dilution, by the mean ratios (%,
SD) of absorbances in virus-infected wells (n = 6)
to those in control (only virus-infected) wells (n = 6).
The minimal inhibitory concentration (IC50) of the
tested compounds was obtained from the concentra-
tion–effect curve.
ESI-MS (m/z): Found 251.22 (M+H+).
4.2. Methods for measurement of biological activity
4.2.1. Cells and culture conditions. The human colon
adenocarcinoma cell line Caco-2 was a generous gift of
Dr. Rene L’Harridon, INRA, VIM, Jouy-en-Josas,
France. Rhesus monkey kidney cell line MA104 and
human fetal small intestine cell line H4 were used. Cells
were grown in Dulbecco’s modified Eagle’s medium
(DMEM) (Sigma–Aldrich, Grand Island, USA), supple-
mented with 5% fetal calf serum (Cambrex, Verviers,
Belgium), L-glutamine (2 mmol/L, Sigma, St. Louis,
USA), penicillin (100 U/mL, Sigma, St. Louis, USA), and
streptomycin (1 mg/mL, Fluka, Buchs, Switzerland) at
37 ꢁC in 5% CO2 atmosphere in tissue culture flasks until
confluent. Cell culture medium was regularly changed.
4.2.2. Pyranonucleosides. Compounds 7, 8, 10, and 11
were freshly prepared in sterile dimethylsulfoxide
(DMSO) at the concentration of 0.5 mg/mL. The final
4.2.5. Growth inhibition assay. It was performed on
Caco-2 cell line by modified method described previ-
ously.59 Briefly, in 96-well plates, six wells of 3-fold

G. Agelis et al. / Bioorg. Med. Chem. 15 (2007) 5448–5456
5455
´ ˇ
11. Manta, S.; Agelis, G.; Botic, T.; Cencic, A.; Komiotis, D.
dilutions of each compound or 5FU (initial concentra-
tion of 0.5 mg/mL) were applied to monolayers of 10
cells/well in Dulbecco’s modified Eagle’s medium
(DMEM)/10% fetal bovine serum. Incubation was per-
formed at 37 ꢁC in the humidified incubator for 10 days.
The colonies were counted in each well and the results
were expressed, for each dilution, by the mean ratios
(%, SD) of colony number in treated wells (n = 2) to
those in control wells (n = 24). The minimal inhibitory
concentration (IC50) of the tested compounds was ob-
tained from the concentration–effect curve.
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Acknowledgments
This work was supported in part by the Greek General
Secretariat for Research and Technology Program No.
3245C and Bilateral Scientific-Technology Project be-
tween Greece and Slovenia. The authors are grateful
to Dr. K. Antonakis (Ec. Nat. Sup. de Chimie de Paris;
CNRS-FRE 2463) for encouraging this work and to Dr.
T. Halmos (Ec. Nat. Sup. de Chimie de Paris) for the
unstinted and fruitful aid, as well as ELPEN pharma-
ceuticals for financial support.
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