Synthesis and molecular modelling of unsaturated exomethylene pyranonucleoside analogues with antitumor and antiviral activities

Article abstract of DOI:10.1016/j.ejmech.2007.10.014

This report describes the total and facile synthesis of the unsaturated keto and exomethylene pyranonucleoside analogues, 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-α-d-glycero-hex-2-enopyranosyl)uracil (10), 1-(2,3-dideoxy-α-d-glycero-hex-2-enopyranosyl-4-ulose)uracil (17) and 1-(2,3,4-trideoxy-4-methylene-α-d-glycero-hex-2-enopyranosyl)uracil (18). Commercially available 1,2,3,4,6-penta-O-acetyl-α-d-mannopyranose (1) was condensed with silylated uracil, deacetylated and acetalated to afford 1-(2,3-O-isopropylidene-α-d-mannopyranosyl)uracil (4). Two different synthetic routes were investigated for the conversion of 4 into the olefinic derivative 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-α-d-glycero-hex-2-enopyranosyl)uracil (10). Although the two procedures are quite similar with respect to yields and final products, the second also leads to the keto-2′,3′-unsaturated analogue (17). The new analogues were evaluated for their anticancer and antiviral activities using several tumor cell lines and gastrointestinal rotavirus. All of the compounds showed direct antiviral effect against rotavirus infectivity in Caco-2 cell line. Moreover, 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-α-d-glycero-hex-2-enopyranosyl)uracil (10) was found to be potent in MCF-7 breast carcinoma cell line.

Full text of DOI:10.1016/j.ejmech.2007.10.014

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1366e1375
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and molecular modelling of unsaturated exomethylene
pyranonucleoside analogues with antitumor and antiviral activities
a
George Agelis , Niki Tzioumaki , Theodore Tselios , Tanja Botic ,
a
b
c
´
a,
ꢀ c,d
*
Avrelija Cencic , Dimitri Komiotis
a Department of Biochemistry & Biotechnology, Laboratory of Organic Chemistry, University of Thessaly, 26 Ploutonos Strasse, 41221 Larissa, Greece
b Department of Chemistry, Laboratory of Organic Chemistry, University of Patras, Patras, Greece
c Department of Microbiology, Biochemistry and Biotechnology, Faculty of Agriculture, University of Maribor, Vrbanska c.30, 2000 Maribor, Slovenia
d Department of Biochemistry, Medical Faculty, University of Maribor, Slovenia
Received 19 June 2007; accepted 12 October 2007
Available online 18 October 2007
Abstract
This report describes the total and facile synthesis of the unsaturated keto and exomethylene pyranonucleoside analogues, 1-(2,3,4-trideoxy-
4-methylene-6-O-trityl-a-D-glycero-hex-2-enopyranosyl)uracil (10), 1-(2,3-dideoxy-a-D-glycero-hex-2-enopyranosyl-4-ulose)uracil (17) and
1-(2,3,4-trideoxy-4-methylene-a-D-glycero-hex-2-enopyranosyl)uracil (18). Commercially available 1,2,3,4,6-penta-O-acetyl-a-D-mannopyra-
nose (1) was condensed with silylated uracil, deacetylated and acetalated to afford 1-(2,3-O-isopropylidene-a-D-mannopyranosyl)uracil (4).
Two different synthetic routes were investigated for the conversion of 4 into the olefinic derivative 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-a-
D-glycero-hex-2-enopyranosyl)uracil (10). Although the two procedures are quite similar with respect to yields and final products, the second
also leads to the keto-20,30-unsaturated analogue (17). The new analogues were evaluated for their anticancer and antiviral activities using several
tumor cell lines and gastrointestinal rotavirus. All of the compounds showed direct antiviral effect against rotavirus infectivity in Caco-2 cell line.
Moreover, 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-a-D-glycero-hex-2-enopyranosyl)uracil (10) was found to be potent in MCF-7 breast carci-
noma cell line.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Unsaturated nucleosides; Exomethylene nucleosides; Ketonucleosides; Antiviral; Cytotoxicity; MCF-7 cell line
1. Introduction
bond cleavage is a frequently encountered degradative pathway
of nucleoside antivirals, especially for the 20,30-dideoxy-nucleo-
sides [15]. On the other hand, the insertion of unsaturation in the
20,30-position of the glycone moiety to provide 20,30-didehydroe
20,30-dideoxy analogues distinguishes this ring system from
common six-membered rings and allows to categorize the cyclo-
hexene ring as a bioisoster of a saturated furanose ring [16].
Among them, the ketonucleosides proved to be key intermedi-
ates in synthetic and biosynthetic processes and a number of
them exhibit interesting antitumor and antiviral properties
[17e21]. It appears that these nucleosides not only exhibit
growth inhibitory activity against a variety of tumor cells
[22,23] in vitro and L1210 leukemia [24,25] in vivo, but they
also may constitute important synthetic intermediates in nucleo-
side chemistry due to their chemical reactivity in various media
[26,27]. Furthermore, the presence of an a,b-unsaturated double
Nucleoside analogues display a wide range of biological
activities, such as antitumor, antiviral and chemotherapeutic
[1e4]. Over the past two decades, nucleoside chemistry has
evolved to facilitate efficient routes to effective agents for
the treatment of AIDS [5], herpes [6] and viral hepatitis [7].
Lately, nucleosides bearing a six-membered carbohydrate
moiety have been evaluated for their potential antiviral [8e11]
and antibiotic [12] properties and as building blocks in nucleic
acid synthesis [13,14]. In particular, six-membered carbocyclic
nucleosides show resistance to hydrolysis, since glycosidic
* Corresponding author. Tel.: þ30 2410 565285; fax: þ30 2410 565290.
E-mail address: dkom@bio.uth.gr (D. Komiotis).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.10.014

G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
1367
bond enhances biological activity [19,24,28], and these unsatu-
rated ketonucleosides are well established for their antineoplastic
activity and immunosuppressive properties [18,29,30].
Furthermore, modified nucleosides bearing an exocyclic
methylene or fluoromethylene group in positions 20, 30 or 40
exhibited noteworthy antitumor and antiviral activities [31e
40]. Their antiviral and anticancer activities were attributed,
at least in part, to the ability of these nucleosides to irrevers-
ibly inactivate ribonucleotide reductase after phosphorylation
via specific nucleoside kinases.
Recently, we reported [41] on a new class of unusual nucle-
osides, both 20-exomethylene and 30,40-unsaturated-20-exo-
methylene pyranonucleosides, with satisfactory rotavirus and
antitumor growth inhibition.
On the basis of these findings, we report herein the synthesis
and pharmacology of a novel series of modified nucleosides, i.e.
the 40-exomethylene and 20,30-unsaturated-40-exomethylene
pyranonucleosides shown in Scheme 1.
(Me3SiOSO2CF3) afforded 1-(2,3,4,6-tetra-O-acetyl-a-D-man-
nopyranosyl)uracil (2). Removal of all O-acetyl protecting
groups of 2, with saturated methanolic ammonia [43], gave
3, which was selectively acetylated, using 2,2-dimethoxypro-
pane [43] in acetone, to give 20,30-O-isopropylidene 4.
Two routes were investigated for the conversion of 1-(2,3-
O-isopropylidene-a-D-mannopyranosyl)uracil (4) into the ole-
finic derivative 10. In the first approach, the primary hydroxyl
group of 4 was selectively tritylated to give 5. Oxidation of the
free hydroxyl group with the pyridinium dichromate (PDC)/
acetic anhydride (Ac2O) [44] led to the formation of the de-
sired 40-ketonucleoside 6, which was isolated as a white solid.
1
The H NMR spectrum of 6 showed a doublet at 4.65 ppm
0
0
0
(J2 ,3 ¼ 7.4 Hz), which is assigned to H-3 confirming the
absence of proton at C-40. Wittig olefination of the keto inter-
mediate 6 with sodium hydride and methyl triphenylphospho-
nium bromide (Ph3PCH3Br) at 0 ꢀC, in the presence of t-amyl
alcohol [38] in tetrahydrofuran (THF), led to 7, which was ob-
1
tained as a white solid in satisfactory yield (73%). The H
NMR spectrum of 7 showed two vinylic protons at 5.40 and
5.26 ppm, each as a broad singlet, which correspond to the
40-exomethylene group. Compound 7 was converted to the
fully unprotected derivative 8 by treatment with trifluoroacetic
acid (TFA) 90% in methanol (MeOH), for 10 min. The primary
hydroxyl group of 8 was selectively tritylated to afford deriva-
tive 9 in 78% yield. The olefination of the 2,3-vicinal diol
2. Results and discussion
2.1. Synthesis
Condensation of commercially available 1,2,3,4,6-penta-O-
acetyl-a-D-mannopyranose (1) with silylated uracil in the
presence of trimethylsilyl trifluoromethane-sulfonate [42]
OAc
OR
OH
O
O
OTr
O
O
OTr
O
O
OAc
O
OR
d
e
a
c
OAc
O
O
OR
O
O
O
Route A
HO
AcO
OAc
RO
b
B
B
HO
B
B
1
2 R = Ac
4
6
5
3 R = H
f
OTr
O
O
OTr
O
OH
O
OR
O
OH
g
l
h
H2C
H2C
H2C
H2C
O
OH
B
B
B
B
7
8 R = H
9 R = Tr
18
10
d
f
OH
O
OTr
O
OTr
O
OR
O
e
l
d
O
O
B
HO
RO
b
B
B
B
13 R = Ac
17
16
15
14 R = H
k
O
OAc
OAc
O
NH
NH
O
O
B
j
i
Route B
B=
4
OH
OH
O
O
AcO
AcO
B
11
12
Scheme 1. (a) Uracil, Me3SiOSO2CF3, HMDS, saccharine, dry CH3CN, 120e80 ꢀC; (b) ammonia/MeOH; (c) acetone, 2,2-dimethoxypropane, p-toluenesulfonic
acid, water; (d) triphenylmethyl chloride, pyridine; (e) PDC, Ac2O, dry CH2Cl2; (f) Ph3PCH3Br, sodium hydride, t-amyl alcohol in THF, 0e25 ꢀC; (g) TFA 90% in
MeOH; (h) dry Tol/DMF (4:1), iodineeimidazoleePh3P, 80 ꢀC; (i) pyridine, Ac2O; (j) formic acid/CH2Cl2 (1:1); (k) dry Tol/DMF (4:1), iodoformeimidazolee
Ph3P, 120 ꢀC; and (l) formic acid/diethyl ether, 1:1.

1368
G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
1
proved to be a crucial step in our synthetic methodology and
several possible synthetic approaches to the olefinic nucleoside
10 were investigated. Thus, we employed the CoreyeWinter
[45,46] on diol 9 by reacting it with thiocarbonyldiimidazole
followed by reductive olefination with trimethylphosphite,
the Barton [47] deoxygenation via the corresponding xanthate
ester (treatment with tributyltin hydride), and the Hanessian
et al. [48] method, which involved treatment with N,N-dime-
thylformamide (DMF) and dimethylacetal/methyl iodide.
None of these methods were successful, as only intractable
were isolated. In contrast, the GareggeSamuelsson method
(iodineetriphenylphosphine (Ph3P)eimidazole [49e52]) led
to the direct conversion of diol 9 to the desired olefinic com-
pound 10 in 54% yield. The 1H NMR spectrum of 10 showed,
besides the two exomethylene protons at 5.15 and 5.04 ppm,
agreement with the H NMR data (Table 1), the base moiety
occupies the equatorial position (energetically favoured).
0
0
The H1 and H2 protons of 8 have an axialeaxial dirꢀection
0
with a dihedral angle of H2 eC2 eC1 eH1 : 178.1 and
0
0
0
0
0
J1 ,2 ¼ 9.5 Hz (Table 1) adopting thus a ‘‘chair’’ conformation.
Moreover, in all analogues the low energy difference revealed
0
0
0
that after rotation around C1 eN1 and C5 eC6 bonds (Grid
Scan simulation) the positions of the base and hydroxy methyl
groups are possibly interconverted at room temperature
(Fig. 1). Finally, computational analysis and 1H NMꢀR data of an-
0
0
0
0
0
0
alogues 10 (dihedral angle H2 eC2 eC1 eH1 : 102.0 , J1 ,2 w 0),
ꢀ
0
0
0
0
0
0
17 (dihedral angle H2 eC2 eC1 eH1 : 86.7 , J1 ,2 w 0) and 18
ꢀ
0
0
0
0
0
0
(dihedral angle H2 eC2 eC1 eH1 : 103.1 , J1 ,2 w 0) suggest
that these compounds adopt the ‘‘sofa’’ conformation with H-10
proton lying perpendicularly to the ring (Fig. 1). Such a confor-
mation, compared to the ‘‘chair’’ conformation of analogue 8,
should be favoured because of the insertion of a double
bond in the 20,30-position of the sugar moiety.
the presence of two vinylic protons a0t 5.69 and 6.62 ppm (dou-
0
0
0
blet, J2 ,3 ¼ 9.1 Hz) ascribed to H-2 and H-3 , respectively.
In the second approach, acetylation of 4 using acetic anhy-
dride in pyridine followed by deisopropylidenation of the re-
sulted derivative 11 in dichloromethane/formic acid, 1:1, led
to the vicinal diol derivative 12 in high yield (88%). Olefination
of 12 with iodoformePh3Peimidazole [53e55] afforded 1-
(4,6-di-O-acetyl-2,3-dideoxy-a-D-erythro-hex-2-enopyrano-
2.3. Biological activity
2.3.1. Antiviral activity
1
The antiviral properties of the nucleoside analogues were
examined using a colon adenocarcinoma Caco-2 cell line in-
fected with gastrointestinal rotavirus as a model virus and
AZT drug as positive control (Table 2). Although the tested
compounds did not show any antiviral activity in neutraliza-
tion assay, they inhibited rotavirus infectivity following virus
attachment. In comparison to AZT, higher concentrations
were needed in order to obtain the same effect against virus
and their CC50/IC50 values were smaller than that found for
AZT.
syl)uracil (13) in 76% yield. The H NMR spectrum of 13
showed the presence of two vinylic protons, H-20 appearing as
0
0
0
0
a doublet of doublets at 5.91 ppm (J1 ,2 ¼ 1.5 Hz, J2 ,3
¼
10.1 Hz) and a doublet at 6.34 ppm, which is assigned to H-30.
Deacetylation of 13 with saturated methanolic ammonia gave
14, which after selective tritylation of the primary hydroxyl
group afforded 15. Oxidation of the free hydroxyl with PDC/
Ac2O and Wittig condensation of 40-ketonucleoside 16 led to
the 1-(2,3,4-trideoxy-4-methylene-6-O-trityl-a-D-glycero-hex-
2-enopyranosyl)uracil (10). Finally, detritylation of 16 and 10
with formic acid/diethyl ether [56], 1:1, for 7 min, afforded
the 40-keto-20,30-unsaturated derivative 17 as well as the title
nucleoside 1-(2,3,4-trideoxy-4-methylene-a-D-glycero-hex-2-
enopyranosyl)uracil (18), respectively (Scheme 1).
2.3.2. Cytotoxic and growth inhibition activity
The cytotoxicity (CC50) of the new compounds was mea-
sured on normal human intestinal cell line (H4) and on a series
of human tumor cells, such as human colonic adenocarcinoma
cell line (Caco-2), skin melanoma cell line, and epithelial
breast cancer cell line (MCF-7). The growth inhibition of
Caco-2 cells induced by the new compounds was measured
by determining the minimal inhibitory concentration, IC50.
The results are summarized in Table 3 and compared with
the values obtained with 5-fluorouracil (5FU).
All of the tested compounds exhibit higher cytotoxicity in
tumor cells than in the normal H4 cell line. Compound 10
showed to be 3-fold more cytotoxic to skin melanoma and
MCF-7 cell line than to Caco-2 cells. Furthermore, compared
to 5FU compound 10 was found to be 16-fold more selective
toward MCF-7 cells, 2.5-fold more selective in Caco-2 cells
and as selective as 5FU in skin melanoma cells. Compounds
8, 17 and 18 in comparison to 5FU exhibit higher selectivity
in all tested tumorgenic cell lines and higher cytotoxicity to-
ward Caco-2 and MCF-7 cell lines. This selective activity of
the tested compounds is of great importance in their potential
use as antitumor drugs, as nucleoside analogues are molecules
that require specialized nucleoside transporter proteins to
In conclusion, the yields and the experimental processes
quoted above are quite similar, although the second approach
is more attractive because it leads not only the olefinic 40-exo-
methylene derivative 10 but also the 40-keto-20,30-unsaturated
nucleoside 17.
2.2. Conformational analysis of synthesized
exomethylene pyranonucleoside analogues using
computational methods
In an effort to decipher the preferred low energy conforma-
tions of the new keto and exomethylene pyranonucleoside an-
alogues and also correlate their 3D-structures with the 1H
NMR spectrum, we studied the conformational preferences
of 8, 10, 17 and 18. The study was mainly focused on the for-
mation of uracil nucleobase in correlation with the sugar moi-
ety using energy minimization methods and systematic Grid
Scan search.
Fig. 1 shows the lowest and the highest energy conformers
of 8, 10, 17 and 18 after Grid Scan. In analogue 8 and in

G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
1369
Fig. 1. The lowest (left side) and the highest energy (right side) conformations of 8, 10, 17 and 18 analogues obtained after energy minimization and Grid Scan
simulation.
enter the cell and there is emerging evidence that the
abundance and tissue distribution of these proteins contributes
to cellular specificity and sensitivity to nucleoside analogues
[57].
Furthermore, the pronounced inhibitory activity of the new
compounds and especially of analogue 10 on cell growth in
Caco-2 cells (2.5-fold higher TSI values than 5FU) identifies
them as potential lead candidates for in vivo experiments.
3. Conclusion
In conclusion, biological assays were performed on the new
compounds using a variety of tumor cell lines. 1-(2,3,4-Tri-
deoxy-4-methylene-6-O-trityl-a-D-glycero-hex-2-enopyrano-
syl)uracil (10) being sufficiently cytotoxic on carcinoma cells
and also highly selective was identified as a lead compound for
further studies. All new molecules inhibited the growth of

1370
G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
Table 1
Torsion angles and J coupling of analogues 8, 10, 17 and 18 obtained from the
Table 3
Cytotoxic effect (CC50, mM) of compounds 8, 10, 17, 18 and 5FU on Caco-2,
H4, MCF-7, and skin melanoma cells, and growth inhibition (IC50, mM) on
Caco-2 cells
3D-structures and 1H NMR, respectively
Analogue
Torsion angles
J Coupling (Hz)
Compound Cytotoxic effect
(CC50, mM)
TSIa
Growth
inhibition
(IC50, mM)
ꢀ
ꢀ
0
C4 eC5 eC6 eO6 : 177.7
0
0
0
8
e
OH
O
ꢀ
0
0
0
0
H3 eC3 eC2 eH2 : 52.0
e
9.5
e
0
0
0
0
H2 eC2 eC1 eH1 : 178.1
ꢀ
0
0
C2 eC1 eN1eC6: ꢁ112.9
H4
Caco-2 Mela MCF-7 Caco-2 Mela MCF-7 Caco-2
noma
H2C
OH
OH
noma
B
8
1850.2 74.0 74.0 74.0 25.0
1044.8 41.7 12.5 12.5 25.0
2099.0 83.9 83.9 83.9 25.0
2116.6 84.6 84.6 84.6 25.0
3843.8 384.4 46.1 768.8 10
25.0 25.0
83.6 83.6
25.0 25.0
25.0 25.0
83.4 5.0
7.4
4.1
8.3
8.4
1.5
ꢀ
ꢀ
10
17
18
5FU
a
0
C4 eC5 eC6 eO6 : 163.7
0
0
0
10
e
9.1
0
OTr
0
0
0
0
H3 eC3 eC2 eH2 : ꢁ6.3
O
ꢀ
0
H2 eC2 eC1 eH1 : 102.0
0
0
0
ꢀ
0
C2 eC1 eN1eC6: 120.0
0
e
H2C
TSI: tumor selectivity index (CC50 on H4 cells/CC50 on the specified host
cells).
B
ꢀ
0
0
0
0
17
C4 eC5 eC6 eO6 : ꢁ179.5
e
8.9
0
OH
ꢀ
0
0
0
0
H3 eC3 eC2 eH2 : ꢁ20.0
O
ꢀ
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 reac-
tions were carried out in dry solvents. CH2Cl2 was distilled
from phosphorous pentoxide and stored over 4E molecular
sieves. Acetonitrile, toluene (Tol) and DMF were distilled
from calcium 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 potas-
sium hydroxide.
0
H2 eC2 eC1 eH1 : 86.7
0
0
0
ꢀ
0
C2 eC1 eN1eC6: 90.0
0
e
O
B
ꢀ
0
C4 eC5 eC6 eO6 : 44.9
0
0
0
18
e
10.1
0
OH
ꢀ
ꢀ
0
0
0
0
H3 eC3 eC2 eH2 : ꢁ7.6
0
H2 eC2 eC1 eH1 : 103.1
0
0
0
O
ꢀ
0
0
C2 eC1 eN1eC6: 100.0
e
H2C
B
Caco-2 cells and showed direct antiviral effect as they were
able to inhibit rotavirus action.
4.1.1. 1-(2,3,4,6-Tetra-O-acetyl-a-D-mannopyranosyl)uracil (2)
A mixture of uracil (4.96 g, 44.25 mmol), hexamethyldisi-
lazane (HMDS) (11 mL, 52.75 mmol), and saccharin (0.37 g,
2.03 mmol) in dry CH3CN (152 mL) was refluxed for
20 min at 120 ꢀC under nitrogen. To this were added
1,2,3,4,6-penta-O-acetyl-a-D-mannopyranose (1) (12.32 g,
31.56 mmol) and Me3SiOSO2CF3 (8 mL, 44.2 mmol). The re-
action mixture was refluxed at 80 ꢀC for 2 h. The mixture was
neutralized with saturated sodium bicarbonate, then diluted
with water, and extracted with ethyl acetate (EtOAc). The ex-
tract was dried with sodium sulfate, filtered and evaporated to
a syrup, which was purified by flash chromatography (EtOAc/
hexane, 7:3) to give 2 (12.56 g, 90%, Rf ¼ 0.44 in EtOAc/hex-
ane, 8:2) as a clear viscous oil. [a]2D2 45.5 (c 0.230, CHCl3);
UV (CHCl3): lmax 261 nm (3 7874); 1H NMR (CDCl3):
4. Experimental
4.1. General procedure
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 sulphuric acid. Flash chromatography was
performed using silica gel (240e400 mesh, Merck).
1H NMR spectra were recorded at room temperature with
a Bruker 400 MHz spectrometer using chloroform-d (CDCl3)
Table 2
Antiviral activity of nucleosides 8, 10, 17, 18 and AZT against rotavirus RF
strain on Caco-2 cells (IC50
Compound Treatment Aa
IC50
)
Treatment Ba
IC50
d 8.42 (br s, 1H, NH), 7.36 (d, 1H, J5,6 ¼ 8.2 Hz, H-6), 6.14
0
b
0
0
(d, 1H, J1 ,2 ¼ 9.6 Hz, H-1 ), 5.78 (d, 1H, H-5), 5.48 (t, 1H,
CC50/IC50
CC50/IC50
0
0
0
0
0
0
J2 ,3 ¼ J3 ,4 ¼ 3.2 Hz, H-3 ), 5.35 (dd, 1H, H-2 ), 4.97 (d,
mg/mL mM
mg/mL mM
1H, H-40), 4.63 (dd, 1H, J5 ,6b ¼ 9.8, J6b ,6a ¼ 13.5 Hz, H-
0
0
00
0
8
n.e.
n.e.c
n.e.
n.e.
0.02
n.e.
n.e.
n.e.
n.e.
e
e
e
e
0.2
0.5
740.00 0.1
1044.8 0.04
6b0), 4.36e4.31 (m, 2H, J5 ,6a ¼ 5.3 Hz, H-5 , H-6a ), 2.21,
2.18, 2.10, 1.99 (4s, 12H, 4OAc); ESI-MS (m/z) 443.45
[M þ H]. Anal. Calcd. for C18H22N2O11: C, 48.87; H, 5.01;
N, 6.33. Found: C, 48.95; H, 5.32; N, 6.21.
0
0
10
17
18
AZT
a
0.2
0.2
839.60 0.1
846.60 0.1
22.45 2.5d
74.84 0.75d
0.006
Treatment A: neutralization of the virus in the solution before its attach-
ment. Treatment B: inhibition of infectivity following virus attachment.
b
4.1.2. 1-(a-D-Mannopyranosyl)uracil (3)
Compound 2 (12.56 g, 28.42 mmol) was treated with am-
monia/MeOH (saturated at 0 ꢀC, 1 L). The solution was stirred
CC50/IC50 values were calculated using CC50 values in Table 3.
n.e. ¼ No effect.
c
d
CC50 for AZT on CaCo-2 cells ¼ 56.1 mM.

G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
1371
0
0
0
overnight at room temperature and then was concentrated un-
der reduced pressure. The residue was purified by flash chro-
matography (EtOAc/MeOH, 8:2) to give 3 (6.80 g, 88%,
Rf ¼ 0.24) as a white foam. [a]2D2 34.7 (c 0.150, MeOH); UV
(MeOH): lmax 260 nm (3 6984); ESI-MS (m/z) 275.38
[M þ H]. Anal. Calcd. for C10H14N2O7: C, 43.80; H, 5.15;
N, 10.22. Found: C, 43.96; H, 5.32; N, 10.01.
(d, 1H, H-5), 5.56 (d, 1H, J1 ,2 ¼ 3.6 Hz, H-1 ), 4.88 (dd, 1H,
0
0
0
0
0
0
J2 ,3 ¼ 7.4 Hz, H-2 ), 4.65 (d, 1H, H-3 ), 4.09 (dd, 1H, J5 ,6a
¼
0
0
0
0
0
2.6, J5 ,6b ¼ 5.6 Hz, H-5 ), 3.64 (dd, 1H, J6a ,6b ¼ 10.5 Hz, H-
6a0), 3.38 (dd, 1H, H-6b0), 1.48, 1.33 (2s, 6H, 2CH3); ESI-MS
(m/z) 555.43 [M þ H]. Anal. Calcd. for C32H30N2O7: C,
69.30; H, 5.45; N, 5.05. Found: C, 69.17; H, 5.21; N, 5.28.
4.1.6. 1-(4-Deoxy-2,3-O-isopropylidene-4-methylene-
6-O-trityl-a-D-lyxo-hexopyranosyl)uracil (7)
4.1.3. 1-(2,3-O-Isopropylidene-a-D-mannopyranosyl)uracil (4)
To a stirred suspension of 3 (6.80 g, 25 mmol) in anhydrous
acetone (50 mL) and 2,2-dimethoxypropane (50 mL) was
added p-toluenesulfonic acid monohydrate (0.98 g,
5.15 mmol). After 30 min, water (100 mL) was added and
the reaction mixture was stirred for 16 h. The resulting solu-
tion was neutralized with saturated sodium bicarbonate so
that pH did not exceed 7, concentrated and the residue was pu-
rified by flash chromatography (EtOAc) to give 4 (6.30 g,
81%, Rf ¼ 0.14) as a white foam. [a]2D2 25.8 (c 0.280,
MeOH); UV (MeOH): lmax 260 nm (3 6755); 1H NMR
To a stirred suspension of Ph3PCH3Br (2.11 g, 5.89 mmol)
and t-amyl alcohol (0.71 mL, 6.44 mmol) in dry THF (17 mL)
was added sodium hydride (0.15 g, 60% in oil, 6.44 mmol) at
0 ꢀC under nitrogen and the reaction mixture was stirred for
2 h at ambient temperature under nitrogen. To this yellow
phosphorous ylide was added a solution of 6 (0.99 g,
1.79 mmol) in dry THF (2.80 mL) dropwise, at 0 ꢀC under ni-
trogen. The mixture was stirred for 30 min at ambient temper-
ature under nitrogen, then the reaction mixture was quenched
with saturated sodium bicarbonate and extracted with EtOAc.
The organic layer was washed with water, dried with sodium
sulfate and evaporated. The residue was purified by flash chro-
matography (EtOAc/hexane, 5:5) to give 7 (0.72 g, 73%,
Rf ¼ 0.34) as a white solid, m.p. 107e112 ꢀC. [a]D22 29.8 (c
0.150, CHCl3); UV (CHCl3): lmax 260 nm (3 12,469); 1H
NMR (CDCl3): d 8.74 (br s, 1H, NH), 7.40 (d, 1H,
(CDCl3): d 8.40 (br s, 1H, NH), 7.35 (d, 1H, J5,6 ¼ 8.1 Hz,
0
0
0
H-6), 5.79 (d, 1H, H-5), 5.69 (d, 1H, J1 ,2 ¼ 6.8 Hz, H-1 ),
4.55e4.47 (m, 2H, 0 H-20, H-30), 4.22 (0t, 1H,
0
0
0
0
0
J3 ,4 ¼ J4 ,5 ¼ 7.1 Hz, H-4 ), 3.87e3.76 (m, 3H, H-5 , H-6a ,
H-6b0), 1.52, 1.39 (2s, 6H, 2CH3); ESI-MS (m/z) 315.34
[M þ H]. Anal. Calcd. for C13H18N2O7: C, 49.68; H, 5.77;
N, 8.91. Found: C, 49.45; H, 5.34; N, 8.63.
J6,5 ¼ 8.1 Hz, H-6), 7.31e7.19 (m, 15H, 3C6H5), 5.80 (d,
0
0
0
1H, J1 ,2 ¼ 6.7 Hz, H-1 ), 5.73 (d, 1H, H-5), 5.40, 5.26 (br s,
0
4.1.4. 1-(2,3-O-Isopropylidene-6-O-trityl-a-D-manno-
pyranosyl)uracil (5)
0
0
2H, methylene), 4.72 (d, 1H, J2 ,3 ¼ 5.8 Hz, H-3 ), 4.56 (dd,
1H, H-20), 4.21 (dd, 1H, J5 ,6a ¼ 5.5, J5 ,6b ¼ 8.1 Hz, H-5 ),
0
0
0
0
0
0
0
To a solution of 4 (1 g, 3.18 mmol) in pyridine (16 mL)
were added triphenylmethyl chloride (1.15 g, 4.13 mmol)
and a catalytic amount of 4-dimethylaminopyridine. The reac-
tion mixture was stirred overnight at room temperature. After
being quenched with MeOH and concentrated, the residue was
purified by flash chromatography (EtOAc/hexane, 7:3) to give
5 (1.44 g, 82%, Rf ¼ 0.6 in EtOAc) as a white solid, m.p. 143e
146 ꢀC. [a]D22 18.2 (c 0.100, CHCl3); UV (CHCl3): lmax
0
0
3.62 (dd, 1H, J6a ,6b ¼ 10.2 Hz, H-6a ), 3.32 (dd, 1H, H-6b ),
1.36, 1.33 (2s, 6H, 2CH3); ESI-MS (m/z) 553.72 [M þ H].
Anal. Calcd. for C33H32N2O6: C, 71.72; H, 5.84; N, 5.07.
Found: C, 71.38; H, 5.65; N, 5.30.
4.1.7. 1-(4-Deoxy-4-methylene-a-D-lyxo-hexo-
pyranosyl)uracil (8)
1
Compound 7 (0.72 g, 1.30 mmol) was dissolved in 5.85 mL
of 90% TFA in MeOH. The solution was stirred for 10 min at
room temperature and then concentrated under reduced pres-
sure, in order to remove traces of TFA. The resulting residue
was crystallized from diethyl ether to give pure 8 (0.30 g,
85%, Rf ¼ 0.4 in EtOAc/MeOH, 9:1) as a white solid, m.p.
178e180 ꢀC. [a]2D2 ꢁ72.7 (c 0.100, MeOH); UV (MeOH):
260 nm (3 8432); H NMR (CDCl3): d 8.71 (br s, 1H, NH),
7.40 (d, 1H, J5,6 ¼ 8.2 Hz, H-6), 7.32e7.21 (m, 15H,
0
0
3C6H5), 5.75 (d, 1H, H-5), 5.70 (d, 1H, J1 ,2 ¼ 6.5 Hz, H-
10), 4.46e4.34 (m, 2H, H-20, H-30), 4.09 (m, 1H, H-40),
3.84e3.77 (m, 1H, H-50), 3.46e3.32 (m, 2H, H-6a0, H-6b0),
1.44, 1.31 (2s, 6H, 2CH3); ESI-MS (m/z) 557.81 [M þ H].
Anal. Calcd. for C32H32N2O7: C, 69.05; H, 5.79; N, 5.03.
Found: C, 69.21; H, 5.52; N, 5.21.
1
lmax 261 nm (3 5418); H NMR (CD3OD): d 7.67 (d, 1H,
0
0
0
J5,6 ¼ 8.1 Hz, H-6), 6.15 (d, 1H, J1 ,2 ¼ 9.5 Hz, H-1 ), 5.72
(d, 1H, H-5), 5.23, 5.15 (br s, 2H, methylene), 4.47e4.43
0
4.1.5. 1-(2,3-O-Isopropylidene-6-O-trityl-a-D-lyxo-hexo-
pyranosyl-4-ulose) uracil (6)
(m, 2H, H-20, H-30), 4.25 (dd, 1H, J5 ,6 b ¼ 8.9,
0
0
0
0
0
0
J6a ,6b ¼ 12.2 Hz, H-6b ), 3.73 (dd, 1H, J5 ,6a ¼ 3.6 Hz, H-
50), 3.53 (dd, 1H, H-6a0); ESI-MS (m/z) 271.32 [M þ H].
Anal. Calcd. for C11H14N2O6: C, 48.89; H, 5.22; N, 10.37.
Found: C, 48.57; H, 5.43; N, 10.21.
A mixture of 5 (1.44 g, 2.61 mmol; dried by co-evaporation
with Tol), PDC (1.17 g, 3.10 mmol) and Ac2O (0.73 mL,
7.74 mmol) was stirred in dry CH2Cl2 (26 mL) for 4 h, under
nitrogen at room temperature. Purification by flash chromatog-
raphy (EtOAc/hexane, 8:2) yielded pure 6 (0.99 g, 70%,
Rf ¼ 0.65) as white solid, m.p. 115e118 ꢀC. [a]D22 4.5 (c
0.150, CHCl3); UV (CHCl3): lmax 260 nm (3 7858); 1H
NMR (CDCl3): d 8.70 (br s, 1H, NH), 7.36 (d, 1H,
J5,6 ¼ 8.2 Hz, H-6), 7.24e7.15 (m, 15H, 3C6H5), 5.72
4.1.8. 1-(4-Deoxy-4-methylene-6-O-trityl-a-D-lyxo-hexo-
pyranosyl)uracil (9)
To a stirred solution of 8 (0.30 g, 1.10 mmol) in pyridine
(5 mL) were added successively triphenylmethyl chloride

1372
G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
(0.40 g, 1.43 mmol) and a catalytic amount of 4-dimethylami-
nopyridine. The reaction mixture was stirred overnight at room
temperature, then was quenched with MeOH and evaporated
under reduced pressure. The residue was purified by flash
chromatography (EtOAc/hexane, 8:2) to give 9 (0.43 g, 78%,
Rf ¼ 0.5 in EtOAc) as a white foam. [a]2D2 13.4 (c 0.200,
CHCl3); UV (CHCl3): lmax 261 nm (3 8658); 1H NMR
(CDCl3): d 8.75 (br s, 1H, NH), 7.44 (d, 1H, J5,6 ¼ 8.1 Hz,
purified by flash chromatography (EtOAc) to give 12 (1.0 g,
88%, Rf ¼ 0.25) as a white solid, m.p. 118e121 ꢀC; [a]D22
24.2 (c 0.250, CHCl3); UV (CHCl3): lmax 261 nm (3 4884);
1H NMR (CDCl3): d 10.60 (br s, 1H, NH), 7.46 (d, 1H,
J5,6 ¼ 8.1 Hz, H-6), 6.73 (d, 1H, H-5), 6.15 (d, 1H,
0
0
0
0
0
0
J1 ,2 ¼ 9.5 Hz, H-1 ), 5.04 (d, 1H, J3 ,4 ¼ 3.4 Hz, H-4 ), 4.98
0
0
0
0
0
(t, 1H, J2 ,3 ¼ 9.5 Hz, H-2 ), 4.28e4.14 (m, 3H, H-5 , H-6a ,
H-6b0), 3.91 (dd, 1H, H-30), 2.14, 2.05 (2s, 6H, 2OAc); ESI-
MS (m/z) 359.42 [M þ H]. Anal. Calcd. for C14H18N2O9: C,
46.93; H, 5.06; N, 7.82. Found: C, 46.72; H, 5.28; N, 7.69.
0
0
H-6), 7.31e7.21 (m, 15H, 3C6H5), 6.21 (d, 1H, J1 ,2 ¼ 9.1 Hz,
H-10), 5.75 (d, 1H, H-5), 5.22, 5.04 (br s, 2H, methylene), 4.54
(d, 1H, H-20), 4.38 (br s, 1H, H-30), 3.59e3.51 (m, 3H, H-50,
H-6a0, H-6b0); ESI-MS (m/z) 513.68 [M þ H]. Anal. Calcd.
for C30H28N2O6: C, 70.30; H, 5.51; N, 5.47. Found: C,
70.12; H, 5.70; N, 5.63.
4.1.12. 1-(4,6-Di-O-acetyl-2,3-dideoxy-a-D-erythro-hex-
2-enopyranosyl)uracil (13)
Imidazole (0.40 g, 5.88 mmol), Ph3P (3.08 g, 11.75 mmol)
and iodoform (2.31 g, 5.88 mmol) were added to the suspen-
sion of 12 (1 g, 2.80 mmol) in 40 mL of dry Tol/DMF (4:1).
The reaction mixture was heated (120 ꢀC, oil bath) under ni-
trogen for 2.5 h, was concentrated in vacuum and the residue
diluted with EtOAc, washed with saturated sodium bicarbon-
ate, sodium thiosulfate and water. The organic phase was dried
with sodium sulfate, the solvent was removed in vacuum, and
purification by flash chromatography (EtOAc /hexane, 6:4)
yielded 13 (0.69 g, 76%, Rf ¼ 0.5 in EtOAc), as a colorless
oil. [a]2D2 23.46 (c 0.115, CHCl3); UV (CHCl3): lmax 261 nm
4.1.9. 1-(2,3,4-Trideoxy-4-methylene-6-O-trityl-a-D-glycero-
hex-2-enopyranosyl)uracil (10)
Imidazole (0.88 g, 1.29 mmol), Ph3P (0.68 g, 2.58 mmol)
and iodine (0.33 g, 1.29 mmol) were added to the suspension
of 9 (0.44 g, 0.86 mmol) in 20 mL of dry Tol/DMF (4:1).
The reaction mixture was heated (80 ꢀC, oil bath) under nitro-
gen for 10 min. The residue was purified by flash chromatog-
raphy (EtOAc/hexane, 5:5) to give 10 (0.22 g, 54%, Rf ¼ 0.6 in
EtOAc/hexane, 7:3) as a yellowish syrup. [a]2D2 10.3 (c 0.125,
CHCl3); UV (CHCl3): lmax 260 nm (3 6030); 1H NMR
(CDCl3): d 8.20 (br s, 1H, NH), 7.44 (d, 1H, J5,6 ¼ 8.1 Hz,
1
(3 5754); H NMR (CDCl3): d 8.43 (br s, 1H, NH), 7.70 (d,
0
0
0
1H, J5,6 ¼ 7.9 Hz, H-6), 6.45 (d, 1H, J1 ,2 ¼ 1.5 Hz, H-1 ),
0
0
0
0
0
0
H-6), 7.32e7.21 (m, 15H, 3C6H5), 6.62 (d, 1H, J2 ,3 ¼ 9.1 Hz,
6.34 (d, 1H, J2 ,3 ¼ 10.1 Hz, H-3 ), 5.91 (dd, 1H, H-2 ), 5.75
H-30), 6.58 (s, 1H, H-10), 5.69 (m, 2H, H-20, H-5), 5.15, 5.04
(d, 1H, J5,6 ¼ 7.9 Hz, H-5), 5.29e5.26 (m, 1H, H-40), 4.31
0
0
0
0
0
0
0
0
0
(br s, 2H, methylene), 4.47 (t, 1H, J5 ,6a ¼ J5 ,6b ¼ 5.1 Hz,
(dd, 1H, J5 ,6b ¼ 5.8, J6a ,6b ¼ 12.2 Hz, H-6b ), 4.21 (dd, 1H,
H-50), 3.49 (dd, 1H, J6a ,6b ¼ 10.2 Hz, H-6a ), 3.35 (dd, 1H,
H-6b0); ESI-MS (m/z) 479.31 [M þ H]. Anal. Calcd. for
C30H26N2O4: C, 75.30; H, 5.48; N, 5.85. Found: C, 75.58;
H, 5.63; N, 5.58.
J5 ,6a ¼ 3.4 Hz, H-6a ), 4.10 (dd, 1H, H-5 ), 2.15, 2.10 (2s,
6H, 2OAc); ESI-MS (m/z) 325.17 [M þ H]. Anal. Calcd. for
C14H16N2O7: C, 51.85; H, 4.97; N, 8.64. Found: C, 51.98;
H, 4.72; N, 8.78.
0
0
0
0
0
0
0
4.1.10. 1-(4,6-Di-O-acetyl-2,3-O-isopropylidene-a-D-manno-
pyranosyl)uracil (11)
4.1.13. 1-(2,3-Dideoxy-a-D-erythro-hex-2-enopyranosyl)
uracil (14)
Compound 4 (1.20 g, 3.82 mmol) was dissolved in a mix-
ture of pyridine (14 mL) and Ac2O (2 mL, 21.20 mmol). The
reaction was carried out at room temperature for 2 h, then
was quenched with MeOH at 0 ꢀC and was concentrated.
The residue was purified by flash chromatography (EtOAc/
hexane, 7:3) to give 11 (1.26 g, 83%, Rf ¼ 0.6 in EtOAc) as
a viscous oil. [a]2D2 14.7 (c 0.140, CHCl3); UV (CHCl3):
Compound 13 (0.69 g, 2.13 mmol) was treated with ammo-
nia/MeOH (saturated at 0 ꢀC, 120 mL). The solution was
stirred overnight at room temperature and then was concen-
trated under reduced pressure. Purification by flash chroma-
tography (EtOAc/MeOH, 9:1) yielded 14 (0.33 g, 64%,
Rf ¼ 0.4), as a viscous oil. [a]2D2 16.6 (c 0.130, MeOH); UV
1
(MeOH): lmax 261 nm (3 6302); H NMR (CD3OD): d 7.69
1
0
0
lmax 261 nm (3 4326); H NMR (CDCl3): d 9.05 (br s, 1H,
(d, 1H, J5,6 ¼ 8.1 Hz, H-6), 6.38 (d, 1H, J2 ,3 ¼ 10.1 Hz, H-
NH), 7.30 (d, 1H, J5,6 ¼ 8.1 Hz, H-6), 5.78 (d, 1H, H-5),
30), 6.30 (s, 1H, H-10), 5.73 (d, 1H, H-20), 5.68 (d, 1H, H-5),
0
5.72 (d, 1H, J1 ,2 ¼ 5.5 Hz, H-1 ), 5.23 (dd, 1H, J2 ,3 ¼ 6.5 Hz,
H-20), 4.55e4.49 (m, 2H, H-30, H-40), 4.46e4.41 (m, 1H, H-
50), 4.18e4.10 (m, 2H, H-6a0, H-6b0), 2.12, 2.08 (2s, 6H,
2OAc), 1.58, 1.36 (2s, 6H, 2CH3); ESI-MS (m/z) 399.51
[M þ H]. Anal. Calcd. for C17H22N2O9: C, 51.26; H, 5.57;
N, 7.03. Found: C, 51.39; H, 5.38; N, 7.22.
4.19e4.16 (m, 1H, H-40), 3.81 (dd, 1H, J5 ,6a ¼ 1.8,
0
0
0
0
0
0
0
0
0
0
0
J6a ,6b ¼ 12.1 Hz, H-6a ), 3.71 (dd, 1H, J5 ,6b ¼ 5.1 Hz, H-
6b0), 3.43 (dd, 1H, H-50), ESI-MS: (m/z) 241.35 [M þ H].
Anal. Calcd. for C10H12N2O5: C, 50.00; H, 5.04; N, 11.66.
Found: C, 50.32; H, 5.17; N, 11.52.
4.1.14. 1-(2,3-Dideoxy-6-O-trityl-a-D-erythro-hex-2-
enopyranosyl)uracil (15)
To a solution of 14 (0.33 g, 1.36 mmol) in pyridine (7 mL)
were added triphenylmethyl chloride (0.49 g, 1.77 mmol) and
a catalytic amount of 4-dimethylaminopyridine. The reaction
mixture was stirred overnight at room temperature. After being
4.1.11. 1-(4,6-Di-O-acetyl-a-D-mannopyranosyl)uracil (12)
Compound 11 (1.26 g, 3.18 mmol) was dissolved in a mix-
ture of CH2Cl2 (11 mL) and formic acid (11 mL, 90%). The
solution was stirred overnight at room temperature and then
was concentrated under reduced pressure. The residue was

G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
1373
quenched with MeOH and concentrated, the residue was puri-
fied by flash chromatography (EtOAc/hexane, 7:3) to give 15
(0.49 g, 75%, Rf ¼ 0.65 in EtOAc/MeOH, 9.5:0.5) as a yellow-
ish solid, m.p. 185e187 ꢀC. [a]D22 10.8 (c 0.150, CHCl3); UV
(CHCl3): lmax 260 nm (3 7244); 1H NMR (CDCl3): d 8.32 (br
s, 1H, NH), 7.48 (d, 1H, J5,6 ¼ 8.1 Hz, H-6), 7.41e7.25 (m,
17 (0.08 g, 80%, Rf ¼ 0.25) as a viscous oil. [a]2D2 ꢁ4.4 (c
0.240, CHCl3); UV (CHCl3): lmax 260 nm (3 9162); 1H
NMR (CDCl3): d 8.40 (br s, 1H, NH), 7.32 (d, 1H,
0
0
0
J5,6 ¼ 8.1 Hz, H-6), 6.91 (d, 1H, J2 ,3 ¼ 8.9 Hz, H-3 ), 6.88
(s, 1H, H-10), 6.47 (d, 1H, H-20), 5.78 (d, 1H, H-5), 4.37
0
0
0
0
0
(dd, 1H, J5 ,6a ¼ 2.9, J5 ,6b ¼ 4.6 Hz, H-5 ), 4.03 (dd, 1H,
0
0
0
0
0
0
0
0
15H, 3C6H5), 6.31 (dt, 1H, J2 ,3 ¼ 10.2, J3 ,4 ¼ 1.8 Hz, H-
J6a ,6b ¼ 12.1 Hz, H-6b ), 3.97 (dd, 1H, H-6a ); ESI-MS (m/
z) 239.44 [M þ H]. Anal. Calcd. for C10H10N2O5: C, 50.42;
H, 4.23; N, 11.76. Found: C, 50.74; H, 4.38; N, 11.42.
0
30), 6.26 (d, 1H, J1 ,2 ¼ 2.1 Hz H-1 ), 5.71e5.67 (m, 2H, H-
0
0
20, H-5), 4.21 (dd, 1H, J4 ,5 ¼ 8.1 Hz, H-4 ), 3.56 (dd, 1H,
0
0
0
0
0
0
0
0
J5 ,6a ¼ 4.5, J6a ,6b ¼ 9.5 Hz, H-6a ), 3.47e3.42 (m, 1H,
0
0
0
0
J5 ,6b ¼ 7.2 Hz, H-5 ), 3.36 (dd, 1H, H-6b ); ESI-MS (m/z)
483.69 [M þ H]. Anal. Calcd. for C29H26N2O5: C, 72.18; H,
5.43; N, 5.81. Found: C, 72.37; H, 5.32; N, 5.97.
4.1.18. 1-(2,3,4-Trideoxy-4-methylene-a-D-glycero-hex-2-
enopyranosyl)uracil (18)
Compound 18 was synthesized from 10 (0.30 g,
0.63 mmol) by the similar procedure as described for 17 and
purified by flash chromatography (EtOAc/hexane, 5:5) to af-
ford pure 18 (0.12 g, 81%, Rf ¼ 0.25), as a colorless oil.
[a]2D2 ꢁ7.3 (c 0.140, CHCl3); UV (CHCl3): lmax 260 nm (3
4.1.15. 1-(2,3-Dideoxy-6-O-trityl-a-D-glycero-hex-2-
enopyranosyl-4-ulose) uracil (16)
A mixture of 15 (0.49 g, 1.02 mmol), PDC (0.46 g,
1.22 mmol) and Ac2O (0.29 mL, 3.06 mmol) was stirred in
dry CH2Cl2 (12 mL) for 4 h, under nitrogen at room tempera-
ture. Purification by flash chromatography (EtOAc/hexane,
6:4) yielded pure 16 (0.41 g, 83%, Rf ¼ 0.55 in EtOAc/hexane,
7:3) as a white solid m.p. 110e113 ꢀC; [a]D22 17.4 (c 0.250,
CHCl3); UV (CHCl3): lmax 260 nm (3 9580); 1H NMR
(CDCl3): d 8.31 (br s, 1H, NH), 7.37 (d, 1H, J5,6 ¼ 8.1 Hz,
H-6), 7.30e7.21 (m, 0 15H, 3C6H5), 7.10 (t, 1H,
1
5246); H NMR (CDCl3): d 8.40 (br s, 1H, NH), 7.36 (d,
0
0
0
1H, J5,6 ¼ 8.1 Hz, H-6), 6.65 (d, 1H, J2 ,3 ¼ 10.1 Hz, H-3 ),
6.55 (s, 1H, H-10), 5.74e5.71 (m, 2H, H-20, H-5), 5.23, 5.15
0
0
(br s, 2H, methylene), 4.50 (dd, 1H, J5 ,6a ¼ 3.6,
0
0
0
0
0
J5 ,6b ¼ 7.5 Hz, H-5 ), 3.91 (dd, 1H, J6a ,6b ¼ 12.1 Hz, H-
6b0), 3.80 (dd, 1H, H-6a0); ESI-MS (m/z) 237.31 [M þ H].
Anal. Calcd. for C11H12N2O4: C, 55.93; H, 5.12; N, 11.86.
Found: C, 55.69; H, 5.28; N, 11.72.
0
0
0
0
0
0
J1 ,2 ¼ J1 ,3 ¼ 1.5 Hz, H-1 ), 6.98 (dd, 1H, J2 ,3 ¼ 10.5 Hz,
H-30), 6.59 (dd, 1H, H-20), 5.77 (d, 1H, J5,6 ¼ 8.1 Hz, H-5),
5. Molecular modelling
0
0
0
0
0
4.41 (dd, 1H, J5 ,6a ¼ 2.5, J5 ,6b ¼ 3.6 Hz, H-5 ), 3.75 (dd,
0
0
0
0
1H, J6a ,6b ¼ 10.4 Hz, H-6b ), 3.47 (dd, 1H, H-6a ); ESI-MS
(m/z) 481.64 [M þ H]. Anal. Calcd. for C29H24N2O5: C,
72.49; H, 5.03; N, 5.83. Found: C, 72.62; H, 5.28; N, 5.61.
Computer calculations were performed on a Silicon
Graphics O2 workstation using Quanta 2005 by Accelrys
Inc. Grid Scan search was performed and the derived confor-
mations were examined for their agreement with the experi-
mental NMR data (J couplings). These conformations
represent local minima identified at the potential energy
surface.
4.1.16. 1-(2,3,4-Trideoxy-4-methylene-6-O-trityl-a-
D-glycero-hex-2-enopyranosyl)uracil (10)
To a stirred suspension of Ph3PCH3Br (0.49 g, 1.37 mmol)
and t-amyl alcohol (0.16 mL, 1.5 mmol) in dry THF (4 mL)
was added sodium hydride (0.04 g, 60% in oil, 1.5 mmol) at
0 ꢀC under nitrogen and the reaction mixture was stirred for
2 h at ambient temperature under nitrogen. To this yellow
phosphorous ylide was added a solution of 16 (0.20 g,
0.42 mmol) in dry THF (0.80 mL) dropwise, at 0 ꢀC under ni-
trogen. After the mixture was stirred for 30 min at ambient
temperature under nitrogen, the reaction mixture was
quenched with saturated sodium bicarbonate and extracted
with EtOAc. The organic layer was washed with water, dried
with sodium sulfate and evaporated. The residue was purified
by flash chromatography (EtOAc/hexane, 5:5) to give 10
(0.13 g, 65%, Rf ¼ 0.4), as a yellowish syrup.
5.1. Generating the starting conformation
The extended structures of 8, 10, 17 and 18 molecules were
built, using standard parameters for all atoms, and then mini-
mized with a succession of three methods [59,60]: Steepest
Descents (SD) algorithm to remove unfavorable steric contacts
then conjugate gradients (CONJ) to find its local minimum,
followed by Powell method to reach the local minimum.
CHARMm [61] force field was employed for all energy min-
imizations. The energy convergence criterion was root mean
˚
square (RMSD) force ꢂ0.001 kcal molꢁ1
A
ꢁ1. The resulting
conformations were used as initial following by systematic
Grid Scan search.
4.1.17. 1-(2,3-Dideoxy-a-D-glycero-hex-2-enopyranosyl-
4-ulose)uracil (17)
5.2. Grid Scan search
Compound 16 (0.20 g, 0.42 mmol) was dissolved in a mix-
ture of formic acid/diethyl ether (11 mL, 1:1). The mixture
was stirred for 7 min at room temperature, diluted with Tol
and co-distilled several times with the same solvent to avoid
ester formation [58]. The concentrated residue was purified
by flash chromatography (EtOAc/hexane, 5:5) to afford pure
The lowest energy conformations, obtained from each clus-
ter, were further explored using systematic Grid Scan search.
Intervals of 10ꢀ were applied for the single bond rotation
0
(C1 eN1 and C5 eC6 ) following by energy minimization us-
0
0
ing 2000 steps of Steepest Descents algorithm. Finally, the

1374
G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
lowest energy conformation for each molecule was further used
in order to calculate the selected torsion angles (Table 1) and
was checked for consistency with NMR J couplings.
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 (%, ꢃSD) of absorbances in virus-
infected wells (n ¼ 6) compared to those in control (only
virus infected) wells (n ¼ 6). The minimal inhibitory con-
centration (IC50) of the tested compounds was obtained
from the concentrationeeffect curve.
6. Methods for measurement of biological activity
6.1. Cells and culture conditions
The human colonic adenocarcinoma Caco-2 cells were
a generous gift of Dr. Rene L’Harridon, INRA, VIM, Jouy-
en-Josas, France; human foetal small intestine cell line H4,
breast carcinoma cell line MCF-7 and skin melanoma cell
line were used. Cells were grown in Dulbecco’s modified Ea-
gle’s medium (DMEM, SigmaeAldrich, Grand Island, USA),
supplemented with 5% foetal calf serum (Cambrex, Verviers,
Belgium), L-glutamine (2 mmol/L, Sigma, St. Louis, USA),
penicillin (100 U/mL, Sigma, St. Louis, USA) and streptomy-
cin (1 mg/mL, Fluka, Buchs, Switzerland) at 37 ꢀC in 5% car-
bon dioxide (CO2) atmosphere in tissue culture flasks until
confluent. Cell culture medium was regularly changed.
(ii) The neutralization of the virus in solution before attach-
ment: 3-fold dilutions of each tested compound (initial
concentration of 0.5 mg/mL) were first pre-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 af-
ter 72 h post-infection for rotavirus. Rotavirus alone was
treated in the same way as the control. After 72 h of in-
cubation, 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 expressed, for each dilution, by the mean ratios
(%, ꢃSD) of absorbances in virus-infected wells
(n ¼ 6) in comparison to those in control (only virus in-
fected) wells (n ¼ 6). The minimal inhibitory concentra-
tion (IC50) of the tested compounds was obtained from
the concentrationeeffect curve.
6.2. Unsaturated exomethylene pyranonucleosides
Stock drug solutions were freshly prepared in sterile
dimethyl sulfoxide (DMSO) at the concentration of 0.5 mg/
mL. The final concentration of DMSO in the cell culture
medium was less than 0.1%. All solutions were protected
against light.
AZT (RetrovirÒ) GlaxoSmithKline, USA, a drug used for
antiretroviral therapy (ART) was used as a standard compound
in antiviral experiments and 5FU as a standard compound in
antitumor experiments. Solutions were prepared in the same
way as those of unsaturated exomethylene pyranonucleosides.
6.5. Growth inhibition assay
It was performed on Caco-2 cell line by modified method
described previously [63]. Briefly, in 96-well plates, six wells of
3-fold dilutions of each compound (initial concentration of
0.5 mg/mL) were applied to monolayers of 10 cells/well in
DMEM/10% foetal bovine serum. Incubation was performed 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 (%, ꢃSD) of colony number in
treated wells (n ¼ 2) in contrast with those in control wells
(n ¼ 24). The minimalinhibitoryconcentration (IC50)ofthetested
compounds was obtained from the concentrationeeffect curve.
6.3. Virus propagation
Rotavirus RF strain was propagated on Caco-2 monolayers
in the presence of trypsin (1 mg/mL of DMEM) as described
previously [62]. Supernatant containing the virus was col-
lected from the flasks when cytopathic effect (CPE) was ob-
served (24e48 h at 37 ꢀC) by microscopy and clarified by
centrifugation. Virus was stored at ꢁ70 ꢀC until used. For
the antiviral assay, virus with 1.5 tissue culture infective
dose 50% U/mL (TCID50/mL) was used (100 mL per well).
6.6. Cytotoxicity assay
6.4. Antiviral assay
Caco-2, H4, MCF-7, and skin melanoma cells (6 ꢄ 106 cells/
plate) were seeded in P 96 plate and treated with the compounds
at 3-fold serial dilutions of each compound (initial concentration
of 0.5 mg/mL). Then, the cells were incubated at37 ꢀC inthe hu-
midified incubator for 72 h. 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 (%, ꢃSD) of absor-
bances in treated wells (n ¼ 2) compared to those in control
wells (n ¼ 24). The minimal inhibitory concentration (CC50)
of the tested compounds was obtained from the concentra-
tioneeffect curve.
The potential antiviral activity of the newly synthesized
compounds was tested against rotavirus by investigating:
(i) The inhibition of infectivity following virus attachment:
Washed monolayer Caco-2 cells were first incubated
with rotavirus for 1 h at 37 ꢀC in the presence of 5%
CO2 (time for virus to attach to cell membrane receptors).
After incubation, the remaining virus was washed off
with DMEM without supplements and monolayer was
treated immediately with the nucleosides added in 3-fold

G. Agelis et al. / European Journal of Medicinal Chemistry 43 (2008) 1366e1375
1375
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Acknowledgments
[28] T. Halmos, D. Komiotis, K. Antonakis, Carbohydr. Res. 145 (1985)
163e168.
This work was supported in part by the Greek General Sec-
retariat for Research and Technology Program No. 3245C and
bilateral scientific-technology project between Greece and
Slovenia. The authors are grateful to Dr. K. Antonakis (Ec.
Nat. Sup. de Chimie de Paris; CNRS-FRE 2463) for encourag-
ing this work and to Dr. T. Halmos (Ec. Nat. Sup. de Chimie
de Paris) for the unstinted and fruitful aid, as well as ELPEN
pharmaceuticals for financial support.
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