Synthesis and biological evaluation of 3′-c-ethynyl and 3′-c-(1,4-disubstituted-1,2,3-triazolo) double-headed pyranonucleosides

Article abstract of DOI:10.2174/157340612800786624

A novel series of 3′-C-ethynyl and 3′-C-(1,4-disubstituted-1,2, 3-triazolo) double-headed pyranonucleosides has been designed and synthesized. Reaction of 3-keto glucoside 1 with ethynyl magnesium bromide gave the desired precursor 3-C-ethynyl-1,2:5,6-di-O-isopropylidene-α-D-allofuranose (2). Hydrolysis followed by acetylation led to the 1,2,4,6-tetra-O-acetyl-3-C- ethynyl-β-D-allopyranose (3). Compound 3 was condensed with silylated 5-fluorouracil, uracil, thymine, N⁴-benzoylcytosine and N ⁶-benzoyladenine, respectively and deacetylated to afford the target 1-(3′-C-ethynyl-β-D-allopyranosyl)nucleosides 5a-c,f,g. Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction was utilized to couple the 3′-C-ethynyl pyranonucleoside derivatives with azidoethyl adenine, 5-fluorouracil and thymine, respectively to afford novel triazole double-headed nucleoside analogues 8a-h. 3′-C-Ethynyl pyranonucleosides and the new doubleheaded analogues were evaluated for their antiviral and cytostatic activities. Although none of the compounds showed pronounced cytostatic activity and were devoid of a significant antiviral potential, the double-headed nucleoside derivatives 8a, 8c and 8e showed a moderate cytostatic activity against human cervix carcinoma HeLa cells which may be the basis for the synthesis of analogous derivatives with improved cytostatic potential.

Full text of DOI:10.2174/157340612800786624

320
Medicinal Chemistry, 2012, 8, 320-329
Synthesis and Biological Evaluation of 3ꢀ-C-Ethynyl and 3ꢀ-C-(1,4-
disubstituted-1,2,3-triazolo) Double-Headed Pyranonucleosides
Christos Kiritsis1, Stella Manta1, Ioannis Papasotiriou2, Evdoxia Coutouli-Argyropoulou3,
Sakellarios Trakossas3, Jan Balzarini4 and Dimitri Komiotis1,*
1Department of Biochemistry and Biotechnology, Laboratory of Bio-organic Chemistry, University of Thessaly, 41221
Larissa, Greece
2Research Genetic Cancer Center (R.G.C.C. Ltd.), Megalou Alexandrou 155 str. GR–53070 Filotas, Greece
3Department of Chemistry, Laboratory of Organic Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124,
Greece
4Rega Institute for Medical Research, Katholieke Universtiteit Leuven, 3000 Leuven, Belgium
Abstract: A novel series of 3ꢀ-C-ethynyl and 3ꢀ-C-(1,4-disubstituted-1,2,3-triazolo) double-headed pyranonucleosides
has been designed and synthesized. Reaction of 3-keto glucoside 1 with ethynyl magnesium bromide gave the desired
precursor 3-C-ethynyl-1,2:5,6-di-O-isopropylidene-ꢀ-D-allofuranose (2). Hydrolysis followed by acetylation led to the
1,2,4,6-tetra-O-acetyl-3-C-ethynyl-ꢁ-D-allopyranose (3). Compound 3 was condensed with silylated 5-fluorouracil, uracil,
thymine, N4-benzoylcytosine and N6-benzoyladenine, respectively and deacetylated to afford the target 1-(3ꢀ-C-ethynyl-ꢁ-
D-allopyranosyl)nucleosides 5a-c,f,g. Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) reaction was utilized to
couple the 3ꢀ-C-ethynyl pyranonucleoside derivatives with azidoethyl adenine, 5-fluorouracil and thymine, respectively to
afford novel triazole double-headed nucleoside analogues 8a-h. 3ꢀ-C-Ethynyl pyranonucleosides and the new double-
headed analogues were evaluated for their antiviral and cytostatic activities. Although none of the compounds showed
pronounced cytostatic activity and were devoid of a significant antiviral potential, the double-headed nucleoside deriva-
tives 8a, 8c and 8e showed a moderate cytostatic activity against human cervix carcinoma HeLa cells which may be the
basis for the synthesis of analogous derivatives with improved cytostatic potential.
Keywords: Branched-chain nucleosides, C-ethynyl pyranonucleosides, click chemistry, double-headed nucleosides.
INTRODUCTION
Last decade, nucleosides bearing pyranosyl ring have
been evaluated for their potential anticancer [14-16], anti-
oxidant [17,18] and antibiotic [19] properties and as building
blocks in nucleic acid synthesis [20]. In seeking to investi-
gate novel biologically active agents, we recently reported
that new classes of uncommon 3ꢀ-fluorinated and 3ꢀ-C-
cyano pyranonucleosides proved to be efficient as tumor cell
growth inhibitors and showed to have a promising potential
in combating rotaviral infections [21-25]. Experimental evi-
dence also disclosed that human poly(A)-specific ribonucle-
ase [26] and glycogen phosphorylase [27] are among the
molecular targets of these compounds.
Modified nucleosides have emerged as important thera-
peutic agents for the development of antiviral and anti-tumor
drugs [1-4]. During the last decades, many modifications of
the base and/or sugar moiety of natural nucleosides have
been attempted, in order to discover novel derivatives en-
dowed with potential biological properties.
Recently, considerable attention has been paid to furano-
nucleosides containing an ethynyl group in their carbohy-
drate backbone as a result of their significant biological ac-
tivity. Branched C-ethynyl nucleoside analogues showed
antiviral properties against herpes simplex virus type-1
(HSV-1) and human immunodeficiency virus type-1 (HIV-1)
[5-8], while 3ꢀ-C-ethynyl-branched nucleoside analogues
have been reported to exhibit potent and broad spectrum
anti-cancer activities [9-11]. Among them, the 1-(3ꢀ-C-
With the above applications in mind and as an extension
of our studies, we decided to synthesize novel pyranonucleo-
sides in which the fluorine atom or the cyano group will be
emplaced by the biologically promising ethynyl group.
Therefore, we report herein on the preparation and biological
evaluation of 3ꢀ-C-ethynyl-ꢁ-D-allopyranonucleoside ana-
logues, containing 5-fluorouracil, uracil, thymine, cytosine
and adenine as base moieties. The presence of the triple bond
of C-ethynyl-pyranonucleosides, provided an excellent op-
portunity to further synthesize a novel class of double-
headed pyranonucleosides, via CuAAC reaction [28-31] with
various purine or pyrimidine azides. Thus, in the present
study, we also describe the utilization of 3ꢀ-C-ethynyl-ꢁ-D-
ethynyl-ꢁ-D-ribo-pentofuranosyl)cytosine,
RNA-directed nucleoside anti-metabolite, is currently under-
going clinical trials [12,13].
a
promising
*Address correspondence to this author at the University of Thessaly, De-
partment of Biochemistry & Biotechnology, Laboratory of Bio-organic
Chemistry, 26 Ploutonos Str., 41221 Larissa, Greece; Tel: +30 2410
565285; Fax: +30 2410 565290; E-mail: dkom@bio.uth.gr
1875-6638/12 $58.00+.00
© 2012 Bentham Science Publishers

Synthesis and Biological Evaluation of 3ꢀ-C-Ethynyl and 3ꢀ-C-(1,4-disubstituted-1,2,3-triazolo)
Medicinal Chemistry, 2012, Vol. 8, No. 3 321
CH
OAc
OR
O
O
O
O
O
O
O
O
AcO
HC
Me2C
RO
HC
Me2C
ii
iii
i
OAc
B
O
O
O
O
O
OAc
OR
OH
CMe2
CMe2
OH
OH
1
4a-e:
R=Ac
2
3
iv
5a-c,f,g: R=H
v
CH
OH
O
O
vi
O
O
O
O
O
B
HO
Me2C
Me2C
O
O
O
O
OH
O
MeOCC-O
O
CMe2
CMe2
CH
II
CH
I
III
B=a: 5-fluorouracil, b: uracil, c: thymine, d: N4-benzoylcytosine, e: N6-benzoyladenine, f: cytosine, g: adenine
Scheme 1. (i) HCꢁCMgBr, THF; (ii) a) H2O, MeOH, Amberlite IR 120(H+); b) Ac2O, pyridine; (iii) Silylated base, CH3CN,
Me3SiOSO2CF3; (iv) Methanolic ammonia. (v) Methoxalyl chloride, triethylamine, 4-Dimethylaminopyridine, CH2Cl2; (vi) tributyltin hy-
dride, 2,2ꢀ-azobis(isobutyronitrile), toluene, 100 oC.
allopyranonucleosides as synthons for newly uncommon
double-headed nucleosides and supply data on their biologi-
cal activity.
5a-c,f,g, in good yields (72-78%). The IR spectra of the
newly synthesized nucleosides 5 exhibited characteristic
absorption bands at 2115-2130 cm-1 due to the presence of
the ethynyl group.
CHEMISTRY
3ꢀ-C-Ethynyl-ꢀ-D-allopyranonucleosides 5a-c,f,g were
prepared according to the synthetic route outlined in Scheme
1. Reaction of known furanose 1 [32] with HCꢁCMgBr in
tetrahydrofuran (THF) afforded 3-C-ethynyl-1,2:5,6-di-O-
isopropylidene-ꢁ-D-allofuranose (2), with high stereoselec-
tivity [33]. The assignment of its configuration was further
supported by NOE measurements, as depicted in Fig. (1).
The mutual NOE enhancements observed between ethynyl
proton with both H-2 and H-5 show that all these protons are
in the ꢀ face of the furanose ring. Similarly, the positive
NOE observed between the free OH group with H-4 shows
also that these protons are on the same side of the ring sys-
tem. Hydrolysis of 2 using Amberlite IR 120 (H+) resin fol-
lowed by direct standard acetylation gave the 1,2,4,6-tetra-
4%
3%
H
2%
H
2%
H
H
O
O
O
H
O
OH O
2%
5%
2
Fig. (1). NOE enhancements measured on compound 2.
To further explore structure-activity relationships, the
synthesis of a new class of 3ꢀ-deoxy-3ꢀ-C-ethynyl pyranonu-
cleosides III was undertaken (Scheme 1). Although alcohol
2 was easily converted to methyloxalate ester I, all attempts
to deoxygenate the latter were unsuccessful. It seems that the
presence of the ethynyl group destabilizes the planar radical
intermediate formed and deoxygenation reaction leads to
intractable materials.
1
O-acetyl-3-C-ethynyl-ꢀ-D-allopyranose (3). H NMR spectra
of analogue 3 –in accordance with mass spectroscopy data–
revealed the presence of only four acetoxy groups, suggest-
ing that its tertiary hydroxyl was not acetylated [34]. Com-
pound 3 was readily converted into the protected 1-(2ꢀ,4ꢀ,6ꢀ-
tri-O-acetyl-3ꢀ-C-ethynyl-ꢀ-D-allopyranosyl) nucleosides 4a-
e, upon reaction with silyl-protected 5-fluorouracil, uracil,
thymine, N4-benzoylcytosine and N6-benzoyladenine, respec-
tively, in the presence of trimethylsilyl trifluoromethane-
Compounds 4a-e and 5a were used as key intermediates
to prepare 1,2,3-triazole double-headed nucleoside analogues
8a-h (Scheme 2). CuAAC reaction between azidoethyl ade-
nine 6a and C-ethynyl pyranonucleosides 4b-e, performed
with catalytic amounts of CuSO4·5H2O and sodium ascor-
bate in refluxing THF/H2O (1:1), afforded the protected tria-
zole double-headed nucleoside analogues 7a-d. Removal of
all O-acetyl protecting groups of 7a-d with saturated metha-
nolic ammonia gave 8a-d, respectively. By the same manner,
1
sulfonate (Me3SiOSO2CF3) as catalyst [35]. As expected, H
NMR spectra of 4a-e showed a large coupling between pro-
tons H-1ꢀ and H-2ꢀ (J1ꢀ,2ꢀ = 9.2-9.3 Hz), indicating an axial
orientation of both protons and an equatorially oriented base
ring. Removal of all protecting groups of 4a-e, with satu-
rated methanolic ammonia [36], furnished the target products

322 Medicinal Chemistry, 2012, Vol. 8, No. 3
Kiritsis et al.
NH2
N
N
O
N
N
NH
OR
OH
N
O
O
RO
N
N
OR
NH2
NH2
N
ii
7a: R=Ac
N
N
O
N
N
N
N
O
N
N
8a: R=H
F
NH
OH
OH
N
NH
OR
OH
N
O
O
4b
i
HO
O
O
RO
N
OH
N
OR
N
N
5a
4c
i
N
N
8e
NH2
N
7b: R=Ac
8b: R=H
ii
iii
N
N
N
N3
4e
6a
4d
NH2
i
NH2
i
N
NHR2
N
N
N
NHR2
NH
N
N
N
N
N
OR1
OH
OR1
OH
N
N
O
O
R1O
N
O
R1O
N
N
N
OR1
OR1
N
N
N
N
7c: R1= Ac ,R2= COPh
8c: R1= R2= H
7d: R1=Ac ,R2=COPh
ii
ii
8d R1= R2= H
O
N
O
N
O
N
F
F
F
O
N
O
HN
HN
NH
F
4a
i
4b
i
NH
NH
O
O
OR
OR
O
O
O
O
N
O
RO
RO
N3
N
N
OR
OH
OR
6b
OH
N
N
N
N
7f: R=Ac
8f: R=H
7g: R=Ac
8g: R=H
ii
ii
O
N
O
O
N
NH
HN
4b
NH
O
O
N
OR
OH
O
i
O
RO
N3
N
OR
6c
N
N
7h: R=Ac
8h: R=H
ii
Scheme 2. (i) THF, H2O, CuSO4·5H2O, sodium ascorbate; ii) Methanolic ammonia; iii) CH3CN, H2O, copper wire.
CuAAC reaction of azidoethyl fluorouracil 6b with protected
C-ethynyl derivatives 4a,b and of azidoethyl thymine 6c
with uracil derivative 4b, respectively followed by subse-
quent deacetylation of the nucleosides formed 7f,g,h, yielded
the target compound 8f,g,h, in very good yields (83-86%). In
the case of triazole derivative 8e, a modified click cycloaddi-

Synthesis and Biological Evaluation of 3ꢀ-C-Ethynyl and 3ꢀ-C-(1,4-disubstituted-1,2,3-triazolo)
Medicinal Chemistry, 2012, Vol. 8, No. 3 323
Table 1. Cytostatic Activity of 5a-c,f,g and 8a-h Against a Panel
of Tumor Cell Lines
lymphocyte CEM tumor cell cultures (Table 1). Compounds
8a, 8c and 8e showed a moderate cytostatic activity against
human cervix carcinoma HeLa cells (IC50: 89-115 ꢁM)
which may be the basis for preparing closely related deriva-
tives with improved cytostatic potential.
a
IC50 (ꢀM)
Compound
L1210
CEM
HeLa
EXPERIMENTAL SECTION
Chemistry
5a
5b
5c
5f
> 200
> 200
> 200
> 200
> 200
> 200
> 250
> 200
> 250
> 200
> 250
> 250
> 250
> 250
> 200
> 200
> 200
General Procedures
> 200
> 200
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 chromatog-
raphy was performed using silica gel (240-400 mesh,
Merck). Microanalyses were performed on a Perkin-Elmer
2400-II Analyzer. Analyses were within ± 0.4% of the calcu-
lated values. 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 chloroform-d
(CDCl3), methanol-d4 (CD3OD), and dimethyl sulfoxide-d6
(DMSO-d6). Chemical shifts are reported in parts per million
(ꢀ) downfield from tetramethylsilane (TMS) as internal stan-
dard. UV-Vis spectra were recorded on a PG T70 UV-VIS
spectrometer and mass spectra were obtained with a Micro-
mass Platform LC (ESIMS). Optical rotations were meas-
ured using Autopol I polarimeter. Infrared spectra were ob-
tained with a Thermo Scientific Nicolet IR100 FT-IR spec-
trometer.
> 200
> 200
5g
8a
8b
8c
8d
8e
8f
> 250
244 ± 9
89 ± 1
ꢅ 250
> 200
188 ± 22
> 200
115 ± 7
> 250
> 250
129 ± 94
> 250
94 ± 43
182 ± 95
ꢅ 250
8g
8h
> 250
> 200
> 200
a50% inhibitory concentration, or compound concentration required to inhibit cell
proliferation by 50%.
tion method was employed. Thus, reaction of the azide 6a
and unprotected analogue 5a catalyzed by copper wire, in
refluxing CH3CN/H2O (9:1), gave the double-headed nucleo-
side 8e. All triazole cycloadducts were well characterized by
NMR, UV spectroscopies, mass spectrometry and elemental
With the exception of CuAAC all reactions were carried
out in dry solvents. Acetonitrile and toluene were distilled
from calcium hydride and stored over 3E molecular sieves.
N,N-Dimethylformamide (DMF) was stored over 3E mo-
lecular sieves. THF was freshly distilled under nitrogen from
sodium/benzophenone before use. All reactions sensitive to
oxygen or moisture were carried out under nitrogen atmos-
phere.
1
analysis. H NMR data, revealed the resonance of H-C(5) of
the triazole ring in the aromatic region, while 13C NMR spec-
tra showed the C-atom signals corresponding to the triazole
derivatives. Due to the broadening of the peaks some of the
1H NMR spectra were recorded at higher temperatures (45-
o
3-C-Ethynyl-1,2:5,6-di-O-isopropylidene-ꢁ-D-allofuranose
(2)
55 C). The proton assignment was supported in most cases
by H,H-COSY experiments, whereas those of carbon by
DEPT experiments. The characterization of the obtained
cycloadducts as 1,4-regioisomers was made in comparison
with their chemical shifts with other analogous 4-substituted
triazoles [28]. ꢀhe most informative peak is that of the CH-
triazole carbon which appears at a very narrow range (ꢀ
124.5-125.0). It should also be mentioned that in all cases,
transformation into the triazole cycloadducts was quantita-
tive and no byproducts were observed, while copper-
catalyzed click reactions directed the formation of only one
regioisomer (1,4-regioisomer is formed out of the two possi-
bilities) [29, 37].
A THF solution of HCꢂCMgBr (186 mL, 0.5 M in THF,
93 mmol) was added to a solution of 1 (4 g, 15.50 mmol) in
THF (155 mL) at room temperature. The mixture was stirred
for 60 min, and aqueous NH4Cl (1M, 100 mL) was added.
The mixture was diluted with ethyl acetate (EtOAc) (800
mL), washed with brine (400 mL), dried with sodium sul-
phate, and evaporated. The residue was purified by flash
chromatography (EtOAc/hexane, 3:7) to give compound 2
o
(3.74 g, 85%) as a white solid: mp 104-105 C; Rf= 0.51
(EtOAc/hexane 4:6); [ꢃ]D +3 (c 0.5, CHCl3); 1H NMR
22
(CDCl3, 300 MHz): ꢀ 5.83 (d, 1H, J1,2 = 3.5 Hz, ꢄ-1), 4.63
(d, 1H, ꢄ-2), 4.44 (ddd, J4,5 = 8.2 Hz, J5,6a = 4.6 Hz, J5,6b= 6.2
Hz, 1H, ꢄ-5), 4.15 (dd, 1H, J6a,6b = 8.8 Hz, H-6a), 4.04 (dd,
1H, H-6b), 3.87 (d, 1H, H-4), 3.12 (br s, 1H, OH), 2.68 (s,
1H, HCꢂC), 1.61 (s, 3H, CH3), 1.47 (s, 3H, CH3), 1.38 (s,
6H, 2 CH3). ESIMS: m/z 285.3 [M+H+].
BIOLOGICAL EVALUATION
The compounds have been evaluated against a broad
panel of DNA and RNA viruses, but found to be inactive at
the highest tested concentration (100-200 ꢁM). Also, none of
the compounds showed pronounced cytostatic or cytotoxic
activity in the monolayer cell cultures used for the antiviral
assays, as well as in the murine leukemia L1210 and human
1,2,4,6-Tetra-O-acetyl-3-C-ethynyl-ꢂ-D-allopyranose (3)
To a solution of 2 (3.74 g, 13.16 mmol) in methanol
(MeOH) (20.5 mL) and H2O (116.9 mL) was added Amber-

324 Medicinal Chemistry, 2012, Vol. 8, No. 3
Kiritsis et al.
lite IR 120 (H+) resin and the mixture was refluxed over-
night. The reaction mixture was filtered and evaporated to
dryness. The residue was then dissolved in a mixture of
pyridine (46.1 mL) and acetic anhydride (Ac2O) (23.7 mL).
The reaction was carried out at room temperature for 1 hour,
then was quenched with MeOH at 0 oC and was concentrated
in vacuum. The residue was diluted with EtOAc and washed
with saturated sodium bisulfate, sodium bicarbonate and
water. The organic extract was dried over anhydrous sodium
sulfate, filtered and evaporated to dryness to give compound
3 (4.07 g, 83%) as a white foam: Rf= 0.41 (EtOAc/hexane
1:1); [ꢀ]D22 -6 (c 0.5, CHCl3); 1H NMR (CDCl3, 300 MHz): ꢀ
5.99 (d, 1H, J1,2 = 8.3 Hz, ꢁ-1), 5.29 (d, 1H, J4,5 = 9.9 Hz, ꢁ-
6.09 (d, 1H, J1ꢄ,2ꢄ = 9.2 Hz, H-1ꢄ), 5.24-5.18 (m, 2H, H-2ꢄ, H-
4ꢄ), 4.29-4.21 (m, 2H, H-6aꢄ, H-6bꢄ), 4.09-4.07 (m, 1H, H-
5ꢄ), 2.55 (s, 1H, HCꢂC), 2.17, 2.12, 2.10 (3s, 9H, 3OAc),
1.95 (s, 3H, 5-CH3). ESIMS: m/z 439.4 [M+H+].
Data
for
1-(2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-ethynyl-ꢁ-D-
allopyranosyl)N4-benzoylcytosine (4d)
o
Yield 68%; mp 124ꢅ125 C; Rf= 0.32 (EtOAc/hexane,
22
1
1:1); [ꢀ]D +10 (c 0.5, CHCl3); ꢂmax 262 nm (ꢃ 17613); H
NMR (CDCl3, 300 MHz): ꢀ 7.94-7.49 (m, 7H, Bz, H-5 and
H-6), 6.32 (d, 1H, J1ꢄ,2ꢄ = 9.3 Hz, H-1ꢄ), 5.29-5.14 (m, 2H, H-
2ꢄ, H-4ꢄ), 4.30-4.22 (m, 2H, H-6aꢄ, H-6bꢄ), 4.08-4.05 (m,
1H, H-5ꢄ), 2.52 (s, 1H, HCꢂC), 2.15, 2.09, 2.06 (3s, 9H,
3OAc). ESIMS: m/z 528.5 [M+H+].
4), 5.23 (d, 1H, ꢁ-2), 4.30 (dd, 1H, J6a,6b = 12.3 Hz, J5,6a
=
4.2 Hz, H-6a), 4.25 (ddd, 1H, J5,6b = 2.0 Hz, H-5), 4.09 (dd,
1H, H-6b), 2.76 (br s, 1H, OH), 2.47 (s, 1H, HCꢂC), 2.17,
2.16, 2.11, 2.10 (4s, 12H, 4OAc). ESIMS: m/z 373.3
[M+H+].
Data
for
9-(2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-ethynyl-ꢁ-D-
allopyranosyl)N6-benzoyladenine (4e)
22
Yield 59%; Rf= 0.14 (EtOAc/hexane, 1:1); [ꢀ]D -10 (c
0.5, CHCl3); ꢂmax 280 nm (ꢃ 18662); H NMR (CDCl3, 300
1
General Procedure for Preparation of 3ꢀ-C-ethynyl-ꢁ-D-
allopyranonucleosides 4a-e
MHz): ꢀ 9.08 (br s, 1H, NH), 8.89 and 8.25 (2s, 2H, H-2 and
H-8), 8.06-7.53 (m, 5H, Bz), 6.21 (d, 1H, J1ꢄ,2ꢄ = 9.3 Hz, H-
1ꢄ), 5.64 (d, 1H, H-2ꢄ), 5.41 (d, 1H, J4ꢄ,5ꢄ = 9.5 Hz, H-4ꢄ),
4.38-4.28 (m, 2H, H-6aꢄ, H-6bꢄ), 4.14-4.10 (m, 1H, H-5ꢄ),
2.58 (s, 1H, HCꢂC), 2.19, 2.10, 1.87 (3s, 9H, 3OAc).
ESIMS: m/z 552.5 [M+H+].
A mixture of the proper base (2.25 mmol), hexamethyld-
isilazane (HMDS) (2.79 mmol) and saccharine (0.104 mmol)
in anhydrous CH3CN (8.8 mL) was refluxed for 30 min un-
der
nitrogen.
1,2,4,6-Tetra-O-acetyl-3-C-ethynyl-ꢁ-D-
allopyranose (3) (1.61 mmol) and Me3SiOSO2CF3 (2.25
mmol) were then added and the reaction mixture was re-
fluxed for 2 hours, cooled, neutralized with aqueous sodium
bicarbonate, and extracted with CH2Cl2 (400 mL). The or-
ganic extract was dried over anhydrous sodium sulfate, fil-
tered and evaporated to dryness. The residue was purified by
flash chromatography (3:7 EtOAc/hexane) to give com-
pounds 4a-d as white solids and compound 4e as a white
foam.
General Procedure for Preparation of 3ꢀ-C-ethynyl-ꢁ-D-
allopyranonucleosides 5a-c,f,g
The protected nucleosides 4 (1.37 mmol) were treated
with ammonia/MeOH (saturated at 0 °C, 57.3 mL). The solu-
tion was stirred overnight at room temperature and then was
concentrated under reduced pressure. The residue was puri-
fied by flash chromatography (EtOAc) to afford pure 5 as
white foams.
Data
for
1-(2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-ethynyl-ꢁ-D-
Data for 1-(3ꢀ-C-ethynyl-ꢁ-D-allopyranosyl)5-fluorouracil
(5a)
allopyranosyl)5-fluorouracil (4a)
o
22
Yield 85%; mp 108-110 C; Rf= 0.59 (EtOAc/hexane
Yield 72%; Rf= 0.50 (EtOAc/MeOH, 9:1); [ꢀ]D +10 (c
1:1); [ꢀ]D22 -6 (c 0.4, CHCl3); ꢂmax 264 nm (ꢃ 6575); 1H NMR
(CDCl3, 300 MHz): ꢀ 8.37 (br s, 1H, NH), 7.43 (d, 1H, J6,F5
= 5.8 Hz, H-6), 6.03 (dd, 1H, J1ꢄ,2ꢄ = 9.2 Hz, J1ꢄ,F5 = 1.2 Hz,
H-1ꢄ), 5.19 (d, 1H, J4ꢄ,5ꢄ = 9.6 Hz, H-4ꢄ), 5.08 (d, 1H, H-2ꢄ),
4.28ꢅ4.17 (m, 2H, H-6aꢄ, H-6bꢄ), 4.07ꢅ4.04 (m, 1H, H-5ꢄ),
2.54 (s, 1H, HCꢂC), 2.14, 2.10, 2.09 (3s, 9H, 3OAc). ESIMS
m/z 443.4 [M+H+].
0.3, CD3OD); ꢂmax 264 nm (ꢃ 8689); H NMR (CD3OD, 300
MHz): ꢀ 8.00 (d, 1H, J6,F5 = 6.6 Hz, H-6), 5.84 (d, 1H, J1ꢄ,2ꢄ
9.0 Hz, H-1ꢄ), 3.87-3.68 (m, 5H, H-2ꢄ, H-4ꢄ, H-5ꢄ, H-6aꢄ, H-
6bꢄ), 2.89 (s, 1H, HCꢂC); 13C NMR (CD3OD, 75.5 MHz): ꢀ
158.01, 150.12, 140.91, 127.33, 91.29, 84.71, 79.43, 75.06,
73.52, 72.79, 72.28, 61.83; IR (Nujol, cm-1): 2115 (CꢂC).
MS: m/z 317.2 [M+H+].
1
=
Data
for
1-(2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-ethynyl-ꢁ-D-
Data for 1-(3ꢀ-C-ethynyl-ꢁ-D-allopyranosyl)uracil (5b)
allopyranosyl)uracil (4b)
22
Yield 78%; Rf= 0.25 (EtOAc/MeOH, 9:1); [ꢀ]D +26 (c
o
1
Yield 82%; mp 97-99 C; Rf= 0.34 (EtOAc/hexane 1:1);
0.5, CD3OD); ꢂmax 259 nm (ꢃ 5220); H NMR (CD3OD, 300
MHz): ꢀ 7.71 (d, 1H, J5,6 = 8.1 Hz, H-6), 5.82 (d, 1H, J1ꢄ,2ꢄ =
9.3 Hz, H-1ꢄ), 5.72 (d, 1H, H-5), 3.86-3.65 (m, 5H, H-2ꢄ, H-
4ꢄ, H-5ꢄ, H-6aꢄ, H-6bꢄ), 2.85 (s, 1H, HCꢂC); 13C NMR
(CD3OD, 75.5 MHz): ꢀ 164.26, 150.13, 142.48, 102.09,
91.66, 83.42, 81.36, 74.97, 73.67, 72.88, 72.01, 61.03; IR
(Nujol, cm-1): 2120 (CꢂC). ESIMS: m/z 299.3 [M+H+].
22
1
[ꢀ]D +2 (c 0.5, CHCl3); ꢂmax 257 nm (ꢃ 5346); H NMR
(CDCl3, 300 MHz): ꢀ 8.14 (br s, 1H, NH), 7.35 (d, 1H, J5,6
=
8.1 Hz, H-6), 6.07 (d, 1H, J1ꢄ,2ꢄ = 9.3 Hz, H-1ꢄ), 5.78 (d, 1H,
H-5), 5.19 (d, 1H, J4ꢄ,5ꢄ = 9.7 Hz, H-4ꢄ), 5.14 (d, 1H, H-2ꢄ),
4.28-4.17 (m, 2H, H-6aꢄ, H-6bꢄ), 4.06-4.03 (m, 1H, H-5ꢄ),
2.53 (s, 1H, HCꢂC), 2.14, 2.09, 2.08 (3s, 9H, 3OAc).
ESIMS: m/z 425.4 [M+H+].
Data for 1-(3ꢀ-C-ethynyl-ꢁ-D-allopyranosyl)thymine (5c)
Data
for
1-(2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-ethynyl-ꢁ-D-
22
Yield 75%; Rf= 0.39 (EtOAc/MeOH, 9:1); [ꢀ]D +11 (c
allopyranosyl)thymine (4c)
0.6, CD3OD); ꢂmax 262 nm (ꢃ 8776); 1H NMR (CD3OD, 300
MHz): ꢀ 7.56 (s, 1H, H-6), 5.81 (d, 1H, J1ꢄ,2ꢄ = 9.3 Hz, H-1ꢄ),
3.86-3.66 (m, 5H, H-2ꢄ, H-4ꢄ, H-5ꢄ, H-6aꢄ, H-6bꢄ), 2.85 (s,
1H, HCꢂC), 1.94 (s, 3H, CH3); 13C NMR (CD3OD, 75.5
o
Yield 72%; mp 94-96 C; Rf= 0.46 (EtOAc/hexane, 1:1);
22
1
[ꢀ]D -7 (c 0.7, CHCl3); ꢂmax 262 nm (ꢃ 7549); H NMR
(CDCl3, 300 MHz): ꢀ 8.19 (br s, 1H, NH), 7.19 (s, 1H, H-6),

Synthesis and Biological Evaluation of 3ꢀ-C-Ethynyl and 3ꢀ-C-(1,4-disubstituted-1,2,3-triazolo)
Medicinal Chemistry, 2012, Vol. 8, No. 3 325
MHz): ꢀ 163.64, 150.13, 136.74, 110.10, 91.37, 83.47,
80.02, 74.33, 73.91, 72.68, 71.89, 61.53, 12.21; IR (Nujol,
cm-1): 2130 (CꢀC). ESIMS: m/z 313.3 [M+H+].
of sodium ascorbate (0.046 mmol) and CuSO4·5H2O (0.023
mmol) in H2O (2.3 mL). The reaction mixture was refluxed
for 2 hours and then cooled. After being treated with MeOH
and concentrated, the residue was purified by flash chroma-
tography (CH2Cl2/MeOH, 95:5) to give the double-headed
nucleosides 7 as white foams.
Data for 1-(3ꢀ-C-ethynyl-ꢁ-D-allopyranosyl)cytosine (5f)
22
Yield 73%; Rf= 0.23 (EtOAc/MeOH 8:2); [ꢁ]D +16 (c
0.4, CD3OD); ꢁmax 268 nm (ꢂ 7913); 1H NMR (CD3OD, 300
MHz): ꢀ 7.72 (d, 1H, J5,6 = 7.2 Hz, H-6), 5.95-5.88 (m, 2H,
H-1ꢃ, H-5), 3.86-3.64 (m, 5H, H-2ꢃ, H-4ꢃ, H-5ꢃ, H-6aꢃ, H-
6bꢃ), 2.83 (s, 1H, HCꢀC); 13C NMR (CD3OD, 75.5 MHz): ꢀ
165.73, 156.28, 143.03, 94.55, 92.94, 83.81, 79.92, 74.56,
74.13, 73.19, 72.71, 61.26; IR (Nujol, cm-1): 2124 (CꢀC).
ESIMS: m/z 298.3 [M+H+].
Data
for
1-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(adenine-9-
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]uracil (7a)
22
Yield 74%; Rf= 0.30 (CH2Cl2/MeOH, 9:1); [ꢁ]D +10 (c
1
0.5, CHCl3); ꢁmax 260 nm (ꢂ 10786); H NMR (CDCl3, 300
MHz, 45 ºC): ꢀ 9.19 (br s, 1H, NH) 8.49 and 7.70 (2 br s,
2H, H-2 and H-8 Ade), 7.32 (d, 1H, J5,6 = 7.6 Hz, H-6 Ur),
7.10 (s, 1H, Triazole H), 6.20 (d, 1H, J1ꢃ,2ꢃ = 8.7 Hz, H-1ꢃ),
5.89 (br s, 2H, NH2 Ade), 5.74 (d, 1H, H-5 Ur), 5.20 (d, 1H,
H-2ꢃ), 5.05 (d, 1H, J4ꢃ,5ꢃ = 9.8 Hz, H-4ꢃ), 4.91 (br, 2H, CH2),
4.73 (br, 2H, CH2), 4.37ꢄ4.09 (m, 3H, H-5ꢃ, H-6aꢃ, H-6bꢃ),
2.07, 2.00, 1.90 (3s, 9H, 3OAc). ESIMS m/z 629.6 [M+H+].
Data for 9-(3ꢀ-C-ethynyl-ꢁ-D-allopyranosyl)adenine (5g)
22
Yield 75%; Rf= 0.18 (EtOAc/MeOH, 8:2); [ꢁ]D -4 (c
1
0.1, DMSO); ꢁmax 263 nm (ꢂ 12279); H NMR (DMSO-d6,
300 MHz): ꢀ 8.33 and 8.15 (2s, 2H, H-2 and H-8), 7.15 (br s,
2H, NH2), 5.65 (d, 1H, J1ꢃ,2ꢃ = 9.3 Hz, H-1ꢃ), 5.63 (s, 1H, 3ꢃ-
OH), 5.36 (d, 1H, J4ꢃ-OH,4ꢃ = 7.9 Hz, 4ꢃ-OH), 5.16 (d, 1H, J2ꢃ-
OH,2ꢃ = 8.0 Hz, 2ꢃ-OH), 4.48 (t, 1H, J = 5.5 Hz, 6ꢃ-OH), 4.26
(t, 1H, H-2ꢃ), 3.70-3.43 (m, 4H, H-4ꢃ, H-5ꢃ, H-6aꢃ, H-6bꢃ),
3.26 (s, 1H, HCꢀC); 13C NMR (DMSO-d6, 75.5 MHz): ꢀ
156.23, 152.37, 149.76, 140.33, 119.57, 100.66, 83.19,
80.01, 74.23, 73.87, 72.74, 72.43, 61.06; IR (Nujol, cm-1):
2129 (CꢀC). ESIMS: m/z 322.3 [M+H+].
Data
for
1-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(adenine-9-
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]thymine (7b)
22
Yield 69%; Rf= 0.38 (CH2Cl2/MeOH, 9:1); [ꢁ]D +4 (c
1
0.5, CHCl3); ꢁmax 262 nm (ꢂ 15229); H NMR (CDCl3, 300
MHz): ꢀ 9.99 (br s, 1H, NH Thy), 8.43 and 7.51 (2s, 2H, H-2
and H-8 Ade), 7.18 (s, 1H, H-6 Thy), 7.11 (s, 1H, Triazole
H), 6.18 (d, 1H, J1ꢃ,2ꢃ = 9.1 Hz, H-1ꢃ), 6.07 (br s, 2H, NH2
Ade), 5.25 (d, 1H, H-2ꢃ), 5.04 (d, 1H, J4ꢃ,5ꢃ = 9.7 Hz, H-4ꢃ),
4.93 (t, 2H, J = 5.1 Hz, CH2), 4.75 (t, 2H, J = 5.1 Hz, CH2),
4.38-4.07 (m, 3H, H-5ꢃ, H-6aꢃ, H-6bꢃ), 2.08, 2.01, 1.96 (3s,
9H, 3OAc), 1.85 (s, 3H, 5-CH3 Thy). ESIMS m/z 643.6
[M+H+].
Preparation of Azides 6a,b,c
Azides 6a and 6c were prepared according to the proce-
dures described in the literature [38,39].
1-(2-Azidoethyl)5-fluorouracil (6b)
Data
for
1-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(adenine-9-
A mixture of 5-fluorouracil (500 mg, 3.85 mmol), HMDS
(3mL) and trimethylchlorosilane (0.25 mL) was refluxed
under nitrogen. After evaporation of excess HMDS under
reduced pressure, 1,2-dibromoethane (1 mL, 11.6 mmol) and
dry DMF (12 mL) were added and the reaction mixture was
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]N4-
benzoylcytosine (7c)
22
Yield 72%; Rf= 0.45 (CH2Cl2/MeOH, 9:1); [ꢁ]D +12 (c
0.5, CHCl3); ꢁmax 262 nm (ꢂ 26064); H NMR (CDCl3, 300
1
o
heated at 80 C under nitrogen for 20 hours. After evapora-
MHz): ꢀ 8.38 (br s, 1H, NH BzCyt), 7.97-7.49 (m, 9H, Bz,
H-2 and H-8 Ade, H-5 and H-6 BzCyt), 7.11 (s, 1H, Triazole
H), 6.45 (d, 1H, J1ꢃ,2ꢃ = 8.9 Hz, H-1ꢃ), 5.69 (br s, 2H, NH2
Ade), 5.21 (d, 1H, H-2ꢃ), 5.09 (d, 1H, J4ꢃ,5ꢃ = 9.7 Hz, H-4ꢃ),
4.90 (br, 2H, CH2), 4.71 (br, 2H, CH2), 4.43-4.38 (m, 1H, H-
5ꢃ), 4.29 (dd, 1H, J6aꢃ,6bꢃ =12.5 Hz, J5ꢃ,6aꢃ = 4.5 Hz, H-6aꢃ),
4.14 (d, 1H, H-6bꢃ), 2.08, 2.02, 1.90 (3s, 9H, 3OAc). ESIMS
m/z 732.7 [M+H+].
tion of the solvent under reduced pressure the residue was
separated by flash chromatography (EtOAc) to give 1-(2-
bromoethyl)5-fluorouracil (395mg, 40%) as an oily solid
1
which was identified only by its H NMR and used for the
1
next step without further purification. H NMR (CDCl3, 300
MHz): ꢀ 11.25 (br s, 1H, NH), 7.63 (d, 1H, J6,F5 = 6.4 Hz, H-
6), 4.15-4.05 (m, 2H, CH2) 3.62-3.56 (m, 2H, CH2). A solu-
tion of 1-(2-bromoethyl)5-fluorouracil (395 mg, 1.67 mmol)
in dry THF (15 mL), trimethylsilyl azide (564 mg 4.9 mmol)
and tetrabutylammonium fluoride (TBAF) (7 mL, 1M in
Data
for
9-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(adenine-9-
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]N6-
o
benzoyladenine (7d)
THF) was heated at 65 C under nitrogen for 3 hours. After
22
the removal of the solvent the residue was treated with etha-
nol to give 6b (230 mg, 69%), as a white solid: mp 177-179
oC; Rf= 0.49 (CH2Cl2/MeOH, 9:1); ꢁmax 270 nm (ꢂ 3429); 1H
NMR (CDCl3, 300 MHz): ꢀ 8.23 (br s, 1H, NH), 7.31 (d, 1H,
J6,F5 = 5.5 Hz, H-6), 3.85 (t, J = 5.8 Hz, 2H, CH2), 3.72 (t, J =
5.8 Hz, 2H, CH2). 13C NMR (CDCl3/CD3OD, 75.5 MHz): ꢀ
157.87, 149.53, 139.8, 129.22, 49.27, 48.01. ESIMS m/z
200.1[M+H+].
Yield 79%; Rf= 0.43 (CH2Cl2/MeOH, 9:1); [ꢁ]D -6 (c
1
0.5, CHCl3); ꢁmax 276nm (ꢂ 22506), H NMR (CDCl3, 300
MHz): ꢀ 9.12 (br s, 1H, NH BzAde), 8.86 and 8.36 (2s, 2H,
H-2 and H-8 BzAde), 8.23 (s, 1H, H-2 Ade), 8.04 (d, 2H, J =
7.4 Hz, Bz), 7.63 (t, 1H, J = 7.4 Hz, Bz), 7.54 (t, 2H, J = 7.4
Hz, Bz), 7.45 (s, 1H, H-8 Ade), 7.24 (s, 1H, Triazole H),
6.29 (d, 1H, J1ꢃ,2ꢃ = 9.1 Hz, H-1ꢃ), 5.93 (br s, 2H, NH2 Ade),
5.64 (d, 1H, H-2ꢃ), 5.23 (d, 1H, J4ꢃ,5ꢃ = 9.8 Hz, H-4ꢃ), 4.90 (t,
2H, J = 5.3, CH2), 4.72 (t, 2H, J = 5.3, CH2), 4.49-4.45 (m,
1H, H-5ꢃ), 4.27 (dd, 1H, J6aꢃ,6bꢂ = 12.5 Hz, J5ꢂ,6aꢃ = 4.8 Hz, H-
6aꢃ), 4.11 (dd, 1H, J5ꢂ ,6bꢃ= 1.0 Hz, H-6bꢃ), 2.04, 2.02, 1.68
(3s, 9H, 3OAc). ESIMS m/z 756.7 [M+H+].
General Procedure for Preparation of Double-Headed Nu-
cleosides 7a-d,f-h
To a stirred mixture of the alkyne 4 (0.23 mmol) and the
azide 6 (0.23 mmol) in THF (2.3 mL), was added a solution

326 Medicinal Chemistry, 2012, Vol. 8, No. 3
Kiritsis et al.
Data for 1-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(5-fluorouracil-1-
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]5-fluoro-
uracil (7f)
101.87, 80.30, 77.50, 75.31, 71.06, 69.69, 61.27, 48.52,
43.21. ESIMS: m/z 503.4 [M+H+].
Data for 1-[3ꢀ-C-[1-{(adenine-9-yl)ethyl}-1,2,3-triazol-4-
yl]-ꢁ-D-allopyranosyl]thymine (8b)
22
Yield 84%; Rf= 0.50 (CH2Cl2/MeOH, 9:1); [ꢀ]D +4 (c
1
0.4, CHCl3); ꢀmax 264 nm (ꢁ 10965); H NMR (CDCl3, 300
Yield 87%; mp 203-205 oC (dec); Rf= 0.38
MHz): ꢁ 9.67 (br s, 1H, NH), 8.84 (br s, 1H, NH), 7.63 (s,
1H, Triazole H), 7.54 (d, 1H, J6,F5 = 6.0 Hz, H-6 FUr), 7.06
(d, 1H, J6,F5 = 5.4 Hz, H-6 FUr), 6.17 (d, 1H, J1ꢂ,2ꢂ = 9.0 Hz,
H-1ꢂ), 5.36 (d, 1H, H-2ꢂ), 5.02 (d, 1H, J4ꢂ,5ꢂ = 9.9 Hz, H-4ꢂ),
4.75 (t, 2H, J = 4.7 Hz, CH2), 4.44-4.38 (m, 1H, H-5ꢂ),
4.32ꢃ4.24 (m, 2H, CH2), 4.18-4.11 (m, 2H, H-6aꢂ, H-6bꢂ),
2.08, 2.05, 1.95 (3s, 9H, 3OAc). ESIMS m/z 642.5 [M+H+].
22
(CH2Cl2/MeOH, 2:8); [ꢀ]D +3 (c 0.2, DMSO); ꢀmax 270 nm
1
(ꢁ 24183), 263 nm (ꢁ 26659); H NMR (DMSO-d6, 300
MHz, 55 °C): ꢁ 10.80 (br s, 1H, NH Thy), 8.16, 7.89 and
7.75 (3s, 3H, H-2 Ade, H-8 Ade and H triazole), 7.48 (s, 1H,
H-6 Thy), 6.72 (br s, 2H, NH2 Ade), 5.83 (d, 1H, J1ꢂ,2ꢂ = 9.0
Hz, H-1ꢂ), 4.84 (t, 2H, J = 6.0 Hz, CH2), 4.63 (t, 2H, J = 5.3
Hz, CH2), 4.50-3.20 (m, 9H, H-2ꢂ,H-4ꢂ, H-5ꢂ, H-6aꢂ, H-6bꢂ
and 4OH), 1.82 (s, 3H, 5-CH3 Thy). 13C NMR (DMSO-d6,
75.5 MHz): ꢁ 164.14, 156.12, 152.84, 151.35, 150.71,
150.33, 141.09, 137.23, 124.72, 121.10, 109.75, 80.53,
77.79, 75.50, 71.30, 70.10, 61.66, 48.96, 43.64, 12.4. E-
SIMS: m/z 517.5 [M+H+].
Data for 1-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(5-fluorouracil-1-
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]uracil (7g)
22
Yield 79%; Rf= 0.45 (CH2Cl2/MeOH, 9:1); [ꢀ]D +5 (c
1
1.1, CHCl3); ꢀmax 264 nm (ꢁ 11784); H NMR (CDCl3, 300
MHz): ꢁ 8.64 (br s, 1H, NH), 8.19 (br s, 1H, NH), 7.44
(s,1H, Triazole H), 7.39 (d, 1H, J5,6 = 8.2 Hz, H-6 Ur), 7.04
(d, 1H, J5,6 = 5.6 Hz, H-6 FUr), 6.14 (d, 1H, J1ꢂ,2ꢂ = 9.1 Hz,
H-1ꢂ), 5.69 (d, 1H, H-5 Ur), 5.27 (d, 1H, H-2ꢂ), 4.99 (d, 1H,
Data for 1-[3ꢀ-C-[1-{(adenine-9-yl)ethyl}-1,2,3-triazol-4-
yl]-ꢁ-D-allopyranosyl]cytosine (8c)
Yield 70%; mp 250-252 oC (dec); Rf= 0.20
J4ꢂ,5ꢂ = 9.7 Hz, H-4ꢂ), 4.61 (t, 2H, J = 5.0 Hz, CH2), 4.32-4.05
22
(CH2Cl2/MeOH, 2:8), [ꢀ]D +3 (c 0.5, DMSO); ꢀmax 263 nm
(m, 5H, H-5ꢂ, CH2, H-6aꢂ, H-6bꢂ), 2.00, 1.95, 1.88 (3s, 9H,
1
(ꢁ 23401);): H NMR (DMSO-d6, 300 MHz, 50 °C): ꢁ 8.17
3OAc). ESIMS m/z 624.5 [M+H+].
and 7.91 (2s, 2H, H-2 Ade and H-8 Ade), 7.74 (s, 1H, Tria-
zole H), 7.59 (d, 1H, J5,6 = 7.5 Hz, H-6 Cyt), 7.00-6.82 (br,
4H, NH2 Ade and Cyt), 5.95 (d, 1H, J1ꢂ,2ꢂ = 9.5 Hz, H-1ꢂ),
5.79 (d, 1H, H-5 Cyt), 4.90 (br s, 1H, OH), 4.83 (t, J = 6.0
Hz, 2H, CH2), 4.64 (t, J = 6.0 Hz, 2H, CH2), 4.52-4.22 (m,
3H, H-2ꢂ and 2OH), 3.92-3.45 (m, 5H, ꢄ-4ꢂ, H-5ꢂ, H-6aꢂ, H-
6aꢂ and OH). 13C NMR (DMSO-d6, 75.5 MHz): ꢁ 165.83,
156.35, 156.23, 153.01, 151.12, 149.94, 142.45, 141.20,
124.91, 119.00, 94.80, 80.89, 77.75, 75.90, 72.06, 70.26,
61.71, 48.92, 43.63. ESIMS: m/z 502.5 [M+H+].
Data
for
1-[2ꢀ,4ꢀ,6ꢀ-tri-O-acetyl-3ꢀ-C-[1-{(thymine-1-
yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-allopyranosyl]uracil (7h)
22
Yield 67%; Rf= 0.48 (CH2Cl2/MeOH, 9:1); [ꢀ]D +2 (c
1
0.5, CHCl3); ꢀmax 261 nm (ꢁ 12865); H NMR (CDCl3, 300
MHz): ꢁ 9.75 (br s, 1H, NH), 9.46 (br s, 1H, NH), 7.45 (d,
1H, J5,6 = 7.8 Hz, H-6 Ur), 7.44 (s, 1H, Triazole H ), 6.75
(s,1H, H-6 Thy), 6.21 (d, 1H, J1ꢂ,2ꢂ = 8.7 Hz, H-1ꢂ), 5.79 (d,
1H, H-5 Ur), 5.36 (d, 1H, H-2ꢂ), 4.92 (d, 1H, J4ꢂ,5ꢂ = 9.8 Hz,
H-4ꢂ), 4.73-4.67 (m, 2H, CH2), 4.41-4.36 (m, 1H, H-5ꢂ),
4.29ꢃ4.09 (m, 4H, CH2, H-6aꢂ, H-6bꢂ), 1.92, 1.90, 1.81 (3s,
9H, 3OAc), 1.69 (s, 3H, 5-CH3 Thy). ESIMS m/z 620.5
[M+H+].
Data for 9-[3ꢀ-C-[1-{(adenine-9-yl)ethyl}-1,2,3-triazol-4-
yl]-ꢁ-D-allopyranosyl]adenine (8d)
Yield 84%; mp 204-206 oC (dec); Rf= 0.17
22
General Procedure for Preparation of Double-Headed Nu-
cleosides 8a-d
(CH2Cl2/MeOH, 2:8); [ꢀ]D -3 (c 0.4, DMSO); ꢀmax 263 nm
1
(ꢁ 23342); H NMR (DMSO-d6, 300 MHz, 50 °C): ꢁ 8.24,
8.17, 7.92 and 7.76 (4s, 4H, H-2 and H-8 Ade), 7.76 (s, 1H,
The protected double-headed nucleosides 7a-d (0.193
mmol,) were treated with ammonia/MeOH (saturated at 0 oC,
8 mL). The solution was stirred overnight at room tempera-
ture and then was concentrated under reduced pressure. The
residue was recrystallized from MeOH/H2O to afford 8a-d as
white solids respectively.
Triazole H), 6.74 (br s, 4H, NH2 Ade), 5.88 (d, 1H, J1ꢂ,2ꢂ
=
9.4 Hz, H-1ꢂ), 4.99 (br s, 1H, OH), 4.83 (t, 2H, J = 6.0 Hz,
CH2), 4.66 (t, 2H, J = 6.0 Hz, CH2), 4.48-4.18 (m, 3H, H-2ꢂ
and 2OH), 3.92-3.50 (m, 5H, H-4ꢂ, H-5ꢂ, H-6aꢂ, H-6aꢂ and
OH). 13C NMR (DMSO-d6, 75.5 MHz): ꢁ 156.16, 156.13
154.5, 152.86, 150.67, 150.20, 149.80, 141.10, 140.25,
124.80, 118.99, 118.98, 80.98, 77.70, 75.56, 71.56, 70.09,
61.51, 48.81, 43.47. ESIMS: m/z 526.5 [M+H+].
Data for 1-[3ꢀ-C-[1-{(adenine-9-yl)ethyl}-1,2,3-triazol-4-
yl]-ꢁ-D-allopyranosyl]uracil (8a)
Yield 77%; mp 225-227 oC (dec); Rf= 0.33
1-[3ꢀ-C-[1-{(Adenine-9-yl)ethyl}-1,2,3-triazol-4-yl]-ꢁ-D-
allopyranosyl]5-fluorouracil (8e)
22
(CH2Cl2/MeOH, 2:8); [ꢀ]D +4 (c 0.3, DMSO); ꢀmax 263 nm
1
(ꢁ 20310), 270 nm (ꢁ 23650) ; H NMR (DMSO-d6, 300
A mixture of the alkyne 5a (80 mg, 0.25 mmol) and the
azide 6a (51 mg, 0.25 mmol) was stirred in CH3CN/H2O
(9:1) in the presence of a copper wire. The reaction mixture
was moderate heated for 18 hours and then cooled. After
being treated with MeOH and concentrated, the residue was
recrystallized from (MeOH/H2O) to afford pure 8e (100 mg,
77%) as a white solid: mp 238-240 oC (dec); Rf= 0.40
MHz, 50 ºC): ꢁ 11.18 (br s, 1H, NH), 8.18 and 7.91 (2s, 2H,
H-2 and H-8 Ade), 7.76 (s, 1H, Triazole H), 7.65 (d, 1H, J5,6
= 7.1 Hz, H-6 Ur), 6.83 (br s, 2H, NH2 Ade), 5.84 (d, 1H,
J1ꢂ,2ꢂ = 9.2 Hz, H-1ꢂ), 5.62 (d, 1H, H-5 Ur), 5.01 (br s, 1H,
OH), 4.85 (t, J = 5.9 Hz, 2H, CH2), 4.76 (br s, 1H, OH), 4.65
(t, J = 5.9 Hz, 2H, CH2), 4.45 (br s, 1H, OH), 4.28 (br s, 1H,
OH), 3.99-3.45 (m, 5H, H-2ꢂ,H-4ꢂ,H-5ꢂ, H-6aꢂ and H-6bꢂ).
13C NMR (DMSO-d6, 75.5 MHz): ꢁ 163.17, 155.94, 152.60,
151.04, 150.45, 149.35, 141.58, 140.76, 124.51, 118.55,
22
(CH2Cl2/MeOH, 2:8); [ꢀ]D +4 (c 0.2, DMSO); ꢀmax 266 nm
1
(ꢁ 17627); H NMR (DMSO-d6, 300 MHz, 50 °C): ꢁ 11.50

Synthesis and Biological Evaluation of 3ꢀ-C-Ethynyl and 3ꢀ-C-(1,4-disubstituted-1,2,3-triazolo)
Medicinal Chemistry, 2012, Vol. 8, No. 3 327
5-CH3 Thy); 13C NMR (DMSO-d6, 75.5 MHz): ꢀ 164.35,
163.16, 151.04, 150.89, 150.42, 141.58, 141.06, 124.66,
108.82, 101.86, 80.29, 77.51, 75.33, 71.08, 69.78, 61.29,
47.72, 47.61, 11.70. ESIMS: m/z 494.4 [M+H+].
(br s, 1H, NH), 8.16 (s, 1H, H-2 Ade), 8.01-7.83 (m, 2H, 6-H
FUr and 8-H Ade), 7.73 (s, 1H, Triazole H), 6.80 (br s, 2H,
NH2), 5.82 (d, 1H, J1ꢀ,2ꢀ = 9.3 Hz, H-1ꢀ), 4.97 (br s, 1H, OH),
4.83 (t, 2H, J = 6.1 Hz, CH2), 4.64 (t, 2H, J = 6.1 Hz, CH2),
4.48-4.20 (m, 3H, H-2ꢀ and 2OH), 3.98-3.48 (m, 5H, H-
4ꢀ,H-5ꢀ, H-6aꢀ, H-6aꢀ and OH). 13C NMR (DMSO-d6, 75.5
MHz): ꢀ 157.20, 156.21, 154.30, 152.85, 150.47, 149.93,
141.09, 139.80, 126.10, 124.57, 119.10, 81.30, 77.70, 75.48,
71.35, 69.94, 61.63, 48.84, 43.43. ESIMS: m/z 521.4
[M+H+].
BIOLOGICAL ASSAYS
Antiviral Assays
The antiviral assays [except anti-human immunodefici-
ency virus (HIV) assays] were based on inhibition of virus-
induced cytopathicity in HEL [herpes simplex virus type 1
(HSV-1), HSV-2 (G), vaccinia virus, vesicular stomatitis
virus, varicella-zoster virus (OKA and 07-1) and human cy-
tomegalovirus (AD169 and Davis)], Vero (parainfluenza-3,
reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus),
HeLa (vesicular stomatitis virus, Coxsackie virus B4, and
respiratory syncytial virus), CrFK (feline herpes virus and
feline corona (FIPV) virus) and MDCK (influenza virus A
(H1N1 and H3N2) and influenza virus B) cell cultures. Con-
fluent cell cultures in microtiter 96-well plates were inocu-
lated with 100 cell culture inhibitory dose-50 (CCID50) of
virus (1 CCID50 being the virus dose to infect 50% of the cell
cultures) and the cell cultures were incubated in the presence
of varying concentrations (100, 20, 5, … μM) of the test
compounds. Viral cytopathicity was recorded as soon as it
reached completion in the control virus-infected cell cultures
that were not treated with the test compounds. The method-
ology of the anti-HIV assays was as follows: human CEM
(3 ꢃ 105 cells/ml) cells were infected with 100 CCID50 of
HIV-1(IIIB) or HIV-2(ROD)/mL and seeded in 200 μL 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 micro-
scopically
General Procedure for Preparation of Double-Headed Nu-
cleosides 8f-h
The protected double-headed nucleosides 7f-h (0.193
mmol,) were treated with ammonia/MeOH (saturated at 0 oC,
8 mL). The solution was stirred overnight at room tempera-
ture and then was concentrated under reduced pressure. The
residue was recrystallized from MeOH/CH2Cl2 to afford 8f-h
as white solids respectively.
Data for 1-[3ꢀ-C-[1-{(5-fluorouracil-1-yl)ethyl}-1,2,3-
triazol-4-yl]-ꢁ-D-allopyranosyl]5-fluorouracil (8f)
o
Yield 83%; mp 184-186 C; Rf= 0.28 (CH2Cl2/MeOH,
22
1
7:3); [ꢁ]D +9 (c 0.3, CH3OH); ꢁmax 269 nm (ꢂ 13761); H
NMR (DMSO-d6, 300 MHz): ꢀ 11.8 (br s, 2H, NH), 8.15 (d,
1H, J6,F5 = 7.0 Hz, H-6 FUr), 7.93 (s, 1H, Triazole H), 7.65
(d, 1H, J6,F5 = 6.5 Hz, H-6 FUr), 5.81 (d, 1H, J1ꢀ,2ꢀ = 9.0 Hz,
H-1ꢀ), 5.43 (s, 1H, OH), 5.09 (d, 1H, H-2ꢀ), 4.78-4.56 (m,
4H, 2CH2), 4.20-3.30 (m, 7H, H-4ꢀ, H-5ꢀ, H-6aꢀ, H-6bꢀand
3OH). 13C NMR (DMSO-d6, 75.5 MHz): ꢀ 157.75, 157.35,
150.55, 149.91, 149.87, 140.33, 139.81, 130.12, 126.22,
124.99, 81.00, 77.62, 75.53, 71.22, 69.78, 61.51, 48.27,
47.56. ESIMS: m/z 516.4 [M+H+].
Data
for
1-[3ꢀ-C-[1-{(5-fluorouracil-1-yl)ethyl}-1,2,3-
triazol-4-yl]-ꢁ-D-allopyranosyl]uracil (8g)
o
Cytostatic Assays
Yield 85%; mp 190-192 C; Rf= 0.25 (CH2Cl2/MeOH,
22
7:3); [ꢁ]D +2 (c 0.5, CH3OH); ꢁmax 258 nm (ꢂ 19021), 263
To each well of a 96-well microtiter plate were added 5-
7.5 x 104 cells and a given amount of the test compound. The
cells were allowed to proliferate for 48 hours (murine leu-
kemia L1210) or 72 hours (human lymphocyte CEM and
human cervix carcinoma HeLa) at 37 °C in a humidified
CO2-controlled atmosphere. At the end of the incubation
period, the cells were counted in a Coulter Counter (Coulter
Electronics Ltd., Harpenden Herts, United Kingdom). The
IC50 (50% inhibitory concentration) was defined as the con-
centration of compound that reduced the number of cells by
50%.
nm (ꢂ 18757); 1H NMR (DMSO-d6, 300 MHz): ꢀ 11.75 (br s,
1H, NH), 11.25 (br s, 1H, NH), 8.01 (s, 1H, Triazole H),
7.75 (d, 1H, JH-6,F = 6.6 Hz, H-6 FUr), 7.74 (d, 1H, J5,6 = 8.1
Hz, H-6 Ur), 5.84 (d, 1H, J1ꢀ,2ꢀ = 9.5 Hz, H-1ꢀ), 5.63 (d, 1H,
J
5,6 = 8.1 Hz, H-5 Ur), 5.34 (s, 1H, OH), 4.97 (d, 1H, H-2ꢀ),
4.73-4.47 (m, 4H, H-4ꢀ, CH2 and OH), 4.06 (t, 2H, J = 5.6
Hz, CH2), 3.93 (br, 1H, OH), 3.82-3.42 (m, 4H, H-5ꢀ, H-6aꢀ,
H-6bꢀand OH). 13C NMR (DMSO-d6, 75.5 MHz): ꢀ 163.30,
157.75, 151.29, 150.65, 149.89, 141.81, 139.85, 130.16,
125.02, 102.09, 80.55, 77.80, 75.61, 71.37, 70.02, 61.58,
48.18, 47.51. ESIMS: m/z 498.4 [M+H+].
ACKNOWLEDGEMENTS
Data for 1-[3ꢀ-C-[1-{(thymine-1-yl)ethyl}-1,2,3-triazol-4-
yl]-ꢁ-D-allopyranosyl]uracil (8h)
This work was supported in part by the Postgraduate
Programmes ‘‘Biotechnology-Quality assessment in Nutri-
tion and the Environment”, ‘‘Application of Molecular Biol-
ogy-Molecular Genetics-Molecular Markers”, Department of
Biochemistry and Biotechnology, University of Thessaly and
the K.U.Leuven (GOA 10/014). The authors thank Mrs.
Lizette van Berckelaer, Mrs. Leentje Persoons, Mrs. Frieda
De Meyer, Mrs. Anita Camps, Mrs. Lies Van den Heurck,
Mr. Steven Carmans and Mrs. Leen Ingels for excellent
technical assistance.
o
Yield 86%; mp 146-148 C; Rf= 0.26 (CH2Cl2/MeOH,
22
7:3); [ꢁ]D +2 (c 0.2, CH3OH); ꢁmax 258 nm (ꢂ 21045), 269
(ꢂ 17681); H NMR (DMSO-d6, 300 MHz, 50 °C): ꢀ 11.30
1
(br s, 2H, NH), 7.95 (s, 1H, Triazole H), 7.67 (d, 1H, J5,6
8.1 Hz, H-6 Ur), 7.05 (s, 1H, H-6 Thy), 5.81 (d, 1H, J1ꢀ,2ꢀ
9.3 Hz, H-1ꢀ), 5.62 (d, 1H, H-5 Ur), 5.31 (br s, 1H, OH),
4.75 (br s, 1H, OH), 4.62 (t, 2H, J = 6.1 Hz, CH2), 4.50-4.22
(m, 2H, H-2ꢀ and OH), 4.08 (t, 2H, J = 6.1 Hz, CH2), 3.98-
3.48 (m, 5H, H-4ꢀ,H-5ꢀ, H-6aꢀ, H-6aꢀ and OH). 1.64 (s, 3H,
=
=

328 Medicinal Chemistry, 2012, Vol. 8, No. 3
Kiritsis et al.
Antioxidant Properties. Pharmacol. Pharmacy, 2011, 2(3), 122-
126.
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Received: August 26, 2011
Revised: March 09, 2012
Accepted: March 14, 2012