Rapid microwave-enhanced synthesis of C5-alkynyl pyranonucleosides as novel cytotoxic antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2012.12.092

A microwave-assisted, one-pot, coupling reaction for the synthesis of C5-alkynyl-uracil and cytosine glucopyranonucleosides has been developed. The reaction is carried out under standard Sonogashira coupling conditions from glucopyranonucleosides of 5-iodouracil or 5-iodocytosine and various terminal alkynes. All compounds were evaluated for their cytostatic and antiviral activity. The 5-phenylethynyluracil pyranonucleoside derivative 6a showed the most promising cytostatic activity (50% inhibitory concentration in the lower micromolar range). No meaningful antiviral activity was recorded.

Full text of DOI:10.1016/j.bmcl.2012.12.092

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1330–1333
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rapid microwave-enhanced synthesis of C5-alkynyl
pyranonucleosides as novel cytotoxic antitumor agents
Athina Dimopoulou a, Stella Manta a, Christos Kiritsis a, Dimitra-Niki Gkaragkouni a, Ioannis Papasotiriou b,
Jan Balzarini c, Dimitri Komiotis a,
⇑
a Department of Biochemistry and Biotechnology, Laboratory of Bio-Organic Chemistry, University of Thessaly, 26 Ploutonos Str., 41221 Larissa, Greece
b Research Genetic Cancer Center (R.G.C.C. Ltd), Megalou Alexandrou 155 Str., GR-53070 Filotas, Greece
c Rega Institute for Medical Research, KU Leuven, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
A microwave-assisted, one-pot, coupling reaction for the synthesis of C5-alkynyl-uracil and cytosine
glucopyranonucleosides has been developed. The reaction is carried out under standard Sonogashira cou-
pling conditions from glucopyranonucleosides of 5-iodouracil or 5-iodocytosine and various terminal
alkynes. All compounds were evaluated for their cytostatic and antiviral activity. The 5-phenylethynyl-
uracil pyranonucleoside derivative 6a showed the most promising cytostatic activity (50% inhibitory con-
centration in the lower micromolar range). No meaningful antiviral activity was recorded.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 13 November 2012
Revised 22 December 2012
Accepted 27 December 2012
Available online 7 January 2013
Keywords:
C5-Alkynyl pyranonucleosides
Microwave-irradiation
Sonogashira coupling reaction
Cytotoxic activity
Nucleoside analogues, as potential inhibitors of nucleic acid
metabolism, play a significant role in the treatment of patients
with devastating cancer and viral infections.1–3 Extensive varia-
tions have been made to both the heterocyclic base and the sugar
moiety in the search for effective and selective derivatives with a
benign profile. Due to the need for the base moiety to preserve
the base pairing functionalities, only minor modifications of the
base are usually found in biologically active nucleoside ana-
logues.4,5 On the contrary, a lot of variations have been made in
the sugar part by introducing pyranosyl, carbocyclic or heterocyclic
rings and acyclic moieties.5
During recent years and in connection with our interest on syn-
thetic nucleoside mimetics, we reported novel series of sugar-
modified pyranonucleosides as anti-rotavirus,6 antioxidant7 and
cytotoxic agents8 against various cancer cell lines. Nearly at the
same time, we became interested in base-modified nucleosides,
since C5-substituted pyrimidine nucleosides are known to have
interesting biological properties and have been investigated as
antiviral and anticancer agents.9–11 Thus, we performed the syn-
thesis of C5-halogen- and C5-alkynyl-uracil glucopyranonucleo-
sides, which have been discovered as some of the most potent
inhibitors of the active site of glycogen phosphorylase.12,13
As a continuation of our long-term interest in the chemical and
pharmacological properties of modified pyranonucleosides, we
hereby report an efficient, extremely rapid and high-yield method-
ology for the synthesis of various C5-alkynyl-uracil and cytosine
glucopyranonucleosides under microwave-assisted Sonogashira
coupling conditions. The biological activity of the synthesized com-
pounds was also examined.
The starting nucleoside synthons, 1-(b-D-glucopyranonucleo-
sides) of 5-iodouracil 3a12 and 5-iodocytosine 3b (Scheme 1) were
obtained by performing the two-step Vorbruggen coupling reac-
tion,14 in one-pot, within 3 min reaction, using a domestic micro-
wave oven, followed by subsequent deacetylation by the action
of saturated methanolic ammonia.15
A common approach for the synthesis of the C5-alkynyl gluco-
pyranonucleosides (compounds 4–10) and alkylfurano[2,3-d]pyr-
imidino glucopyranonucleosides (compounds 11a and 12a) is
based upon the attachment of the active units via the ethynyl arm
on C5 of the uracil and cytosine bases. Aiming at more detailed
structure-activity relationship studies, a variety of alkyne substitu-
ents was chosen, which include linear alkyl chains (4a,b, R = C3H7,
5a,b, R = C5H11), aromatic rings (6a,b, R = C6H5) substituted with
linear (7a,b, R = p-pentylphenyl) and branched alkyl groups (8a,b,
R = p-tert-butylphenyl) and the trimethylsilylo-functionality
(9a,b). The nucleosides containing a terminal alkyne (10a,b, R = H)
were prepared by desilylating 9a,b with n-Bu4NF/CH3CN.16
In a typical experiment, the unprotected nucleosides of 5-iodo-
uracil 3a or 5-iodocytosine 3b were mixed with N,N-dimethyl-
formamide (DMF), the appropriate alkyne, triethylamine (base),
copper (I) iodide (CuI) (cocatalyst) and tetrakis(triphenylphos-
phine)palladium (0) (Pd(PPh3)4) (catalyst) and were irradiated for
3 min with the microwave power of ꢀ120 W. After removing all
⇑
Corresponding author. Tel.: +30 2410 565285; fax: +30 2410 565290.
E-mail addresses: dkom@bio.uth.gr, komiotisd@gmail.com (D. Komiotis).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.092

A. Dimopoulou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1330–1333
1331
X
I
OAc
OR
N
O
O
AcO
AcO
i
RO
RO
N
O
OAc
O
OAc
OR
1
2a: R = Ac, X = OH 2b: R = Ac, X = NH2
3a: R = H, X = OH 3b: R = H, X = NH2
ii
iii
R
iii
OH
NH
O
HO
HO
N
O
OH
R
X
N
11a R = C3H7
12a R = C5H11
OH
N
O
HO
HO
O
OH
v
O
N
4a: X = OH, 4b: X = NH2, R = C3H7
5a: X = OH, 5b: X = NH2, R = C5H11
6a: X = OH, 6b: X = NH2, R = Ph
7a: X = OH, 7b: X = NH2, R = p-CH3(CH2)4C6H4
8a: X = OH, 8b: X = NH2, R = p-(CH3)3CC6H4
9a: X = OH, 9b: X = NH2, R = Me3Si
10a: X = OH, 10b: X = NH2, R = H
OH
NH
O
HO
HO
O
OH
iv
13a
Scheme 1. Reagents and conditions: (i) sillylated base, Me3SiOSO2CF3, CH3CN, HMDS, Saccharine, MW (ꢀ120 W), 3 min; (ii) ammonia/MeOH; (iii) R-C„CH, CuI, Pd(PPh3)4,
Et3N, DMF, MW (ꢀ120 W), 3 min or 8 min; (iv) n-Bu4NF, CH3CN; (v) Pd/C, H2, MeOH, 48 h.
the volatile materials in vacuo, the solid obtained was purified by
flash chromatography to provide C5-alkynyl pyranonucleosides
4–1017,18 as pure compounds, in high yields 72–83%. When the
reaction time is longer (irradiation for 8 min), 5-pentynyl 4a and
5-heptynyl 5a uracil pyranonucleosides undergo intramolecular
cyclization to the bicyclic furanopyrimidine derivatives13 11a and
12a, respectively. Identical results were obtained when the reac-
tions were proceeded at the same wattage in different domestic
ovens.
vix carcinoma HeLa) (Table 1). The 5-substituted uracil pyranonu-
cleosides showed superior antiproliferative activity to their
cytosine counterparts. The most striking data were obtained for
the phenylethynyl uracil pyranonucleoside derivative 6a, which
effectively inhibited tumor cell proliferation (IC50 of 5.2–6.2
whereas its cytosine congener showed no appreciable cytostatic ac-
tion (IC50 201–>250 M). It was interesting to observe that the
lM),
l
phenylethyl uracil pyranonucleoside derivative 13a23 was devoid
of significant cytostatic activity pointing to the necessity to have a
rather bulky but rigid substituent at the 5-position of uracil. Besides
5a (a heptynyluracil derivative) and 7a (a p-pentylphenylethynyl
uracil derivative), none of the other 5-substituted uracil and cyto-
sine derivatives showed appreciable antiproliferative action. It is
interesting to note that 10a, which is an unsubstituted 5-ethynyl-
uracil pyranonucleoside derivative, is virtually devoid of cytostatic
activity, whereas the corresponding 5-ethynyl-20-deoxyuridine
derivative ranks among the most potent thymidylate synthase
(TS) inhibitors, and is endowed with a pronounced cytostatic ac-
tion.24 This may indicate that compound 10a (and the other uracil
derivatives in this study) is not phosphorylated to the monophos-
phate derivative (to allow for an interaction with thymidylate syn-
thase, TS) and/or the presence of a hydroxyl entity at the 2 or 3
position of the pyranose ring prevents efficient interaction with TS.
Given the interesting activity of 6a, the phenyl moiety was re-
placed by a pyridine (15a,b) or a pyridinium (17a,b) entity, but
these modifications resulted in a marked loss of cytostatic poten-
tial of 6a. Also 11a and 12a, containing an alkyl-substituted fused
ethenouracil ring, did not show any appreciable activity. When the
compounds were evaluated against a broad variety of DNA and
RNA viruses, no action was recorded.
All new compounds were characterized by NMR and UV
spectroscopy, mass spectrometry and elemental analysis. The
characteristic NMR signals for 4–10 include the 1H H-6 signal
(8.17–7.78 ppm) and 13C signals of C5 (101.70–97.30 ppm) and
C„C (99.95–72.28 ppm). The 1H NMR spectra of 11a and 12a
provided conclusive evidence of cyclization, as the signal of a
new olefinic proton appeared at 6.39 and 6.30 ppm,13 respectively.
Among the pyranonucleosides tested, the phenyluracil deriva-
tive 6a exhibited the highest cytostatic activity. This observation
prompted us to explore the possibility of replacing the phenyl ring
with pyridine, by linking pyridine moieties to 5-iodouracil nucleo-
side 2a (pyridinyl analogues 15a,b) and then converting them into
pyridiniumylo-conjugates 17a,b (Scheme 2), since the latter
molecules could function as reversible electron acceptors when
incorporated into DNA duplexes.19 Thus, 2- and 3-ethynylpyridines
were efficiently coupled with acetylated 5-iodouracil nucleoside
2a under microwave irradiation, giving conjugates 14a,b.20
Subsequently, these analogues were quaternized readily in dry
acetonitrile containing iodomethane to give the protected 5-(N-
methylpyridinium) nucleosides 16a,b.21 Deacetylation of 14a,b
and 16a,b, performed with saturated methanolic ammonia, led to
the target pyridinyl 15a,b22 and pyridiniumyl 17a,b22 nucleosides,
respectively.
In conclusion, an efficient microwave-assisted, one-pot, cou-
pling reaction for the synthesis of C5-alkynyl-uracil and cytosine
glucopyranonucleosides has been developed. Among the various
5-substituted pyrimidine pyranonucleosides tested, the 5-pheny-
lethynyluracil derivative 6a showed appreciable cytostatic activity.
The cytostatic activity was determined, for the uracil (4a–10a)
and cytosine (4b–10b) series, against three tumor cell lines
(murine leukemia L1210, human lymphocyte CEM and human cer-

1332
A. Dimopoulou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1330–1333
Y
X
Y
X
I
O
O
O
OAc
OH
OR
NH
NH
O
O
NH
O
AcO
AcO
HO
HO
N
N
i
ii
RO
RO
N
O
O
OAc
OH
O
OR
14a: X = N, Y = C
14b: X = C, Y = N
2a R = Ac
3a R = H
15a: X = N, Y = C
15b: X = C, Y = N
ii
iii
iii
N
N
O
O
OR
OR
NH
NH
O
N
O
RO
RO
RO
RO
N
O
O
OR
16b: R = Ac
17b: R = H
OR
16a: R = Ac
17a: R = H
ii
ii
Scheme 2. Reagents and conditions: (i) 2-or 3-ethynyl pyridine, CuI, Pd(PPh3)4, Et3N, DMF, MW (ꢀ120 W), 3 min; (ii) ammonia/MeOH; (iii) CH3I, CH3CN.
Table 1
grammes ‘Biotechnology-Quality assessment in Nutrition and the
Cytostatic activity of 4–13a and 14–17 against a panel of tumor cell lines
Environment’, ‘Application of Molecular Biology-Molecular Genet-
ics-Molecular Markers’, Department of Biochemistry and Biotech-
nology, University of Thessaly, and the KU Leuven (GOA 10/14).
We thank Lizette van Berckelaer, Leen Ingels, Leentje Persoons
and Frieda De Meyer for excellent technical assistance in the bio-
logical assays.
a
Compound
IC50
(lM)
HeLa
L1210
CEM
4a
5a
6a
7a
115
22
1
123 32
26 22
5.2 2.6
81 30
>250
>250
173 10
>250
95 17
0
29
2
6.2 0.4
70 23
185 91
141 67
132 29
P250
5.4 0.0
31 12
8a
37
5
Supplementary data
10a
11a
12a
13a
4b
5b
6b
7b
8b
10b
14a
15a
16a
17a
14b
15b
16b
17b
231 27
125
>250
5
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
12.092.
300
0
244
0
43
7
P250
P250
P250
132
>250
P250
115
>250
>250
>250
115 15
>250
191 25
P250
201 68
2
4
43
9
References and notes
140 63
P250
>250
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90
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´
´
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Acknowledgments
This work was supported by the Research Programme Aristeia I
‘Structure-assisted Design, Synthesis, and Evaluation of Bioactive
compounds for type 2 Diabetes mellitus’ and by Postgraduate Pro-

A. Dimopoulou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1330–1333
1333
}
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(20,30,40,60-tetra-O-acetyl-b-
-glucopyranosyl) nucleosides of 5-(N-methyl-
15. Experimental details of the general procedure for the preparation of 1-(b-
D-
D
glucopyranosyl)pyrimidine nucleosides 3a,b are provided in Supplementary
data.
16. Escuret, V.; Aucagne, V.; Joubert, N.; Durantel, D.; Rapp, K. L.; Schinazi, R. F.;
Zoulim, F.; Agrofoglio, L. A. Bioorg. Med. Chem. 2005, 13, 6015.
17. Experimental details of the general procedure for the preparation of C5-alkynyl
pyranonucleosides 4–9 are provided in Supplementary data.
pyridiniumyl)uracil (16a, b) are provided in Supplementary data.
22. Experimental details of the general procedure for the preparation of 1-(b-
glucopyranosyl)-5-(2-and 3-ethynylpyridinyl)uracil (15a, b) and 1-(b-
glucopyranosyl)-5-(N-methyl-pyridiniumyl)uracil nucleosides (17a, b) are
provided in Supplementary data.
23. Experimental details of the general procedure for the preparation of 1-(b-D-
D
-
-
D
18. Experimental details of the general procedure for the preparation of 1-(b-
D-
glucopyranosyl)-5-phenylethyluracil (13a) are provided in Supplementary
glucopyranosyl) nucleosides of 5-ethynyluracil (10a) and of 5-ethynylcytosine
(10b) are provided in Supplementary data.
data.
24. Balzarini, J.; De Clercq, E.; Mertes, M. P.; Shugar, D.; Torrence, P. F. Biochem.
Pharmacol. 1982, 31, 3673.
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20. Experimental details of the general procedure for the preparation of 1-
(20,30,40,60-tetra-O-acetyl-b-
D-glucopyranosyl)-5-(2-and 3-ethynylpyridinyl)-
uracil nucleosides (14a, b) are provided in Supplementary data.