Efficient synthesis and in vitro cytostatic activity of 4-substituted triazolyl-nucleosides

Article abstract of DOI:10.1016/j.bmcl.2007.08.077

We report herein an efficient synthesis of 4-substituted triazolyl-nucleosides and their in vitro cytostatic activity. The synthesis is based on a straightforward 1,3-dipolar cycloaddition between 1-azido-ribose 2 and terminal alkynes under a cooperative

Full text of DOI:10.1016/j.bmcl.2007.08.077

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6656–6659
Efficient synthesis and in vitro cytostatic activity
of 4-substituted triazolyl-nucleosides
Khalid El Akri,a,b Khalid Bougrin,b Jan Balzarini,c
Abdesslem Farajd and Rachid Benhidaa,*
a
ˆ
Laboratoire de Chimie des Mole´cules Bioactives et des Aromes, UMR-CNRS 6001, Institut de Chimie de Nice,
Universite´ de Nice-Sophia Antipolis, Parc Valrose, F-06108 Nice Ce´dex 2, France
b
`
Laboratoire de Chimie des Plantes et de Synthese organique et Bioorganique, Universite Mohammed V-Agdal,
´
Faculte´ des Sciences B.P., 1014 Rabat, Morocco
cRega Institute for Medical Research, Katholieke Univesiteit Leuven, Minderbroedersstraat 10, BE-3000 Leuven, Belgium
dTheravia, EFS, 240 Avenue E. Jeanbrau, 34095 Montpellier, France
Received 13 July 2007; revised 3 August 2007; accepted 4 August 2007
Available online 29 September 2007
Abstract—We report herein an efficient synthesis of 4-substituted triazolyl-nucleosides and their in vitro cytostatic activity. The syn-
thesis is based on a straightforward 1,3-dipolar cycloaddition between 1-azido-ribose 2 and terminal alkynes under a cooperative
effect of microwave activation and copper (I) catalysis. All cycloadducts were obtained in nearly quantitative yield after a short reac-
tion time (1 to 2 min). After removal of acetyl protecting groups, the free nucleosides were evaluated against L1210, Molt4/C8, and
CEM tumor cell lines. Structure–activity relationship study shows that the substituent on the triazole ring has a major effect since
nucleosides 4c and 4g, containing, respectively, a long alkyl chain and an aryl donor group are the most active compounds in this
series.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Nucleosides, which are the genomic building blocks,
interact with major constituents of living cells such as
nucleic acids, enzymes, and proteins. Naturally occur-
ring and synthetic analogues of nucleosides have been
the cornerstone of antiviral therapy over the last dec-
Figure 1. Examples of five-membered antitumor nucleosides, tiazofu-
rin and Eicar.
ades. The growing interest in such analogues arises from
their high potential value not only as therapeutic agents
but also as biochemical probes and as building blocks in
oligonucleotide synthesis following the well-known
phosphoramidite chemistry.1
Eicar is an other five-membered N-nucleoside with a
potent antiviral and antitumor activity (Fig. 1).3 These
encouraging results prompted us to investigate the
synthesis of new five-membered nucleosides in the
triazolyl series.
Among antitumor nucleosides, those anchoring a five-
membered heterocyclic ring are of great interest. Thus,
tiazofurin (Fig. 1) is a synthetic C-nucleoside recently
approved as orphan drug for treatment of chronic
myelogenous leukemia in accelerated phase or blast
crisis. It is a potent inhibitor of inosine monophos-
We recently found that azido-deoxyribose and terminal
alkynes undergo fast 1,3-dipolar cycloaddition under the
cooperative effect of microwave activation and cooper(I)
catalysis to give high yield of 20-deoxy-triazolyl-nucleo-
sides cycloadducts.4 To get further insight into such a
process, we describe herein a straightforward two-step’s
synthesis of 4-substituted triazolyl-nucleosides 4, in ribo
series, and their cytostatic activity on L1210, Molt4/C8,
and CEM cell lines. The role of SiO2 support or
phate dehydrogenase (IMPDH),
a
rate-limiting
enzyme of the de novo guanylate pool synthesis.2
Keywords: Nucleosides; Microwave; Antitumor; Cycloaddition.
*
Corresponding author. Tel.: +33 (0) 4 92 07 61 74; fax: +33 (0) 4 92
07 61 51; e-mail: benhida@unice.fr
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.08.077

K. El Akri et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6656–6659
6657
SiO2 as support (entries 4 and 5). By contrast, in solu-
tion, the best results were obtained with SiO2 (entries
6 and 7) instead of Al2O3 (entry 8) or sand (entry 9).
Moreover, the reaction proceeded in solution more
slowly when carried out with Al2O3 or sand (entries 8
and 9, respectively) than with SiO2.
Scheme 1. Synthesis of azido-tri-O-acetylribose 2.
Brønsted acid in the activation of the key 1,3-dipolar
cycloaddition step will also be discussed.
These differences are likely attributable to an acid cata-
lytic role of the SiO2 support for this 1,3-dipolar cyclo-
addition.8 The importance of this acid catalytic property
was unambiguously proven by the use of acetic acid (1
equiv/mmol of alkyne) instead of SiO2, which resulted
in a fast reaction and high cycloaddition yield (20 min,
95%, entry 10). This cycloaddition could also be carried
out efficiently in protic solvents and aqueous media as
shown in entries 11 and 12, respectively. To our knowl-
edge, this is the first report of such acid catalysis in
azide-alkyne 1,3-dipolar cycloaddition. It offers further
new perspectives in the well-known click chemistry field.
However, the mechanism by which Brønsted acid is
involved in the catalytic cycloaddition process is not
very clear and further experiments are required for its
elucidation.
First, the protected azido-ribose starting material
(2) was obtained in high yield from commercially
available tetra-O-acetylribose by using TMSN3 and
BF3Et2O as a catalyst (Scheme 1). The reaction was
completely stereoselective since only the b-anomer 2
was obtained.5
The survey of the 1,3-dipolar cycloaddition conditions
was next examined by using azide 2 and ethyl propiol-
ic ester as a model (Table 1). We found that the reac-
tion, when performed under solvent free microwave
activation, Cu(I) catalysis and supported on SiO2, pro-
vided the desired cycloadduct 3a stereo- and regio-
selectively and almost quantitatively (entry 1). In this
cycloaddition, only the b-anomer-4-substituted-triazole
3a was obtained within a very short reaction time
(1 min).6
The synthesis of the various protected triazolyl-nucleo-
sides 3 listed in Table 2 was performed applying the
most efficient procedure indicated in entry 1 of Table
1.9 It should be noted that all these nucleosides were ob-
tained with high yields whatever the chemical nature of
the starting alkyne. The different alkynes were chosen in
order to increase the molecular diversity around the tri-
azole nucleobase (long chain, polar, and aromatic
alkynes).
The reaction proceeded also efficiently in solution since
high yields were obtained in CH2Cl2 or toluene at reflux
(entry 3). However, poor yields were obtained at room
temperature even with increased reaction times (entry 2).
To get more information about the role of silica gel, we
tested Al2O3 and sand,7 a poorly hydroxylated silicate,
as support. When the reaction was performed directly
on Al2O3 or sand and under microwave, 3a was ob-
tained with yields comparable to those obtained with
The acetyl groups of the protected nucleosides 3a–i were
then cleaved using NH3/MeOH solution to afford in
high yields the corresponding free analogues 4a–i,
respectively (Table 2).
Table 1. Survey of cycloaddition reaction and SiO2 effect
Entrya
Additives
Activation
Time
T (ꢁC)
Yieldb (%)
1
SiO2
No
MW
1 min
24 h
16 h
110
rt
96
2
CH2Cl2 or toluene
CH2Cl2 or toluene
MW
20c
85
3
No
Reflux
110
110
rt
4
Sand
Al2O3
SiO2
1 min
1 min
16 h
95
95
5
MW
CH2Cl2
6
90
7
SiO2
Toluene
CH2Cl2
236 h
16 h
16 h
rt
65c
37c
35c
95
8
Al2O3
Sand
AcOH
AcOHd
AcOHd
rt
9
CH2Cl2
CH2Cl2
rt
10
11
12
20 min
8 h
8 h
rt
EtOH
H2O/t-BuOH
rt
82
79
rt
a Azide (1 mmol), alkyne (1.2 equiv), CuI (2 equiv), DIEA (5 equiv), support (100 mg/mmol of azide).
b Yield based on the isolated product.
c The starting material 2 was recovered.
d 1 equiv/mmol of alkyne.

6658
K. El Akri et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6656–6659
Table 2. Two-steps synthesis of free nucleosides 4a–i
Entrya
1
Alkyne
Time (min)
1
Products 3a–p (yieldb %)
3a (97)
Products 4a–p (yieldb %)
4a (85) (R = CONH2)
2
2
3b (95)
4b (87)
3
4
5
2
1
1
3c (95)
3d (93)
3e (96)
4c (90)
4d (94)
4e (92)
6
7
2
2
3f (93)
3g (95)
4f (90)
4g (88)
8
9
2
2
3h (94)
3i (93)
4h (95)
4i (92)
a Conditions: azide (1 mmol), alkyne (2 equiv), CuI (2 equiv), and DIEA (5 equiv) were adsorbed on silica gel (1 g/mmol of azide) and irradiated
(95 ꢁC < T < 115 ꢁC, reaction temperature was digitally measured).
b Yields of isolated products.
The newly synthesized free nucleosides 4a–i were evalu-
ated for their in vitro inhibitory effects on the prolifera-
tion of murine leukemia cells (L1210) and human
T-lymphocyte cells (Molt4/C8 and CEM) as described
elsewhere.10 5-Fluorouracil (5FU) was used as a control.
The results are listed in Table 3.
three cell lines being p-methoxyphenyltriazolyl-nucleo-
side 4g.
In summary, we reported an efficient microwave-assisted
eco-friendly synthesis of 4-substituted triazolyl-nucleo-
sides together with their cytostatic activity on L1210,
Mol4/C8, and CEM tumor cell lines. All cycloadducts
were obtained in high yields by using 1,3-dipolar cyclo-
addition between azido-ribose and a range of terminal
alkynes when proceeded in the presence of a Brønsted
acid such as SiO2 and AcOH. Of the various target
nucleosides tested, we found that nucleosides 4c and
4g bearing a C8 alkyl chain or p-methoxyphenyl on
the triazolyl ring are the most active compounds. They
can be used as lead compounds in the search for more
active and potent nucleosides to be applied in cancer
chemotherapy.
Among all the nucleosides tested, 4c with a C8 alkyl
chain was the most active one on the three cell lines
though its activity was much lower than that of 5-FU.
It is surprising that its structurally close analogue 4b
with a C6 alkyl chain did not show any cytostatic activ-
ity (at least at a concentration below 200 lM). Further-
more, the aromatic substituted triazoles 4f–i showed
moderate to low activities, the most active one on the
Table 3. Inhibitory effects of triazolyl-nucleosides 4a–i on L1210,
Molt4/C8, and CEM cells
Compound
R
IC50 (lM)
Acknowledgments
L1210
Molt4/C8
CEM
´
We are grateful to the CNRS, Universite de Nice-So-
phia-Antipolis and Region PACA for financial support
(bourse Med-Accueil to K.E.).
4a
4b
–CONH2
>200
>200
56
>200
>200
50
>200
>200
44
´
–(CH2)5–CH3
–(CH2)7–CH3
–CH2OH
4c
4d
>200
>200
236
>200
>200
170
36
>200
>200
245
73
4e
–CH(OH)CH3
–2-Pyridine
–p-Ph–OMe
–p-Ph–F
4f
4g
References and notes
152
209
4h
4i
5-Fluorouracil
171
126
23
217
139
9.0
1. For reviews on the chemistry, biochemistry, and synthesis
of nucleoside analogues, see: (a) Watanabe, K. A. Chem-
istry of Nucleosides and Nucleotides; Townsend, L. B., Ed.;
–3-Thiophenyl
182
0.28

K. El Akri et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6656–6659
6659
Plenum Press: New York, 1994, Vol. 3, p 421; Hacksell,
U., ; Daves, G. D., Jr. Prog. Med. Chem. 1985, 22, 1;
Buchanan, J. G. Prog. Chem. Org. Nat. Prod. 1983, 44,
243.
(ppm) 3.45–3.70 (m, 2H, 2· H-50), 3.98 (m, 1H, H-40), 4.12
(dd, 1H, J = 9.6 and 4.8 Hz, H-30), 4.37 (dd, 1H, J = 10.3
and 4.9 Hz, H-20), 5.07 (t, 1H, J = 5.3 Hz, OH-50), 5.27 (d,
1H, J = 5.3 Hz, OH-30), 5.65 (d, 1H, J = 5.9 Hz, OH-20),
5.98 (d, 1H, J = 4.5 Hz, H-10), 7.52 (br s, 1H, NH), 7.92
(br s, 1H, NH), 8.76 (s, 1H, H-5). 13C NMR (DMSO-d6,
50 MHz) d (ppm) 70.56, 79.70, 84.89, 95.53, 101.93,
134.53, 152.72, 170.93. MS (ESI+) m/z = 267 (MNa)+.
Anal. Calcd for C8H12N4O5: C, 39.35; H, 4.95. Found: C,
2. Recent reviews on IMPDH inhibition: Pankiewicz, K.;
Patterson, S. E.; Black, P. L.; Jayaram, H. N.; Risal, D.;
Goldstein, B. M.; Stuyver, L. J.; Schinazi, R. F. Curr.
Med. Chem. 2004, 11, 887; Christopherson, R. I.; Lyons,
S. D.; Wilson, P. K. Acc. Chem. Res. 2002, 35, 961.
1
`
3. Moya, J.; Pizzaro, H.; Jashes, M.; De Clercq, E.; Sandino,
A. M. Antiviral Res. 2000, 48, 125; Jashes, M.; Mlynarz,
39.56; H, 4.73; 3c H NMR (CDCl3, 200 MHz) d (ppm)
`
0.80 (t, 3H, J = 7.1 Hz, CH3), 1.10–1.40 (br m, 8H,
(CH2)4), 1.62 (br m, 2H, CH2), 2.00 (s, 3H, Ac), 2.10 (s,
6H, 2· Ac), 2.62 (t, 2H, J = 7.3 Hz, CH2), 4.15 (dd, 1H,
J = 12.2 and 4.4 Hz, H-50), 4.35 (dd, 1H, J = 12.7 and
3.2 Hz, H-50), 4.40 (m, 1H, H-40), 5.55 (t, 1H, J = 5.3 Hz,
H-30), 5.74 (dd, 1H, J = 5.2 and 3.8 Hz, H-20), 6.05 (d, 1H,
J = 3.8 Hz, H-10), 7.37 (s, 1H, H-5); 13C NMR (CDCl3,
50 MHz) d (ppm) 14.15, 20.48, 20.55, 20.73, 22.70, 25.66,
29.24, 29.34, 3.89, 62.97, 70.80, 74.34, 80.87, 119.80,
149.00, 158.15, 169.39, 169.54, 170.44. MS (ESI+) m/z =
440 (MH)+; 4c 1H NMR (CD3OD, 200 MHz) d (ppm)
0.80 (t, 3H, J = 6.6 Hz, CH3), 1.10–1.40 (br m, 8H,
(CH2)4), 1.52 (m, 2H, CH2), 2.59 (t, 2H, J = 7.8 Hz, CH2),
3.58 (dd, 1H, J = 12.2 and 4.2 Hz, H-50), 3.72 (dd, 1H,
J = 12.1 and 3.2 Hz, H-50), 4.01 (dd, 1H, J = 4.2 and
3.3 Hz, H-40), 4.23 (t, 1H, J = 5 Hz, H-30), 4.37 (t, 1H, J =
4.2 Hz, H-20), 5.90 (d, 1H, J = 4.0 Hz, H-10), 7.91 (s, 1H,
H-5).1H NMR (acetone-d6, 50 MHz) d (ppm) 14.01, 22.92,
25.80, 32.20, 62.28, 71.31, 76.44, 86.44, 93.30, 120.63,
148.21, 158.62. MS (ESI+) m/z = 314 (MH)+, 336 (MNa)+,
649 (2M+Na)+. Anal. Calcd for C15H27N3O4: C, 57.49; H,
8.68; 3g 1H NMR (CDCl3, 200 MHz) d (ppm) 1.94 (s, 3H,
Ac), 2.02 (s, 6H, 2· Ac), 3.72 (s, 3H, OMe), 4.14 (dd, 1H,
J = 12.1 and 4.2 Hz, H-50), 4.34 (dd, 1H, J = 12.2 and
3.2 Hz, H-50), 4.41 (m, 1H, H-40), 5.56 (t, 1H, J = 5.3 Hz,
H-30), 5.82 (dd, 1H, J = 3.6 and 3.4 Hz, H-20), 6.11 (d,
1H, J = 3.3 Hz, H-10), 6.85 (d, 2H, J = 8.8 Hz, Ph), 7.65
(d, 2H, J = 8.8 Hz, Ph), 7.83 (s, 1H, H-5). 13C NMR
(CDCl3, 50 MHz) d (ppm) 20.32, 20.37, 20.56, 55.21,
62.77, 70.63, 74.15, 80.74, 89.93, 113.94, 114.23, 118.11,
122.62, 127.02, 128.89, 147.88, 159.72, 169.26, 169.42,
170.31. MS (ESI+) m/z = 456 (MNa)+; 4g 1H NMR
(CD3OD, 200 MHz) d (ppm) 3.75 (dd, 1H, J = 12.2 and
4.2 Hz, H-50), 3.84 (s, 3H, OMe), 3.88 (dd, 1H, J = 12.1
and 3.1 Hz, H-50), 4.19 (br dd, 1H, J = 4.6 and 4.0 Hz, H-
40), 4.39 (t, 1H, J = 5.0 Hz, H-30), 4.58 (t, 1H, J = 4.2 Hz,
H-20), 6.10 (d, 1H, J = 4.0 Hz, H-10), 7.00 (d, 2H,
J = 8.8 Hz, Ph), 7.75 (d, 2H, J = 8.8 Hz, Ph), 8.49 (s, 1H,
H-5). 13C NMR (CD3OD, 50 MHz) d (ppm) 55.77, 62.82,
71.87, 77.03, 87.14, 94.42, 115.02, 115.34, 119.98, 124.02,
128.05, 130.19, 148.90, 159.70. MS (ESI+) m/z = 30
(MNa)+. Anal. Calcd for C14H17N3O5: C, 54.72; H,
5.58. Found: C, 54.85; H, 5.67.
G.; De Clercq, E.; Sandino, A. M. Antiviral Res. 2000, 45,
17 and references therein.
4. Guezguez, R.; Bougrin, K.; El Akri, K.; Benhida, R.
Tetrahedron Lett. 2006, 47, 4807.
5. 1-Azido-tri-O-acetylribose(2), selected spectral data: 1H
NMR (CDCl3, 200 MHz) d (ppm) 1.99 (s, 3H, Ac), 2.04 (s,
6H, 2· Ac), 4.06 (dd, 1H, J = 11.6 and 2.1 Hz, H-5), 4.24–
4.40 (m, 2H, H-4 and H-5), 4.25 (m, 1H, H-4), 5.05 (dd,
1H, J = 4.8 and 2.0 Hz, H-2), 5.25 (dd, 1H, J = 6.6 and
4.7 Hz, H-3), 5.27 (d, 1H, J = 2.0 Hz, H-1). 13C NMR
(CDCl3, 50 MHz) d (ppm) 20.27, 20.32, 20.48, 62.85,
70.32, 74.31, 79.23, 92.51, 169.30, 169.45, 170.41. MS
(ESI+) m/z = 324 (MNa)+. The b-configuration of azide 2
was attested by 1H 2D NOESY experiment and by
comparison to the previously reported data Stimac, A.;
Kobe, J. Carbohydr. Res. 1992, 232, 359; Camarasa, M.-J.;
Alonso, R.; De Las Heras, F. G. Carbohydr. Res. 1980, 83,
152.
6. The stereo- and regio-chemistry of 3a (b-configuration and
phenyl in position 4) was unambiguously attested by
NOESY and HMBC experiments. Indeed, the 1H 2D
0
NOESY spectrum shows correlations between H1 –H4 and
0
1
13
0
the H– C HMBC experiment shows C1 –H5 cross cou-
pling, in accordance with the proposed structure for 3a.
7. The silica gel catalysis effect was mainly reported. Recent
examples: Wang, L.; Jing, H.; Bu, X.; Chang, T.; Jin, L.;
Liang, Y. Catalysis Commun. 2007, 8, 80; Bandgar, B. P.;
Patil, A. V. Tetrahedron Lett. 2007, 48, 173; Firouzabadi,
H.; Iranpoor, N.; Jafarpour, M.; Ghaderi, A. J. Mol.
Catalysis 2006, 249, 98.
8. Fontainebleau Sand was purchased from VWR. It was
previously reported for such a comparison with other
silicates: Ben-Alloum, A.; Bakkas, S.; Soufiaoui, M.
Tetrahedron Lett. 1998, 39, 4481; Villemin, D.; Ricard,
M. Tetrahedron Lett. 1984, 25, 1059.
9. Analytical and spectral data for selected active nucleo-
1
sides. 3a : H NMR (CDCl3, 200 MHz) d (ppm) 1.34 (t,
3H, J = 7.1, CH3), 2.05 (s, 3H, Ac), 2.06 (s, 3H, Ac), 2.07
(s, 3H, Ac), 4.19 (dd, 1H, J = 12.6 and 3.5 Hz, H-50), 4.37
(q, J = 7.2 Hz, CH2 ester), 4.30–4.50 (m, 2H, H-40 and H-
50), 5.49 (t, J = 5.5 Hz, H-30), 5.74 (dd, J = 5.1 and 3.6 Hz,
H-20), 6.16 (d, 1H, J = 3.6, H-10), 8.29 (s, 1H, H-5
triazole). 13C NMR (CDCl3, 50 MHz) d (ppm) 14.37,
20.45, 20.53, 20.77, 61.57, 62.51, 70.29, 74.54, 81.14, 90.52,
126.77, 140.65, 160.41, 169.27, 169.51, 170.36. MS (ESI+)
m/z = 423 (MNa)+; 4a 1H NMR (DMSO-d6, 200 MHz) d
10. Balzarini, J.; Pannecouque, C.; De Clercq, E.; Pavlov, A.
Y.; Printsevskaya, S. S.; Miroshnikova, O. V.; Reznikova,
M. I.; Preobrazhenskaya, M. N. J. Med. Chem. 2003, 46,
2755.