Stereospecific synthesis of two novel cytotoxic pyrazole C-nucleosides from D-glucose

Article abstract of DOI:10.1016/S0040-4039(00)00894-7

A multistep stereospecific synthesis of two novel pyrazole C-nucleosides 12 and 21 has been achieved starting from D-glucose, by utilizing the 2,5- anhydro-D-glucose ethylene acetal derivative 1 as a divergent intermediate. The C-nucleoside 12 was shown to be a moderate inhibitor of the in vitro growth of N2a and BHK 21 tumor cell lines, whereas 21 showed a moderate cytotoxic activity only against N2a cells. (C) 2000 Published by Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00894-7

Tetrahedron Letters 41 (2000) 5737±5740
Stereospeci®c synthesis of two novel cytotoxic pyrazole
C-nucleosides from d-glucose
Mirjana Popsavin,a,* Ljilja Torovic,a Sasa Spaic,a Srdjan Stankovb
and Velimir Popsavina
aInstitute of Chemistry, Faculty of Sciences, University of Novi Sad, Trg D. Obradovica 3, 21000 Novi Sad, Yugoslavia
bPasteur Institute, Hajduk Veljkova 1, 21000 Novi Sad, Yugoslavia
Received 10 May 2000; accepted 2 June 2000
Abstract
A multistep stereospeci®c synthesis of two novel pyrazole C-nucleosides 12 and 21 has been achieved
starting from d-glucose, by utilizing the 2,5-anhydro-d-glucose ethylene acetal derivative 1 as a divergent
intermediate. The C-nucleoside 12 was shown to be a moderate inhibitor of the in vitro growth of N2a and
BHK 21 tumor cell lines, whereas 21 showed a moderate cytotoxic activity only against N2a cells. # 2000
Published by Elsevier Science Ltd.
Keywords: C-nucleosides; isosteres; pyrazoles; cytotoxic activity.
Introducing diversity into the carbohydrate and/or base subunit of nucleosides represents a
promising strategy for the design of novel chemotherapeutic agents.1 Signi®cant antitumor and
antiviral activity displayed by certain pyrazole-related nucleosides2 prompted us to prepare the
related C-nucleoside analogues. The synthesis of several 4-(b-d-ribofuranosyl)pyrazoles,3 includ-
ing a pyrazole homo-C-nucleoside, has already been reported.4 Conversely, the synthesis of
C-nucleosides containing the pyrazole-5-carboxamide moiety and a modi®ed sugar segment has
not been described in the literature so far. Herein we report a divergent synthesis of two novel
pyrazole-related C-nucleosides 12 and 21 from d-glucose, along with preliminary results related
to their in vitro cytotoxic activity against mouse neuroblastoma (N2a) and baby hamster kidney
(BHK 21) tumor cell lines.
The crucial problem in the synthesis of both targets 12 and 21 was the establishment of the
requisite b-con®guration at the anomeric position. In a previous paper we have described5 the
conversion of d-glucose to the 2,5-anhydro-d-glucose ethylene acetal derivative 1 (Scheme 1), a
compound already containing the desired b-C-glycosidic bond, as well as the functionalities suitable
* Corresponding author. Fax: +38 121 54 065; e-mail: popsavin@ih.ns.ac.yu
0040-4039/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0040-4039(00)00894-7

5738
Scheme 1. (a) BnBr, Ag2O, Et2O, re¯ux, 22 h, 87%; (b) KOBz, DMF, 100ꢀC, 48 h, 83%; (c) NaN3, DMSO, 120ꢀC, 47
h, 77%; (d) H2, Pd/C, CHCl3 (cat.), EtOH, rt, 120 h; (e) BzCl, Py, rt, 96 h, 71% from 4; (f) 4:1 CF3CO2H:6 M HCl,
for further introduction of diversity into the nucleoside carbohydrate segment. The 2,5-anhydro-
d-glucose derivative 1 reacted with benzyl bromide in ether, in the presence of silver(I)-oxide as a
catalyst, to a€ord the expected 4-O-benzyl derivative 2 in 87% yield. Reaction of 2 with potas-
sium benzoate in N,N-dimethylformamide gave the corresponding 6-O-benzoyl derivative 3
(83%), which was further treated with sodium azide in dimethyl sulfoxide to a€ord the 3-azido-3-
deoxy derivative 4 in 77% yield.
Catalytic reduction of 4 over 10% Pd/C, in ethanol containing a catalytic amount of chloro-
form, gave the amine hydrochloride 5 as a product of a sequential azide reduction/benzyl ether
hydrogenolysis process.6 Treatment of crude 5 with benzoyl chloride in pyridine yielded the key
intermediate 6 as the major reaction product (71% from 4). Hydrolytic removal of the dioxolane
protective group in 6 was achieved with a 4:1 mixture of tri¯uoroacetic acid and 6 M hydro-
chloric acid at +4ꢀC, whereupon the corresponding aldehyde 7 was obtained. Due to its instability
it was not characterized further, but was immediately treated with (carbomethoxymethylene)-
triphenylphosphorane in dichloromethane to a€ord a 2:1 mixture of the corresponding Z and E
unsaturated esters 8 and 9 in 85% combined yield.
The unsaturated esters 8 and 9 were readily separated by column chromatography and converted
to the C-nucleoside 12 according to the methodology developed by Mo€att et al.7 Both 8 and 9
readily underwent 1,3-dipolar cycloaddition with an excess of diazomethane in ether at 0ꢀC to
yield the 2-pyrazoline 10 as the only reaction product. The transformation of 8 to 10 was com-
pleted within 2 h, while the conversion of 9 to 10 required 15 h for completion. The enhanced

5739
reactivity of the Z-isomer 8 is presumably due to the activation of its a,b-unsaturated system by
the intramolecular hydrogen bond between H (amide) and O (ester carbonyl). Indeed, the H
1
NMR spectrum of 8 showed a signi®cant down®eld shift of the amide proton signal (ꢀNH 7.67) in
contrast to the corresponding signal in the spectrum of 9, which appeared at a high®eld position
(ꢀNH 6.78). This result is consistent with the presence of an intramolecular hydrogen bond in 8.
Without puri®cation or further characterization the intermediate 10 was treated with a saturated
solution of chlorine in carbon tetrachloride to give the pyrazole derivative 11. Finally, treatment
of 11 with methanolic ammonia a€orded the O-deprotected C-nucleoside 12,8 ready for biological
testing. Under these reaction conditions the intermediates 8 and 9 were converted into the target
12 in 97 and 85% overall yields, respectively.
Synthesis of the pyrazole C-nucleoside 21 bearing the 30-mesyloxy group as an isostere is out-
lined in Scheme 2. Solvolysis of 135 in wet N,N-dimethylformamide (5% of water), in the pre-
sence of calcium carbonate as a proton acceptor, gave a mixture of 4,6- and 3,6-di-O-benzoyl
derivatives 14 and 15 in 86% combined yield. The regioisomers 14 and 15 could not be separated
by column chromatography, presumably due to their rapid inter-conversion a€ected by silica gel.
However, direct crystallization of crude reaction mixture from benzene±hexane a€orded pure 15,
which was subsequently mesylated to give the key intermediate 16. The addition of mesyl chloride
in pyridine to the mother liquor, which remained after crystallization of 15, a€orded an addi-
tional quantity of 16. The total yield of 16 was 41% with respect to 13. The intermediate 16 was
converted to the target molecule 219 by using the same ®ve-step sequence already applied for the
conversion of 6 into the C-nucleoside 12. In this way the 4-O-mesyl derivative 16 has been
transformed into the target 21 in 38% overall yield.
Scheme 2. (a) DMF, H2O (5%), CaCO3, 155±160ꢀC, 20 h, 86%; (b) MsCl, Py, +4ꢀC, 48 h, 41% from 13; (c) 4:1
CF3CO2H-6M HCl, +4ꢀC, 24 h; (d) Ph3P:CHCO2Me, CH2Cl2, rt, 24 h, 48% from 16; (e) CH2N2, Et2O, 0ꢀC, 5 h; (f)
Cl2/CCl4, CH2Cl2, rt, 3 h, 91% from 18; (g) NH3, MeOH, rt, 7 days, 86%.
The C-nucleosides 12 and 21 were preliminarily tested in vitro for cytotoxic activity against
mouse neuroblastoma (N2a) and baby hamster kidney (BHK 21) tumor cell lines. As shown in
Table 1, the C-nucleoside 12 showed a moderate cytotoxic e€ect against both N2a and BHK 21
cells. Compound 21 displayed no signi®cant cytotoxicity towards BHK 21, but showed some
cytostatic activity against N2a cells. Studies on further biological evaluation of these compounds
will be reported in due course.

5740
Table 1
Cytotoxic activity of synthesized C-nucleosides 12 and 21
In summary, a stereospeci®c synthesis of cytotoxic pyrazole-related C-nucleosides bearing the
2-benzamido (12) or the 3-mesyloxy group (21) as isosteres has been achieved starting from
d-glucose, via the 2,5-anhydro-d-glucose derivative 1 as a divergent intermediate. Molecules of type
12 might be of a wider medicinal interest because they are partly related to certain 20-benzamido-
20-deoxy adenosines, the potential agents for treatment of sleeping sickness.10
Acknowledgements
This work was supported by a research grant from the Ministry of Science and Technology of
the Republic of Serbia.
References
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6. Standard conditions for this transformation are: 1 atm, 10 mmol of 4, 146 mL of absolute EtOH, 7.2 mL of
CHCl3, and 9.5 g of 10% Pd/C. Decreasing the quantity of catalyst (214 mg/1 mmol) resulted in complete
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8. Compound 12: m.p. 133ꢀC (from MeOH EtOAc); [ꢁ]D ^116.6 (c, 1.08 in MeOH); H NMR (250 MHz, DMSO-
1
0
0
0
0
0
0
0
0
0
0
0
d6): ꢀ 3.54 (d, 2H, J4 ,5 =4.6 Hz, 2ÂH-5 ), 3.82±3.95 (m, 2H, J1 ,2 =10.4, J2 ,3 =5.2, J3 ,4 =1.5 Hz, H-4 and H-2 ),
3.40±4.60 (bs, 2H, 2ÂOH), 4.25 (dd, 1H, H-30), 5.39 (d, 1H, H-10), 7.38±7.84 (m, 5H, Ph), 7.60 and 7.90 (2Âbs, 1H
each, CONH2), 7.85 (s, 1H, H-3), 8.54 (d, 1H, J2 ,NH=6.1 Hz, NHBz), 13.26 (bs, 1H, NH); 13C NMR (62.5 MHz,
0
DMSO-d6): ꢀ 61.17 (C-20), 62.06 (C-50), 70.77 (C-30), 72.34 (C-10), 86.85 (C-40), 121.30 (C-3), 127.11, 128.41, 131.46
and 133.83 (ArC), 130.20 and 142.82 (C-4 and C-5), 164.97 (CONH2), 166.24 (PhCO).
9. Compound 21: m.p. 110±111ꢀC (from MeOH±iPr2O); [ꢁ]D +26.4 (c, 1.03 in MeOH); 1H NMR (250 MHz, DMSO-d6):
0
0
0
0
0
0
0
0
ꢀ 3.21 (s, 3H, CH3SO2), 3.51 (dd, 1H, J5a ,5b =12.1, J4 ,5a =4.1 Hz, H-5a ), 3.56 (dd, 1H, J4 ,5b =4.0 Hz, H-5b ),
0
0
0
0
0
0
0
0
0
4.05 (m, 1H, J3 ,4 =3.2 Hz, H-4 ), 4.13 (dd, 1H, J1 ,2 =7.7, J2 ,3 =5.2 Hz, H-2 ), 4.88 (dd, 1H, H-3 ), 5.11 (d, 1H,
H-10), 3.42 and 5.84 (2Âbs, 1H each, 2ÂOH), 7.38 and 7.64 (2Âbs, 1H each, CONH2), 7.86 (s, 1H, H-3), 13.28 (bs,
1H, NH); 13C NMR (62.5 MHz, DMSO-d6): ꢀ 38.21 (CH3SO2), 60.92 (C-50), 74.96 (C-20), 75.44 (C-10), 81.21 (C-
30), 82.51 (C-40), 120.44 (C-3), 130.15 and 143.05 (C-4 and C-5), 164.47 (CONH2).
10. Van Calenbergh, S.; Verlinde, C. L. M. J.; Soenens, J.; De Bruyn, A.; Callens, M.; Blaton, N. M.; Peeters, O. M.;
Rozenski, J.; Hol, W. G. J.; Herdewijn, P. J. Med. Chem. 1995, 38, 3838±3849.