A new C-nucleoside analogue of tiazofurin: Synthesis and biological evaluation of 2-β-D-ribofuranosylimidazole-4-carboxamide (imidazofurin)

Article abstract of DOI:10.1016/S0960-894X(00)00594-1

2-β-D-Ribofuranosylimidazole-4-carboxamide, an imidazole analogue of the antitumor agent tiazofurin, was synthesized and evaluated for the growth inhibitory activity of human myelogenous leukemia K562 cells. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0960-894X(00)00594-1

Bioorganic & Medicinal Chemistry Letters 11 (2001) 67±69
A New C-Nucleoside Analogue of Tiazofurin:
Synthesis and Biological Evaluation of
2-ꢀ-D-Ribofuranosylimidazole-4-carboxamide (Imidazofurin)
Palmarisa Franchetti,a,* Stefano Marchetti,a Loredana Cappellacci,a Joel A. Yalowitz,b
Hiremagalur N. Jayaram,b Barry M. Goldsteinc and Mario Grifantinia
a
Á
Dipartimento di Scienze Chimiche, Universita di Camerino, 62032 Camerino, Italy
bDepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA
cDepartment of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA
Received 5 June 2000; revised 20 October 2000; accepted 23 October 2000
AbstractÐ2-b-d-Ribofuranosylimidazole-4-carboxamide, an imidazole analogue of the antitumor agent tiazofurin, was synthesized
and evaluated for the growth inhibitory activity of human myelogenous leukemia K562 cells. # 2000 Elsevier Science Ltd. All
rights reserved.
Inosine 50-monophosphate dehydrogenase (IMPDH)
catalyzes the conversion of IMP to XMP and it is the
rate limiting enzyme in de novo guanylate biosynthesis.1
The activity of this enzyme was shown to be signi®cantly
increased in tumor cells and therefore considered to be a
potential target for cancer chemotherapy.2 The inhibi-
tion of IMPDH and subsequent reduction in guanine
nucleotides interrupts DNA and RNA synthesis in
rapidly-dividing tumor cells. Tiazofurin (2-b-d-ribofur-
anosylthiazole-4-carboxamide) is an oncolytic C-nucleo-
side with potent inhibitory activity against IMPDH.3 In
Phase I/II clinical trials tiazofurin showed a signi®cant
reduction in leukemic cell burden in acute myelogenous
leukemia patients.4 6
Structural studies carried out by us and other authors
on tiazofurin, selenazofurin and a number of their
C-nucleoside analogues, have pointed out that con®g-
urational and conformational e€ects are important for
IMPDH inhibition.12 15 In particular, it was found that
the active compounds are those with an electrophilic
sulfur or selenium in a delocalized environment adjacent
to the C-glycosidic bond.15 Compounds in which the
sulfur or selenium atom is replaced by a negatively
charged oxygen show minimal or no activity. It has
been suggested that this requirement may result from
either an intermolecular interaction between the sulfur
or selenium and a negatively charged side chain in the
active site of IMPDH, and/or an intramolecular inter-
action between the sulfur or selenium and the furanose
oxygen (O40).12 An intramolecular S/Se±O interaction
might be expected to constrain rotation around the
C-glycosidic bond, both in the parent compounds tiazo-
and selenazofurin and in their anabolites TAD and
SAD. The speci®city of TAD and SAD for the target
enzyme would be either enhanced or diminished,
depending upon the degree to which the heteroatom±
oxygen interaction is maintained by the binding
Tiazofurin requires metabolic activation to the corre-
sponding nicotinamide adenine dinucleotide (NAD)
analogue, thiazole-4-carboxamide adenine dinucleotide
(TAD). This NAD analogue inhibits IMPDH activity
through competition for the NAD cofactor-binding site
of the enzyme.7
Several analogues of tiazofurin and TAD have been
studied. The selenium analogue, selenazofurin (2-b-d-
ribofuranosylselenazole-4-carboxamide), is more cytotoxic
than tiazofurin,8 10 and its corresponding anabolite,
SAD, binds more tightly to IMPDH than TAD does.11
*Corresponding author. Tel.: +39-0737-402228; fax: +39-0737-
637345; e-mail: frapa@camserv.unicam.it
0960-894X/01/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00594-1

68
P. Franchetti et al. / Bioorg. Med. Chem. Lett. 11 (2001) 67±69
enzyme.16 Further support for the electrostatic hypo-
thesis is provided by the inactive tiazo- and selenazo-
furin analogue oxazofurin (2-b-d-ribofuranosyloxazole-
4-carboxamide).17 In this C-nucleoside the positive sul-
fur or selenium heteroatom is replaced by a more elec-
tronegative oxygen. Ab initio computations suggest that
the positive charge in the region of the former sulfur or
selenium position is replaced by a negative charge.
Thus, this analogue is expected to demonstrate a C-gly-
cosidic angle higher than those observed in the thiazole
and selenazole nucleosides, a prediction con®rmed in
the crystal structure of oxazofurin.18 These ®ndings
were further con®rmed by the synthesis of thio-
phenfurin and furanfurin, two C-nucleoside isosteres of
tiazofurin, in which the thiazole ring was replaced by a
thiophene and furan heterocycle, respectively.13 While
thiophenfurin was found active as an antitumor agent
both in vitro and in vivo, furanfurin proved to be inac-
tive. In 1997, Makara and Keseruuhave questioned the
electrostatic hypothesis and suggested that the ¯exibility
of the C-glycosydic bond in this type of C-nucleosides is
ultimately determined by steric interactions of the het-
eroatoms with the C20-H and O40 of the ribose.19
Application of this theory led these authors to design
2-b-d-ribofuranosylimidazole-4-carboxamide (imidazo-
furin, 1) as an analogue that exhibits an almost identical
behavior to tiazofurin in ab initio computations. This
hypothesis prompted us to synthesize imidazofurin and
to test its antitumor activity.
Chemistry
Imidazofurin was synthesized through two slightly dif-
ferent approaches. The ®rst approach is summarized in
Scheme 1. The reaction of b-d-ribofuranosyl-1-carboxi-
midic acid methyl ester (2)20 with ethyl 2-amino-3,3-
diethoxypropionate hydrochloride (3)21 in anhydrous
methanol gave a mixture of ethyl 2-b-d-ribofur-
anosylimidazole-4-carboxylate (4) and by-product 5, a
1,50-cycloimidazole C-nucleoside, as hydrochlorides.
Treatment of this mixture with methanolic ammonia
gave a mixture of imidazofurin22 (1, 30.6%) and pro-
duct 623 (5%) which were separated by column chro-
matography using CHCl3±MeOH (85:15) as eluent.
Compound 6 was converted into the amide 7 by reac-
tion with 30% aqueous ammonia. In order to avoid the
formation of 5 we developed the method reported in
Scheme 2. The reaction of 2,3,5-tri-O-benzyl-b-d-ribo-
furanosyl cyanide (8)24 with sodium methoxide in
methanol gave the methyl imidate 9 which, by reaction
with 3 in methanol, followed by treatment of inter-
mediate 10 with methanolic ammonia, was converted
into the amide 11. Debenzylation of 11 with ammonium
Scheme 1. (i) CH3OH (27 h, rt); (ii) NH3/CH3OH (6 h, rt); (iii) 30% NH4OH (48 h, 60 ꢀC).
Scheme 2. (i) CH3ONa/CH3OH (5 h, rt); (ii) CH3OH (24 h, rt); (iii) NH3/CH3OH (2 h, rt); (iv) Pd/C, HCOONH4, CH3OH (1.5 h, re¯ux).

P. Franchetti et al. / Bioorg. Med. Chem. Lett. 11 (2001) 67±69
69
formate and Pd/C (10%) in methanol a€orded
imidazofurin in 35% yield. All compounds were char-
acterized by mass spectrometry, 1H NMR spectroscopy,
and elemental analysis. Two tautomeric forms of imi-
dazofurin (1a and 1b) were observed in solution by H
NMR in DMSO-d6 as well as in D2O in the ratio 2:1.
5. Jayaram, H. N.; Lapis, E.; Tricot, G. J.; Kneebone, P.;
Paulik, E.; Zhen, W.; Engeler, G. P.; Ho€man, R.; Weber, G.
Int. J. Cancer 1992, 51, 182.
6. Wright, D. G.; Boosalis, M. S.; Waraska, K.; Osrly, L. J.;
Weeintraub, L. R.; Vosburgh, E. Anticancer Res. 1996, 16,
3349.
7. Lui, M. S.; Faderan, M. A.; Liepnieks, J. J.; Natsumeda,
Y.; Olah, E.; Jayaram, H. N.; Weber, G. J. Biol. Chem. 1984,
259, 5078.
1
8. Boritzki, T. J.; Berry, D. A.; Besserer, J. A.; Cook, P. D.;
Fry, D. W.; Leopold, W. R.; Jackson, R. C. Biochem. Phar-
macol. 1985, 34, 1109.
9. Streeter, D.; Robins, R. K. Biochem. Biophys. Res. Com-
mun. 1983, 115, 544.
10. Jayaram, H. N.; Ahluwalia, G. S.; Dion, R. L.; Gebeyehu,
G.; Marquez, V. E.; Kelley, J. A.; Robins, R. K.; Cooney, D.
A.; Johns, D. G. Biochem. Pharmacol. 1983, 32, 2633.
11. Marquez, V. E.; Tseng, C. K.; Gebeyehu, G.; Cooney,
D. A.; Ahluwalia, G. S.; Kelley, J. A.; Dalal, M.; Fuller, R.
W.; Wilson, Y. A.; Johns, D. G. J. Med. Chem. 1986, 29,
1726.
The structure of compound 6 was con®rmed by nuclear
Overhauser enhancement (NOE) e€ects. In fact, when
H50 protons were irradiated a NOE e€ect was observed
on H5, indicating that these protons are proximate.
12. Franchetti, P.; Grifantini, M. Curr. Med. Chem. 1999, 6,
457 and references therein.
13. Franchetti, P.; Cappellacci, L.; Grifantini, M.; Barzi, A.;
Nocentini, G.; Yang, H.; O'Connor, A.; Jayaram, H. N.;
Carrell, C.; Goldstein, B. M. J. Med. Chem. 1995, 38, 3829.
14. Franchetti, P.; Cappellacci, L.; Abu Sheikha, G.;
Jayaram, H. N.; Gurudutt, V. V.; Sint, T.; Schneider, B. P.;
Jones, W. D.; Goldstein, B. M.; Perra, G.; De Montis, A.; Loi,
A. G.; La Colla, P.; Grifantini, M. J. Med. Chem. 1997, 40,
1731.
15. Franchetti, P.; Marchetti, S.; Cappellacci, L.; Jayaram, H.
N.; Yalowitz, J. A.; Goldstein, B. M.; Barascut, J.-L.;
Dukhan, D.; Imbach, J.-L.; Grifantini, M. J. Med. Chem.
2000, 43, 1264.
Biological Evaluation
Imidazofurin was evaluated for its ability to inhibit the
growth of human myelogenous leukemia K562 cells.
Tumor cell proliferation was evaluated by incubating
the cells continuously with either the compound or sal-
ine for 48 h.14 Thiophenfurin was used as a reference
compound. Contrary to what Makara and Keseruuhave
predicted,19 imidazofurin proved to be nontoxic to cell
growth (no growth inhibition observed at 100 mM) as
compared with thiophenfurin (IC50=4.6 mM). The poor
activity of imidazofurin might be due to its inability to
be phosphorylated by cellular kinases and nucleo-
tidases, inability to be converted to the dinucleotide
analogue of NAD, or failure of the dinucleotide to bind
to the target. We believe that the presence of imidazole
annular prototropic tautomerism could destabilize the
conformation of imidazofurin suitable to bind the enzyme.
Makara and Keseruucarried out their ab initio computa-
tions taking into consideration only the tautomeric form
1b. Thus, the contribution of tautomer 1a was neglected.
16. Burling, F. T.; Goldstein, B. M. J. Am. Chem. Soc. 1992,
114, 2313.
17. Franchetti, P.; Cristalli, G.; Grifantini, M.; Cappellacci,
L.; Vittori, S.; Nocentini, G. J. Med. Chem. 1990, 33, 2849.
18. Goldstein, B. M.; Hallows, W.; Langs, D.; Franchetti, P.;
Cappellacci, L.; Grifantini, M. J. Med. Chem. 1994, 37, 1684.
19. Makara, G. M.; Keseruu, G. M. J. Med. Chem. 1997, 40,
4154.
20. Poonian, M. S.; Nowoswiat, E. F. J. Org. Chem. 1980, 45, 203.
21. DiMaio, J.; Belleau, B. J. Chem. Soc., Perkin Trans. 1
1989, 1687.
22. Selected data for 1: TLC (CHCl3±MeOH, 8:2): Rf 0.12; 1H
NMR (200 MHz, DMSO-d6): d 3.52 (m, 1H, H50b); 3.62 (m,
1H, H50a); 3.75 (m, 1H, H40); 3.90 (m, 2H, H20 and H30); 4.35
(two overlapping d, J=5.5 Hz, 1H, H10); 4.90 (m, 1H, OH);
5.15 (m, 1H, OH); 5.25 (pseudo d, 1H, OH); 7.10, 7.39 (2 s,
1H, H5), 7.32, 7.58 (2 br s, 2H, NH2); 10.55, 10.70 (2 br s, 1H,
NH). Positive ion electrospray MS (M+H)=244.1. Anal.
calcd for C9H13N3O5: C, 44.45; H, 5.39; N, 17.28. Found: C,
44.32; H, 5.13; N, 17.15.
Acknowledgements
Work supported by Ministero dell'Universita e della
Ricerca Scienti®ca e Tecnologica (40% funds) and by
CNR, Italy.
23. Selected data for 6: TLC (CHCl3±MeOH, 9:1): Rf 0.56; 1H
NMR (200 MHz, DMSO-d6): d 1.25 (t, J=7.0 Hz, 3H, CH3);
3.49 (dd, J=4.3, 11.9 Hz, 1H, H50b); 3.63 (dd, J=3.7, 11.9 Hz,
1H, H50a); 3.81 (pseudo q, J=4.2 Hz, 1H, H40); 3.95 (pseudo
q, J=5.5 Hz, 1H, H30); 4.15 (pseudo q, J=5.0 Hz, 1H, H20);
4.22 (q, J=7.2 Hz, 2H, CH2CH3); 4.69 (d, J=5.5 Hz, 1H,
H10); 4.98 (d, J=5.3 Hz, 1H, OH30); 5.22 (d, J=5.6 Hz, 1H,
OH20); 7.80 (br s, 1H, H5). Positive ion electrospray MS
(M+H)=226.0. Anal. calcd for C11H14N2O5: C, 51.97; H,
5.55; N, 11.02. Found: C, 51.92; H, 5.85; N, 10.98.
24. De las Heras, F. G.; Fernandez-Resa, P. J. Chem. Soc.,
Perkin Trans. 1 1982, 903.
References and Notes
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2. Weber, G. Cancer Res. 1983, 43, 3466.
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Pharmacol. 1982, 31, 2371.
4. Tricot, G. J.; Jayaram, H. N.; Lapis, E.; Natsumeda, Y.;
Nichols, C. R.; Kneebone, P.; Heerema, N.; Weber, G.; Ho€-
man, R. Cancer Res. 1989, 49, 3696.