Synthesis and biological evaluation of two novel 2′-substituted tiazofurin analogues

Article abstract of DOI:10.1016/j.tetlet.2004.07.104

Two novel tiazofurin analogues, 2-(2-benzamido-2-deoxy-β-d- ribofuranosyl)thiazole-4-carboxamide 4 and 2-(2-azido-2-deoxy-β-d- ribofuranosyl)thiazole-4-carboxamide 5, have been synthesized starting from d-glucose and evaluated for their in vitro cytotoxicity against several human leukaemia and solid tumour cell lines.

Full text of DOI:10.1016/j.tetlet.2004.07.104

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 7125–7128
Synthesis and biological evaluation of two novel
20-substituted tiazofurin analogues
a
b
b
Mirjana Popsavin,a, Ljilja Torovic, Vesna Kojic, Gordana Bogdanovic and
*
´
´
´
Velimir Popsavina
aDepartment of Chemistry, Faculty of Sciences, University of Novi Sad,
´
Trg D. Obradovica 3, 21000 Novi Sad, Serbia and Montenegro
bInstitute of Oncology Sremska Kamenica, Institutski put 4, 21204 Sremska Kamenica, Serbia and Montenegro
Received 7 June 2004; revised 13 July 2004; accepted 22 July 2004
Abstract—Two novel tiazofurin analogues, 2-(2-benzamido-2-deoxy-b-D-ribofuranosyl)thiazole-4-carboxamide 4 and 2-(2-azido-2-
deoxy-b-D-ribofuranosyl)thiazole-4-carboxamide 5, have been synthesized starting from D-glucose and evaluated for their in vitro
cytotoxicity against several human leukaemia and solid tumour cell lines.
ꢀ 2004 Elsevier Ltd. All rights reserved.
C-Nucleosides have received much attention in recent
years due to their interesting biological activities.1,2
Among them, tiazofurin (1, Fig. 1) has recently been
approved as an orphan drug for treatment of chronic
myelogenous leukaemia in accelerated phase or blast cri-
sis.3 It inhibits the enzyme inosine 50-monophosphate
dehydrogenase (IMPDH) inducing shutdown of guanine
nucleotide synthesis and causing apoptosis of certain
malignant cells.4 Tiazofurin is intracellularly converted
to the NAD analogue, thiazole-4-carboxamide adenine
dinucleotide (TAD), which binds to the NAD cofac-
tor-binding site of the enzyme and thus inhibits IMPDH
activity.5,6 Because of its critical role in purine de novo
synthesis, IMPDH represents an important target for
anticancer and antiviral chemotherapy.7 Despite the
availability of IMPDH inhibitors, lack of specificity re-
mains a problem in their clinical use. A significant effi-
cacy of tiazofurin was achieved in phase II clinical
trials. However, hospitalization due to neuro- and cardi-
ovascular toxicities, including their aggressive treatment
is required.8 It is believed that modification of the ribose
moiety of tiazofurin may provide an access to analogues
with reduced toxicity and enhanced antitumour activ-
ity. Accordingly, a number of tiazofurin analogues with
a modified sugar moiety have been reported,9 including a
recent preparation of 2-(3-azido- and 3-amino-3-deoxy-
b-D-ribofuranosyl)thiazole-4-carboxamides.10 However,
these substances were devoid of any significant biologi-
cal activity.
We have recently reported on the synthesis and biologi-
cal evaluation of two novel tiazofurin analogues 2 and
3.11 Both C-nucleosides 2 and 3 show in vitro cytotoxic-
ity against certain malignant cells, but against normal
foetal lung cell line, MRC-5, the fluoro derivative 2 was
found to be completely inactive, while the acetamido
Figure 1. Tiazofurin (1) and analogues.
Keywords: 2,5-Anhydro sugars; in vitro Cytotoxicity; de Novo synthesis; Tiazofurin analogues; Thiazoles.
*
Corresponding author. Tel.: +381 21 350 122; fax: +381 21 454 065; e-mail: mpopsavin@ih.ns.ac.yu
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.07.104

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M. Popsavin et al. / Tetrahedron Letters 45 (2004) 7125–7128
derivative 3 exhibited weak cytotoxicity.11 Continuing
our work in this field, we present herein the synthesis
and preliminary biological evaluation of two new tia-
zofurin analogues 4 and 5 bearing the 20-benzamido
and 20-azido groups. In view of the interesting biological
activities of some 20-arylamido-12 or 20-azido-20-deoxy-
ribonucleosides,13 it was of interest to put these groups
at the C-20 position of tiazofurin in order to compare
the biological activity of the resulting analogues with
that observed for the parent compound 1.
in our laboratory14,15 starting from commercially avail-
able 1,2-O-isopropylidene-b-D-glucofuranose
6
via
seven14 and eight15 synthetic steps, respectively.
Treatment of 7 with sodium azide (DMSO, 108–110ꢁC)
afforded predominantly the 3-azido-3-deoxy derivative 9
(61%) as a result of nucleophilic displacement at C-3
(with Walden inversion). The reaction mixture also con-
tained a minor amount of the regioisomeric 3-O- and 4-
O-benzoyl derivatives 10, earlier obtained as a major
reaction product from the solvolysis of 7 in N,N-dimethyl-
formamide.14 The stereochemistry of 9 was elucidated
Our synthetic strategy to the target C-nucleosides 4 and
5 was to synthesize the ribofuranosyl thioamides 15 and
21 (Scheme 1) as key intermediates and then to cyclo-
condense them with ethyl bromopyruvate to form the
thiazole ring. Two independent five-step sequences to-
wards the key intermediates 15 and 21 were therefore de-
vised starting from the 2,5-anhydro sugar derivatives 7
and 8. Both 7 and 8 have been synthesized previously
1
by means of NOE differential H NMR spectroscopy.
Thus, irradiation at 5.08ppm (d, 1H, J1,2 = 2.8Hz,
H-1) gave a strong NOE with H-3 and H-4 thus indicat-
ing the close spatial arrangement of these protons and
consequently the D-allo configuration of product 9.
The structure of 9 was additionally confirmed by its
independent synthesis starting from the 4-O-benzyl
Scheme 1. Reagents and conditions: (a) NaN3, DMSP, 108–110ꢁC, 70h, 61%; (b) TiCl4, CH2Cl2, rt, 1h, 59% of 11, 0ꢁC, 1h, 67% of 21; (c) BzCl, Py,
rt, 72h, 84%; (d) 4:1 TFA-6M HCl, 4ꢁC, 11days for 9, 3days for 8; (e) NH2OH · HCl, NaOAc, EtOH, CH2Cl2, rt, 2h; (f) MsCl, Py, ꢀ15ꢁC, 0.5h,
then 0ꢁC, 1h, then rt, 1h, 59% of 14 (from 9), 39% of 19 (from 8); (g) H2S, Py, rt, 1.5h for 14, 71% of 15, 25% of 16, 2h for 19, 34% of 20; (h) H2S,
DMAP, EtOH, rt, 8h, 82% of 15.
Scheme 2. Reagents and conditions: (a) BrCH2COCO2Et, EtOH, reflux, 80min for 15, 55% of 22, 50min for 21, 57% of 23, 22% of 24; (b) NH3,
MeOH, rt, 7days for 22, 56% of 4, 6days for 23, 75% of 5.

M. Popsavin et al. / Tetrahedron Letters 45 (2004) 7125–7128
7127
derivative 8. Debenzylation of 8 with titanium tetrachlo-
ride in dry dichloromethane gave a 59% yield of the cor-
responding azido derivative 11, which was subsequently
converted to 9 (84%) by treatment with benzoyl chloride
the presence of several additional components of higher
polarity, with one of them presumably being the amino
derivative. However, none of these by-products could be
isolated in pure form due to their similar chromato-
graphic properties. Moreover, O-debenzylation of 20
with titanium tetrachloride gave the corresponding
azido derivative 21 in 67% yield.
1
in pyridine. The IR, H and 13C NMR spectra of the
product 9 thus obtained displayed the same signals as
the material 9 prepared from the 2,5-anhydride 7.
Hydrolytic removal of the acetal protective group in 9
gave the corresponding aldehyde 12, which was not
purified but was further treated with hydroxylamine
hydrochloride and sodium acetate immediately giving
the oxime derivatives 13 as an inseparable mixture of
the corresponding E- and Z-isomers. The mixture 13
was used in the next step without purification and was
reacted with mesyl chloride in pyridine to give the corre-
sponding nitrile 14 (59% from 9). Exposure of 14 to
hydrogen sulphide gas gave the thioamide 15 as a result
of the expected addition of hydrogen sulphide to the nit-
rile group, followed by subsequent reduction of the
azido group to an amino function, as well as a final O!N
acyl migration. Depending on the reaction conditions
and reagents used, the major product 15 was accompa-
nied with a variable amount of 16. Thus when 14 was re-
acted with hydrogen sulphide in dry pyridine for 1.5h at
room temperature both 15 and 16 were obtained in 71%
and 25% yields, respectively. However, on prolonging
the reaction time to 8h, the benzamido derivative 15
was obtained as the only reaction product, but in some-
what lower yield (69%). This result indicates that the
addition of hydrogen sulphide to the nitrile function in
14 precedes the reduction of the azido group. Finally,
when the reaction was carried out with hydrogen sul-
phide and N,N-dimethylaminopyridine in ethanol for
8h at room temperature, the key intermediate 15 was
obtained as the only reaction product in a yield of
82%. To the best of our knowledge, the transformation
of 14 to 15 represents the first example of a direct con-
version of a 2-azido-3-O-acyl-ribofuranosyl cyanide to
the corresponding 2-acylamido thiocarboxamide. If this
reaction is general it may provide access to a variety of
2-acylamido thiocarboxamides and consequently to
novel tiazofurin analogues bearing acylamido functio-
nalities at C-20.
Having obtained the key intermediates 15 and 21, we
next focused on their further transformations in order
to elaborate the thiazole-4-carboxamide heterocylic sys-
tem using a modified Hantzsch thiazole synthesis.16
Accordingly, treatment of 15 with ethyl bromopyruvate
in refluxing ethanol produced the corresponding thia-
zole 22 in 55% yield. The cyclocondensation of 21 under
the same reaction conditions gave the required thiazole
23 (57%), but with a minor amount of the known17 fur-
an 24 (22%). Finally, exposure of 22 and 23 to metha-
nolic ammonia provided the tiazofurin analogues 418
and 519 in 56% and 75% yields, respectively (Scheme 2).
The newly synthesized tiazofurin analogues 4 and 5 were
preliminarily evaluated for their antiproliferative activ-
ity against human myelogenous leukaemia K562, pro-
myelocytic leukaemia HL60, colon adenocarcinoma
HT29, estrogen receptor positive breast adenocarci-
noma MCF7, cervix carcinoma HeLa and normal foetal
lung MRC-5 cells. Tiazofurin 1 was used as a reference
compound. In vitro cytotoxicity was evaluated after
24h cell treatment by the MTT assay.20 The results are
presented in Table 1.
Compound 4 was inactive against the MCF7 and HeLa
cells, but showed significant cytotoxic activity against
K562, HL-60, HT-29 and MRC-5 cell lines. The activity
of this compound towards K562 and HT-29 cells was
comparable to that observed for tiazofurin. However
the analogue 4 was found to be significantly more active
against the HL-60 and MRC-5 cell lines, being almost
80- and 8-fold more potent than tiazofurin 1, respec-
tively. Conversely, the C-nucleoside 5 was completely
inactive against K562, HL-60, HT-29 and MRC-5 cells,
but exhibited strong cytotoxicity against the MCF7 cell
line, being approximately 200-fold higher than observed
for the reference compound 1. This analogue was also
active against HeLa cells, but was more than 3-fold less
active with respect to tiazofurin 1. These results indicate
that the incorporation of 20-nitrogen functionalities into
the tiazofurin sugar moiety may result in an improve-
ment of the lead compounds cytotoxity against some
neoplastic cells, and therefore is a process, which may
The 2,5-anhydride 8 was used as a convenient starting
compound for the preparation of 21, a key intermediate
in a planned synthesis of the 20-azido-substituted tiazo-
furin analogue 5. Compound 8 was converted to the
glycosyl cyanide 19 using the same three-step sequence
already applied for the conversion of 9 into the nitrile
14. In this way, the dioxolane derivative 8 was converted
into the glycosyl cyanide 19 in 39% overall yield. Treat-
ment of 19 with hydrogen sulphide gas in dry pyridine
for 2h at room temperature gave a moderate yield of
the desired thioamide 20 (34%). All attempts to improve
the yield of 20 by varying the reaction conditions were
unsuccessful. The relatively low yield of the reaction
might be the result of a competitive H2S-mediated
reduction process that involved further conversion of
20 to the corresponding 2-amino-2-deoxy derivative
(not shown in the Scheme). Indeed, apart from the iso-
lated product 20, TLC of the reaction mixture showed
Table 1. In vitro cytotoxicity of compounds 1, 4 and 5
Compds
IC50, lMa
K562 HL-60 HT-29 MCF7 HeLa MRC-5
1
4
5
5.29
9.84
9.32
0.12
1.01
0.93
>100
7.98
>100
0.04
4.76
>100
0.85
0.11
>100 >100
16.43 >100
a IC50 is the concentration of compound required to inhibit the cell
growth by 50% compared to the untreated control.

7128
M. Popsavin et al. / Tetrahedron Letters 45 (2004) 7125–7128
961–971; (c) Pankiewicz, K. W. Exp. Opin. Ther. Patents
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be of use in the search for new anticancer agents derived
from the parent molecule 1.
In conclusion, we have synthesized two novel tiazofurin
analogues 4 and 5, which have shown potent cytotoxic
activity against some human leukaemia and solid
tumour cell lines, whereupon the 20-azido derivative 5
did not exhibit any significant cytotoxicity towards nor-
mal foetal lung MRC-5 cells. In addition, this approach
provided a new and convenient one-step procedure for
efficient H2S-mediated conversion of 2-azido-2-deoxy-
3,6-di-O-benzoyl-b-D-ribofuranosyl cyanide 14 to the
corresponding 2-benzamido thiocarboxamide derivative
15, thus enabling a facile access to a key intermediate for
preparation of the corresponding tiazofurin analogue 4.
Further work on the scope and limitations of this reac-
tion is currently underway and the results will be
reported elsewhere.
´
7. Weber, G.; Shen, F.; Orban, T. I.; Ko¨keny, S.; Olah, E.
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Acknowledgements
This work was supported by a research grant from the
Ministry of Science, Technologies and Development of
the Republic of Serbia (Grant No. 1896). The authors
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14. Popsavin, M.; Popsavin, V.; Vukojevic, N.; Csanadi, J.;
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´
´
are grateful to Mr. D. Djokovic (Faculty of Chemistry,
´
University of Belgrade, S & M) and to Mrs. T. Marinko-
Covell (Department of Chemistry, University of Leeds,
UK) for recording the mass spectra.
17. Ramasamy, K. S.; Banderu, R.; Averett, D. J. Org. Chem.
2000, 65, 5849–5851.
23
18. Compound 4: mp 225ꢁC (from MeOH-iPr2O); ½aꢁD ꢀ 96:2
References and notes
(c 1.05, MeOH); mmax 3455 (OH), 1641 (C@O); 1H
0
0
NMR (methanol-d4): d 3.78 (pseudo d, 2H, J4 ,5 = 4.5Hz,
1. For recent reviews see: (a) Pankiewicz, K. W.; Watanabe,
K. A.; Lesiak-Watanabe, K.; Goldstein, B. M.; Jayaram,
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F.; Kool, E. T. Synlett 1997, 341–347; (f) Watanabe, K.A.,
Chemistry of Nucleosides and Nucleotides; Townsend,
L. B., Ed.; Plenum: New York, 1994; Vol. 3, pp. 421–535.
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2199–2202, and references cited therein.
2 · H-50), 4.17 (td, 1H, J3 ,4 = 2.6, J4 ,5 = 4.5Hz, H-4 ),
0
0
0
0
0
4.36 (dd, 1H, J2 ,3 = 5.5, J3 ,4 = 2.6Hz, H-30), 4.71 (dd,
0
0
0
0
1 H, J1 ,2 = 8.8, J2 ,3 = 5.5Hz, H-20), 5.30 (d, 1 H,
0
0
0
0
J1 ,2 = 8.8Hz, H-10), 7.40–7.89 (m, 5H, Ph), 8.20 (s, 1H,
0
0
13
H-5); C NMR (DMSO-d6): d 59.2 (C-20), 62.2 (C-50),
71.4 (C-30), 78.6 (C-10), 87.7 (C-40), 125.1 (C-5), 127.8,
128.6, 131.8 and 134.4 (Ph), 150.0 (C-4), 162.8 (C-2), 167.1
(PhC@O), 171.2 (CONH2); HR MS (ES+): m/z 364.0959
(M++H); calcd for C16H18N3O5S: 364.0967.
23
19. Compound 5: mp 160ꢁC (from MeOH-iPr2O); ½aꢁD ꢀ 48:2
(c 0.85 in MeOH); mmax 3441 (OH), 2119 (N3), 1683
(C@O); 1H NMR (methanol-d4):
d
3.69 (dd, 1H,
J4 ,5 a = 4.6, J5 a,5 b = 12.1Hz, H-50a), 3.79 (dd, 1H,
0
0
0
0
3. For a brief review on the chemistry, biochemistry and
pharmacology of tiazofurin see: Grifantini, M. Curr. Opin.
Invest. Drugs 2000, 1, 257–262.
4. Franchetti, P.; Cappellacci, L.; Grifantini, M. Farmaco
1996, 51, 457–469.
J4 ,5 b = 3.3, J5 a,5 b = 12.1Hz, H-50b), 4.05 (ddd, 1H,
0
0
0
0
J3 ,4 = 5.1, J4 ,5 a = 4.6, J4 ,5 b = 3.3Hz, H-40), 4.17 (t,
0
0
0
0
0
0
1H, J1 ,2 = J2 ,3 = 5.5Hz, H-20), 4.33 (dd, 1H,
0
0
0
0
0
0
0
0
J2 ,3 = 5.5, J3 ,4 = 5.1Hz,
H-30), 5.16 (d, 1H,
J1 ,2 = 5.5Hz, H-10), 8.22 (s, 1H, H-5); 13C NMR (meth-
anol-d4): d 63.0 (C-50), 69.3 (C-20), 73.7 (C-30), 81.4 (C-10),
87.0 (C-40), 126.2 (C-5), 150.9 (C-4), 165.5 (C-2), 172.6
(CONH2); CI MS: m/z 286 (M++H). Anal. Found: C,
37.84; H, 3.95; N, 24.89; S, 10.78; calcd for C9H11N5O4S:
C, 37.89; H, 3.89; N, 24.55; S, 11.24.
0
0
5. Lui, M. S.; Faderan, M. A.; Liepnieks, J. J.; Natsumeda,
Y.; Olah, E.; Jayaram, H. N.; Weber, G. J. Biol. Chem.
1984, 259, 5078–5082.
6. (a) For recent reviews on inhibition of IMPDH and
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Med. Chem. 2004, 11, 887–900; (b) Christopherson, R. I.;
Lyons, S. D.; Wilson, P. K. Acc. Chem. Res. 2002, 35,
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