De novo synthesis of two new cytotoxic tiazofurin analogues with modified sugar moieties

Article abstract of DOI:10.1016/S0960-894X(03)00712-1

A divergent synthesis of two novel tiazofurin analogues, 2-(3-deoxy-3-fluoro-β-D-xylofuranosyl)thiazole-4-carboxamide (2) and 2-(3-acetamido-3-deoxy-β-D-xylofuranosyl)thiazole-4-carboxamide (3), has been achieved starting from D-glucose. Both nucleoside a

Full text of DOI:10.1016/S0960-894X(03)00712-1

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3167–3170
De Novo Synthesis of Two New Cytotoxic Tiazofurin Analogues
with Modified Sugar Moieties
Mirjana Popsavin,a,* Ljilja Torovic,a Vesna Kojic,b Gordana Bogdanovic,b Sasa Spaica
and Velimir Popsavina
a
´
Department of Chemistry, Faculty of Sciences, University of Novi Sad, Trg D. Obradovica 3, 21000 Novi Sad,
Serbia and Montenegro, Yugoslavia
bInstitute of Oncology Sremska Kamenica, Institutski put 4, 21204 Sremska Kamenica, Serbia & Montenegro, Yugoslavia
Received 19March 2003; revised 4 May 2003; accepted 4 July 2003
Abstract—A divergent synthesis of two novel tiazofurin analogues, 2-(3-deoxy-3-fluoro-b-d-xylofuranosyl)thiazole-4-carboxamide
(2) and 2-(3-acetamido-3-deoxy-b-d-xylofuranosyl)thiazole-4-carboxamide (3), has been achieved starting from d-glucose. Both
nucleoside analogues were evaluated for their in vitro cytotoxicity against several human leukaemia and solid tumour cell lines.
# 2003 Elsevier Ltd. All rights reserved.
Tiazofurin (1) is a thiazole C-nucleoside that exhibits a
potent cytotoxic activity against a number of malignant
cells.1 Tiazofurin is currently in phase II/III clinical
trials, but it has been recently approved as an orphan-
drug for the treatment of chronic myelogenous leukae-
mia in accelerated phase or blast crisis. The biological
activity of tiazofurin is attributed to its ability to reduce
intracellular guanine nucleotide pools via inhibition of
inosine monophosphate dehydrogenase (IMPDH, EC
1.1.1.205).2 The inhibition of IMPDH induced by tia-
zofurin is due to the tiazofurin metabolite, thiazole-4-
carboxamide adenine dinucleotide (TAD). This active
metabolite inhibits IMPDH activity through competi-
tion for the NAD cofactor-binding site of the enzyme.3
Two IMPDH isoforms (type I and II) were identified, of
which type II is up-regulated in human leukaemia cells,4
as well as in some solid tumour cell lines.5 These find-
ings were prompted studies towards the design of iso-
form selective inhibitors,6 and renewed interest in the
synthesis of tiazofurin7 and its analogues, particularly
those with modified heterocyclic aglycons.8 Structural
modifications of the ribofuranosyl moiety have also
been reported including preparation of 50-, 30-, or 20-
substituted derivatives,9 xylo- and arabinofuranosyl,10
pyranosyl,11 acyclic,12 carbocyclic13 and 40-aza-sugar14
analogues. However, none of these substances was
shown any significant biological activity. In this paper,
we wish to report on the synthesis of two novel tiazo-
furin analogues 2 and 3, along with their effects on the
proliferation of some malignant and normal cell lines.
Chemistry
The 2,5-anhydro-d-glucose derivative 4 (Scheme 1),
earlier prepared in our laboratory starting from d-glu-
cose,15,16 has been used as a divergent intermediate for
the synthesis of both tiazofurin analogues 2 and 3.
Reaction of 4 with triflic anhydride in pyridine and di-
chloromethane gave the corresponding triflic ester 5,
which was isolated in pure form (TLC, 1H and 13C
NMR) after the usual workup and used in the next step
without further purification. Subsequent treatment of 5
with tetrabutylammonium fluoride in THF afforded the
corresponding 4-fluoro derivative 617 as the only reac-
tion product (67% from 4). Hydrolytic removal of the
dioxolane protective group in 6, followed by subsequent
reaction of the unstable aldehyde 7 with hydroxyl amine
hydrochloride and sodium acetate, gave the oximino
*Corresponding author. Tel.:+38-121-350-122; fax:+38-121-454-065;
e-mail: popsavin@ih.ns.ac.yu
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00712-1

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M. Popsavin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3167–3170
Scheme 1. Reagents and conditions: (a) Tf2O, Py, CH2Cl2, À10 ꢀC, 0.5 h, 0 ꢀC, 0.5 h, rt, 0.5 h, 98%; (b) Bu4NF, THF, À18 ꢀC, 22 h, 68%; (c) 9:1
TFA–6 M HCl, 4 ꢀC, 6 days; (d) NH2OHÂHCl, NaOAc, EtOH, rt, 2 h, 75% from 6; (e) MsCl, Py, À17 ꢀC, 0.5 h then rt, 2 h, 78%; (f) H2S, DMAP,
EtOH, rt, 8 h, 89%; (g) BrCH2COCO2Et, EtOH, 80 ꢀC, 50 min, 28.5%; (h) NH3, MeOH, rt, 9days, 91%.
Scheme 2. Reagents and conditions: (a) NaN3, DMSO, THF, rt, 24 h, 50%; (b) PtO2, Ac2O, AcOH, rt, 21 h, 56%; (c) 9:1 TFA–6 M HCl, 4 ꢀC, 6
days; (d) NH2OHÂHCl, NaOAc, EtOH, rt, 2 h, then +4 ꢀC, 70 h; (e) MsCl, Py, À15 ꢀC, 0.5 h, then rt, 1.5 h, 77% from 14; (f) H2S, DMAP, EtOH,
rt, 4 h, 81%; (g) BrCH2COCO2Et, EtOH, 80 ꢀC, 50 min, 37%; (h) NH3, MeOH, rt, 8 days, 93%.
derivative 8 as a mixture of the corresponding E- and Z-
isomers (75% from 6). Due to their similar chromato-
graphic properties, the E- and Z-isomers 8 could not be
separated by column chromatography, but were in the
mixture further treated with mesyl chloride in pyridine
to afford the corresponding nitrile 9. Treatment of 9
with hydrogen sulphide, in the presence of DMAP as a
proton acceptor furnished the corresponding thioamide
10 (69% from 8). The Hantzsch reaction of 10 with
ethyl bromopyruvate gave the expected thiazole 11
(29%), accompanied with a similar amount of the aro-
matised product 12. The pure product 11 was isolated
by silica gel column chromatography and treated with
saturated ammonia in dry methanol to afford the cor-
responding nucleoside 218 (91%) ready for biological
testing.
The triflic ester 5 reacted with sodium azide in dimethyl
sulfoxide and tetrahydrofuran to afford the known16
azido derivative 13, with physical and spectral data in
excellent agreement with those already reported.16 Cat-
alytic hydrogenation of 13 over PtO2 in a mixture of
glacial acetic acid and acetic anhydride gave the acet-
amido derivative 14,19 a key intermediate in the planned
synthesis of 3. The intermediate 14 was converted to the
final product 320 by using the same six-step sequence
already applied for the conversion of 6 into the C-
nucleoside 2. In this way the 2,5-anhydro derivative 14
has been transformed into the target molecule 3 in
21.5% overall yield.
Biological Evaluation
Synthesis of the tiazofurin analogue 3 bearing the 30-
acetamido group as an isostere is outlined in Scheme 2.
The synthesized tiazofurin analogues 2 and 3 were
evaluated for their ability to inhibit the growth of

M. Popsavin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3167–3170
3169
human myelogenous leukaemia K562, promyelocytic
leukaemia HL60, colon adenocarcinoma HT29, breast
adenocarcinoma MCF7, and normal foetal lung MRC-
5 cells. Tiazofurin (1) was used as a reference com-
pound. In vitro cytotoxicity was evaluated by the MTT
assay21 after incubation for 24 h. The results are pre-
sented in Table 1.
tiazofurin analogues lacking the ribofuranosyl moiety,
which is thought to be crucial for biological activity.
Further work directed towards the synthesis of novel
tiazofurin derivatives bearing modified sugar segments
is currently underway.
Acknowledgements
Compound 3 showed a potent cytotoxic activity against
human myelogenous leukaemia K562, which was 44-
fold higher than that observed for tiazofurin. Both C-
nucleosides 2 and 3 exhibited a potent cytotoxic activity
against human colon adenocarcinoma HT29, essentially
the same as that exhibited by tiazofurin itself. More-
over, the 30-fluoro derivative 2 showed a significant
cytotoxicity against human promyelocytic leukaemia
HL60 cell line, comparable to that of tiazofurin. Both
C-nucleoside analogues 2 and 3 were inactive against
breast adenocarcinoma MCF7 until a maximum tested
concentration of 1000 mM. Compound 2 was shown to
be inactive against normal foetal lung MRC-5 cell line,
while the acetamido derivative 3 devoid of any sig-
nificant cytotoxicity towards leukaemia HL60. Finally,
compound 2 showed a weak cytotoxicity against K562
cells comparable to that exhibited by compound 3
against MRC-5 cell line. The biological data are some-
what surprising as there is large variation in the results
for each cell line. Both 2 and 3 are generally either
equipotent with tiazofurin (1), or completely inactive,
depending on the cell line with the exception of com-
pound 3 which is significantly more potent than tiazo-
furin (1), but only against K562. These data might
suggest that compounds 2 and 3 are not acting at the
same biological target as tiazofurin (1). If the com-
pounds are acting on the same biological target as tia-
zofurin (IMPDH), then one should observe similar
activity to tiazofurin for each cell line. In order to
resolve this problem more work should be done includ-
ing further biological evaluation of 2 and 3 for their
ability to inhibit IMPDH activity.
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
are grateful to Mr. D. Djokovic (Faculty of Chemistry,
University of Belgrade, S&M) and to Mrs. T. Marinko-
Covell (School of Chemistry, University of Leeds, UK)
for recording the mass spectra.
References and Notes
1. For a brief overview of the molecular, biological and clin-
ical impact of tiazofurin, see: Grifantini, M. Curr. Opin. Invest.
Drugs, 2000, 1, 257.
2. Weber, G.; Prajada, N.; Abonyi, M.; Look, K. Y.; Tricot,
G. Anticancer Res. 1996, 16, 3313.
3. Lui, M. S.; Faderan, M. A.; Liepnieks, J. J.; Natsumeda,
Y.; Olah, E.; Jayaram, H. N.; Weber, G. J. Biol. Chem. 1984,
259, 5078.
4. (a) Konno, Y.; Natsumeda, Y.; Nagai, M.; Yamaji, Y.;
Ohno, S.; Suzuki, K.; Weber, G. J. Biol. Chem. 1991, 266, 506.
(b) Natsumeda, Y.; Ohno, S.; Kawasaki, H.; Konno, Y.;
Weber, G.; Suzuki, K. J. Biol. Chem. 1990, 265, 5292.
5. Jayaram, H. N.; Grusch, M.; Cooney, D. A.; Krupitza, G.
Curr. Med. Chem. 1999, 6, 561.
6. Colby, T. D.; Vanderveen, K.; Strickler, M. D.; Markham,
G. D.; Goldstein, B. M. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 3531.
7. (a) For recent approaches to tiazofurin see: Brown, R. S.;
Dowden, J.; Moreau, C.; Potter, B. V. L. Tetrahedron. Lett.
2002, 43, 6561. (b) Ramasamy, K. S.; Banderu, R.; Averett, D.
J. Org. Chem. 2000, 65, 5849. (c) Ramasamy, K. S.; Averett,
D. Nucleosides Nucleotides 1999, 18, 2425.
8. Franchetti, P.; Marchetti, S.; Cappellacci, L.; Yalowitz,
J. A.; Jayaram, H. N.; Goldstein, B. M.; Grifantini, M.
Bioorg. Med. Chem. Lett. 2001, 11, 67, and references therein.
9. (a) 50-Subsituted analogues: Srivastava, P. C.; Pickering,
M. V.; Allen, L. B.; Streeter, D. G.; Campbell, M.T; Wit-
kowski, J. T.; Sidwell, R. W.; Robins, R. K. J. Med. Chem.
1977, 20, 256. (b) Hanna, N. B.; Upadhya, K. G.; Petrie, C. R.;
Robins, R. K.; Revankar, G. R. Nucleosides Nucleotides 1986,
5, 343. (c) Andres, J. I.; Garcia-Lopez, M. T.; De las Heras,
F. G.; Mendez-Castrilon, P. P. Nucleosides Nucleotides 1986,
5, 423. 30-Substituted analogues: Baur, R. H.; Baker, D. C.
Nucleosides Nucleotides 1984, 3, 77. 20-Substituted analogues:
Jayaram, H. N.; Dion, R. L.; Glazer, R. I.; Johns, D. G.;
Robins, R. K.; Srivastava, P. C.; Cooney, D. A. Biochem.
Pharmacol. 1982, 31, 2371.
In conclusion, we have synthesized two novel cytotoxic
tiazofurin analogues bearing the 30-fluoro (2), as well as
the 30-acetamido (3) function as an isostere. Compound
3 showed a potent in vitro cytotoxicity against K562
leukaemia cells, which was 44-fold higher than that
observed for tiazofurin, an approved orphan-drug for
treatment of certain human leukaemia. In addition,
both 2 and 3 exhibited a potent cytotoxic activity
against human colon adenocarcinoma HT29, compar-
able to that of tiazofurin. To the best of our knowledge
compounds 2 and 3 represent the first biologically active
Table 1. In vitro cytotoxicity of compounds 1–3
IC50, mMa
Compd
10. Mao, D. T.; Marquez, V. E. Tetrahedron Lett. 1984, 25,
2111.
K562
HL-60
HT-29MCF7
MRC-5
11. Kovacs, L.; Herczegh, P.; Batta, G.; Farkas, I. Tetra-
hedron 1991, 47, 5539.
12. Kovacs, L.; Herczegh, P.; Batta, G.; Farkas, I. Hetero-
cycles 1987, 26, 947.
13. Dishington, A. P.; Humber, D. C.; Stoodley, R. J. J.
Chem. Soc., Perkin Trans. 1 1993, 57.
1
2
3
4.16
96.60
0.095
9.32
10.50
1000
1.01
0.91
1.00
8.49
>1000
>1000
0.85
>1000
89.70
aIC50 is a compound concentration required to inhibit the cell growth
by 50% compared to an untreated control.

3170
M. Popsavin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3167–3170
1
0
14. Kini, G. D.; Hennen, W. J. J. Org. Chem. 1986, 51, 4436.
15. Popsavin, M.; Popsavin, V.; Vukojevic, N.; Csanadi, J.;
Miljkovic, D. Carbohydr. Res. 1994, 260, 145.
10), 97.86 (d, J3 ,F=184.8 Hz, C-30), 125.62 (C-5), 150.88 (C-
4), 165.72 (C-2), 173.73 (C¼O); CI MS: m/z 263 (M++1).
19. Selected data for 14 (syrup): [a]D +24.93 (c 4.35 in
1
16. Popsavin, M.; Torovic, Lj.; Spaic, S.; Stankov, S.; Kapor,
A.; Tomic, Z.; Popsavin, V. Tetrahedron 2002, 58, 569.
17. Compound 6: mp 72.5–73 ꢀC (from CH2Cl2–pentane);
CHCl3); H NMR (CDCl3): d 2.04 (s, 3H, NHAc), 4.01–4.20
(m, 4H, 2ÂCH2-dioxolane), 4.31 (t, 1H, J1,2=J2,3=1.2 Hz, H-
2), 4.44–4.63 (m, 3H, J4,5=3.4 Hz, H-5 and 2ÂH-6), 4.88
(ddd, 1H, J3,4=0.6, J4,5=3.4, J4,NH=9.5 Hz, H-4), 5.31 (d,
1H, J1,2=1.2 Hz, H-1), 6.89(d, 1H, J4,NH=9.5 Hz, NH),
7.39–8.17 (m, 10H, 2ÂPh); 13C NMR (CDCl3): d 23.24
(MeCO), 54.85 (C-4), 62.92 (C-6), 65.63 and 65.67 (2ÂCH2-
dioxolane), 78.57 (C-3), 79.07 (C-5), 84.02 (C-2), 102.08 (C-1),
128.30, 128.45, 128.96, 129.72, 133.03 and 133.56 (2ÂPh),
165.38 and 166.22 (2ÂPhC¼O), 169.25 (MeCO); HR MS: m/z
478.1472 (M++Na); calcd for C24H25NO8Na: 478.1478.
20. Selected data for 3 (syrup): [a]D À3.93 (c 1.3 in MeOH);
1H NMR (methanol-d4): d 1.97 (s, 3H, MeCO), 3.68 (dd, 1H,
1
[a]D À22.19( c 2.3 in CHCl3); H NMR (CDCl3): d 3.95–4.09
(m, 4H, 2ÂCH2-dioxolane), 4.14 (dd, 1H, J1,2=5.2, J2,3=3
Hz, H-2); 4.49(dddd, 1H, J4,5=2.7, J5,6a=5.8, J5,6b=5.6,
J5,F=28.9Hz, H-5), 4.64–4.74 (m, 2H, J6a,6b=11.7, J5,6a=5.8,
J5,6b=5.6, J6a,F=1 Hz, 2ÂH-6), 5.16 (d, 1H, J1,2=5.2 Hz, H-
1), 5.19(ddd, 1H, J3,4=0.5, J4,5=2.7, J4,F=50.4 Hz, H-4),
5.69(ddd, 1H, J3,4=0.5, J3,F=16.2, J2,3=3 Hz, H-3), 7.43–
8.15 (m, 10H, 2ÂPh); 13C NMR (CDCl3): d 61.53 (d,
3J6,F=10.6 Hz, C-6), 65.42 and 65.52 (2ÂCH2-dioxolane),
2
2
77.39(d, J3,F=31.4 Hz, C-3), 79.28 (d, J5,F=19.5 Hz, C-5),
1
84.22 (C-2), 94.64 (d, J4,F=188.5 Hz, C-4), 102.27 (C-1),
J5 a,5 b=12.3, J4 ,5 a=4.6 Hz, H-50a), 3.79(dd, 1H,
0 0 0 0
0
0
0
0
0
0
128.33, 128.51, 128.84, 129.56, 129.72, 129.77, 133.16 and
133.66 (2ÂPh), 164.96 and 166.14 (2ÂC¼O); CI MS: m/z 417
(M++1). Anal. found: C, 63.21; H, 5.05; calcd for
C22H21FO7: C, 63.46; H, 5.08.
J5 a,5 b=12.3, J4 ,5 b=3.5 Hz, H-50b), 4.36 (t, 1H, J1 ,2 =5.8,
0
0
0
0
0
0
0
0
J2 ,3 =5.7 Hz, H-2 ), 4.40 (ddd, 1H, J4 ,5 a=4.6, J4 ,5 b=3.5,
J3 ,4 =6.6 Hz, H-4 ), 4.47 (dd, 1H, J2 ,3 =5.7, J3 ,4 =6.6 Hz,
0
0
0
0
0
0
0
H-30), 4.98 (d, 1H, J1 ,2 =5.8 Hz, H-1 ), 8.23 (s, 1H, H-5); 13C
NMR (methanol-d4): d 22.52 (MeCO), 59.96 (C-30), 61.88 (C-
50), 81.22 (C-40), 81.81 (C-20), 83.83 (C-10), 125.86 (C-5), 150.80
(C-4), 165.66 (C-2), 172.60 (CONH2), 173.75 (MeCO). Anal.
Found: C, 44.02; H, 5.18; N, 13.67; S, 10.43; calcd for
C11H15N3O5S: C, 43.85; H, 5.02; N, 13.95; S, 10.64.
0
0
18. Selected data for 2 (syrup): [a]D À35.91 (c 1.04 in MeOH);
1H NMR (methanol-d4): d 3.91 (pseudo d, 2H, J4 ,5 =6.4 Hz,
0
0
2ÂH-50), 4.38 (dtd, 1H, J4 ,F=30.7, J3 ,4 =2.4, J4 ,5 =6.4 Hz,
0
0
0
0
0
H-40), 4.57 (ddd, 1H, J1 ,2 =1.2, J2 ,3 =0.6, J2 ,F=13.9Hz, H-
0
0
0
0
0
20), 4.97 (ddd, 1H, J2 ,3 =0.6, J3 ,4 =2.4, J3 ,F=50.7 Hz, H-30),
0
0
0
0
0
5.13 (d, 1H, J1 ,2 Hz,=1.2, H-10), 8.15 (s, 1H, H-5); 13C NMR
21. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks,
A.; Tierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.;
Boyd, M. R. Cancer Res. 1988, 48, 4827.
0
0
(methanol-d4): d 60.21 (d, J5 ,F=10.7 Hz, C-50), 81.72 (d,
3
0
J4 ,F=27 Hz, C-40), 84.04 (d, J2 ,F=19.4 Hz, C-20), 86.56 (C-
2
2
0
0