Synthesis and antiproliferative activity of two new tiazofurin analogues with 2′-amido functionalities

Article abstract of DOI:10.1016/j.bmcl.2006.02.001

Two novel tiazofurin analogues 2 and 3 were synthesized starting from d-glucose. The key step of the synthesis was the efficient one-step hydrogen sulfide-mediated conversion of 2-azido-3-O-acyl-ribofuranosyl cyanides to the corresponding 2-amido thiocarb

Full text of DOI:10.1016/j.bmcl.2006.02.001

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2773–2776
Synthesis and antiproliferative activity of two new
tiazofurin analogues with 20-amido functionalities
a
a
b
Mirjana Popsavin,a, Ljilja Torovic, Milosˇ Svircˇev, Vesna Kojic,
*
´
´
b
a
´
Gordana Bogdanovic and Velimir Popsavin
aDepartment of Chemistry, Faculty of Sciences, University of Novi Sad, Trg D. Obradovic´a 3, 21000 Novi Sad, Serbia and Montenegro
bInstitute of Oncology Sremska Kamenica, Institutski put 4, 21204 Sremska Kamenica, Serbia and Montenegro
Received 17 January 2006; revised 1 February 2006; accepted 1 February 2006
Available online 21 February 2006
Abstract—Two novel tiazofurin analogues 2 and 3 were synthesized starting from D-glucose. The key step of the synthesis was the
efficient one-step hydrogen sulfide-mediated conversion of 2-azido-3-O-acyl-ribofuranosyl cyanides to the corresponding 2-amido
thiocarboxamides. Compounds 2 and 3 were evaluated for their in vitro antiproliferative activity against certain human tumour cell
lines. Remarkably, compound 2 was found to be 570-fold more potent than tiazofurin against MCF-7 cells, while compound 3
showed the most powerful cytotoxicity against HT-29 cancer cells, being almost 100-fold more active than tiazofurin.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Tiazofurin (1, Fig. 1) is a synthetic1 C-nucleoside with a
potent antitumour activity.2 It is a prodrug as once with-
in the cell it is converted to thiazole adenine dinucleotide
(TAD) which blocks the key step in the de novo synthe-
sis of GTP.3 In phase I/II clinical trials, it induced com-
plete haematological remissions in patients with end-
stage acute nonlymphocytic leukaemia, or in myeloblas-
tic crisis of chronic myeloid leukaemia.4 Despite the
Figure 1. Tiazofurin (1) and analogues 2 and 3.
remarkable efficacy of tiazofurin, lack of specificity
dodecanamido functions at C-20, along with their effects
and a significant neurotoxicity remains a problem in
its clinical use.2 In the search for new antineoplastic
agents of improved therapeutic effects, many tiazofurin
derivatives were synthesized, including a number of
on the proliferation of some malignant cell lines.
Our strategy for the synthesis of both targets 2 and 3 as-
sumes previous transformation of D-glucose into the
ribofuranosyl thioamides 10 and 16 (Scheme 1), fol-
those with a modified sugar segment.5 However, none
of these compounds showed favourable biological prop-
erties. We have recently reported on the synthesis of sev-
lowed by their subsequent cyclocondensation with ethyl
eral tiazofurin analogues with modified sugar moieties
that showed increased antitumour activities with respect
bromopyruvate to form the thiazole ring. It was further
assumed that the key intermediates 10 and 16 could be
accessible from the 2-azido-2-deoxy-D-ribofuranosyl
cyanides 9 and 15, through a one-step H2S-mediated
to the lead compound 1.6,7 The sub-micromolar activity
of 20-benzamido tiazofurin derivative7 directed our fur-
ther work in this field to the synthesis and biological
cascade similar to that recently observed in our labora-
tory.7 As the previous conversion included a single aro-
evaluation of related molecules bearing different amide
functions at C-20. Herein, we describe the synthesis of
two new tiazofurin analogues 2 and 3 with hexan- and
matic ester derivative, further evaluation of this method
in the saturated ester series represents an important task
of the present work.
Keywords: 2,5-Anhydro sugars; C-Nucleosides; Antitumour agents;
De novo synthesis; Tiazofurin analogues; Thiazoles.
The 2,5-anhydro-D-glucose derivative 4, readily avail-
able from D-glucose,8 was used as a convenient starting
material for the synthesis of both targets 2 and 3. Com-
*
Corresponding author. Tel.: +381 21 6350 122; fax: +381 21 454
065; e-mail: mpopsavin@ih.ns.ac.yu
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.001

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M. Popsavin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2773–2776
Scheme 1. Reagents and conditions: (a) C5H11COCl, Py, CH2Cl2, 0 ꢁC ! rt, 24 h, 87% of 5, 22 h, 99% of 11; (b) KOBz, DMF, 100 ꢁC, 12 h, 86%
of 6, 8 h, 77% of 12; (c) NaN3, DMSO, 110 ꢁC, 24 h, 47% of 7, 70 h, 44% of 13; (d) i—4:1 TFA/6 M HCl, 4 ꢁC, 5 days for 7, 8 days for 13,
ii—NH2OHÆHCl, NaOAc, EtOH, rt, 4 h; (e) MsCl, Py, ꢀ15 ꢁC ! rt, 2 h, 46% of 9, 36% of 15; (f) H2S, Py, rt, 12 h, 99% of 10, or H2S, DMAP,
EtOH, rt, 8 h, 92% of 16.
pound 4 readily reacted with hexanoyl chloride under
the standard acylation conditions to afford the corre-
sponding 4-O-hexanoyl derivative 5 in 87% yield. Reac-
tion of 5 with potassium benzoate in DMF gave the
corresponding 6-O-benzoyl derivative 6 (86%), which
was subsequently treated with sodium azide in dimethyl-
sulfoxide to afford the 3-azido derivative 7 in 47% yield.
Hydrolytic removal of the dioxolane protective group in
7 was achieved in a mixture of trifluoroacetic acid and
6 M hydrochloric acid at +4 ꢁC. The resulting unstable
aldehyde (not shown in the scheme) was not purified,
but was rather immediately treated with hydroxylamine
hydrochloride to yield the corresponding oxime 8 (68%)
as a mixture of the corresponding E- and Z-isomers. The
mixture was not separated (except for the characterisa-
tion purpose), but was rather further treated with mesyl
chloride to give a 68% yield of the corresponding nitrile
9. Moreover, 9 was treated with hydrogen sulfide in pyr-
idine,7 to provide an almost quantitative yield of the de-
sired thioamide 10,9 a key intermediate in the synthesis
of analogue 2.
nitrile 15 was treated with hydrogen sulfide gas and 4-
dimethylaminopyridine in ethanol, whereby the desired
thioamide 1610 was obtained in 92% yield.
The last H2S-mediated conversion of 2-azido-3-O-acyl-
2-deoxy-D-ribofuranosyl cyanides 9 and 15 into the cor-
responding thioamides 10 and 16 deserves some addi-
tional comments. This cascade process is presumably
comprised of an initial addition of hydrogen sulfide to
the nitrile group,11 followed by the azide reduction
and spontaneous O,N-acyl rearrangement. This se-
quence already gave good results in the aromatic ester
series,7 but the present work reveals that this method
is even more efficient when applied to the saturated ester
derivatives. The transformation is likely to be a general
method that provides an access to a variety of 2-amido-
ribofuranosyl-thiocarboxamides, which may be further
converted to an array of tiazofurin analogues with dif-
ferent amido functions at the C-20 position.
With the requisite intermediates 10 and 16 in hands, we
next focused on their transformation to the target C-nu-
cleosides 2 and 3, by using a modified Hantzsch thiazole
synthesis,12 as outlined in Scheme 2. Treatment of both
10 and 16 with ethyl bromopyruvate in refluxing ethanol
gave the required thiazoles 17 and 18 in 54% and 56%
yield, respectively. Final exposure of 17 and 18 to
methanolic ammonia provided the tiazofurin analogues
213 and 314 in 66% and 80% yield, respectively.
After the synthesis of thioamide 10, we were also inter-
ested in preparation of its six-carbon homologue 16, a
key intermediate in the synthesis of dodecan-amido
derivative 3. By applying essentially the same multistep
sequence as that used for the preparation of 9, com-
pound 4 was converted to the nitrile 15. The conversion
of 13 to 15 was carried out without purification of both
aldehydo and oximino intermediates to afford the nitrile
15 in 36% overall yield (three steps). Although the nitrile
15 can be converted to the thioamide 16 under the reac-
tion conditions similar to those already used for the con-
version of 9 to 10 (H2S, Py, rt), at this point we wanted
to explore alternative reagent system for the preparation
of 16 (H2S/DMAP in EtOH), which was also successful-
ly applied in the aromatic ester series.7 Accordingly, the
The newly synthesized tiazofurin analogues 2 and 3 were
evaluated for their antiproliferative activity against hu-
man myelogenous leukaemia K562, promyelocytic leu-
kaemia HL-60, colon adenocarcinoma HT-29, estrogen
receptor positive breast adenocarcinoma MCF7, cervix
carcinoma HeLa and normal foetal lung fibroblasts
MRC-5. In vitro cytotoxicity was evaluated after 24-h

M. Popsavin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2773–2776
2775
Scheme 2. Reagents and conditions: (a) BrCH2COCO2Et, EtOH, 80 ꢁC, 50 min, 54% of 17, 56% of 18; (b) NH3, MeOH, rt, 7 days, 66% of 2, 80%
of 3.
cell treatment by using the MTT assay.15 The results,
including the data for the reference compound tiazofu-
rin (1), are presented in Table 1.
their amide moiety. Such a study is currently underway
in our laboratory the results will be reported elsewhere.
In conclusion, two novel tiazofurin derivatives bearing a
hexan- (2), or a dodecanamido function (3) in the C-20
position, were synthesized starting from D-glucose. The
key intermediates for this approach, that is, 2-amido-
D-ribofuranosyl thiocarboxamides 10 and 16, were easi-
ly accessible from the 2-azido-2-deoxy-D-ribofuranosyl
cyanides 9 and 15, through the one-step H2S-mediated
cascade, comprised of an initial addition of hydrogen
sulfide to the nitrile group, followed by the azide reduc-
tion and spontaneous O,N-acyl rearrangement. The se-
quence represents a general and particularly versatile
approach to a variety of 2-amido-D-ribofuranosyl thio-
carboxamides, which can be easily converted to the cor-
responding ribofuranosyl thiazole-4-carboxamides, as
exemplified by the conversion of 10 and 16 to 2 and 3,
respectively. Analogues 2 and 3 have shown a potent
cytotoxic activity against some human leukaemia and
solid tumour cell lines, but did not exhibit any signifi-
cant cytotoxicity towards normal foetal lung MRC-5
cells. These results, along with our previous findings,7
confirmed that the introduction of 20-nitrogen function-
alities into the tiazofurin sugar moiety may provide ana-
logues of improved antiproliferative effects against some
neoplastic cells, and therefore, it may be of use in the
search for new anticancer agents derived from the lead
compound 1.
Compound 2 was inactive against the K562, HL-60 and
HT-29 malignant cell lines, but showed a powerful cyto-
toxic activity against MCF-7 cells being 570-fold more
potent than the reference compound 1. This analogue
was also active against HeLa cells, but it was almost
2-fold less potent with respect to the parent molecule
1. However, compound 3 exhibited a significant cytotox-
icity against HL-60, HT-29 and HeLa cell lines, but de-
void of any activity towards the MCF-7 neoplastic cells.
This analogue showed the most pronounced cytotoxicity
against the HT-29 cells, being almost 100-fold more po-
tent than tiazofurin. Compound 3 was approximately 2-
and 1.5-fold more active than tiazofurin against HL-60
and HeLa cells, respectively. It also showed a moderate
antiproliferative activity against K562 cells, but it was
more than 6-fold less active than the reference com-
pound 1. Remarkably, both analogues were devoid of
any cytotoxicity against the normal foetal lung fibro-
blasts MRC-5. These results do suggest that 2 and 3
are more selective than tiazofurin, but such a generaliza-
tion should be supported by additional in vitro experi-
ments with a larger number of cell lines.
Due to such a powerful cytotoxicity against certain
malignant cell lines, and their nontoxicity towards the
normal MRC-5 cells, the analogues 2 and 3 may serve
as important leads in the synthesis of more potent and
selective anticancer agents. However, the difference in
their cytotoxic activity is large and suggests that com-
pounds 2 and 3 are not acting at the same biological tar-
get (IMPDH) as tiazofurin.2,3 In order to explain the
biological activity, structure–activity relationship should
be performed that would include the synthesis and a
more detailed biological evaluation of a series of 20-ami-
do tiazofurin analogues bearing different R groups in
Acknowledgments
Financial support from the Ministry of Science and
Environment Protection of the Republic of Serbia (Pro-
ject No. 142005) is gratefully acknowledged. The
´
authors thank Mr. D. Djokovic (Faculty of Chemistry,
University of Belgrade, S&M) for recording the mass
spectra.
References and notes
Table 1. In vitro cytotoxicity of 1, 2 and 3
a
Compound
IC50 (lM)
1. For recent syntheses of tiazofurin, see: (a) Brown, R. S.;
Dowden, J.; Moreau, C.; Potter, B. V. L. Tetrahedron
Lett. 2002, 43, 6561; (b) Ramasamy, K. S.; Lau, J. Y. N.
Nucleosides Nucleotides Nucleic Acids 2001, 20, 1329; (c)
Ramasamy, K. S.; Banderu, R.; Averett, D. J. Org. Chem.
2000, 65, 5849; (d) Ramasamy, K. S.; Averett, D.
Nucleosides Nucleotides 1999, 18, 2425.
K562 HL 60 HT-29 MCF-7 HeLa MRC-5
1
2
3
5.29 9.32
>100 >100
35.62 4.82
1.01
>100
0.011
7.98
0.014
>100
4.76
8.75
3.22
0.85
>100
>100
a IC50 is the concentration of compound required to inhibit the cell
growth by 50% compared to an untreated control.
2. Grifantini, M. Curr. Opin. Invest. Drugs 2000, 1, 257.

2776
M. Popsavin et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2773–2776
3. (a) Pankiewicz, K. W.; Patterson, S. E.; Black, P. L.;
s, 1H, after treatment with D2O changed to d,
J3,NH = 5.5 Hz, CONH), 7.36–8.04 (m, 5H, Ph), 8.21 and
8.47 (2· br s, 1H each, CSNH2); 13C NMR (62.9 MHz,
CDCl3): d 14.00 (Me), 22.56, 25.43, 29.20, 29.22, 29.41,
29.50, 29.52, 29.5 and 31.78 (9· CH2), 36.59 (CH2CO),
58.99 (C-3), 64.76 (C-6), 71.90 (C-4), 84.05 (C-5), 84.63 (C-
2), 128.50, 129.17, 129.61 and 133.43 (Ph), 166.87
(PhC@O), 174.74 (C11H23C@O), 203.65 (C@S). CI MS:
m/z 479 (M++H).
Jayaram, H. N.; Risal, D.; Goldstein, B. M.; Stuyver, L.
J.; Schinazi, R. F. Curr. Med. Chem. 2004, 11, 887; (b)
Szekeres, T.; Fitzer, M.; Pillwein, K.; Felzmann, T.;
Chiba, P. Life Sci. 1992, 51, 1309.
4. Malek, K.; Boosalis, M.; Waraska, K.; Mitchell, B. S.;
Wright, D. G. Leuk. Res. 2004, 28, 1125, and references
therein.
5. For recent syntheses of tiazofurin analogues with modified
sugar moieties, see: (a) Chun, M. W.; Kim, M. J.; Shin, J.
H.; Jeong, L. S. Nucleosides Nucleotides Nucleic Acids
2005, 24, 975; (b) Nair, V.; Wenzel, T. ARKIVOC 2004,
14, 128, <http://arkat-usa.alfahosting.net/ark/journal/
2004/I14_General/1184/04-1184B.pdf>; (c) Liang, C. W.;
Kim, M. J.; Jeong, L. S.; Chun, M. W. Nucleosides
Nucleotides Nucleic Acids 2003, 22, 2039; (d) Cappellacci,
L.; Barboni, G.; Franchetti, P.; Martini, C.; Jayaram, H.
N.; Grifantini, M. Nucleosides Nucleotides Nucleic Acids
2003, 22, 869; (e) Zhang, H. Y.; Yu, H. W.; Ma, L. T.;
Min, J. M.; Zhang, L. H. Tetrahedron: Asymmetry 1998, 9,
141.
11. After shorter reaction time, the main reaction product was
accompanied with the thiocarboxamide bearing an
unchanged azido group at C-2. This proves that addition
of hydrogen sulfide to the nitrile group precedes reduction
of the azido function.
12. Aguilar, E.; Meyers, A. I. Tetrahedron Lett. 1994, 35,
2773.
13. Selected data for 2: mp 155–156 ꢁC (from MeOH–iPr2O),
25
½aꢁD ꢀ16.44 (c 1.11, MeOH). 1H NMR (250 MHz,
methanol-d4): d 0.85 (t, 3H, J = 7.1 Hz, Me), 1.16–1.36
(m, 4H, 2· CH2), 1.56 (m, 2H, CH2), 2.24 (t, 2H,
J = 7.7 Hz, CH2CO), 3.74 (d, 2H, J4 ,5 = 4.6 Hz, 2· H-50),
0
0
4.13 (m, 1H, H-40), 4.26 (dd, 1H, J2 ,3 = 5.4,
0 0
´
´
´
6. Popsavin, M.; Torovic, Lj.; Kojic, V.; Bogdanovic, G.;
Spaic, S.; Popsavin, V. Bioorg. Med. Chem. Lett. 2003, 13,
J3 ,4 = 2.4 Hz, H-30), 4.53 (dd, 1H, J1 ,2 = 8.7,
0
0
0
0
´
0
J2 ,3 = 5.4 Hz, H-20), 5.10 (d, 1H, J1 ,2 = 8.7 Hz, H-1 ),
7.56 and 7.88 (2· br s, 0.4H each, CONH2), 8.12 (d, 0.3H,
J = 8.9 Hz, NH), 8.21 (s, 1H, H-5); 13C NMR (62.9 MHz,
methanol-d4): d 14.30 (Me), 23.35, 26.59 and 32.30 (3·
CH2), 36.89 (CH2CO), 60.02 (C-20), 63.44 (C-50), 72.70 (C-
30), 80.61 (C-10), 88.79 (C-40), 126.32 (C-5), 150.33 (C-4),
165.67 (C-2), 172.23 and 176.78 (2· C@O); CI MS: m/z
358 (M++H); Anal. Calcd for C15H23N3O5S: C, 50.40; H,
6.49, N, 11.76; S, 8.97. Found: C, 50.12; H, 6.21, N, 11.96;
S, 8.66.
0
0
0
0
3167.
7. Popsavin, M.; Torovic, Lj.; Kojic, V.; Bogdanovic, G.;
´
´
Popsavin, V. Tetrahedron. Lett. 2004, 45, 7125.
´
´
´
8. Popsavin, M.; Popsavin, V.; Vukojevic, N.; Csanadi, J.;
´
Miljkovic, D. Carbohydr. Res. 1994, 260, 145.
25
9. Selected data for 10 (syrup): ½aꢁD +15.36 (c 1.34, CHCl3).
1H NMR (250 MHz, CDCl3+D2O): d 0.84 (t, 3H,
J = 7.1 Hz, Me), 1.27 (m, 4H, 2· CH2), 1.60 (m, 2H,
CH2), 2.24 (m, 2H, CH2CO), 4.05 (ddd, 1H, J2,3 = 9.1,
J3,4 = 5.2, J3,NH = 5.4 Hz, H-3), 4.29 (br s, 1H, exchange-
able with D2O, OH), 4.36 (m, 1H, H-5), 4.45 (dd, 1H,
J5,6a = 3.1, J6a, 6b = 12.1 Hz, H-6a), 4.50–4.62 (m, 2H, H-4
and H-6b), 4.68 (d, 1H, J2,3 = 9.1 Hz, H-2), 6.86 (d, 1H,
exchangeable with D2O, J3,NH = 5.4 Hz, CONH), 7.33–
8.10 (m, 5H, Ph), 8.24 and 8.47 (2· br s, 1H each,
CSNH2); 13C NMR (62.9 MHz, CDCl3): d 13.81 (Me),
22.22, 25.05 and 31.25 (3· CH2), 36.48 (CH2CO), 58.95
(C-3), 64.74 (C-6), 71.79 (C-4), 83.99 (C-5), 84.56 (C-2),
128.53, 129.15, 129.62 and 133.47 (Ph), 166.92 (PhC@O),
174.74 (C5H11C@O), 203.46 (C@S). CI MS: m/z 395
(M++H).
14. Selected data for 3: mp 133.5–134.5 ꢁC (from MeOH–iPr2O),
25
½aꢁD ꢀ17.03 (c 0.88, MeOH). 1H NMR (250 MHz,
methanol-d4): d 0.87 (t, 3H, J = 7.0 Hz, Me), 1.18–1.62
(m, 18H, 9· CH2), 2.25 (t, 2H, J = 7.3 Hz, CH2CO),
3.74 (d, 2H, J4 ,5 = 4.5 Hz, 2· H-50), 4.12 (m, 1H, H-
0
0
40), 4.26 (dd, 1H, J2 ,3 = 5.1, J3 ,4 = 2.2 Hz, H-3 ), 4.52
0
0
0
0
0
(dd, 1H, J1 ,2 = 8.7, J2 ,3 = 5.1 Hz, H-20), 5.09 (d, 1H,
0
0
0
0
J1 ,2 = 8.7 Hz, H-10), 8.21 (s, 1H, H-5); 13C NMR
(62.9 MHz, methanol-d4): d 14.41 (Me), 23.61, 26.86,
30.10, 30.32, 30.46, 30.59 and 32.93 (9· CH2), 36.94
(CH2CO), 59.92 (C-20), 63.42 (C-50), 72.65 (C-30), 80.66
(C-10), 88.74 (C-40), 126.27 (C-5), 150.36 (C-4), 165.59
(C-2), 172.17 and 176.69 (2· C@O); CI MS: m/z 442
(M++H); Anal. Calcd for C21H35N3O5S: C, 57.12; H,
7.99, N, 9.52; S, 7.26. Found: C, 56.92; H, 7.77, N,
9.86; S, 6.90.
0
0
10. Selected data for 16: mp 110.5–111 ꢁC (from CH2Cl2–
25
hexane), ½aꢁD +15.36 (c 1.34, CHCl3). 1H NMR
(250 MHz, CDCl3): d 0.87 (t, 3H, J = 6.7 Hz, Me), 1.24
(m, 16H, 8· CH2), 1.60 (m, 2H, CH2), 2.24 (dd, 2H,
J = 7.3, J = 8.2 Hz, CH2CO), 4.06 (ddd, 1H, J2,3 = 9.0,
J3,4 = 5.2, J3,NH = 5.5 Hz, H-3), 4.13 (br s, 1H, exchange-
able with D2O, OH), 4.36 (m, 1H, H-5), 4.41–4.61 (m, 3H,
2· H-6 and H-4), 4.65 (d, 1H, J2,3 = 9.0 Hz, H-2), 6.85 (br
15. 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.