2-Substituted thiazole-4-carboxamide derivatives as tiazofurin mimics: Synthesis and in vitro antitumour activity

Article abstract of DOI:10.1016/j.tet.2014.02.035

Tiazofurin analogues bearing a 5-hydroxymethyl-2-methyl-tetrahydrofuro[2,3- d][1,3]dioxol-6-ol moiety as a sugar mimic (2 and 3), and two novel thiazole-based acyclo-C-nucleosides 4 and 16 have been synthesized in multistep sequences starting from d-xylose (compounds 2 and 3) or from d-arabinose (compounds 4 and 16). All synthesized analogues showed potent in vitro antitumour activities against a panel of human tumour cell lines. Flow cytometry data suggest that cytotoxic effects of analogues 2-4 and 16 in the culture of K562 cells might be mediated by apoptosis. It was also found that these analogues induced changes in cell cycle distribution of K562 cells. Results of western blot analysis (upregulation of Bax and downregulation of Bcl-2, activation of caspase-3 and the presence of a PARP cleavage product) suggest that tiazofurin mimics (2-4 and 16) in K562 cells induced apoptosis in a caspase-dependent way.

Full text of DOI:10.1016/j.tet.2014.02.035

Tetrahedron 70 (2014) 2343e2350
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
2-Substituted thiazole-4-carboxamide derivatives as tiazofurin
mimics: synthesis and in vitro antitumour activity
,
a
ꢀ b
Mirjana Popsavin a , Vesna Kojic , Sasa Spaic , Milos Svircev , Gordana Bogdanovic ,
ꢁ
ꢀ a
ꢁ
a
ꢁ
ꢀ b
*
b
ꢀ b
Dimitar Jakimov , Lidija Aleksic , Velimir Popsavin
a
ꢀ
Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3,
21000 Novi Sad, Serbia
b Oncology Institute of Vojvodina, Put doktora Goldmana 4, 21204 Sremska Kamenica, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
Tiazofurin analogues bearing a 5-hydroxymethyl-2-methyl-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol moiety
as a sugar mimic (2 and 3), and two novel thiazole-based acyclo-C-nucleosides 4 and 16 have been
synthesized in multistep sequences starting from D-xylose (compounds 2 and 3) or from D-arabinose
(compounds 4 and 16). All synthesized analogues showed potent in vitro antitumour activities against
a panel of human tumour cell lines. Flow cytometry data suggest that cytotoxic effects of analogues 2e4
and 16 in the culture of K562 cells might be mediated by apoptosis. It was also found that these ana-
logues induced changes in cell cycle distribution of K562 cells. Results of western blot analysis (upre-
gulation of Bax and downregulation of Bcl-2, activation of caspase-3 and the presence of a PARP cleavage
product) suggest that tiazofurin mimics (2e4 and 16) in K562 cells induced apoptosis in a caspase-
dependent way.
Received 24 December 2013
Received in revised form 7 February 2014
Accepted 17 February 2014
Available online 21 February 2014
Keywords:
Tiazofurin
Thiazole-4-carboxamide
Analogue synthesis
Antitumour activity
Apoptosis
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
The development of practical and efficient routes for the syn-
thesis of drug-like small molecules is of considerable interest for
medicinal chemistry and chemical biology.1 Compounds containing
the thiazole moiety are of particular importance for their potential
application as antifungal,2 antimicrobial,3 antitumour,4 anti-in-
flammatory,5 anticonvulsant6 and antitubercular7 agents. The
antitumour activity of 2,4-disubstituted thiazole derivatives has
been reported and well documented,8,9 including the pronounced
antitumour activity of the thiazole C-nucleoside tiazofurin (1,
Fig. 1).10 Tiazofurin is converted in vivo into the active metabolite
TAD (thiazole-4-carboxamide adenine dinucleotide), an analogue
of NAD that prevents de novo guanine nucleotide synthesis via
inhibition of the enzyme inosine monophosphate dehydrogenase
(IMPDH, EC 1.1.1.205).11 Although tiazofurin proved effective in
reducing the leukaemic cell burden in acute myelogenous leukae-
mia patients, it was found to be too toxic for general clinical ap-
plication.12 Subsequent discovery of two IMPDH isoforms, of which
type II is upregulated in human leukaemia cell lines,13 has
prompted studies towards the design of isoform selective
Fig. 1. Tiazofurin (1) and the corresponding mimetics 2, 3 and 4.
inhibitors14 and has renewed interest in tiazofurin and its
analogues.15
In order to improve therapeutic properties, a number of struc-
tural modifications of the tiazofurin sugar moiety have been re-
ported.16 However, the most of these compounds did not show
favourable biological effects, although a number of recently syn-
thesized analogues have not been assayed for their antitumour
activity. A series of novel tiazofurin derivatives were recently re-
ported by our group and some of them exhibited a more potent
* Corresponding author. Tel.: þ381 21 485 27 68; fax: þ381 21 454 065; e-mail
address: mirjana.popsavin@dh.uns.ac.rs (M. Popsavin).
0040-4020/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.02.035

2344
M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
antitumour activity than lead 1.17 The majority of these analogues
were devoid of any toxicity towards normal human cells. In a recent
communication, we reported an approach to tiazofurin analogues 2
and 3 with substituted tetrahydrofurodioxol moiety as a sugar
mimic.18 We herein describe in detail the synthesis of 2 and 3, along
with the extension of our work to a new thiazole-based acyclo-C-
nucleoside 4, which formally represents a ring opened analogue of
1 that lacks the C-40 hydroxymethyl and the C-20 hydroxyl group.
The effects of the synthesized compounds on the proliferation of
selected human tumour cell lines, including their apoptosis-
inducing properties in the K562 cell culture, were also studied in
this work.
workup of the reaction mixture, it was used in further steps
without any purification. Exposure of 6 to an excess of cyano-
trimethylsilane and boron trifluoride etherate, at room tempera-
ture for 2 h, resulted in cyanoethylidene derivatives 7 and 8,
which were obtained in a 7:1 ratio and 64% combined yield. A
minor amount of intermediate 6 (26%) was also isolated as the pure
a
-anomer and fully characterized by physical and spectroscopic
data.
The stereochemistry of cyanoethylidenes 7 and 8 was resolved
by NOE differential 1H NMR spectroscopy. It was found that when
the methyl group hydrogens of 7 were irradiated, the H-4 peak was
enhanced, suggesting that the methyl group and the H-4 has a syn
orientation that means the nitrile group is exo-oriented. This effect
was not observed in 8, thus implying the opposite stereochemistry
at the quaternary carbon atom. However, when the methyl group
hydrogens of 8 were irradiated, the H-2 proton signal was en-
hanced, suggesting that the methyl group and the H-2 are syn-
oriented. Such an arrangement is only possible if the isomer 8 has
the nitrile group endo-positioned.
2. Results and discussion
2.1. Chemical synthesis
The preparation of tiazofurin mimics 2 and 3 is summarized in
Scheme 1. The sequence started from 3,5-di-O-benzoyl-1,2-O-iso-
Compound 7 was allowed to react with cysteine ethyl ester
hydrochloride, in the presence of Et3N at room temperature, to
afford thiazoline 9 as an inseparable mixture of C-4 epimers. The
newly formed stereocenter will be destroyed in the next reaction,
so no attempts were made to analyze the diastereomeric ratio of
this mixture. A clean conversion of the thiazolines 9 was achieved
in the presence of DBU and bromotrichloromethane.20 Thiazole 10
was thus prepared from 7 in a non-optimized yield of 54%, over the
last two steps and a single chromatographic separation. Ester
aminolysis followed by global deprotection, which were effected by
stirring 10 in methanolic ammonia, provided target 2 in 89% yield.
The same synthetic methodology was then applied for the prepa-
ration of analogue 3 starting from 8. The corresponding tiazofurin
mimics 2 and 3 were thus obtained in 48 and 45% respective yields
(calculated to reacted cyanoethylidene derivatives 7 and 8), over
three synthetic steps.
propylidene-
a
-
D
-xylofuranose (5), which is prepared from D-xylose
in three synthetic steps.19 An acetolysis of 5 afforded the mixture of
anomeric glycosyl acetates 6 (w100%) in an anomeric ratio of 2:1 in
favour of the
b-anomer, as established by integration of the
anomeric proton signals in 1H NMR spectrum. As the 1H and 13C
NMR spectra confirmed a high purity of 6 isolated after the usual
The preparation of thiazole-based acyclo-C-nucleoside 4 is
shown in Scheme 2. The sequence commenced from tetra-O-ben-
zoyl-
D
-arabinonitrile 13, which is readily available from D-arabinose
in five steps.19 Treatment of nitrile 13 with cysteine ethyl ester
hydrochloride in the presence of Et3N, afforded an inseparable
mixture of C-4 epimeric thiazolines 14a. In the first experiments,
the epimers 14a were not purified, but were immediately treated
with DBU and BrCCl3 to give the unsaturated thiazole 15. We are not
aware of any precedent for this transformation. Hence, in order to
get a better insight into a possible mechanism of this process, the
reaction with cysteine ethyl ester was repeated, the resulting
mixture of stereoisomers 14a was purified by flash column chro-
matography and characterized by 1H and 13C NMR spectra. The
stereomeric ratio of 9:1 was determined. Although these isomers
could not be separated chromatographically, both spectra of the
dominant isomer were fully designated and the resulting data were
consistent with structure 14a. Again, no attempts were made to
determine the stereochemistry at C-4 since this stereocenter will be
destroyed in the next reaction. It was assumed that the next step of
the process involves the elimination of benzoyloxy groups from C-
10 and C-20 in 14a, presumably over the tribenzoate 14b as an in-
termediate. This assumption suggests that the previous trans-
formation could be realized even in the absence of BrCCl3. Indeed,
when the purified 14a was treated only with DBU, the unsaturated
thiazole 15 was obtained in 81% yield. Subsequent treatment of 15
with a saturated solution of ammonia in MeOH gave the corre-
sponding acyclo-C-nucleoside 16 in 87% yield. Catalytic reduction
of 15 over 10% Pd/C, gave the expected saturated derivative 17 (54%)
accompanied with a minor amount of deoxygenated side-product
18 (31%). Final exposure of 17 to methanolic ammonia provided
the target 4 in 71% yield.
Scheme 1. Reagents and conditions: (a) glacial AcOH, Ac2O, concd H2SO4, rt, 40 h,
100%; (b) 4.5 equiv Me3SiCN, BF3$OEt2, CH2Cl2, rt, 2 h, 56% of 7, 8% of 8; (c) cysteine
ethyl ester hydrochloride, Et3N, MeOH, rt, 2 h for 7, 3.5 h for 8; (d) BrCCl3, DBU, CH2Cl2,
0 ꢀC, 5 h, than 4 ꢀC, 4 days for 9, 54% of 10 (from 7), 18 h for 8, 50% of 12 (from 8); (e)
NH3, MeOH, rt, 7 days for 10, 89% of 2, 6 days for 12, 90% of 3.

M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
2345
normal foetal lung fibroblasts (MRC-5). These results do suggest
that analogues 2, 3, 4 and 16 are more selective than lead 1, but this
assumption should be verified by additional in vitro experiments
with different normal cell lines. Remarkably, all four analogues (2,
3, 4 and 16) exhibited a potent cytotoxicity against K562 malignant
cells, with respective IC50 values in the range from 0.02 to 1.02 mM.
The most active compound against this cell line was mimic 3, which
was over 90- and 10-fold more potent than both control com-
pounds 1 and DOX, respectively. Compound 2 also inhibited the
growth of K562 cell line being over 17- and 2-fold more active than
1 and DOX, respectively. The same compound demonstrated the
similar and powerful antiproliferative activity towards HL-60 (IC50
0.05 mM) and HeLa (IC50 0.06 mM) cells being 4- and 64-fold more
potent than tiazofurin (1), respectively. Analogue 4 demonstrated
a low submicromolar activity against Jurkat cells, with an IC50 value
same as that recorded for tiazofurin (0.04
recorded for DOX (0.03 M) in the same cell line. Molecule 4
demonstrated the same and powerful activity towards MCF-7 cells
(IC50 0.04 M) being 44- and 5-fold more potent than tiazofurin
mM) and similar to that
m
m
and DOX, respectively. MCF-7 cells were the most sensitive to
mimic 16. This analogue was 90-fold more active than control
compound 1 and 10-fold more active than the commercial cyto-
static DOX in the same cell line.
2.3. Cell cycle analysis
The cells passes through the series of events (cell cycle) leading
to cell division and duplication. Cells that actively pass through the
cell cycle are the targets in the therapy of cancer. We studied effects
of tiazofurin and its analogues 2, 3, 4 and 16 on cell cycle pertur-
bations by staining of DNA with propidium iodide. As shown in
Fig. 2 both tiazofurin and analogues increased the S phase and
decreased the G2/M phase of K562 cells after 24 h compared to
untreated cells. However, longer exposure of K562 cells to ana-
logues resulted in different cell cycle perturbations, which depends
Scheme 2. Reagents and conditions: (a) see Ref. 19; (b) cysteine ethyl ester hydro-
chloride, Et3N, MeOH, rt, 2 h; (c) BrCCl3, DBU, CH2Cl2, 0 ꢀC for 5 h, then 4 ꢀC for 17 days,
47% from 13; or DBU, CH2Cl2, rt for 24 h, 81% from 14a; (d) H2, 10% Pd/C, EtOH, rt, 72 h,
54% of 17, 31% of 18; (e) NH3, MeOH, rt, 6 days for 15, 87% of 16, 16 days for 17, 71% of 4.
2.2. In vitro antitumour activity
The cytotoxic activities of the synthesized tiazofurin mimics 2,
3 and 4 were evaluated after 72 h by MTT assay against six human
cancer cell lines (K562, HL-60, Jurkat, Raji, MCF-7 and HeLa) and
one normal human cell line (MRC-5). Unsaturated acyclo-C-
nucleoside 16 was also included in the testing for the sake of
comparison. Tiazofurin (1) and the commercial antitumour agent
doxorubicin (DOX) were used as the positive controls in this assay.
The results are presented in Table 1.
As shown in Table 1, all synthesized tiazofurin mimics (2e4 and
16) exhibited diverse cytotoxic activity towards all tumour cell lines
under evaluation, but were fully devoid of any toxicity against the
Table 1
In vitro cytotoxicity of tiazofurin (1), analogues 2e4, 16 and DOX
a
Compd
IC50
(m
M)
HL-60
K562
Jurkat
Raji
5.28
2.64
5.61
14.02
4.01
2.98
MCF-7
HeLa
MRC-5
0.36
>100
>100
>100
>100
1
2
3
4
16
DOX
1.89
0.11
0.02
0.95
1.02
0.25
0.19
0.05
0.34
1.01
1.11
0.92
0.04
0.59
0.64
0.04
0.61
0.03
1.78
9.21
11.67
0.04
0.02
0.20
3.82
0.06
0.64
2.51
1.51
0.07
0.10
a
IC50 is the concentration of compound required to inhibit the cell growth by 50%
compared to an untreated control. Values are means of three independent experi-
ments. Coefficients of variation were less than 10%.
Fig. 2. The effects of tiazofurin (1) and the corresponding mimetics 2, 3, 4 and 16 on
the cell cycle of K562 cells. (A) After 24 h. (B) After 72 h. ‘Con’ denotes an untreated
control.

2346
M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
on type of analogue. All analogues increased the percent of cells in
the subG1 phase from 2.5- to 4.5-fold compared to the control. The
subG1 peak consisted of cells with hypodiploid DNA content, which
is characteristic of cells undergoing apoptosis. Tiazofurin induced
the most prominent subG1 peak in K562 cells after 72-h treatment.
The obtained results showed that tiazofurin analogues induced
changes in cell cycle distribution of K562 cells, which depends on
both type of analogue and exposure time.
2.4. Detection of apoptosis
Cell cycle analysis indicated the proapoptotic effect of tiazofurin
analogues through the formation of subG1 peak. Therefore we
further analyzed apoptotic cell death using double staining with
Annexin V-FITC and propidium iodide. Double staining enables
detection of cells in the early phase of apoptosis and clearly dis-
criminates truly necrotic cells from the Annexin V positive cells.
K562 cell death was evaluated after 24 and 72 h of cells treatment
with tiazofurin (1) and analogues 2e4 and 16, at their IC50 con-
centrations. The results are summarized in Fig. 3. Apoptotic re-
sponse presented as a percent of specific apoptosis showed that all
tiazofurin analogues after 24 and 72 h induced several-fold more
Annexin V positive cells compared to parent compound 1. Com-
pared to the 24-h treatment, longer cell treatment with analogues 2
and 3, and 4 and 16, increased the percentage of Annexin V positive
cells 2- and 3-fold, respectively, which is in accordance with results
obtained during cell cycle analysis (subG1 peak).
Fig. 4. Western blot analysis of the protein expression of PARP caspase-3, Bcl-2 and
Bax after 24- and 72-h treatment: untreated control (A), tiazofurin (B), analogue 16 (C),
analogue 4 (D), analogue 2 (E), analogue 3 (F).
and flow cytometry data are in a good agreement with the results of
MTT cytotoxicity assay.
3. Conclusion
In conclusion, two interesting tiazofurin analogues bearing the
5-hydroxymethyl-2-methyl-tetrahydrofuro[2,3-d][1,3]dioxol-6-ol
moiety as a sugar mimic (2 and 3), have been synthesized in eight
steps starting from D-xylose. The pivotal steps in the synthesis were
the preparation of cyanoethylidene derivatives 7 and 8, followed by
elaboration of the thiazole carboxamide aglycon at the nitrile
moiety. In addition, two novel acyclo-C-nucleosides 4 and 16
bearing 2,4-disubstituted thiazole moiety have been prepared
Fig. 3. Percentage of specific apoptosis of K562 cells induced by tiazofurin and ana-
logues 2e4 and 16 after 24- and 72-h treatment.
starting from
D-arabinose. The key steps of the synthesis were the
To evaluate the mechanisms underlying the apoptosis, we in-
vestigated how tiazofurin analogues (2e4 and 16) modulated ex-
pression of key proteins Bax, Bcl-2, caspase-3 and PARP, involved in
apoptotic signalling cascade. Western blot analysis (Fig. 4) revealed
that tiazofurin and its mimetics 2, 3 and 4 decreased Bcl-2 ex-
pression after 24-h treatment while analogue 16 increased it,
compared to 72-h treatment. All compounds induced a significantly
higher expression of proapoptotic protein Bax after 72-h
treatment that was in accordance with an increase of specific ap-
optosis at the same time point. Also, all tiazofurin analogues in-
creased expression of caspase-3 in both time points indicating its
involvement in apoptotic process. Activation of caspase-3 is fol-
lowed by cleavage of downstream targets including PARP. Our ex-
periments clearly showed proteolysis cleavage of PARP in K562 cells
by both tiazofurin and all investigated analogues.
initial condensation of tetra-O-benzoyl-
D
-arabinonitrile 13 with
cysteine ethyl ester hydrochloride, followed by the subsequent
reaction of resulting C-4 epimeric thiazolines 14a with DBU and
bromotrichloromethane, to give the unsaturated thiazole 15. Then
it was discovered that treatment of 14a with DBU in the absence of
bromotrichloromethane gave the same reaction product 15, thus
implying that this transformation included two successive E2 pro-
cesses over the intermediates 14a and 14b, as shown in Scheme 2.
An in vitro antitumour assay revealed that all of the synthesized
compounds showed selective cytotoxic effects on specific cancer
cell lines (with IC50 values ranging from 0.02 to 14.2 mM), whereas
none showed any inhibitory effect on the normal cell line, MRC-5.
In contrast, the lead 1 and the commercial drug DOX showed
a potent cytotoxic activity towards all cell lines under evaluation,
with respective IC50 values of 0.36 and 0.10 mM. The most potent
Upregulation of Bax and downregulation of Bcl-2, activation of
caspase-3, and the presence of PARP cleavage product suggest that
cytotoxic effects of tiazofurin analogues in K562 cells might be
mediated by caspase-dependent apoptosis. Western blot analysis
antiproliferative activity of synthesized tiazofurin mimics (2e4 and
16) was observed in the culture of K562 cells (the recorded IC50
values were in the range from 0.02 to 1.02
mM). A comparison be-
tween the bicyclic tiazofurin mimics (2 and 3) and acyclo-C-

M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
2347
nucleosides (4 and 16) revealed either similar (e.g., compounds 2
and 16 on Jurkat) or rather different (e.g., 3 and 16 on MCF-7) ac-
tivity against the same cell line. However, acyclo-C-nucleosides
were generally more active. The results obtained by studying the
effects of tiazofurin and analogues 2e4, and 16 on cell cycle per-
turbations showed that synthesized mimics induced changes in cell
cycle distribution of K562 cells, which depends on both structure of
the analogue and the exposure time. Upregulation of Bax and
downregulation of Bcl-2, activation of caspase-3, and the presence
of the cleavage product of PARP suggest that cytotoxic effects of
tiazofurin mimetics 2e4, and 16 in K562 cells might be mediated by
apoptosis in a caspase-dependent way.
CH2Cl2 (15 mL) were added Et2O$BF3 (0.02 mL, 0.18 mmol) and
Me3SiCN (1.03 mL, 8.23 mmol). The mixture was stirred at room
temperature for 2 h, in an atmosphere of N2, and then rendered
alkaline (to pH w9) by the addition of a saturated solution of
aq NaHCO3 (30 mL). The resulting emulsion was extracted with
ether (4ꢁ30 mL), the combined organic solutions were washed
with brine (1ꢁ30 mL), dried and evaporated. The residue was
purified by preparative TLC (38 preparative plates, 19:1 toluene/
EtOAc, three successive developments, eluted with EtOAc). Pure 7
(0.31 g, 56% calculated to reacted 6) was isolated as colourless
crystals, mp 127 ꢀC (CH2Cl2/hexane), ½a D20
ꢃ46.2 (c 0.2, CHCl3),
ꢂ
Rf¼0.66 (19:1 toluene/EtOAc, three successive developments). IR
(film): nmax 2233 (C^N), 1726 (C]O, Bz). 1H NMR (CDCl3):
d 1.85 (s,
4. Experimental section
4.1. General
3H, CH3), 4.59e4.72 (m, 3H, H-4 and 2ꢁH-5), 4.91 (d, 1H,
J1,2¼4.1 Hz, H-2), 5.64 (d, 1H, J3,4¼2.6 Hz, H-3), 6.24 (d, 1H,
J1,2¼4.1 Hz, H-1), 7.33e8.07 (m, 10H, 2ꢁPh). NOE contact: CH3 and
H-4. 13C NMR (CDCl3):
d 24.2 (CH3), 61.1 (C-5), 75.4 (C-3), 77.8 (C-4),
€
Melting points were determined on a Buchi 510 apparatus and
84.2 (C-2), 100.0 (Cq), 105.5 (C-1), 116.4 (CN), 128.2, 128.3, 128.4,
129.1, 129.5, 129.6, 133.1, 133.7 (2ꢁPh), 164.8 and 165.8 (2ꢁPhC]
O). HRMS (ESI): m/z 432.1041 (MþþNa), calcd for C22H19NO7Na:
432.1054. Anal. Found: C, 64.65; H, 4.67; N, 3.43. Calcd for
were not corrected. Optical rotations were measured on P 3002
(Kruss) and Autopol IV (Rudolph Research) polarimeters at 20 ꢀC.
€
1H (250 MHz) and 13C (62.9 MHz) NMR spectra were recorded on
a Bruker AC 250 E instrument and chemical shifts are expressed in
parts per million (ppm) downfield from TMS. IR spectra were
recorded with an FTIR Nexus 670 spectrophotometer (Thermo-
Nicolet). High resolution mass spectra (ESI) of synthesized com-
pounds were acquired on an Agilent Technologies 1200 series in-
strument equipped with Zorbax Eclipse Plus C18 (100 mmꢁ2.1 mm
C
22H19NO7: C, 64.54; H, 4.68; N, 3.42. Pure minor product 8
(0.045 g, 8%, calculated to reacted 6) was isolated as a colourless
oil, ½a 2D0
ꢃ9.1 (c 0.4, CHCl3), Rf¼0.58 (19:1 toluene/EtOAc, three
ꢂ
successive developments). IR (film): nmax 2235 (C^N), 1725 (C]O,
Bz). 1H NMR (CDCl3):
d
1.82 (s, 3H, CH3), 4.57 (dd, 1H, J5a,5b¼11.7 Hz,
J4,5a¼4.9 Hz, H-5a), 4.66 (dd, 1H, J5a,5b¼11.7 Hz, J4,5b¼6.4 Hz, H-5b),
4.86 (d, 1H, J1,2¼3.8 Hz, H-2), 5.06e5.23 (m, 1H, H-4), 5.83 (d, 1H,
J3,4¼4.2 Hz, H-3), 6.20 (d, 1H, J1,2¼3.8 Hz, H-1), 7.38e8.07 (m, 10H,
i.d. 1.8 mm) column and DAD detector (190e450 nm) in combina-
tion with a 6210 time-of-flight LC/MS instrument (ESI) in the pos-
itive ion mode. Flash column chromatography was performed using
Kieselgel 60 (0.040e0.063, E. Merck). Self-made preparative TLC
plates were prepared using Kieselgel 60 G (E. Merck) with fluo-
rescent indicator F254 as additive. The corresponding bands were
scraped and eluted with a convenient solvent. All organic extracts
were dried with anhydrous Na2SO4. Organic solutions were con-
centrated in a rotary evaporator under reduced pressure at a bath
temperature below 35 ꢀC.
2ꢁPh). NOE contact: CH3 and H-2. 13C NMR (CDCl3):
d 26.9 (CH3),
62.2 (C-5), 75.6 (C-3), 79.4 (C-4), 86.1 (C-2), 101.6 (Cq), 106.1 (C-1),
117.7 (CN), 128.4, 128.6, 129.6, 129.7, 133.2, 133.3, 133.9 (2ꢁPh),
165.0 and 165.9 (2ꢁPhC]O). HRMS (ESI): m/z 432.1049 (MþþNa),
calcd for C22H19NO7Na: 432.1054. Unreacted starting compound 6
was recovered as pure
a
-anomer (0.21 g, 26%), ½a D20
þ90.6 (c 2.3,
ꢂ
CHCl3), Rf¼0.27 (19:1 toluene/EtOAc, three successive de-
velopments). IR (film): nmax 1759 (C]O, Ac), 1727 (C]O, Bz). 1H
NMR (CDCl3):
d
2.09 and 2.11 (2ꢁs, 3H each, 2ꢁCH3C]O), 4.46 (dd,
4.2. 3,5-Di-O-benzoyl-1,2-O-[(1S)-1-cyanoethylidene]-
lofuranose (7) and 3,5-di-O-benzoyl-1,2-O-[(1R)-1-
a-
D-xy-
1H, J5a,5b¼12.2 Hz, J4,5a¼4.8 Hz, H-5a), 4.54 (dd, 1H, J5a,5b¼12.2 Hz,
J4,5b¼4.5 Hz, H-5b), 4.84e4.93 (m, 1H, H-4), 5.59 (dd, 1H,
J1,2¼4.6 Hz, J2,3¼6.2 Hz, H-2), 5.89 (t, 1H, J¼6.6 Hz, H-3), 6.54 (d, 1H,
J1,2¼4.6 Hz, H-1), 7.30e8.01 (m, 10H, 2ꢁPh). 13C NMR (CDCl3):
cyanoethylidene]-a-D-xylofuranose (8)
To a stirred solution of 5 (0.866 g, 2.17 mmol) in glacial AcOH
(8.4 mL) were added Ac2O (2.1 mL) and concentrated H2SO4
(0.59 mL). The mixture was stirred at room temperature for 40 h.
The mixture was cooled to 0 ꢀC, and quenched by the addition of
a saturated solution of aq NaHCO3 (100 mL) in portions, followed
by the addition of solid NaHCO3 until pH w7. The suspension was
extracted with CH2Cl2 (4ꢁ30 mL), the combined organic layers
were washed with brine (1ꢁ50 mL), dried and evaporated. The
residue was dried under high vacuum to afford crude 6, which was
pure enough for the next step of the synthesis. Anomeric ratio
d
20.2 and 20.8 (2ꢁCH3C]O), 62.0 (C-5), 74.2 (C-3), 75.1 (C-2), 75.4
(C-4), 92.7 (C-1), 128.2, 128.4, 129.4, 129.6, 133.1, 133.6 (2ꢁPh),
165.5, 165.7 (2ꢁPhC]O), 169.2 and 169.5 (2ꢁCH3C]O). HRMS
(ESI): m/z 465.1153 (MþþNa), calcd for C23H22O9Na: 465.1156.
4.3. General procedure for the synthesis of thiazole
derivatives 10 and 12
To a stirred solution of 7 or 8 (1 equiv) in 4:1 mixture of absolute
MeOH and dry CH2Cl2 (0.03 M) were added L-cysteine ethyl ester
(from 1H NMR):
nmax 1759 (C]O, Ac), 1727 (C]O, Bz). 1H NMR (CDCl3):
2.12 (3ꢁs, CH3 a and ), 4.46 (dd, J5a,5b¼12.2 Hz, J4,5a¼4.8 Hz, H-5a
), 4.54 (dd, J5a,5b¼12.2 Hz, J4,5b¼4.5 Hz, H-5b ), 4.60 (pseudo d,
), 4.82e4.97 (m, H-4 and ), 5.41 (s, 1H, H-2
), 5.58 (dd, J1,2¼4.6 Hz, J2,3¼6.2 Hz, H-2 ), 5.72 (d, J3,4¼4.9 Hz, H-3
b
/
a
w2:1; Rf¼0.15 (19:1 toluene/EtOAc). IR (film):
hydrochloride (1.5 equiv) and anhydrous Et3N (1.5 equiv). The
mixture was stirred until the starting materials were consumed
(TLC, 2 h for 7, 3.5 h for 8) and evaporated. The residue was dis-
solved in CH2Cl2 and the organic solution was washed with water,
saturated aq NaHCO3 and brine. The organic solution was dried,
filtered and evaporated to give crude thiazolines 9 or 11 as in-
separable mixtures of stereoisomers. To a stirred solution of crude
thiazoline 9 or 11 (1 equiv) in anhydrous CH2Cl2 (0.1 M) was added
DBU (2 equiv). The solution was cooled to 0 ꢀC and BrCCl3
(1.2 equiv) was added. The reaction mixture was stirred in an at-
mosphere of N2, for 5 h at 0 ꢀC and then stored at þ4 ꢀC overnight.
The mixture was evaporated and the residue was purified by pre-
parative TLC (4:1 toluene/EtOAc, eluted with EtOAc) to give pure
thiazoles 10 or 12.
d
2.07, 2.11,
b
a
a
2H, J4,5¼5.8 Hz, H-5
b
a
b
b
b
a
a
), 5.89 (t, J¼6.6 Hz, H-3
a
), 6.24 (s, H-1
b
), 6.54 (d, J1,2¼4.5 Hz, H-1
), 7.30e8.13 (m, 10H, 2ꢁPh). 13C NMR (CDCl3):
d
20.2, 20.2, 20.7,
), 74.1 (C-3
), 79.7 (C-4 ),
), 128.1, 128.2, 128.3, 128.4, 128.6, 129.15,
and ), 164.7,
), 168.9, 169.1, 169.4 (2ꢁMeC]O
). To a stirred solution of 6 (0.811 g, 1.83 mmol) in dry
20.8 (2ꢁMe from 2ꢁAc
a
and
b
), 61.9 (C-5
a
), 62.6 (C-5
b
a
), 74.3 (C-3
b
), 75.0 (C-2
a
), 75.3 (C-4
a
), 79.4 (C-2
b
b
92.6 (C-1
a
), 98.6 (C-1 b
129.2, 129.4, 129.4, 129.5, 132.95, 133.0, 133.5 (2ꢁPh
165.4, 165.7 (2ꢁPhC¼O and
and
a
b
a
b
a
b

2348
M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
4.3.1. 3,5-Di-O-benzoyl-1,2-O-{(1S)-1-[4-(ethoxycarbonyl)-1,3-
HRMS (ESI): m/z 303.0648 (MþþH), calcd for C11H15N2O6S:
thiazol-2-yl]ethylidene}-
a
-
D
-xylofuranose (10). Yield: 54% (calcu-
303.0645.
lated to reacted 7). Colourless oil, ½a D20
ꢃ16.8 (c 1.3, CHCl3), Rf¼0.22
ꢂ
(19:1 toluene/EtOAc, three successive developments). IR (film):
4.5. Ethyl 2-[(10E,30S)-30,40-bis(benzoyloxy)but-10-en-10-yl]-
1,3-thiazole-4-carboxylate (15)
nmax 1725 (C]O, Bz). 1H NMR (CDCl3):
d
1.33 (t, 3H, J¼7.1 Hz,
CO2CH2CH3), 1.97 (s, 3H, CH3), 40.35 (q, 2H, J¼7.1 Hz, CO2CH2CH3),
0
0
0
0
0
4.61 (d, 2H, J4 ,5 ¼6.0 Hz, 2ꢁH-5 ), 4.73 (d, 1H, J1 ,2 ¼3.8 Hz, H-2 ),
Procedure A. To a stirred solution of 13 (0.210 g, 0.37 mmol) in
absolute MeOH (5 mL) was added L-cysteine ethyl ester hydro-
4.82 (td, 1H, J3 ,4 ¼2.9 Hz, J4 ,5 ¼5.9 Hz, H-400), 5.69 (d, 1H,
0
0
0
0
0
0
0
0
0
J3 ,4 ¼2.9 Hz, H-3 ), 6.24 (d, 1H, J1 ,2 ¼3.8 Hz, H-1 ), 7.31e8.05 (m,
chloride (0.192 g, 1.04 mmol) followed by Et3N (0.14 mL,
1.04 mmol). The reaction mixture was stirred for 2 h at room
temperature and evaporated. The residue was dissolved in CH2Cl2
and the organic solution was washed with water, saturated
aq NaHCO3 and brine. The organic layer was dried, filtered and
evaporated to give crude 14a (0.316 g). To a stirred solution of crude
14a (0.259 g, 0.37 mmol) in anhydrous CH2Cl2 (4.3 mL) was added
DBU (0.11 mL, 0.74 mmol). The solution was cooled to 0 ꢀC and
BrCCl3 (0.04 mL, 0.44 mmol) was added over 10 min. The reaction
mixture was stirred in an atmosphere of N2, for 5 h at 0 ꢀC and then
stored at þ4 ꢀC for 17 days. The mixture was evaporated and the
residue was purified by preparative TLC (9:1 toluene/EtOAc, eluted
with EtOAc) to give pure 15 (0.079 g, 47% from two steps) as a col-
10H, 2ꢁPh), 8.10 (s, 1H, H-5). NOE contact: CH3 and H-40. 13C NMR
(CDCl3): d 14.1 (CO2CH2CH3), 25.7 (CH3), 61.2 (CO2CH2CH3), 61.4 (C-
50), 75.9 (C-30), 77.6 (C-40), 84.0 (C-20), 105.7 (C-10), 109.2 (Cq), 127.8
(C-5), 128.1, 128.4, 128.5, 129.2, 129.5, 133.9, 133.5 (2ꢁPh), 147.9 (C-
4), 160.8 (C-2), 164.7, 165.8 (PhC]O), 171.4 (EtOC]O). HRMS (ESI):
m/z 540.1319 (MþþH), calcd for C27H26NO9S: 540.1323.
4.3.2. 3,5-Di-O-benzoyl-1,2-O-{(1R)-1-[4-(ethoxycarbonyl)-1,3-
thiazol-2-yl]ethylidene}-a-D-xylofuranose (12). Yield: 50% (calcu-
lated to reacted 8). Colourless oil, ½a D20
ꢃ28.0 (c 1.0, CHCl3), Rf¼0.41
ꢂ
(4:1 cyclohexane/Me2CO, four successive developments), Rf¼0.51
(4:1 toluene/EtOAc). IR (film): nmax 1725 (C]O, Bz). 1H NMR
a 20
(CDCl3):
d
1.37 (t, 3H, J¼7.1 Hz, CO2CH2CH3), 1.86 (s, 3H, CH3), 4.37
ourless oil, ½ ꢂD ꢃ21.9 (c 0.8, CHCl3), Rf¼0.38 (9:1 toluene/EtOAc).
0
0
(q, 2H, J¼7.1 Hz, CO2CH2CH3), 4.54 (dd, 1H, J5a ,5b ¼5.6 Hz,
Procedure B. To a stirred solution of 13 (0.544 g, 0.97 mmol) in
0
0
0
0
J4 ,5a ¼2.7 Hz, H-5a ), 4.59e4.62 (m, 1H, H-4 ), 4.63 (dd, 1H,
a mixture of absolute MeOH (13 mL) and anhydrous CH2Cl2 (2 mL)
0
0
0
0
0
0
0
0
J5a ,5b ¼5.8 Hz, J4 ,5b ¼2.7 Hz, H-5b ), 4.91 (d, 1H, J1 ,2 ¼1.4 Hz, H-20),
was added
L-cysteine ethyl ester hydrochloride (0.499 g,
0
0
0
0
0
5.70 (d, 1H, J3 ,4 ¼2.8 Hz, H-3 ), 6.25 (d, 1H, J1 ,2 ¼2.8 Hz, H-1 ),
2.69 mmol) and dry Et3N (0.37 mL, 2.65 mmol). The mixture was
stirred for 2 h at room temperature and evaporated. The residue
was dissolved in CH2Cl2 and the organic solution was washed with
water, saturated aq NaHCO3 and brine. The organic layer was dried,
filtered and evaporated to give crude 14a (0.629 g). Flash column
chromatography (19:1 toluene/EtOAc) of the residue gave purified
mixture of thiazoline 14a (0.303 g, 45%) as a colourless oil. 1H NMR
7.33e8.04 (m, 10H, 2ꢁPh), 8.15 (s, 1H, H-5). 13C NMR (CDCl3):
d 14.2
(CO2CH2CH3), 25.5 (CH3), 61.3 (CO2CH2CH3), 61.7 (C-50), 75.9 (C-30),
77.9 (C-40), 84.4 (C-20), 105.6 (C-10), 110.2 (Cq), 128.2, 128.5, 128.6,
129.3, 129.5, 129.6, 133.0, 133.6 (C-5 and 2ꢁPh), 147.7 (C-4), 161.0
(C-2), 164.9, 165.8 (2ꢁPhC]O), 170.5 (EtOC]O). HRMS (ESI): m/z
540.1318 (MþþH), calcd for C27H26NO9S: 540.1323.
(CDCl3):
d
1.28 (t, 5.9H, J¼7.2 Hz, CO2CH2CH3), 3.25e3.64 (m, 2.6H,
0
0
4.4. General procedure for the preparation of thiazole-4-
carboxamide derivatives 2 and 3
2ꢁH-5), 4.03e4.30 (q, 2H, CO2CH2CH3), 4.55 (dd, 1H, J3 ,4 a¼5.8 Hz,
J4 a,4 b¼12.8 Hz, H-40a), 4.86 (dd, 1H, J3 ,4 b¼3.1 Hz, J4 a,4 b¼12.5 Hz,
0
0
0
0
0
0
H-40b), 5.02 (t, 1H, J4,5b¼10.1 Hz, H-4), 5.95 (m, 1H, H-300), 6.24e6.44
(m, 2H, H-10 and H-20), 7.17 (d, 1H, J1 ,2 ¼16.2 Hz, H-1 ), 7.31e8.20
0
0
A solution of 10 or 12 (1 equiv) in saturated methanolic am-
monia (0.03 M) was kept at room temperature (7 days for 10, 6 days
for 12) and then evaporated. The residue was purified by means of
preparative TLC (5:1 CHCl3/MeOH, eluted with 1:1 EtOAc/iPrOH) to
afford pure 2 or 3.
(m, 20H, 4ꢁPh). 13C NMR (CDCl3):
d 13.9 (CO2CH2CH3), 34.3 and
34.5 (C-5), 61.6 (CO2CH2CH3), 62.3 (C-40), 68.9 and 69.1 (C-30), 71.4
and 71.6 (C-10 and C-20), 128.2, 128.4, 128.6, 128.8, 128.9, 129.2,
129.55, 129.6, 129.7, 129.8, 132.9, 133.2, 133.4, 133.5 (4ꢁPh), 164.6,
164.7, 164.95, 165.0, 165.8, 169.8, 171.8 (4ꢁPhC]O and CO2CH2CH3).
To a stirred solution of purified thiazoline 14a (0.133 g, 0.19 mmol)
in anhydrous CH2Cl2 (2.23 mL) was added DBU (0.056 mL,
0.38 mmol). The mixture was stirred at room temperature for 24 h,
and then evaporated. The residue was purified by flash column
chromatography (19:1 toluene/EtOAc), to afford pure 15 (0.070 g,
4.4.1. 1,2-O-[(1S)-1-(4-Carbamoyl-1,3-thiazol-2-yl)ethylidene]-a-D-
xylofuranose (2). Yield: 89%. Colourless needles, mp 165 ꢀC (from
iPr2O/MeOH), ½a 2D0
ꢃ13.0 (c 0.3, MeOH), Rf¼0.44 (5:1 CHCl3/MeOH).
ꢂ
IR (KBr): nmax 1672 (C]O, amide I), 1590 (NH, amide II). 1H NMR
0
0
(methanol-d4):
d
1.88 (s, 3H, CH3), 3.78 (dd, 1H, J4 ,5a ¼6.6 Hz,
J5a ,5b ¼11.7 Hz, H-5a ), 3.86 (dd,1H, J4 ,5b ¼4.9 Hz, J5a ,5b ¼11.6 Hz, H-
81%) as a colourless oil, ½a D20
ꢃ21.9 (c 0.8, CHCl3), Rf¼0.38 (9:1
ꢂ
0
0
0
0
0
0
0
5b0), 4.23 (d,1H, J3 ,4 ¼2.8 Hz, H-3 ), 4.24e4.29 (m,10H, H-4 ), 4.48 (d,
toluene/EtOAc). IR (film): nmax 1722 (PhC]O). 1H NMR (CDCl3):
0
0
0
0
0
0
0
0
0
1H, J1 ,2 ¼3.8 Hz, H-2 ), 6.09 (d, 1H, J1 ,2 ¼3.8 Hz, H-1 ), 8.21 (s, 1H, H-
d
1.33 (t, 3H, J¼7.2 Hz, CO2CH2CH3), 4.33 (q, 2H, CO2CH2CH3), 3.73
5). NOE contact: CH3 and H-20, CH3 and H-40. 13C NMR (methanol-
(dd, 1H, J3 ,4 a¼6.6 Hz, J4 a,4 b¼12.0 Hz, H-40a), 3.68 (dd, 1H,
0
0
0
0
d
26.2 (CH3), 60.8 (C-50), 75.4 (C-30), 83.2 (C-40), 87.6 (C-20),
J3 ,4 b¼3.7 Hz, J4 a,4 b¼12.0 Hz, H-40b), 6.000e6.14 (m, 1H, H-30), 6.99
0
0
0
0
d4):
107.2 (C-10), 109.9 (Cq), 126.4 (C-5), 151.5 (C-4), 165.4 (C-2), 173.9
(dd, 1H, J2 ,3 ¼6.4 Hz, J1 ,2 ¼16.0 Hz, H-2 ), 7.17 (d, 1H, J1 ,2 ¼16.2 Hz,
0
0
0
0
0
0
(CONH2). HRMS (ESI): m/z 303.0636 (MþþH), calcd for
H-10), 7.40e8.16 (m, 10H, 2ꢁPh), 8.33 (s, 1H, H-5). 13C NMR (CDCl3):
C
11H15N2O6S: 303.0645.
d
14.2 (CO2CH2CH3), 61.5 (CO2CH2CH3), 64.7 (C-40), 71.0 (C-30), 126.0
(C-10), 127.0 (C-5), 128.3, 128.4, 129.2, 129.3, 129.6, 129.7, 129.8,
131.5,133.2,133.4 (2ꢁPh),131.5 (C-20),147.6 (C-4),161.0 (C-2),165.2,
165.7, 166.0 (2ꢁPhCO and CO2Et). HRMS (ESI): m/z 452.1161
(MþþH), calcd for C24H22NO6S: 452.1162.
4.4.2. 1,2-O-[(1R)-1-(4-Carbamoyl-1,3-thiazol-2-yl)ethylidene]-a-D-
xylofuranose (3). Yield: 90%. Colourless oil, ½a D20
þ0.01 (c 0.45,
ꢂ
MeOH), Rf¼0.44 (5:1 CHCl3/MeOH). IR (film): nmax 1669 (C]O,
amide I), 1589 (NH, amide II). 1H NMR (methanol-d4):
d 1.78 (s, 3H,
0
0
0
0
0
CH3), 3.70 (dd,1H, J4 ,5a ¼6.5 Hz, J5a ,5b ¼11.7 Hz, H-5a ), 3.78 (0dd,1H,
4.6. 2-[(10E,30S)-30,40-Dihydroxybut-10-en-10-yl]-1,3-thiazole-
4-carboxamide (16)
0
0
0
0
0
J4 ,5b ¼5.7 Hz, J5a ,5b ¼11.7 Hz, H-5b ), 4.02e4.11 (m, 1H, H-4 ), 4.23
0
0
0
0
0
0
(d, 1H, J3 ,4 ¼2.9 Hz, H-3 ), 4.71 (d, 1H, J1 ,2 ¼3.7 Hz, H-2 ), 6.12 (d, 1H,
J1 ,2 ¼3.7 Hz, H-1 ), 8.23 (s, 1H, H-5). 13C NMR (methanol-d4):
d
26.0
A solution of 15 (0.068 g, 0.15 mmol) in saturated methanolic
ammonia (5 mL) was kept at room temperature for 6 days, and then
evaporated. The residue was purified by preparative TLC (4:1
0
0
0
(CH3), 60.9 (C-50), 75.3 (C-30), 83.5 (C-40), 88.1 (C-20), 107.1 (C-10),
110.6 (Cq), 126.7 (C-5), 151.0 (C-4), 165.4 (C-2), 172.9 (CONH2).

M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
2349
EtOAc/iPrOH, eluted with 1:1 EtOAc/iPrOH) to afford pure 16
were obtained from R&D Systems (Minneapolis, MN). Anti- poly
(ADP-ribose) polymerase (PARP) was purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence (ECL
Plus) kit and Hyperfilm were purchased from Amersham Bio-
sciences (Arlington Heights, IL). All other chemicals used in the
experiments were commercial products of reagent grade. Stock
solution (10 mM) was prepared in DMSO and diluted to various
concentrations with serum-free culture medium.
(0.028 g, 87%) as a pale yellow oil, ½a D20
ꢃ3.82 (c 1.02, CH3OH),
ꢂ
Rf¼0.40 (4:1 EtOAc/iPrOH). 1H NMR (methanol-d4):
d 3.62 (dd, 1H,
J3 ,4 a¼6.3 Hz, J4 a,4 b¼11.2 Hz, H-40a), 3.68 (dd, 1H, J3 ,4 b¼5.2 Hz,
0
0
0
0
0
0
J4 a,4 b¼11.2 Hz, H-40b), 4.35e4.41 (m, 1H, H-30), 6.80 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
J2 ,3 ¼4.9 Hz, J1 ,2 ¼16.2 Hz, H-2 ), 6.96 (d, 1H, J1 ,2 ¼16.2 Hz, H-1 ),
8.14 (s, 1H, H-5). 13C NMR (methanol-d4):
d
66.7 (C-30), 73.1 (C-40),
124.3 (C-10), 124.8 (C-5), 139.5 (C-20), 151.0 (C-4), 165.6 (C-2), 168.3
(CONH2). HRMS (ESI): m/z 237.0317 (MþþNa), calcd for
C8H10N2NaO3S: 237.0304.
4.10. Cell lines
4.7. Ethyl 2-[(30S)-30,40-bis(benzoyloxy)butyl]-1,3-thiazole-4-
carboxylate (17) and ethyl 2-butyl-1,3-thiazole-4-carboxylate
(18)
Human chronic myelogenous leukaemia (K562), human pro-
myelocytic leukaemia (HL-60), human T cell leukaemia (Jurkat) and
Burkitt’s lymphoma (Raji) were grown in RPMI 1640, while breast
adenocarcinoma (MCF-7), cervix carcinoma (HeLa) malignant
cells and normal foetal lung fibroblasts (MRC-5) were grown in
DMEM medium. Both media were supplemented with 10% of foetal
calf serum (FTS, NIVNS) and antibiotics (100 IU/mL of penicillin and
To a stirred solution of 15 (0.135 g, 0.3 mmol) in 96% EtOH (7 mL)
was added 10% Pd/C (0.065 g). The suspension was hydrogenated at
room temperature and normal pressure of H2 for 72 h, then filtered
through a Celite pad, washed with EtOH and evaporated. The res-
idue was purified by preparative TLC (9:1 toluene/EtOAc, eluted
with 1:1 CH2Cl2/EtOAc) to give pure 17 (0.073 g, 54%) as a colourless
100
mg/mg of streptomycin). Cell lines were cultured in flasks
(Costar, 25 mL) at 37 ꢀC in the atmosphere of 100% humidity and 5%
of CO2 (Heraeus). Exponentially growing viable cells were used
throughout the assays.
a 20
oil, ½ ꢂD ꢃ24.2 (c 0.4, CHCl3), Rf¼0.29 (9:1 toluene/EtOAc). 1H NMR
(CDCl3):
d
1.38 (t, 3H, J¼7.2 Hz, CO2CH2CH3), 2.28e2.47 (m, 2H, CH2-
20), 3.12e3.38 (m, 2H, CH2-10), 4.40 (q, 2H, J¼7.2 Hz, CO2CH2CH3),
4.51 (dd, 1H, J3 ,4 a¼6.0 Hz, J4 a,4 b¼12.0 Hz, H-40a), 4.61 (dd, 1H,
4.11. Cells treatment
0
0
0
0
J3 ,4 b¼3.7 Hz, J4 a,4 b¼12.0 Hz, H-40b), 5.59 (m, 1H, H-30), 7.35e8.08
0
0
0
0
(m, 10H, 2ꢁPh), 8.03 (s, 1H, H-5). 13C NMR (CDCl3):
d
14.3
The cells were seeded in six-well plates at a concentration of
5ꢁ105 cells/well. Cells were treated for 24 and 72 h with tiazofurin
(1) and mimics (2, 3, 4 and 16) at their IC50/24 and 72 h concen-
trations. Untreated cells were used as control. Viable cells of treated
and control samples were used for apoptosis detection and West-
ern blot analysis. Viability was determined using trypan blue dye-
exclusion assay.
(CO2CH2CH3), 29.4 (C-10), 31.0 (C-20), 61.4 (CO2CH2CH3), 65.2 (C-40),
71.1 (C-30), 127.0 (C-5),128.4,129.5,129.5,129.6,133.1, 133.2 (2ꢁPh),
145.0 (C-4), 161.2 (C-2), 165.9, 166.1, 170.0 (3ꢁPhC]O and CO2Et).
HRMS (ESI): m/z 454.1320 (MþþH), calcd for C24H24NO6S: 454.1319.
The side-product 18 (0.02 g, 31%) was also isolated as a colourless
syrup, Rf¼0.40 (9:1 toluene/EtOAc). 1H NMR (CDCl3):
d 0.95 (t, 3H,
(CH2)3CH3), 1.34e1.53 (m, 2H, CH2 from Bu), 1.40 (t, 3H, J¼7.2 Hz,
CO2CH2CH3), 1.70e1.89 (m, 2H, CH2 from Bu), 3.07 (t, 2H,
CH2(CH2)2CH3), 4.43 (q, 2H, J¼7.2 Hz, CO2CH2CH3), 8.05 (s, 1H, H-5).
4.12. MTT assay
13C NMR (CDCl3):
d 13.6 [(CH2)3CH3], 14.3 (CO2CH2CH3), 22.2, 32.1,
Cells were harvested, counted by trypan blue and plated into
96-well microtiter plates (Costar) at optimal seeding density of
5ꢁ103 cells per well to assure logarithmic growth rate throughout
33.2 (3ꢁCH2 from Bu), 61.3 (CO2CH2CH3), 126.7 (C-5), 146.6 (C-4),
161.4 (C-2), 172.5 (CO2CH2CH3).
the assay period. Viable cells were placed in a volume of 90 mL per
4.8. 2-[(30S)-30,40-Dihydroxybutyl]-1,3-thiazole-4-carboxamide
(4)
well, and preincubated in complete medium at 37 ꢀC for 24 h to
allow cell stabilization prior to the addition of substances. Tested
substances, at 10-fold the required final concentration, were
A solution of 17 (0.035 g, 0.08 mmol) in saturated methanolic
ammonia (5 mL) was left at room temperature for 16 days and then
evaporated. After purification by preparative TLC (5:1 CHCl3/MeOH,
eluted with 1:1 EtOAc/iPrOH) pure 4 (0.012 g, 71%) was isolated as
added (10
mL/well) to all wells except to the control ones and
microplates were incubated for 72 h. The wells containing cells
without tested substances were used as control. Three hours be-
fore the end of incubation period MTT solution (10 mL) was added
to all wells. MTT was dissolved in medium at 5 mg/mL and filtered
to sterilize and remove a small amount of insoluble residue
an amorphous solid, ½a D20
ꢃ46.25 (c 0.2, CH3OH), Rf¼0.37 (5:1
ꢂ
CHCl3/MeOH). 1H NMR (methanol-d4):
d 1.78e1.97 and 1.99e2.18
(2ꢁm, 2H, 2ꢁH-200), 3.01e3.31 (m, 2H, 2ꢁH-10), 3.50 (d, 2H,
present in some batches of MTT. Acidified 2-propanol (100 mL of
0
0
0
0
0
J3 ,4 ¼5.5 Hz, 2ꢁH-4 ), 3.62e3.74 (m, 1H, J3 ,4 ¼5.5 Hz, H-3 ), 8.07 (s,
0.04 M HCl in 2-propanol) was added to all wells and mixed
thoroughly to dissolve the dark blue crystals of formazan. The
samples were left for a few minutes at room temperature, to en-
sure that all crystals were dissolved and the plates were read on
a plate reader (Multiscan MCC340, Labsystems) at 540 and
690 nm. The wells without cells containing complete medium and
MTT acted as blank.
1H, H-5). 13C NMR (methanol-d4):
d
30.26 and 34.3 (C-20 and C-10),
67.1 (C-40), 72.1 (C-30), 125.0 (C-5), 150.4 (C-4), 165.7 (C-2), 173.1
(CONH2). HRMS (ESI): m/z 217.0634 (MþþH), calcd for C8H13N2O3S:
217.0641.
4.9. Biological materials
Rhodamine B, RPMI 1640 medium, foetal calf serum, 3-(4,5-
dimethylthiazol)-2,5-diphenyltetrazolium bromide, propidium
iodide and RNase A, were purchased from Sigma (St. Louis, MO,
USA). Penicillin and streptomycin were purchased from ICN Gale-
nika (Belgrade, Serbia). Annexin V-FLUOS apoptosis detection kit
was purchased from BD Biosciences Pharmingen (Belgium). Pro-
teins were detected by Western blotting using the following
monoclonal: the antibodies against human Bcl-2 and caspase-3
4.13. Flow cytometry
4.13.1. Cell cycle analysis. After treatment K562 cells were washed
in cold PBS, fixed and incubated for 30 min in 70% ethanol on ice,
centrifuged and incubated with 500
500 L propidium iodide (400
g/mL) for 30 min at 37 ꢀC. Cell cycle
was analyzed by FACS Calibur E440 (Becton Dickinson) flow
mL RNase A (100 units/mL) and
m
m

2350
M. Popsavin et al. / Tetrahedron 70 (2014) 2343e2350
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m
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m
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15 min at 25 ꢀC. After incubation, 400
mL of binding buffer was
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Acknowledgements
This work was supported by research grants from the Provincial
Secretariat of Science, Technological Development and Higher Ed-
ucation of the Autonomous Province of Vojvodina (Grant No. 114-
451-2743/2012-03), and from the Ministry of Education, Science
and Technological Development of the Republic of Serbia (Grant No.
172006).
Supplementary data
Modified experimental procedures for preparation of starting
compounds, alternative presentations of flow cytometry and
western blot data obtained after treatment of K562 cells with tia-
zofurin and analogues, copies of 1H and 13C NMR spectra of final
products. Supplementary data associated with this article can be
found in the online version, at http://dx.doi.org/10.1016/
j.tet.2014.02.035. These data include MOL files and InChiKeys of the
most important compounds described in this article.
ꢁ
ꢀ
ꢀ
ꢀ
17. (a) Popsavin, M.; Svircev, M.; Torovic, Lj; Bogdanovic, G.; Kojic, V.; Jakimov, D.;
ꢀ
ꢀ
Spaic, S.; Aleksic, L.; Popsavin, V. Tetrahedron 2011, 67, 6847e6858; (b) Popsavin,
M.; Spaic, S.; Svircev, M.; Kojic, V.; Bogdanovic, G.; Pejanovic, V.; Popsavin, V.
ꢀ
ꢁ
ꢀ
ꢀ
ꢀ
Tetrahedron 2009, 65, 7637e7645 and references cited therein.
ꢀ
ꢁ
ꢀ
ꢀ
18. Popsavin, M.; Spaic, S.; Svircev, M.; Kojic, V.; Bogdanovic, G.; Popsavin, V. Bio-
References and notes
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