Antitumour tiazofurin analogues embedded with an amide moiety at the C-2′ position

Article abstract of DOI:10.1016/j.tet.2011.06.090

Synthesis of four new tiazofurin analogues has been accomplished starting from d-glucose. The key step of the synthesis was the three-step cascade that enabled an efficient hydrogen sulfide mediated one-pot conversion of 2-azido-3-O-acyl-ribofuranosyl cya

Full text of DOI:10.1016/j.tet.2011.06.090

Tetrahedron 67 (2011) 6847e6858
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Antitumour tiazofurin analogues embedded with an amide moiety at the C-20
position
,
a
ꢁ a
Mirjana Popsavin a , Milos Svircev , Ljilja Torovic , Gordana Bogdanovic , Vesna Kojic ,
ꢁ b
ꢁ b
*
ꢀ
ꢀ
b
a
ꢀ
ꢁ a
ꢁ b
Dimitar Jakimov , Sasa Spaic , Lidija Aleksic , Velimir Popsavin
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, Institutski put 4, 21204 Sremska Kamenica, Serbia
a
ꢁ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 April 2011
Synthesis of four new tiazofurin analogues has been accomplished starting from D-glucose. The key step
of the synthesis was the three-step cascade that enabled an efficient hydrogen sulfide mediated one-pot
conversion of 2-azido-3-O-acyl-ribofuranosyl cyanides to the corresponding 2-alkylamido ribofuranosyl
thiocarboxamides. The resulting key intermediates were first converted to protected tiazofurin de-
rivatives by cyclocondensation with ethyl bromopyruvate, and finally to target C-nucleosides by treat-
ment with ammonia in methanol. In vitro cytotoxicities of tiazofurin analogues against a number of
human tumour cell lines were recorded and compared with those observed for the parent molecule
(tiazofurin), as well as the commercial antitumour agent doxorubicin (DOX). Analogues 2bed have
shown a potent in vitro cytotoxic activity against human myelogenous leukaemia K562. Among solid
tumour cell lines, HT29 was sensitive only to 2d, while HeLa cells were sensitive to 2a, 2b and 2d. Only
analogue 2a was highly cytotoxic against MCF-7 cells. No tiazofurin analogue exhibits any significant
cytotoxicity towards normal foetal lung MRC-5 cells. Downregulation of Bcl-2, activation of caspase-3
and presence of cleavage product of PARP suggest that the cytotoxic effects of tiazofurin analogues 2aed
in K562 might be mediated by apoptosis in a caspase-dependent way. On the contrary, tiazofurin did not
induce apoptosis of K562 cells, which suggests a different mechanism of action, most probably through
the inhibition of IMPDH. Flow cytometry and Western blot analysis data agreed well with the results of
MTT assay, and enabled identification of analogue 2c as the most promising antitumour agent that
preferentially target cancer cells over normal cells and thus represents a new lead for further
optimization.
Received in revised form 17 June 2011
Accepted 24 June 2011
Available online 30 June 2011
Keywords:
C-nucleosides
Tiazofurin
Ribofuranosyl-thioamides
Antitumour activity
Apoptosis
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
efficacy of tiazofurin, lack of specificity and a significant neuro-
toxicity3 limits widespread use of this drug and it is not currently
Among the many classes of nucleoside analogues, C-nucleosides
represent popular synthetic targets due to their potential value as
therapeutic agents and biochemical probes.1 Remarkable among
them is tiazofurin (1, Fig. 1), a synthetic2 C-nucleoside that shows
potent antitumour activity in a variety of tumour systems.3 Tiazo-
furin has been extensively studied both in preclinical4 and clinical
studies,5 and has been approved as an orphan drug for the treat-
ment of patients with acute myeloid leukaemia in blast crisis.3 It
exhibits at least two different mechanisms of action. The first one is
the non-competitive inhibition of inosine 50-monophosphate de-
hydrogenase, a rate-limiting enzyme for guanylate synthesis,6 and
the second is the induction of apoptosis.4,7 Despite the remarkable
marketed. In the search for new antineoplastic agents with im-
proved therapeutic effects, many tiazofurin derivatives have been
synthesized, including a number of those with a modified sugar
* Corresponding author. Tel.: þ381 21 485 27 68; fax: þ381 21 454 065; e-mail
address: mirjana.popsavin@dh.uns.ac.rs (M. Popsavin).
Fig. 1. Structures of tiazofurin (1) and analogues 2aed.
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.06.090

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M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
segment.8 However, some of these compounds did not show
favourable biological properties, although a number of recently
synthesized analogues have not been assayed for their antitumour
activity. We have recently reported the synthesis of a number of
tiazofurin analogues (TAs) that showed increased antitumour ac-
tivities with respect to the lead compound 1.9 Most of these ana-
logues were devoid of any toxicity towards normal mammalian
cells. We have also found that some tiazofurin derivatives induced
apoptosis in the C6 rat glioma cells.10 Our previous findings11 that
the incorporation of 20-nitrogen functionalities into the tiazofurin
sugar moiety may result in an improvement of the lead compounds
cytotoxity against some neoplastic cells, prompted us to study
other variations in the structure of the C-20 portion of the tiazofurin
sugar moiety in order to determine the structureeactivity re-
lationships associated with this portion of the molecule, as well as
to possibly get a better insight into the mechanism of action of TAs
in certain tumour cell lines. In particular, we wished to investigate
the biological activity of TAs bearing a saturated amide function at
the C-20 position.
precursor 3, as shown in Scheme 1 and Table S1 in the
Supplementary data. The corresponding 4-O-alcanoyl derivatives
4aed were thus obtained in 87, 93, 69 and 99% respective yields.
Regioselective displacement of the primary mesyloxy groups of
4aed with potassium benzoate in DMF then produced the 6-O-
benzoyl derivatives 5aed, in 86, 70, 86 and 77% yields, respectively.
Subsequent treatment of 5aed with sodium azide in DMSO pro-
ceeded with configurational inversion at C-3 to afford moderate
yields of 6aed. Presumably the lower yields in these reactions are
due to a number of side processes, that may give elimination and/or
neighbouring group participation products, as observed in our
preceding work.14 However, in the present work neither of the side-
products could be isolated in pure form, due to their similar chro-
matographic properties.
In the next stage of the synthesis the protected 2,5-anhydro-3-
azido-3-deoxy-derivatives 6aed were converted to the corre-
sponding glycosyl cyanides 9aed. Hydrolytic removal of the diox-
olane protective group in 6aed was achieved in a mixture of
trifluoroacetic acid and 6 M hydrochloric acid at þ4 ꢀC. The
resulting unstable aldehydes 7aed were not purified, but were
immediately treated with hydroxylamine hydrochloride to yield
the corresponding oximes 8aed as mixtures of the corresponding
E- and Z-isomers. An attempted separation of E-6a and Z-6a by flash
column chromatography provided only pure E-6a (35%), while the
corresponding Z-isomer could not be obtained free of E-6a. At this
point we decided to avoid the purification step of the remaining
oximino derivatives 8bed. Accordingly, the mixtures of E- and Z-
isomers 8bed were not separated but were further treated with
mesyl chloride in pyridine. The expected ribofuranosyl cyanides
9aed were thus obtained in respective overall yields of 46, 57, 47
and 36% (from three synthetic steps).
Herein, we disclose in detail the synthesis of new TAs 2aed,
with hexan-, octan-, decan- and dodecanamido functions at C-20,
along with their effects on the proliferation of a number of malig-
nant cell lines.12 In addition, the purpose of the present study was
to examine the apoptotic signalling induced by tiazofurin and an-
alogues in the human K562 malignant cell line.
2. Results and discussion
2.1. Chemical synthesis
Our strategy for the synthesis of target C-nucleosides of type 2 is
It was assumed that the 2-azido-2-deoxy-D-ribofuranosyl cya-
based on initial multi-step chemical transformations of D-glucose to
nides 9aed may be converted to the corresponding thioamides
through the one-step H2S-mediated cascade that was recently used
for the conversion of 2-azido ribofuranosyl cyanide 9e to the
ribofuranosyl thioamide 10e11 (Scheme 2).
the ribofuranosyl-thioamides 10aed (Scheme 1), followed by their
subsequent cyclocondensation with ethyl bromopyruvate to form
the thiazole ring. The 2,5-anhydro-
D
-glucose derivative 3, readily
available from
D
-glucose13 was used as a convenient starting ma-
This efficient one-pot process is comprised of an initial addition
of hydrogen sulfide to the nitrile group, followed by the azide re-
duction and spontaneous O,N-shift of the acyl group. Depending on
the reaction conditions and reagents used, the major product 10e
was accompanied with a variable amount of 2-azido ribofuranosyl
terial in this work. The synthesis commenced with a three-step
sequence for the conversion of 3 to the key azido intermediates
6aed.
Thus, by use of the appropriate acyl chlorides, the corresponding
4-O-acyl derivatives 4aed were first prepared from the common
Scheme 1. Reagents and conditions: (a) RCOCl, Py, CH2Cl2, rt; (b) KOBz, DMF, 100 ꢀC; (c) NaN3, DMSO, 110e112 ꢀC; (d) 6 M HCl, TFA, þ4 ꢀC; (e) NaOAc, HONH2ꢁHCl, EtOH, CH2Cl2, rt;
(f) MsCl, Py, 0 ꢀC to rt; (g) H2S, Py, rt; (h) H2S, DMAP, EtOH, rt; (i) BrCH2COCO2Et, EtOH, reflux; (j) NH3, MeOH, rt.

M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
6849
Scheme 2. Conversion of 2-azido ribofuranosyl cyanide 9e to the thioamide 10e: (a) addition of H2S to the nitrile function to give thioamide 9f; (b) H2S reduction of azide 9f to
amine 9g; (c) O,N-shift of the acyl group to give amide 10e.
Table 1
thioamide 9f. Thus, when 9e was reacted with hydrogen sulfide in
In vitro cytotoxicity of tiazofurin (1), analogues 2aed and DOX
dry pyridine for 1.5 h at room temperature both 10e and 9f were
M)a
HL-60
obtained in 71% and 25% yields, respectively. However, on pro-
longing the reaction time to 8 h, the benzamido derivative 10e was
obtained as the only reaction product, but in somewhat lower yield
(69%). This result indicates that the addition of hydrogen sulfide to
the nitrile function in 9e precedes the reduction of the azido group.
An alternative conversion of 9e was carried out with hydrogen
sulfide in ethanol and in the presence of DMAP. The reaction also
proceeds cleanly at room temperature for 8 h to afford 10e as the
only reaction product in 82% yield.
As both of these H2S-mediated transformations were previously
carried out only with the aromatic ester derivative 9e, in the
present work we wanted to extend the application of this pro-
cedure to the saturated esters 9aed. Hence, the ribofuranosyl cy-
anides 9aec were treated with hydrogen sulfide in pyridine, under
the conditions presented in Scheme 1 and Table S1 in the
Supplementary data, to provide the corresponding thioamides
10aec in 99, 92 and 87% yields, respectively. Although the nitrile 9d
may also be converted to the thioamide 10d under the same re-
actions conditions (H2S, Py, rt), at this point we wanted to explore
alternative reagent system for the preparation of 10d (H2S, DMAP,
EtOH, rt), which was also successfully applied in the aromatic ester
series.11 Accordingly, the nitrile 9d was treated with hydrogen
sulfide gas and DMAP in ethanol for 8 h at room temperature,
whereby the desired thioamide 10d was obtained in 92% yield.
The synthesis of the final products 2aed began with the mod-
ified Hantzsch thiazole synthesis.15 Accordingly, the intermediates
10aed were treated with ethyl bromopyruvate in refluxing ethanol
to give the protected thiazoles 11aed in 54, 60, 47 and 56% yield,
respectively. Final exposure of 11aed to methanolic ammonia
provided the tiazofurin analogues 2aed in 66, 49, 81 and 80% re-
spective yields.
Compd
IC50 (m
K562
Raji
HT-29
MCF-7
HeLa
MRC-5
0.33
>100
>100
>100
>100
1
2.06
2.69
1.02
0.15
0.94
0.37
0.67
3.64
1.02
5.97
0.87
1.06
4.61
3.96
3.54
6.87
5.34
3.09
0.56
89.34
93.67
91.34
0.012
0.23
2.03
0.099
16.78
69.64
78.37
0.32
3.26
1.99
2.11
6.94
2.06
0.12
2a
2b
2c
2d
DOX
0.23
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%.
cells. Analogues 2aed exhibited notable antiproliferative effects on
Raji cells, with IC50 values in the low, micro-molar range. The most
active molecules against this cell line were analogues 2a and 2b
that displayed the similar activities as both control compounds (1
and DOX). Analogues 2a, 2b and 2c showed a weak activity against
HT-29 cells. Meanwhile, compound 2d exhibited a strong anti-
proliferative activity against this cell line being 47-fold more potent
than the parent compound 1. In the same time, analogue 2d
demonstrated 19-fold higher potency than DOX in the same cell
line. Only moderate to weak activities of analogues 2b, 2c and 2d
were recorded in MCF-7 tumour cell line. However, compound 2a
exhibited 20- and 3-fold higher potency in this cell line with re-
spect to tiazofurin (1) and DOX, respectively. The most active
molecules against HeLa cells were analogues 2a, 2b and 2d that
exhibited 1.5-fold higher potency when compared to the control
compound 1. The most interesting results were obtained in ex-
periments with K562 cells, whereupon all tiazofurin analogues
2aed exhibited remarkable antiproliferative effects with IC50
values in the range 0.15e2.69 mM. The most active molecule against
this cell line was derivative 2c being 14-fold more potent than
tiazofurin (1), while analogues 2b and 2d demonstrated 2-fold
grater cytotoxicity than the parent compound 1. Moreover, it is
noteworthy that compound 2c displayed 2.5-fold higher potency
than the commercial cytotoxic agent doxorubicin (DOX) in the
same cell line. Unfortunately, SAR for the inhibition of tumour cells
growth does not indicate a correlation between the length of car-
bon chain in the amide functionality and the corresponding in-
hibitory potencies.
When the cytotoxic effect of analogues 2aed was expressed in
relation to tiazofurin cytotoxicity, it was obvious that analogue 2c
was the most active towards K562 cells in the range of concentra-
tions from 10ꢂ8 M to 10ꢂ4 M, while other tiazofurin mimetics
showed similar or smaller activity compared to tiazofurin (Fig. 2).
These results indicate that the introduction of 20-alkylamido
functions into the tiazofurin sugar moiety may improve anti-
proliferative effects of the resulting analogues against some human
neoplastic cells. Remarkably, the synthesized tiazofurin mimics
2aed are fully non-toxic towards human normal MRC-5 cells while
lead compound 1, as well as commercial antitumour agent DOX
exhibited sub-micromolar toxicity to both malignant and normal
cells.
2.2. In vitro antitumour activity
After completion of the synthesis, tiazofurin analogues 2a, 2b,
2c and 2d were evaluated for their in vitro cytotoxic activity against
a panel of human malignant cell lines, including chronic myelog-
enous leukaemia (K562), promyelocytic leukaemia (HL-60), Bur-
kitt’s lymphoma (Raji), colon adenocarcinoma (HT-29), breast
adenocarcinoma (MCF-7), cervix carcinoma (HeLa), as well as
normal foetal lung fibroblasts (MRC-5). Cytotoxic activity was
evaluated by using the standard MTT assay, after exposure of cells
to the tested compounds for 72 h. Both tiazofurin (1) and the
commercial antitumour agent doxorubicin (DOX) were used as
reference compounds. The results are presented in Table 1.
According to the resulting IC50 values of the cytotoxic assay
(Table 1) the synthesized tiazofurin analogues 2a, 2b, 2c and 2d
exhibited the similar growth inhibition pattern as tiazofurin (1)
against human K562, HL-60, Raji and HeLa malignant cells, with
IC50 values in the range 0.15e6.94 mM. The most active compounds
against HL-60 cell line were analogues 2b and 2d, which demon-
strated almost the same potency as doxorubicin (DOX), although
tiazofurin remains the most potent agent towards these malignant

6850
M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
2b caused a similar level of specific apoptotic response after 24 h-
treatment, but their activity was significantly lower after longer cell
treatment.
Fig. 2. Cytotoxicity (%) of tiazofurin mimics 2aed against K562 cells compared to
tiazofurin. K562 cells were treated with 2aed in a concentration range from 10ꢂ8 M to
10ꢂ4 M. Cytotoxicity was evaluated after 72 h by MTT assay. Each point represents
a mean value of quadruplicate from two independent experiments.
Fig. 3. Percentage of specific apoptosis of K562 cells induced by tiazofurin and ana-
logues 2aed after 24 and 72 h-treatment. Cells were stained with Annexin-V-FLUOS
and propidium iodide and analyzed by flow cytometry: Percentage of specific apo-
ptosis of tiazofurin (1) and tiazofurin mimetics (2aed) was calculated according to
ref 20.
2.3. Detection of apoptosis
Intact apoptosis pathways are critical for chemotherapy-
induced cytotoxicity. The majority of chemotherapeutic agents
trigger the mitochondrial (intrinsic) pathway, but the cell-death
receptor dependent (extrinsic) pathway is also involved in
chemotherapy-induced apoptosis. Recent findings that tiazofurin
induces apoptosis in SiHa, Hep2 and Ca Ski cancer cells7a encour-
aged us to examine the possible apoptotic signalling induced by
tiazofurin and analogues in the K562 cell line. The type of cell death
induced by tiazofurin (1), and analogues 2aed in K562 human
leukaemia cells, was determined by flow cytometric analysis after
annexin V-FITC and propidium iodide staining. Flow cytometric
analysis clearly differentiates normal, living cells (low Annexin and
low PI staining), apoptotic cells (high Annexin and low PI staining),
and necrotic cells (low Annexin and high PI staining). Translocation
of phosphatidylserine from the inner part of the plasma membrane
to the outer layer occurs in the early stages of apoptosis.16 Necrotic
cells also expose PS according to the loss of membrane integrity.
Annexin V is a Ca2þ-dependent phospholipid-binding protein with
high affinity for PS. As Annexin-V-Fluos stains both apoptotic and
necrotic cells, simultaneous application of PI that stains DNA of
leaky necrotic cells only, allows us to discriminate truly necrotic
cells from the Annexin V positively stained cells. Cells were treated
with 1 and TAs (2aed) for 72 h and then stained with the com-
ponents of the Annexin-V-FLUOS Staining Kit and analyzed by flow
cytometry (for representative dot plots see Fig. S1 in the
Supplementary data).
To elucidate the mechanisms underlying the TAs-induced apo-
ptosis, we investigated how compounds 2aed modulate expression
of Bcl-2, Bax, caspase-3 and Poly (ADP-ribose) polymerase (PARP),
which are critically involved in the apoptosis pathway. Members of
the Bcl-2 protein family are the key regulators of apoptosis. Apo-
ptosis depends on the balance between pro- and anti-apoptotic
proteins of the Bcl-2 family. Anti-apoptotic member Bcl-2 plays
a crucial role in apoptosis and conferring cancer cell drug re-
sistance.18 It interferes with cytochrom C release and suppresses
apoptosis progression. Bax, the pro-apoptotic protein of the Bcl-2
family, undergoes conformational changes in response to apopto-
tic stimuli, and thus counteracts Bcl-2 activity, stimulates the re-
lease of cytochrom c and other apoptogenic proteins into the
cytoplasm, and ultimately initiates apoptosis. Western blot analysis
(Fig. 4) revealed that analogues 2aed reduced expression of Bcl-2
compared to control and tiazofurin suggesting downregulation of
the anti-apoptotic Bcl-2 gene. Compound 2c induced the highest
reduction of Bcl-2 expression. Bax is commonly over-expressed in
apoptotic process initiated by various chemotherapeutics. Tiazo-
furin analogues 2aed did not significantly influence Bax protein
expression. Several literature reports also showed that apoptosis in
various human solid tumours can be induced in the absence of any
change in Bax protein level.19 However, activation of Bax may occur
before its upregulation.20
Exposure of K562 cells to tiazofurin at the concentrations of 8.7
and 2 mM for 24 h and 72 h, respectively, did not induce apoptosis
compared to control (untreated cells). This is in accordance with
the results of De Abreu et al.17 who found that tiazofurin, at similar
concentrations, does not induce apoptosis in the Molt-4 cell line
within the first 24 h. Longer cell treatment resulted in a slight in-
crease of Annexin-V positive cells. These findings do suggest that
tiazofurin, after initial treatment for 24 h at the IC50 concentration,
mainly acts as an IMPDH inhibitor. Apoptotic response presented as
a percentage of specific apoptosis (Fig. 3) showed that all tiazofurin
analogues after 24 or 72 h induced several-fold more Annexin-V
positive K562 cells compared to the parent compound. Among
them, analogue 2c was the most active in both time points, and
percentage of specific apoptosis was doubled (increased 2-fold)
after longer treatment. This compound was also the most active
after 72 h of cell treatment in the MTT assay, when compared to
both tiazofurin and other analogues (Table 1). Compound 2d also
induced a higher specific apoptotic response after 72 h-treatment
(4.11% vs 14.76% after 24 and 72 h, respectively). Compounds 2a and
The expression level of the precursor and active subunit of ef-
fector caspase-3 in the cells exposed to tiazofurin and analogues
were measured in order to determine if TAs-induced apoptosis is
associated with the activation of caspases. Caspases are important
mediators in initiation and execution of apoptotic signal.21 How-
ever, apoptotic morphology can be achieved either by activation of
caspases or by other families of proteases.21b Cell death induced by
antitumour agents is commonly associated with an increase in
apoptosis by the caspase-dependent pathway, but caspase-in-
dependent pathway can be also involved,18d,22 in other words ap-
optosis can be lethal without caspase activation, and caspase
activation does not necessarily cause cell death.23 Western blot data
showed that caspase-3 precursor protein was expressed in TAs-
treated K562 cells; however, expression level of caspase-3 active
subunit was different and depended on the particular compound.
Caspase-3 activation is followed by cleavage of different down-
stream targets including Poly (ADP-ribose) polymerase (PARP)

M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
6851
cleavage product of PARP suggest that cytotoxic effects of tiazo-
furin analogues 2aed in K562 might be mediated by apoptosis
mostly in caspase-dependent way. On the contrary, tiazofurin did
not induce apoptosis of K562 cells after 24 h, which suggests
a different mechanism of its action, most probably through the
inhibition of IMPDH. Flow cytometry and Western blot analysis
data agreed well with the results of MTT assay and enabled
identification of analogue 2c as the most promising antitumour
agent. These results, along with our previous findings11 confirmed
that the introduction of 20-nitrogen functionalities into the tiazo-
furin sugar moiety may provide analogues of significant anti-
tumour activity, but without any significant cytotoxicity towards
normal cells. Therefore we believe that this approach may be of
use in the search for new, more potent and selective anticancer
agents derived from lead 1.
4. Experimental section
4.1. General
€
Melting points were determined on a Buchi 510 apparatus and
were not corrected. Optical rotations were measured on P 3002
€
(Kruss) and Polamat A (CarleZeiss) polarimeters at room temper-
ature. NMR spectra were recorded on a Bruker AC 250 E instrument
and chemical shifts are expressed in ppm downfield from TMS. IR
spectra were recorded with an FTIR Nexus 670 spectrophotometer
(Thermo-Nicolet). Low resolution mass spectra (CI) were recorded
on Finnigan-MAT 8230 and on an Agilent Technologies HPLC/MS
3Q system (ESI), series 1200/6410. High resolution mass spectra
(ESI) of synthesized compounds were acquired on a Agilent Tech-
nologies 1200 series instrument equipped with Zorbax Eclipse Plus
C18 (100 mmꢁ2.1 mm i.d. 1.8
mm) column and DAD detector
Fig. 4. Western blot analysis of the protein expression of Bcl-2, Bax, caspase-3, and
PARP after 72 h-treatment: control (A), tiazofurin (B), analogue 2a (C), analogue 2d (D),
analogue 2c (E), analogue 2b (F).
(190e450 nm) in combination with a 6210 time-of-flight LC/MS
instrument (ESI) in the positive ion mode. Column chromatography
was performed on Kieselgel 60 (<0.063 mm, E. Merck). Flash col-
umn 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 Fluorescent Indicator
F254 as additive. The corresponding bands were scraped and eluted
with EtOAc. All organic extracts were dried with anhydrous Na2SO4.
Organic solutions were concentrated in a rotary evaporator under
reduced pressure at a bath temperature below 35 ꢀC.
protein. The enzyme PARP is involved in short-patch base excision
repair and its cleavage is one of the biochemical hallmarks of ap-
optosis.24 Western blot analysis showed proteolysis cleavage of
PARP in K562 cells after treatment with tiazofurin mimics (2aed)
but it depended on the particular compound. Compared to other
analogues, 2b induced the weakest cleavage of PARP.
3. Conclusion
4.2. General procedure for the preparation of ester
derivatives 4aed
In conclusion, four new tiazofurin mimics bearing a hexan- (2a),
octan- (2b), decan- (2c) and dodecanamido functionality (2d) in the
To a cooled (0 ꢀC) and stirred solution of 3 (1 equiv) in dry
CH2Cl2 (0.2 M) were added anhydrous pyridine (9 equiv) and the
appropriate acyl chloride (1.5e2 equiv). The mixture was first
stirred at 0 ꢀC for 15 min and then at room temperature until the
starting materials were consumed (TLC). The mixture was poured
onto ice and acidified with 6 M HCl (to pH 1e2) and the resulting
suspension was extracted with CH2Cl2 (4ꢁ50 mL). The combined
extracts were washed with water (40 mL), saturated aq NaHCO3
(40 mL) and again with water (40 mL), dried and evaporated. The
residue was then purified by silica gel column chromatography
and/or by crystallization from the appropriate solvents.
C-20 position, were synthesized starting from
D
-glucose. The key
intermediates for this approach, that is, 2-amido- -ribofuranosyl
thiocarboxamides 10aed were easily accessible from the 2-azido-
2-deoxy- -ribofuranosyl cyanides 9aed, through the one-step H2S-
D
D
mediated cascade, comprised of an initial addition of hydrogen
sulfide to the nitrile group, followed by the azide reduction and
spontaneous O,N-acyl shift. This efficient one-pot sequence repre-
sents a general and particularly versatile approach to a variety of 2-
amido-D-ribofuranosyl thiocarboxamides, which can be easily
converted to the corresponding ribofuranosyl thiazole-4-
carboxamides, and finally to a series of new tiazofurin mimics for
biological testing.
4.2.1. 2,5-Anhydro-4-O-hexanoyl-3,6-di-O-methanesulfonyl-D-glu-
Analogues 2bed have shown potent in vitro cytotoxic activity
against human myelogenous leukaemia K562. Among solid tu-
mour cell lines, HT29 was sensitive only to 2d, while HeLa cells
were sensitive to 2a, 2b and 2d. Only analogue 2a was highly cy-
totoxic against MCF-7 cells. Neither analogues exhibit any signif-
icant cytotoxicity towards normal foetal lung MRC-5 cells.
Downregulation of Bcl-2, activation of caspase-3, and presence of
cose ethylene acetal (4a). Alcohol 3 (4.8 g, 13.2 mmol) was con-
verted into crude 4a after treatment with hexanoyl chloride
(3.8 mL, 27 mmol) for 24 h at room temperature according to the
above general procedure. Double crystallization from MeOH gave
pure 4a (5.3 g, 87%) as colourless crystals, mp 94 ꢀC; ½a D20
þ33.8 (c
ꢃ
1.3, CHCl3); IR (KBr): nmax 1724, 1369, 1179; 1H NMR (250 MHz,
CDCl3):
d
0.86 (t, 3H, J¼7.1 Hz, CH3CH2), 1.28 (m, 4H, 2ꢁ CH2), 1.59

6852
M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
(m, 2H, CH2), 2.32 (t, 2H, CH2CO), 3.06 and 3.14 (2ꢁ s, 3H each, 2ꢁ
CH3SO2), 3.84e4.04 (m, 4H, 2ꢁ CH2edioxolane), 3.93 (dd, 1H,
J1,2¼6.3, J2,3¼3.6 Hz, H-2), 4.12 (m, 1H, H-5), 4.43 (pseudo d, 2H,
J5,6¼4.6 Hz, 2ꢁ H-6), 5.07 (dd, 1H, J2,3¼3.7, J3,4¼1 Hz, H-3), 5.10 (d,
1H, J1,2¼6.3 Hz, H-1), 5.20 (dd, 1H, J3,4¼1, J4,5¼2.6 Hz, H-4); 13C
65.3 (2ꢁ CH2edioxolane), 67.7 (C-6), 77.5 (C-4), 80.7 (C-2), 81.9 (C-
3), 82.4 (C-5), 100.9 (C-1), 172.4 (C11H23CO); LRMS (CI): m/z 545
(MþþH). Anal. Calcd for C22H40O11S2: C, 48.51; H, 7.40; S, 11.77.
Found: C, 48.52; H, 7.43; S, 11.50.
(62.9 MHz, CDCl3):
d
13.65 (CH3CH2), 22.0, 24.1 and 30.9 (3ꢁ CH2),
4.3. General procedure for the synthesis of 6-O-benzoyl ester
derivatives 5aed
33.6 (CH2CO), 37.5 and 38.4 (2ꢁ CH3SO2), 65.2 and 65.3 (2ꢁ
CH2edioxolane), 67.8 (C-6), 77.6 (C-4), 80.8 (C-2), 81.9 (C-3), 82.4
(C-5), 100.9 (C-1), 172.4 (C5H11CO). Anal. Calcd for C16H28O11S2: C,
41.73; H, 6.13; S, 13.93. Found: C, 41.42; H, 6.52; S, 13.97.
To a solution of disulfonate 4 (1 equiv) in DMF (0.1e0.15 M) was
added KOBz (1.2e4.2 equiv), and the reaction mixture was heated
at 100 ꢀC until no further reaction was evident by TLC (12 h for
4aec, 8 h for 4d). The mixture was allowed to cool down to room
temperature, poured into cold water and extracted with a 1:1
mixture of benzene/light petroleum. The combined extracts were
washed with water, dried and evaporated. The residue was then
purified by silica gel column chromatography.
4.2.2. 2,5-Anhydro-4-O-octanoyl-3,6-di-O-methanesulfonyl-D-glu-
cose ethylene acetal (4b). Alcohol 3 (2.08 g, 5.7 mmol) was con-
verted into crude 4b after treatment with octanoyl chloride (1.5 mL,
8.8 mmol) for 72 h at room temperature according to the above
general procedure. Flash column chromatography (97:3, 19:1, to 9:1
CH2Cl2/EtOAc) followed by recrystallization from MeOH gave pure
4b (2.61 g, 93%) as colourless crystals, mp 58e59 ꢀC; ½a D20
ꢃ
þ24.6 (c
4.3.1. 2,5-Anhydro-6-O-benzoyl-4-O-hexanoyl-3-O-meth-
anesulfonyl-D-glucose ethylene acetal (5a). Disulfonate 4a (4.75 g,
7.5, CHCl3); IR (KBr): nmax 1744, 1360, 1178; 1H NMR (250 MHz,
CDCl3):
d
0.87 (t, 3H, J¼6.8 Hz, CH3CH2), 1.27 (m, 8H, 4ꢁ CH2), 1.60
10.3 mmol) was treated with KOBz (7.1 g, 44.2 mmol) according to
the above general procedure to give crude 5a as an oil. Pure 5a
(4.32 g, 86%) was obtained by flash column chromatography (2:1
cyclohexane/Me2CO) followed by recrystallization from MeOH, as
(m, 2H, CH2), 2.34 (t, 2H, CH2CO), 3.08 and 3.17 (2ꢁ s, 3H each, 2ꢁ
CH3SO2), 3.87e4.05 (m, 5H, H-2 and 2ꢁ CH2edioxolane), 4.14 (m,
1H, H-5), 4.47 (d, 2H, J5,6¼4.4 Hz, 2ꢁ H-6), 5.10 (dd, 1H, J2,3¼4,
J3,4¼1 Hz, H-3), 5.13 (d, 1H, J1,2¼6.4 Hz, H-1), 5.23 (dd, 1H, J3,4¼1,
colourless crystals, mp 98e99 ꢀC; ½a D20
þ34.3 (c 2.0, CHCl3); IR
ꢃ
J4,5¼2.6 Hz, H-4); 13C NMR (62.9 MHz, CDCl3):
d
14.0 (CH3CH2), 22.5,
(neat): nmax 1743, 1719, 1353, 1185; 1H NMR (250 MHz, CDCl3):
24.5, 28.7, 28.8 and 31.5 (5ꢁ CH2), 33.7 (CH2CO), 37.6 and 38.5 (2ꢁ
CH3SO2), 65.3 and 65.4 (2ꢁ CH2edioxolane), 67.7 (C-6), 77.6 (C-4),
80.9 (C-2), 82.0 (C-3), 82.5 (C-5), 101.0 (C-1), 172.5 (C7H15CO); LRMS
(ESI): m/z 489 (MþþH). Anal. Calcd for C18H32O11S2: C, 44.25; H,
6.60; S, 13.13. Found: C, 44.08; H, 6.44; S, 13.42.
d
0.89 (t, 3H, J¼7.1 Hz, CH3CH2), 1.22e1.38 (m, 4H, 2ꢁ CH2), 1.62 (m,
2H, CH2), 2.34 (m, 2H, CH2CO), 3.10 (s, 3H, CH3SO2), 3.88e4.08 (m,
5H, H-2 and 2ꢁ CH2edioxolane), 4.26 (m, 1H, H-5), 4.56 (dd, 1H,
J5,6a¼4.3, J6a,6b¼12.1 Hz, H-6a), 4.62 (dd, 1H, J5,6b¼4.7,
J6a,6b¼12.1 Hz, H-6b), 5.15 (d, 1H, J2,3¼3.7 Hz, H-3), 5.18 (d, 1H,
J1,2¼6.3 Hz, H-1), 5.35 (d, 1H, J4,5¼3.1 Hz, H-4), 7.38e8.15 (m, 5H,
4.2.3. 2,5-Anhydro-4-O-decanoyl-3,6-di-O-methanesulfonyl-
D
-glu-
Ph); 13C (62.9 MHz, CDCl3):
d
13.8 (CH3CH2), 22.2, 24.3 and 31.1 (3ꢁ
cose ethylene acetal (4c). Alcohol 3 (4.02 g, 11.1 mmol) was con-
verted into crude 4c after treatment with decanoyl chloride
(4.7 mL, 22.7 mmol) for 48 h at room temperature according to the
above general procedure. Flash column chromatography (19:1, to
23:2 CH2Cl2/EtOAc) followed by recrystallization from MeOH gave
CH2), 33.77 (CH2CO), 38.5 (CH3SO2), 63.5 (C-6), 65.4 and 65.5 (2ꢁ
CH2edioxolane), 78.5 (C-4), 80.8 (C-2), 82.6 (C-5), 82.9 (C-3), 101.2
(C-1), 128.3, 129.6, 129.8 and 133.1 (Ph), 166.2 (PhC]O), 172.4
(C5H11CO); LRMS (CI): m/z 545 (MþþH). Anal. Calcd for
C22H30O10S: C, 54.31; H, 6.22; S, 6.59. Found: C, 54.10; H, 6.46; S,
6.97.
pure 4c (3.93 g, 69%) as colourless crystals, mp 55e57 ꢀC; ½a D20
ꢃ
þ20.5 (c 1.3, CHCl3); IR (KBr): nmax 1742, 1360, 1177; 1H NMR
(250 MHz, CDCl3):
d
0.86 (t, 3H, J¼6.8 Hz, CH3CH2), 1.15e1.67 (m,
4.3.2. 2,5-Anhydro-6-O-benzoyl-3-O-methanesulfonyl-4-O-octa-
noyl-D-glucose ethylene acetal (5b). Disulfonate 4b (2.76 g,
14H, 7ꢁ CH2), 2.33 (t, 2H, CH2CO), 3.08 and 3.16 (2ꢁ s, 3H each, 2ꢁ
CH3SO2), 3.85e4.06 (m, 5H, 2ꢁ CH2edioxolane and H-2), 4.14 (m,
1H, H-5), 4.46 (pseudo d, 2H, J5,6¼4.5 Hz, H-6), 5.07 (dd, 1H,
J2,3¼4.0, J3,4¼0.8 Hz, H-3), 5.12 (d, 1H, J1,2¼6.4 Hz, H-1), 5.22 (dd,1H,
5.3 mmol) was treated with KOBz (1.06 g, 6.6 mmol) according to
the above general procedure to give crude 5b as an oil. Pure 5b
(1.9 g, 70%) was obtained after purification on a column of flash
silica (9:1 toluene/EtOAc), followed by recrystallization from
J4,5¼2.2, J3,4¼0.8 Hz, H-4); 13C NMR (62.9 MHz, CDCl3):
d 14.0
(CH3CH2), 22.5, 24.5, 28.9, 29.0, 29.1 29.2, and 31.4 (7ꢁ CH2), 33.8
(CH2CO), 37.6 and 38.5 (2ꢁ CH3SO2), 65.3 and 65.4 (2ꢁ
CH2edioxolane), 67.7 (C-6), 77.6 (C-4), 80.9 (C-2), 82.0 (C-3), 82.5
(C-5), 101.0 (C-1), 172.4 (C9H19CO); LRMS (CI): m/z 517 (MþþH).
Anal. Calcd for C20H36O11S2: C, 46.50; H, 7.02; S, 12.41. Found: C,
46.77; H, 7.19; S, 12.63.
MeOH, as colourless crystals, mp 88e89 ꢀC; ½a D20
þ32.4 (c 1.0,
ꢃ
CHCl3); IR (neat): nmax 1750, 1724, 1367, 1180; 1H NMR (250 MHz,
CDCl3):
d
0.87 (t, 3H, J¼6.8 Hz, CH3CH2), 1.27 (m, 8H, 4ꢁ CH2), 1.60
(m, 2H, CH2), 2.34 (t, 2H, CH2CO), 3.08 (s, 3H, CH3SO2), 3.81e4.07
(m, 5H, H-2 and 2ꢁ CH2edioxolane), 4.24 (m, 1H, H-5), 4.53 (dd,
1H, J5,6a¼4.9, J6a,6b¼11.5 Hz, H-6a), 4.59 (dd, 1H, J5,6b¼4.7,
J6a,6b¼11.8 Hz, H-6b), 5.12 (d, 1H, J2,3¼3.7 Hz, H-3), 5.15 (d, 1H,
J1,2¼6.4 Hz, H-1), 5.33 (d, 1H, J4,5¼2.7 Hz, H-4), 7.34e8.12 (m, 5H,
4.2.4. 2,5-Anhydro-4-O-dodecanoyl-3,6-di-O-methanesulfonyl-D-
glucose ethylene acetal (4d). Alcohol 3 (3.3 g, 9.2 mmol) was con-
verted into crude 4d after treatment with dodecanoyl chloride
(4.2 mL, 18.4 mmol) for 22 h at room temperature according to the
above general procedure. Silica gel column chromatography (7:3, to
3:2, toluene/EtOAc) gave pure 4d (4.96 g, 99%) as a colourless syrup,
Ph); 13C NMR (62.9 MHz, CDCl3):
d 13.9 (CH3CH2), 22.4, 24.5, 28.6,
28.7 and 31.4 (5ꢁ CH2), 33.64 (CH2CO), 38.3 (CH3SO2), 63.4 (C-6),
65.2 and 65.3 (2ꢁ CH2edioxolane), 78.3 (C-4), 80.7 (C-2), 82.5 (C-
5), 82.8 (C-3), 101.0 (C-1), 128.2, 129.4, 129.7 and 133.0 (Ph), 166.0
(PhC]O), 172.3 (C7H15CO); LRMS (ESI): m/z 537 (MþþNa). Anal.
Calcd for C24H34O10S: C, 56.02; H, 6.66; S, 6.23. Found: C, 55.88; H,
6.60; S, 6.10.
½
a 2D0
ꢃ
þ11.8 (c 1.0, CHCl3); IR (neat): nmax 1747, 1360, 1178; 1H NMR
(250 MHz, CDCl3):
d
0.83 (t, 3H, CH3CH2), 1.16e1.68 (m, 18H, 9ꢁ
CH2), 2.35 (t, 2H, CH2CO), 3.09 and 3.17 (2ꢁ s, 3H each, 2ꢁ CH3SO2),
3.89e4.07 (m, 5H, 2ꢁ CH2edioxolane, and H-2), 4.15 (m, 1H, H-5),
4.47 (d, 2H, J5,6¼4.5 Hz, 2ꢁ H-6), 5.12 (d, 1H, J2,3¼4.5 Hz, H-3), 5.14
(d, 1H, J1,2¼6.5 Hz, H-1), 5.24 (d, 1H, J4,5¼2.7 Hz, H-4); 13C NMR
4.3.3. 2,5-Anhydro-6-O-benzoyl-4-O-decanoyl-3-O-meth-
anesulfonyl- -glucose ethylene acetal (5c). Disulfonate 4c (1.9 g,
D
3.7 mmol) was treated with KOBz (4.7 g, 4.6 mmol) according to the
above general procedure to give crude 5c as an oil. After purifica-
tion on a column of flash silica (9:1 toluene/EtOAc), followed by
(62.9 MHz, CDCl3): d 13.9 (CH3CH2), 22.4, 24.4 28.8, 29.0, 29.1, 29.2,
29.4, 31.7 and 33.6 (10ꢁ CH2), 37.4 and 38.3 (2ꢁ CH3SO2), 65.2 and

M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
6853
preparative TLC (4:1 toluene/EtOAc) of several impure fractions
(CH2CO), 60.5 (C-3), 63.6 (C-6), 65.3 and 65.5 (2ꢁ CH2ediox-
olane), 73.5 (C-4), 79.2 (C-5), 82.1 (C-2), 102.1 (C-1), 128.3, 129.6,
and 133.0 (Ph), 166.0 (PhC]O), 172.9 (C5H11CO); LRMS (CI): m/z
434 (MþþH). Anal. Calcd for C21H27N3O7: C, 58.19; H, 6.28; N,
9.69. Found: C, 58.41; H, 6.33; N, 10.01.
pure 5c (1.72 g, 86%) was isolated as a colourless oil, ½a D20
þ32.3 (c
ꢃ
1.1, CHCl3); IR (neat): nmax 1750, 1724, 1367, 1180; 1H NMR
(250 MHz, CDCl3):
d
0.88 (t, 3H, J¼6.8 Hz, CH3CH2), 1.26 (m, 12H, 6ꢁ
CH2), 1.61 (m, 2H, CH2), 2.34 (t, 2H, J¼7.7 Hz, CH2CO), 3.11 (s, 3H,
CH3SO2), 3.88e4.08 (m, 5H, 2ꢁ CH2edioxolane and H-2), 4.26 (m,
1H, H-5), 4.56 (dd, 1H, J5,6a¼4.8, J6a,6b¼11.8 Hz, H-6a), 4.62 (dd, 1H,
J5,6b¼4.8, J6a,6b¼11.8 Hz, H-6b), 5.15 (d, 1H, J2,3¼3.8 Hz, H-3), 5.18 (d,
1H, J1,2¼6.3 Hz, H-1), 5.35 (d, 1H, J4,5¼3.0 Hz, H-4), 7.40e8.14 (m,
4.4.2. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-4-O-octanoyl-D-
allose ethylene acetal (6b). Sulfonate 5b (1.37 g, 2.7 mmol) was
treated with NaN3 (1.51 g, 23.2 mmol) according to the above
general procedure to give crude 6b as a yellow oil. Pure 6b (0.49 g,
40%) was isolated by flash column chromatography (17:3 cyclo-
5H, Ph); 13C NMR (62.9 MHz, CDCl3):
d 13.9 (CH3CH2), 22.4, 24.5,
28.8, 28.98, 29.0, 29.1 and 31.6 (7ꢁ CH2), 33.68 (CH2CO), 38.3
(CH3SO2), 63.4 (C-6), 65.2 and 65.3 (2ꢁ CH2edioxolane), 78.3 (C-4),
80.7 (C-2), 82.5 (C-5), 82.3 (C-3), 101.1 (C-1), 128.2, 129.5, 129.7 and
133.0 (Ph), 166.0 (PhC]O), 172.2 (C9H19CO); LRMS (CI): m/z 543
(MþþH). Anal. Calcd for C26H38O10S: C, 57.55; H, 7.06; S, 5.91.
Found: C, 57.28; H, 7.28; S, 5.97.
hexane/Me2CO) as a colourless oil, ½a D20
ꢃ
þ1.6 (c 0.6, CHCl3); IR
(neat): nmax 2110, 1726, 1604; 1H NMR (250 MHz, CDCl3):
d
0.88 (t,
3H, J¼6.8 Hz, CH3CH2), 1.29 (m, 8H, 4ꢁ CH2), 1.65 (m, 2H, CH2),
2.40 (m, 2H, CH2CO), 3.84e4.07 (m, 4H, 2ꢁ CH2edioxolane), 4.10
(dd, 1H, J1,2¼2.8, J2,3¼5.2 Hz, H-2), 4.22 (t, 1H, J3,4¼5.8 Hz, H-3),
4.28e4.46 (m, 2H, H-5 and H-6a), 4.56 (dd, 1H, J5,6b¼3.5,
J6a,6b¼11.9 Hz, H-6b), 5.02 (d, 1H, J1,2¼2.8 Hz, H-1), 5.28 (t, 1H,
J3,4¼5.8 Hz, H-4), 7.40e8.11 (m, 5H, Ph); 13C NMR (62.9 MHz,
4.3.4. 2,5-Anhydro-6-O-benzoyl-4-O-dodecanoyl-3-O-meth-
anesulfonyl- -glucose ethylene acetal (5d). Disulfonate 4c (4.9 g,
D
9 mmol) was treated with KOBz (6.2 g, 38.7 mmol) according to the
above general procedure to give crude 5d as an oil. Pure 5d (3.97 g,
77%) was obtained after purification on a column of silica gel (17:3
CDCl3):
d
13.9 (CH3CH2), 22.4, 24.6, 28.7, 28.8 and 31.5 (5ꢁ CH2),
33.64 (CH2CO), 60.5 (C-3), 63.6 (C-6), 65.3 and 65.5 (2ꢁ
CH2edioxolane), 73.5 (C-4), 79.3 (C-5), 82.1 (C-2), 102.1 (C-1),
128.3, 129.6 and 133.0 (Ph), 166.0 (PhC]O), 172.9 (C7H15CO);
HRMS (ESI): m/z 462.2222 (MþþH), calcd for C23H32N3O7:
462.2235.
toluene/EtOAc) as a colourless oil, ½a D20
ꢃ
þ24.8 (c 1.2, CHCl3); IR
(neat): nmax 1724, 1369, 1179; 1H NMR (250 MHz, CDCl3):
d
0.86 (t,
3H, CH3CH2), 1.24 (br s, 16H, 8ꢁ CH2), 1.59 (m, 2H, CH2CH2CO), 2.32
(t, 2H, CH2CO), 3.09 (s, 3H, CH3SO2), 3.84e4.06 (m, 5H, 2ꢁ
CH2edioxolane, and H-2), 4.24 (m, 1H, H-5), 4.54 (dd, 1H, J5,6a¼4.9,
J6a,6b¼11.9 Hz, H-6a), 4.60 (dd, 1H, J5,6b¼4.8, J6a,6b¼11.9 Hz, H-6b),
5.13 (dd, 1H, J2,3¼3.7, J3,4¼0.6 Hz, H-3), 5.16 (d, 1H, J1,2¼6.3 Hz, H-1),
5.34 (dd, 1H, J3,4¼0.6, J4,5¼3.0 Hz, H-4), 7.34e8.11 (m, 5H, Ph); 13C
4.4.3. 2,5-Anhydro-3-azido-6-O-benzoyl-4-O-decanoyl-3-deoxy-D-
allose ethylene acetal (6c). Sulfonate 5c (1.74 g, 3.2 mmol) was
treated with NaN3 (1.81 g, 27.8 mmol) according to the above
general procedure to give crude 6c as a yellow oil. After purification
on a column of flash silica (17:3 cyclohexane/Me2CO), followed by
preparative TLC (17:3 cyclohexane/Me2CO) of several impure frac-
NMR (62.9 MHz, CDCl3): d 13.9 (CH3CH2), 22.5, 24.5, 28.8, 29.0, 29.1,
29.2, 29.4 and 31.7 (9ꢁ CH2), 33.7 (CH2CO), 38.4 (CH3SO2), 63.4 (C-
6), 65.26 and 65.3 (2ꢁ CH2edioxolane), 78.4 (C-4), 80.7 (C-2), 82.5
(C-5), 82.3 (C-3),101.1 (C-1),128.2,129.5,129.7 and 133.0 (Ph),166.0
tions, pure 6c (0.76 g, 49%) was isolated as a colourless oil, ½a D20
ꢃ
þ13.3 (c 0.9, CHCl3); IR (neat): nmax 2110, 1750, 1725, 1617; 1H NMR
(PhC]O), 172.3 (C11
Calcd for C28H42O10S: C, 58.93; H, 7.42; S, 5.62. Found: C, 58.56; H,
7.27; S, 5.63.
H
23CO); LRMS (CI): m/z 571 (MþþH). Anal.
(250 MHz, CDCl3):
d
0.87 (t, 3H, J¼6.8 Hz, CH3CH2), 1.25 (m, 12H, 6ꢁ
CH2), 1.64 (m, 2H, CH2), 2.38 (m, 2H, CH2CO), 3.82e4.05 (m, 4H, 2ꢁ
CH2edioxolane), 4.09 (dd, 1H, J1,2¼2.7, J2,3¼5.1 Hz, H-2), 4.20 (t, 1H,
J3,4¼5.8 Hz, H-3), 4.33 (m, 1H, H-5), 4.39 (dd, 1H, J5,6a¼4.5,
J6a,6b¼11.8 Hz, H-6a), 4.55 (dd, 1H, J5,6b¼3.6, J6a,6b¼11.8 Hz, H-6b),
5.00 (d, 1H, J1,2¼2.7 Hz, H-1), 5.28 (t, 1H, J¼5.8 Hz, H-4), 7.39e8.09
4.4. General procedure for the preparation of azido
derivatives 6aed
(m, 5H, Ph); 13C NMR (62.9 MHz, CDCl3):
d 14.0 (CH3CH2), 22.5, 24.6,
To a solution of sulfonate 5 (1 equiv) in DMSO (0.1 M) was added
NaN3 (8.5e10 equiv), and the reaction mixture was heated at 110 ꢀC
until no further reaction was evident by TLC (24 h for 6a, 30 h for
6b, 27 h for 6c, 70 h for 6d). The mixture was allowed to cool down
to room temperature, poured into cold water and extracted with
a 1:1 mixture of benzene/light petroleum. The combined extracts
were washed with water, dried and evaporated. The residue was
purified by flash column chromatography.
29.0, 29.1, 29.3 and 31.7 (7ꢁ CH2), 33.7 (CH2CO), 60.6 (C-3), 63.6 (C-
6), 65.4 and 65.5 (2ꢁ CH2edioxolane), 73.6 (C-4), 79.3 (C-5), 82.2
(C-2), 102.2 (C-1), 128.28, 129.6 and 133.0 (Ph), 166.01 (PhC]O),
172.9 (C9H19CO); LRMS (CI): m/z 490 (MþþH). Anal. Calcd for
C25H35N3O7: C, 61.33; H, 7.21; N, 8.58. Found: C, 61.70; H, 7.44; N,
8.35.
4.4.4. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-4-O-dodecanoyl-
D-allose ethylene acetal (6d). Sulfonate 5d (3.8 g, 6.7 mmol) was
4.4.1. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-4-O-hexanoyl-D-
treated with NaN3 (4.3 g, 66.6 mmol) according to the above general
procedure to give crude 6d as a yellow oil. Pure 6d (1.5 g, 44%) was
isolated after column chromatography on silica gel (19:1, 4:1 tolu-
allose ethylene acetal (6a). Sulfonate 5a (3.2 g, 6.5 mmol) was
treated with NaN3 (3.6 g 55.4 mmol) according to the above
general procedure to give crude 6a as a yellow oil. After purifi-
cation on a column of flash silica (19:1 toluene/EtOAc), followed
by preparative TLC (19:1 toluene/EtOAc) of several impure frac-
ene/EtOAc) as a colourless oil, ½a D20
ꢃ
þ7.5 (c 1.1, CHCl3); IR (neat): nmax
2110, 1735; 1H NMR (250 MHz, CDCl3):
d
0.86 (t, 3H, CH3CH2),
tions, pure 6a (1.33 g, 47%) was isolated as a colourless oil, ½a D20
ꢃ
1.08e1.72 (m, 18H, 9ꢁ CH2), 2.38 (m, 2H, CH2CO), 3.81e4.03 (m, 4H,
2ꢁ CH2edioxolane), 4.08 (dd,1H, J1,2¼2.8, J2,3¼5.2 Hz, H-2), 4.20 (dd,
1H, J2,3¼5.2, J3,4¼5.6 Hz, H-3), 4.25 (m, 1H, H-5), 4.38 (dd, 1H,
J5,6a¼4.5, J6a,6b¼11.9 Hz, H-6a), 4.53 (dd, 1H, J5,6b¼3.7, J6a,6b¼11.9 Hz,
H-6b), 4.99 (d, 1H, J1,2¼2.8 Hz, H-1), 5.28 (dd, 1H, J3,4¼5.6,
J4,5¼5.9 Hz, H-4), 7.37e8.10 (m, 5H, Ph); 13C NMR (62.9 MHz, CDCl3):
þ12.9 (c 7.5, CHCl3); IR (neat): nmax 2111, 1724, 1602; 1H NMR
(250 MHz, CDCl3):
d
0.87 (t, 3H, J¼7.1 Hz, CH3CH2), 1.23e1.36 (m,
4H, 2ꢁ CH2), 1.63 (m, 2H, CH2), 2.38 (m, 2H, CH2CO), 3.81e4.05
(m, 4H, 2ꢁ CH2edioxolane), 4.08 (dd, 1H, J1,2¼2.8 Hz, J2,3¼5.1 Hz,
H-2), 4.20 (t, 1H, J¼5.7 Hz, H-3), 4.31 (m, 1H, H-5), 4.38 (dd, 1H,
J5,6a¼4.5, J6a,6b¼11.8 Hz, H-6a), 4.58 (dd, 1H, J5,6b¼3.6,
J6a,6b¼11.8 Hz, H-6b), 4.99 (d, 1H, J1,2¼2.8 Hz, H-1), 5.27 (t, 1H,
J¼5.8 Hz, H-4), 7.38e8.08 (m, 5H, Ph); 13C NMR (62.9 MHz,
d
13.9 (CH3CH2), 22.5, 24.6, 28.9, 29.0, 29.1, 29.3, 29.4 and 31.7 (9ꢁ
CH2), 33.6 (CH2CO), 60.5 (C-3), 63.6 (C-6), 65.3 and 65.4 (2ꢁ
CH2edioxolane), 73.5 (C-4), 79.2 (C-5), 82.1 (C-2), 102.1 (C-1), 128.2,
129.55, 129.6, and 133.0 (Ph), 165.9 (PhC]O), 172.8 (C11H23CO);
CDCl3):
d
13.8 (CH3CH2), 22.1, 24.2 and 31.0 (3ꢁ CH2), 33.6

6854
M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
LRMS (CI): m/z 518 (MþþH). Anal. Calcd for C27H39N3O7: C, 62.65; H,
LRMS (CI): m/z 387 (MþþH). Anal. Calcd for C19H22N4O5: C, 59.06;
7.59; N, 8.12. Found: C, 62.95; H, 7.74; N, 8.43.
H, 5.74; N, 14.50. Found C, 58.96; H, 5.67; N, 14.31.
4.5.2. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-4-O-octanoyl-D-
4.5. General procedure for the synthesis of ribofuranosyl
cyanides 9aed
allononitrile (9b). Acetal 6b (0.91 g, 2 mmol) was converted into
crude 9b according to the above general procedure. Pure 9b (0.43 g,
57% from three steps) was isolated by flash column chromatogra-
A solution of acetal 6 (1 equiv) in a 4:1 mixture of TFA/6 M HCl
(0.2 M) was kept at þ4 ꢀC for 5e9 days. The mixture was concen-
trated to a third of the initial volume and poured into saturated aq
NaHCO3. The aqueous solution was rendered alkaline with solid
NaHCO3 to pH 8e9 and extracted with CH2Cl2. The combined ex-
tracts were washed successively with saturated aq NaHCO3 and
water, dried and evaporated. The remaining crude aldehyde 7 was
immediately dissolved in a mixture of EtOH/CH2Cl2 (w0.2 M) and
treated with NaOAc (w3 equiv) and NH2OHꢁHCl (w2 equiv) while
stirring at room temperature for 2e4 h. The mixture was evapo-
rated and the residue distributed between water and CH2Cl2. The
organic layer was separated and the aqueous phase extracted with
CH2Cl2. The combined organic solutions were washed with water,
dried and evaporated to afford crude oxime 8 as a mixture of the
corresponding E- and Z-isomers. To a cooled (ꢂ15 ꢀC) and stirred
solution of 8 in anhydrous pyridine (w0.2 M) was added dropwise
during 0.5 h a cold solution of MsCl (w4 equiv) in dry pyridine
(1 M). The mixture was first stirred at 0 ꢀC for 15 min, then at room
temperature for 2 h and then poured into a 1:1 mixture of ice and
concentrated HCl (pH w2). The emulsion was extracted with
CH2Cl2, the combined extracts were washed with water, saturated
aq NaHCO3 and again with water. The extract was dried and
evaporated, and the residue was purified by column
chromatography.
phy (99:1, 9:1 toluene/EtOAc) as a colourless oil, ½a D20
þ5.6 (c 5.4,
ꢃ
CHCl3); IR (neat): nmax 2280, 2117, 1727; 1H NMR (250 MHz, CDCl3):
d
0.86 (t, 3H, J¼6.8 Hz, CH3CH2), 1.25 (m, 8H, 4ꢁ CH2), 1.61 (m, 2H,
CH2), 2.37 (m, 2H, CH2CO), 4.30e4.50 (m, 3H, H-5 and 2ꢁ H-6), 4.57
(d, 1H, J2,3¼5.3 Hz, H-2), 4.60 (t, 1H, J3,4¼5.4 Hz, H-3), 5.46 (t, 1H,
J4,5¼5.4 Hz, H-4), 7.37e8.09 (m, 5H, Ph); 13C NMR (62.9 MHz,
CDCl3):
d
13.7 (CH3CH2), 22.2, 24.3, 28.5, 28.6 and 31.3 (5ꢁ CH2),
33.4 (CH2CO), 62.6 (C-6), 64.2 (C-3), 68.9 (C-2), 72.4 (C-4), 80.5 (C-
5), 115.9 (C-1), 128.2, 129.0, 129.4 and 133.1 (Ph), 165.7 (PhC]O),
172.4 (C7H15CO); LRMS (CI): m/z 415 (MþþH); HRMS (ESI): m/z
415.1967 (MþþH), calcd for C21H27N4O5: 415.1976. Anal. Calcd for
C21H26N4O5: C, 60.86; H, 6.32; N, 13.52. Found: C, 60.68; H, 6.30; N,
13.12. A minor amount of unreacted starting compound 6b (0.075 g,
8%) was recovered.
4.5.3. 2,5-Anhydro-3-azido-6-O-benzoyl-4-O-decanoyl-3-deoxy-D-
allononitrile (9c). Acetal 6c (0.89 g, 1.8 mmol) was converted into
crude 9c according to the above general procedure. Pure 9c (0.25 g,
47% from three steps) was isolated by flash column chromatogra-
phy (99:1, 9:1 toluene/EtOAc) as a colourless oil, ½a D20
ꢂ4.9 (c 1.1,
ꢃ
CHCl3); IR (neat): nmax 2282, 2116, 1725, 1602; 1H NMR (250 MHz,
CDCl3):
d
0.88 (t, 3H, J¼6.8 Hz, CH3CH2), 1.27 (m, 12H, 6ꢁ CH2), 1.64
(m, 2H, CH2), 2.42 (m, 2H, CH2CO), 4.38e4.70 (m, 5H, H-2, H-3, H-5
and 2ꢁ H-6), 5.45 (m, 1H, H-4), 7.42e8.15 (m, 5H, Ph); 13C NMR
(62.9 MHz, CDCl3): d 14.1 (CH3CH2), 22.6, 24.6, 29.0, 29.1, 29.2, 29.3
4.5.1. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-4-O-hexanoyl-
D
-
and 31.8 (7ꢁ CH2), 33.6 (CH2CO), 62.7 (C-6), 64.5 (C-3), 69.2 (C-2),
72.6 (C-4), 80.6 (C-5), 115.9 (C-1), 128.5, 129.0, 129.7 and 133.5 (Ph),
166.0 (PhC]O), 172.7 (C9H19CO); LRMS(CI): m/z 443 (MþþH). Anal.
Calcd for C23H30N4O5: C, 62.43; H, 6.83; N,12.66. Found: C, 62.59; H,
7.19; N, 12.24. A minor amount of unreacted starting compound 6c
(0.04 g, 5%) was recovered.
allononitrile (9a). Acetal 6a (1.31 g, 3 mmol) was converted into
crude aldehyde 7a after treatment with a mixture TFA (11 mL) and
6 M HCl (2.7 mL) for 5 days according to the above general pro-
cedure. Crude 7a was treated with a mixture of NaOAc (0.87 g,
10.6 mmol) and NH2OHꢁHCl (0.49 g, 7 mmol) in EtOH (9 mL) for
2.5 h at room temperature and then evaporated. Flash column
chromatography of the residue (9:1 toluene/EtOAc) gave pure E-8a
(0.4 g) that was characterized by NMR spectral data: 1H NMR
4.5.4. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-4-O-dodecanoyl-
D-allononitrile (9d). Acetal 6d (1.34 g, 2.6 mmol) was converted
(250 MHz, CDCl3):
d
0.89 (t, 3H, J¼7.1 Hz, CH3CH2), 1.23e1.36 (m,
into crude 9d according to the above general procedure. Pure 9d
4H, 2ꢁ CH2), 1.61 (m, 2H, CH2), 2.41 (m, 2H, CH2CO), 4.17 (dd, 1H,
J2,3¼6.8, J3,4¼6.0 Hz, H-3), 4.35 (m, 1H, H-5), 4.43 (dd, 1H,
J6a,6b¼12.0, J5,6a¼4.2 Hz, H-6a), 4.50e4.59 (m, 2H, H-2 and H-6b),
5.35 (dd, 1H, J3,4¼6.0, J4,5¼5.6 Hz, H-4), 7.40e7.50 (m, 3H, H-1 and
2H from Ph), 7.50e8.09 (m, 3H, Ph), 8.79 (br s, 1H, NOH); 13C NMR
(0.41 g, 36% from three steps) was isolated by silica gel column
chromatography (97:3 toluene/EtOAc) as a colourless oil, ½a D20
ꢂ7.3
ꢃ
(c 1.0, CHCl3); IR (neat): nmax 2300, 2130, 1740; 1H NMR (250 MHz,
CDCl3):
d
0.88 (t, 3H, CH3CH2), 1.16e1.72 (m, 18H, 9ꢁ CH2), 2.41 (t,
2H, CH2CO), 4.42 (m, 1H, H-5), 4.48 (dd, 1H, J5,6a¼3.7,
J6a,6b¼12.2 Hz, H-6a), 4.56 (dd, 1H, J5,6b¼3.7, J6a,6b¼12.2 Hz, H-6b),
4.58 (m, 2H, H-2 and H-3), 5.46 (dd, 1H, J3,4¼4.9, J4,5¼5.5 Hz, H-4),
(62.9 MHz, CDCl3):
d
13.7 (CH3CH2), 22.1, 24.2 and 31.0 (3ꢁ CH2),
33.7 (CH2CO), 62.6 (C-3), 63.7 (C-6), 73.4 (C-4), 78.4 (C-2), 80.2 (C-
5), 128.4, 129.2, 129.5 and 133.2 (Ph), 147.6 (C-1), 166.1 (PhC]O),
173.0 (C5H11CO). An inseparable mixture of E- and Z-8a was also
isolated, the total yield of purified 8a was 0.77 g (68% on the basis of
the recovered 6a). A minor amount of unreacted starting com-
pound 6a (0.1 g, 8%) was recovered. Purified 8a (0.77 g, 1.9 mmol)
was converted into crude 9a by treatment with MsCl (0.6 mL,
7.5 mmol) in dry pyridine (8.5 mL) for 2 h, according to the above
general procedure. Pure 9a (0.5 g, 68%) was isolated by flash
7.46e8.12 (m, 5H, Ph); 13C NMR (62.9 MHz, CDCl3):
d 14.1
(CH3CH2), 22.6, 24.6, 29.0, 29.1, 29.2, 29.3, 29.4, 29.5, 31.8 and 33.6
(10ꢁ CH2), 62.8 (C-6), 64.5 (C-3), 69.2 (C-2), 72.6 (C-4), 80.7 (C-5),
115.9 (C-1), 128.5, 129.5, 129.7 and 133.5 (Ph), 166.0 (PhC]O),
172.7 (C11
H
23CO); LRMS (CI): m/z 471 (MþþH). Anal. Calcd for
C25H34N4O5: C, 64.81; H, 7.28; N, 11.91. Found: C, 64.59; H, 7.07; N,
11.86. A minor amount of unreacted starting compound 6d (0.09 g,
7%) was recovered.
chromatography (9:1 toluene/EtOAc) as a colourless oil, ½a D20
ꢂ14.9
ꢃ
(c 3.3, CHCl3); IR (neat): nmax 2230, 2118, 1724; 1H NMR (250 MHz,
CDCl3):
d
0.89 (t, 3H, J¼7.1 Hz, CH3CH2), 1.23e1.66 (m, 6H, 3ꢁ CH2),
4.6. General procedure for the one-pot conversion of 2-azido-
3-O-acyl-ribofuranosyl cyanides 9aec into the corresponding
2-alkylamido-ribofuranosyl thiocarboxamides 10aec
2.40 (m, 2H, CH2CO), 4.41 (dt, 1H, J5,6¼4.3, J4,5¼6.1 Hz, H-5),
4.50e4.65 (m, 4H, H-2, H-3 and 2ꢁ H-6), 5.45 (dd, 1H, J3,4¼4.8,
J4,5¼5.7 Hz, H-4), 7.41e8.12 (m, 5H, Ph); 13C NMR (62.9 MHz,
CDCl3):
d
13.7 (CH3CH2), 22.1, 24.2 and 31.0 (3ꢁ CH2), 33.5 (CH2CO),
Through a solution of nitrile 9 (1 mmol) in anhydrous pyridine
(0.2 M) was passed H2S gas at room temperature. The mixture was
evaporated and the residue was purified on a column of silica gel.
62.7 (C-6), 64.4 (C-3), 69.1 (C-2), 72.6 (C-4), 80.6 (C-5), 115.9 (C-1),
128.4, 129.1, 129.6 and 133.4 (Ph), 165.9 (PhC]O), 172.6 (C5H11CO);

M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
6855
4.6.1. 2,5-Anhydro-6-O-benzoyl-3-deoxy-3-hexanamido-
D
-allono-
toluene/EtOAc) to afford pure 10d (0.1 g, 92%) as a colourless oil.
thioamide (10a). Nitrile 9a (0.5 g, 1.3 mmol) was converted into
crude thioamide 10a after treatment with H2S for 12 h according to
the above general procedure. Pure 10a (0.5 g, 99%) was isolated by
column chromatography (11:9 toluene/EtOAc) as a colourless oil
Crystallization from CH2Cl2/hexane gave colourless crystals, mp
110.5e111 ꢀC; ½a D20
þ15.4 (c 1.3, CHCl3); IR (neat): nmax 3300, 1730,
ꢃ
1630, 1550, 1540, 1320, 1275, 1140; 1H NMR (250 MHz, CDCl3):
d
0.87 (t, 3H, J¼6.7 Hz, CH3CH2),1.24 (br s,16H, 8ꢁ CH2), 1.60 (m, 2H,
½
a 2D0
ꢃ
þ29.0 (c 5.7, CHCl3); IR (neat): nmax 3300, 1716, 1651, 1540,
CH2CH2CO), 2.24 (dd, 2H, J¼7.3, J¼8.2 Hz, CH2CO), 4.06 (ddd, 1H,
J2,3¼9.0, J3,4¼5.2, J3,NH¼5.5 Hz, H-3), 4.13 (br s, 1H, exchangeable
with D2O, OH), 4.36 (m, 1H, H-5), 4.41e4.61 (m, 3H, H-4 and 2ꢁ H-
6), 4.65 (d, 1H, J2,3¼9.0 Hz, H-2), 6.85 (d, 1H, J3,NH¼5.5 Hz,
NHCOC11H23), 7.36e8.04 (m, 5H, Ph), 8.21 and 8.47 (br s, 2H, par-
tially exchangeable with D2O, CSNH2); 13C NMR (62.9 MHz, CDCl3):
1531, 1315, 1275, 1178; 1H NMR (250 MHz, CDCl3):
d
0.84 (t, 3H,
J¼7.1 Hz, CH3CH2), 1.17e1.72 (m, 6H, 3ꢁ CH2), 2.24 (m, 2H, CH2CO),
4.05 (ddd, 1H, J2,3¼9.1, J3,NH¼5.4, J3,4¼5.2 Hz, H-3), 4.29 (br s, 1H,
exchangeable with D2O, OH), 4.36 (m, 1H, H-5), 4.45 (dd, 1H,
J5,6a¼3.1, J6a,6b¼12.1 Hz, H-6a), 4.50e4.62 (m, 2H, H-4 and H-6b),
4.68 (d, 1H, J2,3¼9.1 Hz, H-2), 6.86 (d, 1H, exchangeable with D2O,
J3,NH¼5.4 Hz, NHCOC5H11), 7.38e8.16 (m, 5H, Ph), 8.24 and 8.47 (2ꢁ
br s, 1H each, exchangeable with D2O, CSNH2); 13C NMR (62.9 MHz,
d
14.0 (CH3CH2), 22.6, 25.4, 29.2, 29.22, 29.4, 29.5, 29.52, 29.6 and
31.8 (9ꢁ CH2), 36.6 (CH2CO), 59.0 (C-3), 64.8 (C-6), 71.9 (C-4), 84.0
(C-5), 84.6 (C-2), 128.50, 129.2, 129.6 and 133.4 (Ph), 166.9 (PhC]
O), 174.7 (NHCOC11H23), 203.6 (C-1); LRMS (CI): m/z 479 (MþþH),
477 (MþꢂH). Anal. Calcd for C25H38N2O5S: C, 62.73; H, 8.00; N, 5.85;
S, 6.70. Found: C, 62.59; H, 7.84; N, 6.16; S, 6.52.
CDCl3):
d
13.8 (CH3CH2), 22.2, 25.0 and 31.2 (3ꢁ CH2), 36.5 (CH2CO),
59.0 (C-3), 64.7 (C-6), 71.8 (C-4), 84.0 (C-5), 84.6 (C-2), 128.5, 129.2,
129.6 and 133.5 (Ph), 166.9 (PhC]O), 174.7 (NHCOC5H11), 203.5
(CSNH2); LRMS (CI): m/z 395 (MþþH). Anal. Calcd for C19H26N2O5S:
C, 57.85; H, 6.64; N, 7.10; S, 8.13. Found: C, 57.85; H, 6.83; N, 7.36; S,
8.24.
4.7. General procedure for the synthesis of protected thiazole
derivatives 11aed
4.6.2. 2,5-Anhydro-6-O-benzoyl-3-deoxy-3-octanamido-
D
-allono-
To a solution of thioamide 10 (1 equiv) was added ethyl bro-
mopyruvate (1.8 equiv) in absolute EtOH (0.1 M) and the mixture
was stirred under reflux for 50e180 min. The solvent was evapo-
rated in vacuum and the residue was purified on a column of silica
gel.
thioamide (10b). Nitrile 9b (0.32 g, 0.8 mmol) was converted into
crude thioamide 10b after treatment with H2S for 120 h according
to the above general procedure. Pure 10b (0.3 g, 92%) was isolated
by flash column chromatography (3:2 toluene/EtOAc) as a colour-
less oil, ½a 2D0
ꢃ
þ14.2 (c 3.0, CHCl3); IR (neat): nmax 3300, 1717, 1652,
1533, 1315, 1274, 1177; 1H NMR (250 MHz, CDCl3):
d
0.85 (t, 3H,
4.7.1. Ethyl
2-(5-O-benzoyl-2-deoxy-2-hexanamido-b-D-ribofur-
J¼6.4 Hz, CH3CH2), 1.25 (m, 8H, 4ꢁ CH2), 1.61 (m, 2H, CH2), 2.25 (m,
2H, CH2CO), 3.00 (br s, 1H, exchangeable with D2O, OH), 4.02 (ddd,
1H, J2,3¼9.0, J3,NH¼5.1, J3,4¼5.2 Hz, H-3), 4.38 (m, 1H, H-5), 4.48 (dd,
1H, J5,6a¼3.0, J6a,6b¼12.1 Hz, H-6a), 4.52e4.60 (m, 2H, H-4 and H-
6b), 4.65 (d, 1H, J2,3¼9.0 Hz, H-2), 7.81 (d, 1H, exchangeable with
D2O, J3,NH¼5.1 Hz, NHCOC7H15), 7.37e8.08 (m, 5H, Ph), 8.40 and
8.45 (2ꢁ br s, 1H each, exchangeable with D2O, CSNH2); 13C NMR
anosyl)thiazole-4-carboxylate (11a). Thioamide 10a (0.49 g,
1.3 mmol) was converted into crude thiazole 11a after treatment
with ethyl bromopyruvate (0.3 mL, 2.2 mmol) for 1 h according to
the above general procedure. Pure 11a (0.325 g, 54%) was isolated
by flash column chromatography (1:1 toluene/EtOAc) as a colour-
less oil, ½a 2D0
ꢃ
ꢂ25.0 (c 1.7, CHCl3); IR (neat): nmax 3355, 1722, 1652,
1538,1272; 1H NMR (250 MHz,1:1 CDCl3/benzene-d6):
d
0.87 (t, 3H,
(62.9 MHz, CDCl3):
d
14.0 (CH3CH2), 22.6, 25.5, 29.0, 29.2 and 31.6
J¼6.4 Hz, CH3CH2CH2), 1.05 (t, 3H, J¼7.1 Hz, CO2CH2CH3), 1.20e1.65
(5ꢁ CH2), 36.6 (CH2CO), 58.8 (C-3), 64.9 (C-6), 71.9 (C-4), 84.1 (C-5),
84.8 (C-2), 128.6, 129.2, 129.7 and 133.5 (Ph), 167.0 (PhC]O), 174.9
(NHCOC7H15), 203.7 (CSNH2); HRMS (ESI): m/z 423.1954 (MþþH),
calcd for C21H31N2O5S: 423.1948.
(m, 6H, 3ꢁ CH2), 2.22 (m, 2H, CH2CO), 4.05 (q, 2H, CO2CH2CH3), 4.35
0
0
0
0
0
0
(dd, 1H, J4 ,5a ¼3.2, J5a ,5b ¼12.1 Hz, H-5a ), 4.47 (m, 1H, H-4 ), 4.58
0
0
0
0
0
0
0
(dd, 1H, J2 ,3 ¼4.9, J03 ,4 ¼1.7 Hz, H-3 ),0 4.72 (dd, 1H, J4 ,5b ¼3.7,
0
0
0
0
J5a ,5b ¼12.1 Hz, H-5b ), 4.94 (m, 1H, H-2 ), 5.50 (d, 1H, J1 ,2 ¼9.1 Hz,
H-10), 7.18e8.41 (m, 7H, Ph, NHCOC5H11 and OH), 7.38 (s, 1H, H-5);
4.6.3. 2,5-Anhydro-6-O-benzoyl-3-decanamido-3-deoxy-
D
-allono-
13C NMR (62.9 MHz, CDCl3):
d 13.8 (CH3CH2CH2), 14.2 (CO2CH2CH3),
thioamide (10c). Nitrile 9c (0.25 g, 0.6 mmol) was converted into
crude thioamide 10c after treatment with H2S for 96 h according to
the above general procedure. Pure 10c (0.23 g, 87%) was isolated by
flash column chromatography (3:2 toluene/EtOAc) as a colourless
22.3, 25.0 and 31.3 (3ꢁ CH2), 36.14 (CH2CO), 59.6 (C-20), 61.6
(CO2CH2CH3), 64.6 (C-50), 72.4 (C-30), 79.6 (C-10), 84.3 (C-40), 128.3
(C-5), 128.9, 129.4, 129.7 and 133.2 (Ph), 146.3 (C-4), 161.4 (C-2),
166.3 (PhC]O), 172.0 (CO2Et), 174.4 (NHCOC5H11); LRMS (CI): m/z
491 (MþþH). Anal. Calcd for C24H30N2O7S: C, 58.76; H, 6.16; N, 5.71;
S, 6.54. Found: C, 59.03; H, 5.81; N, 6.03; S, 6.25.
oil, ½a 2D0
ꢃ
þ15.0 (c 1.8, CHCl3); IR (neat): nmax 3295, 1716, 1648, 1532,
1315, 1275, 1177; 1H NMR (250 MHz, CDCl3):
d
0.86 (t, 3H, J¼6.8 Hz,
CH3CH2), 1.12e1.72 (m, 14H, 7ꢁ CH2), 2.25 (t, 2H, J¼7.1 Hz, CH2CO),
3.97 (ddd, 1H, J2,3¼9.2, J3,NH¼4.7, J3,4¼5.4 Hz, H-3), 4.03 (br s, 1H,
exchangeable with D2O, OH), 4.38 (m, 1H, H-5), 4.48 (dd, 1H,
J5,6a¼3.1, J6a,6b¼12.1 Hz, H-6a), 4.50e4.62 (m, 2H, H-4 and H-6b),
4.65 (d, 1H, J2,3¼9.2 Hz, H-2), 6.81 (d, 1H, J3,NH¼4.7 Hz, exchange-
able with D2O, NHCOC9H19), 7.37e8.04 (m, 5H, Ph), 8.07 and 8.47
(2ꢁ br s, 2H, partially exchangeable with D2O, CSNH2); 13C NMR
4.7.2. Ethyl
2-(5-O-benzoyl-2-deoxy-2-octanamido-b-D-ribofur-
anosyl)thiazole-4-carboxylate (11b). Thioamide 10b (0.3 g,
0.7 mmol) was converted into crude thiazole 11b after treatment
with ethyl bromopyruvate (0.2 mL, 1.3 mmol) for 3 h according to
the above general procedure. Pure 11b (0.18 g, 60%) was isolated by
flash column chromatography (1:1 toluene/EtOAc) as a colourless
(62.9 MHz, CDCl3):
d
14.0 (CH3CH2), 22.6, 25.4, 29.17, 29.2, 29.3 and
oil: ½a 2D0
ꢂ4.1 (c 1.0, CHCl3); IR (neat): nmax 3363, 1721, 1651, 1534,
ꢃ
31.7 (7ꢁ CH2), 36.59 (CH2CO), 59.1 (C-3), 64.7 (C-6), 71.9 (C-4), 84.1
(C-5), 84.6 (C-2), 128.5, 129.1, 129.6 and 133.5 (Ph), 166.9 (PhC]O),
174.8 (NHCOC9H19), 203.7 (C-1); HRMS (ESI): m/z 451.2278
(MþþH), calcd for C23H35N2O5S: 451.2261.
1272; 1H NMR (250 MHz, CDCl3):
d
0.85 (t, 3H, J¼6.4 Hz,
CH3CH2CH2), 1.38 (t, 3H, J¼7.1 Hz, CO2CH2CH3), 1.12e1.88 (m, 10H,
5ꢁ CH2), 2.17 (m, 2H, CH2CO), 3.91 (br s, 1H, OH), 4.39 (q, 2H,
CO2CH2CH3), 4.45e4.73 (m, 5H, H-20, H-30, H-40, and 2ꢁ H-50), 5.50
0
0
0
0
(d, 1H, J1 ,2 ¼8.9 Hz, H-1 ), 7.02 (d, 1H, J2 ,NH¼7.3 Hz, NHCOC7H15),
4.6.4. 2,5-Anhydro-6-O-benzoyl-3-deoxy-3-dodecanamido-D-allono-
7.41e8.10 (m, 5H, Ph), 8.11 (s, 1H, H-5); 13C NMR (62.9 MHz, CDCl3):
thioamide (10d). Through a solution of 9d (0.1 g, 0.2 mmol) and
DMAP (0.005 g, 0.04 mmol) in absolute EtOH (2 mL) was passed
H2S gas for 8 h at room temperature. The mixture was evaporated
and the residue was purified on a column of silica gel (7:3, 3:2
d 13.9 (CH3CH2CH2), 14.1 (CO2CH2CH3), 22.5, 25.3, 28.9, 29.2 and
31.3 (5ꢁ CH2), 36.15 (CH2CO), 59.5 (C-20), 61.5 (CO2CH2CH3), 64.6
(C-50), 72.6 (C-30), 79.5 (C-10), 84.4 (C-40), 128.4, 129.4, 129.6 and
133.2 (Ph and C-5), 146.1 (C-4), 161.4 (C-2), 166.3 (PhC]O), 172.1

6856
M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
(CO2Et), 174.2 (NHCOC7H15); HRMS (ESI): m/z 519.2157 (MþþH),
J3 ,4 ¼2.5, Hz, H-3 ), 4.510(dd, 1H, J1 ,2 ¼8.9, J2 ,3 ¼5.4 Hz, H-2 ), 5.10
0
0
0
0
0
0
0
0
0
0
calcd for C26H35N2O7S: 519.2160.
(d, 1H, J1 ,2 ¼8.9 Hz, H-1 ), 7.53 and 7.56 (2ꢁ br s, 0.2H, the minor
NH2 signal remained after partial exchange with methanol-d4), 8.18
(d, 0.2H, the minor NH2 signal remained after partial exchange with
methanol-d4), 8.22 (s, 1H, H-5); 13C NMR (62.9 MHz, methanol-d4):
4.7.3. Ethyl
2-(5-O-benzoyl-2-decanamido-2-deoxy-b-D-ribofur-
anosyl)thiazole-4-carboxylate (11c). Thioamide 10c (0.17 g,
0.4 mmol) was converted into crude thiazole 11c after treatment
with ethyl bromopyruvate (0.1 mL, 0.7 mmol) for 50 min according
to the above general procedure. Pure 11c (0.1 g, 47%) was isolated by
flash column chromatography (1:1 toluene/EtOAc) as a colourless
d
14.3 (CH3CH2CH2), 23.4, 26.6 and 32.3 (3ꢁ CH2), 36.9 (COCH2),
60.0 (C-20), 63.4 (C-50), 72.7 (C-30), 80.6 (C-10), 88.8 (C-40), 126.3 (C-
5), 150.3 (C-4), 165.7 (C-2), 172.2 and 176.8 (CONH2 and
NHCOC5H11); LRMS (CI): m/z 358 (MþþH). Anal. Calcd for
C15H23N3O5S: C, 50.41; H, 6.49; N, 11.76; S, 8.97. Found: C, 50.10; H,
6.40; N, 11.47; S, 8.64.
oil, ½a 2D0
þ4.7 (c 1.0, CHCl3); IR (neat): nmax 3353, 1724, 1652, 1533,
ꢃ
1272; 1H NMR (250 MHz, CDCl3):
d
0.83 (t, 3H, J¼6.8 Hz,
CH3CH2CH2), 1.17 (m, 12H, 6ꢁ CH2), 1.34 (t, 3H, J¼7.1 Hz,
CO2CH2CH3), 1.50 (m, 2H, CH2), 2.12 (m, 2H, CH2CO), 4.33 (q, 2H,
J¼7.1 Hz, CO2CH2CH3), 4.41e4.69 (m, 6H, H-20, H-30, H-40, H-5a0, H-
4.8.2. 2-(2-Deoxy-2-octanamido-b-D-ribofuranosyl)thiazole-4-
carboxamide (2b). Protected thiazole 11b (0.12 g, 0.2 mmol) was
converted into crude 2b after treatment with saturated ammonia in
MeOH (5 mL) according to the above general procedure. Pure 2b
(0.05 g, 49%) was isolated by preparative TLC (9:1 EtOAc/iPrOH).
Recrystallization from a mixture of EtOAc/iPr2O gave pure 2b as
5b0 and OH), 5.29 (d, 1H, J1 2 ¼8.9 Hz, H-10), 7.30 (d, 1H,
0
0
0
J2 ,NH¼8.1 Hz, NHCOC9H19), 7.36e8.11 (m, 5H, Ph), 8.08 (s, 1H, H-5);
13C NMR (62.9 MHz, CDCl3):
d 14.0 (CH3CH2CH2), 14.2 (CO2CH2CH3),
22.5, 25.3, 29.2, 29.26, 29.3 and 31.7 (7ꢁ CH2), 36.1 (CH2CO), 59.5
(C-20), 61.6 (CO2CH2CH3), 64.6 (C-50), 72.5 (C-30), 79.4 (C-10), 84.3
(C-40), 128.4, 129.3, 129.7 and 133.2 (C-5 and Ph), 146.1 (C-4), 161.4
(C-2), 166.3 (PhC]O), 172.2 (CO2Et), 174.3 (NHCOC11H23); LRMS
(CI): m/z 547 (MþþH). Anal. Calcd for C28H38N2O7S: C, 61.52; H,
7.01; N, 5.12; S, 5.87. Found: C, 61.48; H, 6.93; N, 4.89; S, 6.01.
colourless crystals, mp 162 ꢀC; ½a D20
ꢃ
ꢂ24.7 (c 0.6, MeOH); IR (KBr):
nmax 3319, 1649, 1540; 1H NMR (250 MHz, methanol-d4):
d
0.88 (t,
3H, J¼7.1 Hz, CH3CH2), 1.16e1.66 (m, 10H, 5ꢁ CH2), 2.25 (t, 2H,
0
0
0
J¼7.7 Hz, CH2CO), 3.73 (d, 2H, J4 ,5 ¼4.5 Hz, 2ꢁ H-5 ), 4.11 (m, 1H, H-
0
40), 4.24 (dd, 1H, J2 ,3 ¼5.4, J3 ,4 ¼2.4 Hz, H-3 ), 4.52 (dd, 1H, J1 ,2 ¼8.7,
0
0
0
0
0
0
0
0
0
0
0
0
J2 ,3 ¼5.4 Hz, H-2 ), 5.08 (d, 1H, J1 ,2 ¼8.7 Hz, H-1 ), 8.20 (s, 1H, H-5);
4.7.4. Ethyl 2-(5-O-benzoyl-2-deoxy-2-dodecanamido-
b
-D
-ribofur-
13C NMR (62.9 MHz, methanol-d4):
d 15.4 (CH3CH2), 24.6, 28.0, 31.1,
anosyl)thiazole-4-carboxylate (11d). Thioamide 10d (0.14 g,
0.3 mmol) was converted into crude thiazole 11d after treatment
with ethyl bromopyruvate (0.05 mL, 0.4 mmol) for 50 min
according to the above general procedure. Pure 11d (0.09 g, 56%)
was isolated by column chromatography (4:1, 7:3, to 3:2 toluene/
EtOAc) as a bright yellow solid. Recrystallization from a mixture of
CH2Cl2/hexane gave an analytical sample 11d as pale yellow nee-
31.2, and 33.8 (5ꢁ CH2), 38.0 (CH2CO), 61.0 (C-20), 64.5 (C-50), 73.7
(C-30), 81.8 (C-10), 89.9 (C-40), 127.1 (C-5), 151.6 (C-4), 166.6 (C-2),
173.3 and 177.5 (CONH2 and NHCOC7H15); HRMS (ESI): m/z
386.1758 (MþþH), calcd for C17H28N3O5S: 386.1744.
4.8.3. 2-(2-Decanamido-2-deoxy-b-D-ribofuranosyl)thiazole-4-
carboxamide (2c). Protected thiazole 11c (0.09 g, 0.2 mmol) was
converted into crude 2c after treatment with saturated methanolic
ammonia (3.4 mL) according to the above general procedure. Pure
2c (0.05 g, 81%) was isolated by preparative TLC (9:1, 4:1 EtOAc/i-
PrOH, two successive developments). An analytical sample 2c was
obtained after recrystallization from a mixture of EtOAc/iPr2O as
dles, mp: 101e102 ꢀC, ½a D20
ꢃ
ꢂ23.3 (c 1.1, CHCl3); IR (neat): nmax 3452,
1713, 1650, 1546, 1278; 1H NMR (250 MHz, CDCl3):
d
0.87 (t, 3H,
CH3CH2CH2), 1.10e1.62 (m, 18H, 9ꢁ CH2), 1.37 (t, 3H, J¼7.0 Hz,
CO2CH2CH3), 2.15 (t, 2H, CH2CO), 4.36 (q, 2H, CO2CH2CH3),
4.43e4.71 (m, 6H, H-20, H-30, H-40, 2ꢁ H-50 and OH), 5.30 (d, 1H,
J1 ,2 ¼8.2 Hz, H-10), 7.22 (d, 1H, J2 ,NH¼7.9 Hz, NHCOC11H23),
colourless crystals, mp 138 ꢀC; ½a D20
ꢃ
ꢂ15.7 (c 0.8, MeOH); IR (KBr):
0
0
0
7.39e8.12 (m, 5H, Ph), 8.09 (s,1H, H-5); 13C NMR (62.9 MHz, CDCl3):
nmax 3321, 1654, 1541; 1H NMR (250 MHz, methanol-d4):
d
0.89 (t,
d
14.0 (CH3CH2CH2), 14.2 (CO2CH2CH3), 22.6, 25.4, 29.29, 29.3, 29.4,
3H, J¼6.8 Hz, CH3CH2),1.26 (m,12H, 6ꢁ CH2),1.56 (m, 2H, CH2), 2.25
29.44, 29.56, 29.6, 31.8 and 36.2 (10ꢁ CH2), 59.7 (C-20), 61.6
(CO2CH2CH3), 64.63 (C-50), 72.6 (C-30), 79.5 (C-10), 84.34 (C-40),
128.4 (C-5),128.44,129.5,129.7 and 133.2 (Ph),146.3 (C-4),161.4 (C-
2), 166.3 (PhC]O), 172.1 (CO2Et), 174.4 (NHCOC11H23); LRMS (CI):
m/z 575 (MþþH). Anal. Calcd for C30H42N2O7S: C, 62.69; H, 7.37; N,
4.87; S, 5.58. Found: C, 62.89; H, 7.25; N, 5.08; S, 5.28.
(t, 2H, J¼7.4 Hz, C8H17CH2CO), 3.73 (d, 2H, J4 ,5 ¼4.5 Hz, 2ꢁ H-5 ),
0
0
0
0
4.11 (m, 1H, H-40), 4.23 (dd, 1H, J2 ,3 ¼5.4, J3 ,4 ¼2.6 Hz, H-3 ), 4.51
0
0
0
0
0
0
0
0
0
0
0
0
(dd, 1H, J1 ,2 ¼8.5, J2 ,3 ¼5.5 Hz, H-2 ), 5.09 (d, 1H, J1 ,2 ¼8.6 Hz, H-1 ),
8.21 (s, 1H, H-5); 13C NMR (62.9 MHz, methanol-d4):
d 15.0
(CH3CH2), 24.3, 27.5, 30.8, 30.9, 31.0, 31.1 and 33.6 (7ꢁ CH2), 37.5
(CH2CO2), 60.6 (C-20), 64.0 (C-50), 73.2 (C-30), 81.2 (C-10), 89.3 (C-40),
126.6 (C-5), 151.0 (C-4), 166.0 (C-2), 172.9 and 177.1 (CONH2 and
NHCOC9H19); LRMS (CI): m/z 414 (MþþH). Anal. Calcd for
C19H31N3O5S: C, 55.18; H, 7.56; N, 10.16; S, 7.75. Found: C, 55.22; H,
7.53; N, 10.02; S, 7.83.
4.8. General procedure for the preparation of C-nucleosides
2aed
A solution of protected thiazole 11 (1 mmol) in saturated
methanolic ammonia (0.05 M) was kept at room temperature for 7
days, then evaporated and the residue was purified on a column of
silica gel or by preparative TLC.
4.8.4. 2-(2-Deoxy-2-dodecanamido-b-D-ribofuranosyl)thiazole-4-
carboxamide (2d). Protected thiazole 11d (0.06 g, 0.1 mmol) was
converted into crude 2d after treatment with saturated methanolic
ammonia (3 mL) according to the above general procedure. Pure 2d
(0.04 g, 80%) was isolated by silica gel column chromatography (9:1
EtOAc/iPrOH) as a white solid. Recrystallization from a mixture of
MeOH/iPr2O gave an analytical sample 2d as colourless crystals, mp
4.8.1. 2-(2-Deoxy-2-hexanamido-b-D-ribofuranosyl)thiazole-4-
carboxamide (2a). Protected thiazole 11a (0.28 g, 0.6 mmol) was
converted into crude 2a after treatment with saturated methanolic
ammonia (12 mL) according to the above general procedure. Pure
2a (0.13 g, 66%) was isolated by silica gel column chromatography
(9:1, 4:1, to 7:3 EtOAc/iPrOH) as a white solid. Recrystallization from
a mixture of MeOH/iPr2O gave an analytical sample 2a as colourless
133.5e134.5 ꢀC; ½a D20
ꢃ
ꢂ17.0 (c 0.9, MeOH); IR (KBr): nmax 3396,1655,
1541, 1288; 1H NMR (250 MHz, methanol-d4):
d
0.87 (t, 3H,
J¼7.0 Hz, CH3CH2), 1.18e1.62 (m, 18H, 9ꢁ CH2), 2.25 (t, 2H, J¼7.3 Hz,
0
0
0
0
CH2CO), 3.74 (d, 2H, J4 ,5 ¼4.5 Hz, 2ꢁ H-5 ), 4.12 (m, 1H, H-4 ), 4.26
crystals, mp 155e156 ꢀC; ½a D20
ꢃ
ꢂ16.4 (c 1.1, MeOH); IR (KBr): nmax
(dd, 1H, J2 ,3 ¼5.1, J3 ,4 ¼2.2 Hz, H-3 ), 4.52 (dd, 1H, J1 ,2 ¼8.7,
0
0
0
0
0
0
0
3288, 1653, 1271; 1H NMR (250 MHz, methanol-d4):
d
0.85 (t, 3H,
J2 ,3 ¼5.1 Hz, H-2 ), 5.09 (d, 1H, J1 ,2 ¼8.7 Hz, H-1 ), 8.21 (s, 1H, H-5);
0
0
0
0
0
0
CH3CH2CH2), 1.14e1.64 (m, 6H, 3ꢁ CH2), 2.25 (t, 2H, CH2CO), 3.74 (d,
NOE contact: H-5 and H-50; 13C NMR (62.9 MHz, methanol-d4):
0
0
0
0
0
0
2H, J4 ,5 ¼4.5 Hz, 2ꢁ H-5 ), 4.13 (m, 1H, H-4 ), 4.25 (dd, 1H, J2 ,3 ¼5.4,
d
14.4 (CH3CH2), 23.6, 26.9, 30.1, 30.3, 30.5, 30.6 and 32.9 (9ꢁ CH2),

M. Popsavin et al. / Tetrahedron 67 (2011) 6847e6858
6857
36.9 (CH2CO), 59.9 (C-20), 63.4 (C-50), 72.6 (C-30), 80.7 (C-10), 88.7
(C-40), 126.3 (C-5), 150.4 (C-4), 165.6 (C-2), 172.2 and 176.7
(NHCOC11H23 and CONH2); LRMS (CI): m/z 442 (MþþH). Anal. Calcd
for C21H35N3O5S: C, 57.12; H, 7.99; N, 9.52; S, 7.26. Found: C, 57.53;
H, 7.99; N, 9.24; S, 7.21.
ensure that all crystals were dissolved, the plates were read on
a spectrophotometer plate reader (Multiscan MCC340, Labsystems)
at 540 and 690 nm. The wells without cells containing complete
medium and MTT acted as blank.
4.13. Flow cytometry
4.9. Biological materials
Apoptosis of K562 cells was evaluated with an Annexin V-FITC
detection kit. Cells from each sample were collected (800 rpm/
5 min, Megafuge 1.0 R, Heraeus, Thermo Fisher Scientific) and pellet
was re-suspended in 1 mL of phosphate buffer (PBS, pH 7.2). K562
cells were washed twice with cold PBS and then re-suspended in
binding buffer to reach the concentration of 1ꢁ106 cells/mL. The
Rhodamine B, RPMI 1640 medium, foetal calf serum, 3-(4,5-
dimethylthiazol)-2,5-diphenyltetrazolium bromide, propidium io-
dide and RNase A, were purchased from Sigma (St. Louis, MO, USA).
Penicillin and streptomycin were purchased from ICN Galenika
(Belgrade, Serbia). Annexin VeFLUOS apoptosis detection kit was
purchased from BD Biosciences Pharmingen (Belgium). Proteins
were detected by Western blotting using the following monoclonal:
the antibodies against human Bcl-2 and Caspase-3 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 Biosciences (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.
cell suspension (100
mixed with Annexin V (5
were gently vortexed and incubated for 15 min at 25 ꢀC. After in-
cubation, 400 L of binding buffer was added to each tube and
m
L) was transferred to 5 mL culture tubes and
m
L) and propidium iodide (5 L). The cells
m
m
suspension was analyzed after 1 h on FACS Calibur E440 (Becton
Dickinson) flow cytometer. Results were presented as percent of
Annexin V positive gated cells. Percentage of specific apoptosis was
calculated according to Bender et al.20
4.14. Western blot
For the Western blot, 50 mg of proteins per sample were sepa-
4.10. Cell lines
rated by electrophoresis and electro-transferred to a PVDF mem-
brane Hybond-P and then blotted with primary antibodies (Bcl-2,
Caspase-3 and PARP). Proteins were detected by an enhanced
chemiluminescence (ECL Plus) kit that includes peroxidase-labelled
donkey anti-rabbit and sheep anti-mouse secondary antibodies.
Blots were developed with an ECL Plus detection system and
recorded on the Amersham Hyperfilm.
Human chronic myelogenous leukaemia (K562), promyelocytic
leukaemia (HL-60) and Burkitt’s lymphoma (Raji) were grown in
RPMI 1640 while colon adenocarcinoma (HT-29), breast adeno-
carcinoma (MCF-7), cervix carcinoma (HeLa) malignant cells, and
normal foetal lung fibroblasts (MRC-5) were grown in DMEM me-
dium. Both media were supplemented with 10% of foetal calf serum
(FTS, NIVNS) and antibiotics (100 IU/mL of penicillin and 100
mg/mg
Acknowledgements
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.
This work was supported by a research grant from the Ministry
of Science and Technological Development of the Republic of Serbia
(Grant No. 172006).
4.11. Cells treatment
Supplementary data
The cells were seeded in six-well plates at a concentration of
5ꢁ105 cells/well. Cells were treated for 72 h with tiazofurin (1) and
TAs (2aed) at their IC50/72 h concentrations. Untreated cells were
used as control. Viable cells of treated and control samples were
used for apoptosis detection and Western blot analysis. Viability
was determined using trypan blue dye-exclusion assay.
Additional details of experimental procedures, FACS analysis of
apoptotic K562 cells, copies of 1H, and 13C NMR spectra of selected
key compounds. Supplementary data associated with this article
can be found in the online version at doi:10.1016/j.tet.2011.06.090.
References and notes
4.12. MTT assay
ꢀ
ꢁ
ꢀ
ꢁ
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assay period. Viable cells were placed in a volume of 90 mL per 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 added (10 mL/well)
to all wells except to the control ones and microplates were in-
cubated for 72 h. The wells containing cells without tested sub-
stances were used as control. 3 h before the end of incubation
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Felczak, K.; Gao, G.; Bonnac, L.; Yu, J. S.; Bennett, E. M.; Pankiewicz, K. W. Curr.
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period MTT solution (10 mL) was added to all wells. MTT was dis-
solved in medium at 5 mg/mL and filtered to sterilize and remove
a small amount of insoluble residue present in some batches of
MTT. Acidified 2-propanol (100 mL of 0.04 M HCl in 2-propanol) was
added to all wells and mixed thoroughly to dissolve the dark blue
crystals of formazan. After a few minutes at room temperature, to

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~
12. A part of this work recently appeared as a preliminary communication: Pop-
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