Synthesis and in vitro antitumour activity of tiazofurin analogues with nitrogen functionalities at the C-2′ position

Article abstract of DOI:10.1016/j.ejmech.2016.01.037

Synthesis of three tiazofurin (1) isosteres with nitrogen functionalities at the C-2′ position (N₃, NH₂ and NH₃⁺Cl^-) has been achieved, in multistep sequences, starting from monoacetone d-glucose. A n

Full text of DOI:10.1016/j.ejmech.2016.01.037

European Journal of Medicinal Chemistry 111 (2016) 114e125
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and in vitro antitumour activity of tiazofurin analogues with
nitrogen functionalities at the C-20 position
,
*
a
ꢀ b
Mirjana Popsavin a , Vesna Kojic , Ljilja Torovic , Milos Svircev , Sasa Spaic ,
ꢀ a
ꢁ
ꢀ b
ꢁ
ꢁ
ꢀ a
b
a
ꢀ b
Dimitar Jakimov , Lidija Aleksic , Gordana Bogdanovic , Velimir Popsavin
Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi
a
ꢀ
Sad, Serbia
b Oncology Institute of Vojvodina, Put Dr 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
Synthesis of three tiazofurin (1) isosteres with nitrogen functionalities at the C-20 position (N3, NH2 and
Article history:
NHþ3 Clꢀ) has been achieved, in multistep sequences, starting from monoacetone
D-glucose. A number of
Received 27 September 2015
Received in revised form
18 January 2016
Accepted 19 January 2016
Available online 25 January 2016
potential bioisosteres of 1 bearing acylamido functions at the C-20 position have also been synthesized
from the same sugar precursor. In vitro cytotoxicities of target molecules against a number of human
tumour cell lines were recorded and compared with those observed for lead molecule 1. Some of the
synthesized compounds showed potent in vitro antitumour activity, such as 20-azido derivative 2, which
is the most potent of all molecules under evaluation (IC50 0.004 mM against MCF-7 cells). Flow cytometry
Keywords:
Tiazofurin
Analogues synthesis
Antitumour activity
Induction of apoptosis
Cell cycle analysis
data suggest that cytotoxic effects of these compounds in the culture of K562 cells might be mediated by
apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these
cells. Results of Western blot analysis indicate that the synthesized tiazofurin analogues induce apoptosis
in a caspase-dependent way.
© 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction
numerous structural modifications in its sugar residue have been
performed [13e24]. However, most of the resulting tiazofurin
Tiazofurin (1, Fig. 1), 2-(
b
-
D
-ribofuranosyl)thiazole-4-
mimics did not show favourable biological effects, but a number of
synthesized analogues still have not been tested for their anti-
tumour activity. In the last ten years, we have prepared a series of
new tiazofurin mimics, and some of them have demonstrated
stronger antitumour potencies than lead 1 [25e28]. All of these
compounds were completely non-toxic to normal human MRC-
5 cells, indicating the selectivity of analogues against tumour cells.
In continuation of these studies we designed new tiazofurin mimics
bearing the nitrogen functionalities at C-20 position (2e7). Com-
pounds 2e4 may be considered as direct isosteres of 1, while the
analogues 5e7 represent N-acyl derivatives of 3. A reason for the
preparation of amides 5e7 arose from our recent study, which
showed that some 20-acylamido derivatives demonstrate strong
antiproliferative activities against a panel of human tumour cell
lines [27].
carboxamide, is a well-known C-nucleoside with interesting anti-
cancer properties [1,2]. In 2001, the US Food and Drug Adminis-
tration granted orphan-drug status designation to tiazofurin for the
treatment of chronic myelogenous leukaemia in the accelerated
phase of blast crisis, despite its high toxicity [3]. It has been found
that tiazofurin inhibits inosine 50-monophosphate dehydrogenase
(IMPDH), the rate-limiting enzyme of de novo guanine nucleotide
synthesis [1,2]. Subsequent discovery of two IMPDH isoforms, of
which type II is upregulated in human leukaemia cell lines [4,5], has
directed studies toward the design of isoform selective inhibitors
[6] and has renewed interest for the preparation of tiazofurin an-
alogues [7e10]. Inhibitors of IMPDH in patent literature of the last
decade were recently reviewed [11], while the potential of IMP
dehydrogenase inhibitors for the treatment of cancer was reviewed
in 2007 [12]. To improve therapeutic properties of tiazofurin,
Herein, we disclose full details on the synthesis of 2 and 6 [29],
as well as multistep preparations of new tiazofurin mimics 3e5 and
7. The effects of these compounds to the proliferation of certain
human tumour cell lines, including their effects on the K562 cell
cycle and their apoptosis inducing properties in the same cell
* Corresponding author.
E-mail address: mirjana.popsavin@dh.uns.ac.rs (M. Popsavin).
http://dx.doi.org/10.1016/j.ejmech.2016.01.037
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
115
(2% from 8).
Compound 2 was converted to desired 2-amino-2-deoxy de-
rivative 3 (65%) after catalytic reduction in EtOH over 10% Pd/C.
However, when the catalytic reduction of 2 was carried out under
the similar reaction conditions, but in the presence of chloroform
[31], the corresponding amine hydrochloride 4 was obtained in 89%
yield.
2,5-Anhydro derivative 15 has been conveniently used as a
divergent intermediate for the synthesis of 20-acetamido derivative
5 (Scheme 3). Catalytic hydrogenation of 15 over PtO2 in a mixture
of glacial acetic acid and acetic anhydride gave the corresponding
acetamido derivative 19 in 68% yield. Compound 19 was converted
to target 5, by using a six-step sequence similar to that already
applied for the conversion of 15 to target 2 (Scheme 2). Thus, hy-
drolytic removal of the dioxolane protective group in 19 was ach-
ieved in a mixture of trifluoroacetic acid and 6 M hydrochloric acid
at þ4 ꢁC. The resulting crude aldehyde 19a was treated with hy-
droxylamine hydrochloride to yield the corresponding oxime 19b
as a mixture of E- and Z-isomers. An attempted separation of E-19b
and Z-19b by column chromatography provided only pure E-19b
(44%). As the corresponding Z-isomer could not be obtained free of
E-19b the purification step is omitted, and the mixture of E- and Z-
19b was converted to the corresponding ribofuranosyl cyanide 20
in an overall yield of 39% (from the last three steps). Exposure of 20
to hydrogen sulphide gas gave the thioamide 21 (86%). The sub-
sequent cyclocondensation of 21 with ethyl bromopyruvate affor-
ded the corresponding thiazole 22 (73%), which was finally
converted to target 5 (62%) after treatment with methanolic
ammonia.
Fig. 1. Chemical structures of tiazofurin (1) and the corresponding analogues 2e7.
culture, have also been studied in this work.
2. Results and discussion
2.1. Chemistry
Synthesis of 20-azido derivative 2 (Scheme 1), was completed by
using the same ten-step sequence previously reported in the pre-
liminary communication [29]. The most critical step of this
sequence involves the addition of hydrogen sulphide gas to the
nitrile functionality in 10. However, in addition to the expected
reaction with the nitrile functionality H2S may react with the azide
group as well, decreasing the yield of desired thioamide 11e34%. To
avoid the possible low-yielding step we planned an alternative
route (Scheme 2) for the construction of the thiazole heterocyclic
system by avoiding the use of hydrogen sulphide gas [30].
Accordingly, compound 16 was allowed to react with cysteine
ethyl ester hydrochloride, in the presence of Et3N at room tem-
perature, to afford thiazoline 17 as an inseparable mixture of C-4
epimers. As the newly formed stereocenter was to be destroyed
during the formation of the aromatic thiazole ring, no attempts
were made to analyze the diastereomeric ratio of this mixture. The
epimers 17 were subsequently treated with DBU and BrCCl3 to give
the expected thiazole 18 in a non-optimized yield of 64%, over the
last two steps. Ester aminolysis followed by global deprotection,
which was effected by treatment of 18 in methanolic ammonia,
provided target 2 in 93% yield. The latter nine-step sequence
(Scheme 2) represents a more convenient route for the preparation
of analogue 2, since it provided a considerably higher overall yield
(18% from 8) compared to the six-step route presented in Scheme 1
The two independent routes toward the thiazole C-nucleoside 6,
bearing the N-benzoyl functionality at the C-20 position, are out-
lined in Scheme 4.
The first route started from 2,5-anhydride 23, which was con-
verted to the target 6 via the three-step sequence The first route
started from 2,5-anhydride 23, which was converted to the target 6
via the three-step sequence similar to that previously described in
the preliminary account [29]. In this way, the glycosyl cyanide 23
was converted into the target C-nucleoside 6 in 31% overall yield.
The second route for the preparation of 6 started from azido-nitrile
16, which was converted to the required thioamide 26 through the
one-pot H2S-mediated cascade that we recently used for the
preparation of tiazofurin analogues embedded with an amide
moiety at the C-20 position [27]. As expected, exposure of 16 to
hydrogen sulphide gas gave the thioamide 26 as a result of the
Scheme 1. Reagents and conditions: (a) 4:1 TFA/6 M HCl, 4 ꢁC, 72 h; (b) NH2OH$HCl, NaOAc, EtOH, CH2Cl2, st, 2 h; (c) MsCl, Py, ꢀ15 ꢁC, 0.5 h, then rt, 2 h, 39% from 9; (d) H2S, Py, rt,
2 h, 34%; (e) TiCl4, CH2Cl2, 0 ꢁC, 1 h, 67% (f) BrCH2COCO2Et, EtOH, 80 ꢁC, 50 min, 57%; (g) NH3, MeOH, rt, 6 days, 75%.

116
M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
Scheme 2. Reagents and conditions: (a) NaN3, DMSO, 108e110 ꢁC, 24 h, 61%; (b) 4:1 TFA/6 M HCl, 4 ꢁC, 140 h; (c) NH2OH$HCl, NaOAc, EtOH, CH2Cl2, rt, 2 h; (d) MsCl, Py, ꢀ15 ꢁC,
0.5 h, then rt, 2 h, 59% from 15; (e) cysteine ethyl ester hydrochloride, Et3N, MeOH, rt, 2 h, 76%; (f) DBU, BrCCl3, CH2Cl2, 0 ꢁC, 5 h / 4 ꢁC, 17 h, 84%; (g) NH3, MeOH, rt, 7 days, 93% (h)
H2ePd/C, EtOH, rt, 36 h, 65% of 3; (i) H2ePd/C, CHCl3, EtOH, rt, 36 h, 89% of 4.
Scheme 3. Reagents and conditions: (a) H2, PtO2, AcOH, Ac2O, rt, 48 h, 68%; (b) 3:1 TFA/6 M HCl, 4 ꢁC, 10 days; (c) NH2OH$HCl, NaOAc, EtOH, CH2Cl2, rt, 2 h; (d) MsCl, Py, ꢀ15 ꢁC,
0.5 h, then rt, 2 h, 39% from 19; (e) H2S, Py, rt, 8 h, 86%; (f) BrCH2COCO2Et, EtOH, reflux, 50 min, 73%; (g) NH3, MeOH, rt, 8 days, 62%.
Scheme 4. Reagents and conditions: (a) H2S, Py, rt, 8 h for 23, 74% of 24, 1.5 h for 16, 71% of 26, 25% of 16a; (b) BrCH2COCO2Et, EtOH, reflux, 50 min for 24, 78% of 25, 80 min for 26,
55% of 27; (c) NH3, MeOH, rt, 8 days for 25, 53% of 6, 7 days for 27, 56% of 6; (d) H2S, DMAP, EtOH, rt, 8 h, 82%.

M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
117
Scheme 5. Reagents and conditions: (a) H2, Pd/C, CHCl3, EtOH, rt, 168 h, (b) 3-methoxybenzoyl chloride, Py, rt, 72 h, 63% from 9; (c) 3:1 TFA/6 M HCl, 4 ꢁC, 10 days; (d) NH2OH$HCl,
NaOAc, EtOH, CH2Cl2, rt, 2.5 h; (e) MsCl, Py, ꢀ15 ꢁC, 0.5 h, then rt, 2 h, 50% calculated to reacted 28; (f) H2S, Py, rt, 8 h, 93%; (g) BrCH2COCO2Et, EtOH, reflux, 50 min, 60%; (h) NH3,
MeOH, rt, 7 days, 72%.
addition of hydrogen sulphide to the nitrile group, followed by
subsequent reduction of the azido group to an amino function, as
well as a final O / N acyl migration process. Depending on the
reaction conditions and reagents used, the major product 26 was
accompanied with a variable amount of 16a. Thus when 16 was
reacted with hydrogen sulphide in dry pyridine for 1.5 h at room
temperature both 26 and 16a were obtained in 71% and 25% yields,
respectively. However, when the reaction was carried out with
hydrogen sulphide and N,N-dimethylaminopyridine in ethanol (for
8 h at room temperature), the key intermediate 26 was obtained as
the only reaction product in a yield of 82%. Moreover, thioamide 26
was treated with ethyl bromopyruvate in refluxing ethanol to give
the protected thiazole 27 in 55% yield. Final exposure of 27 to
methanolic ammonia provided analogue 6 in 56% yield. This nine-
step sequence, which was carried out over the intermediate 16,
provided the target 6 in 8% overall yield with respect to starting 2,5-
anhydride 8. An alternative sequence, realized over 23, is of same
efficiency regarding the total yield (8% from 8), even though it re-
quires two additional synthetic steps for the completion.
2.2. Antiproliferative activities
Antiproliferative activities of synthesized tiazofurin analogues
were evaluated against seven tumour cell lines (myelogenous
leukaemia K562, promyelocytic leukaemia HL-60, Jurkat T cells
leukaemia, Raji Burkitt's lymphoma, colon adenocarcinoma HT-29,
breast adenocarcinoma MCF-7, cervix carcinoma HeLa), and a single
human normal cell line (MRC-5). The use of foetal lung fibroblasts
(MRC-5) serves to demonstrate the toxicity of synthesized com-
pounds toward normal cells. Cytotoxicity was evaluated by using
the standard MTT assay, after exposure of cells to the tested com-
pounds for 72 h. Tiazofurin (1) was used as a reference compound
in this assay.
As shown in Table 1, of all synthesized compounds, only
analogue 6 has a similar profile of cytotoxicity as tiazofurin,
although it was completely inactive against HeLa cells, toward
which lead 1 showed strong antiproliferative activity (3.82 mM).
Analogue 6 exhibited potent to moderate antiproliferative effects
against seven of eight investigated cell lines. Particularly strong
The synthesis of tiazofurin mimic 7 is outlined in Scheme 5. The
sequence started with catalytic reduction of the azide group in 9
(10% Pd/C, in the presence of CHCl3) to give the amine hydrochlo-
ride 9c as a product of sequential azide reduction/benzyl ether
hydrogenolysis process. Treatment of crude 9c with 3-
methoxybenzoyl chloride in pyridine gave the fully protected
product 28 (63% from 9). Compound 28 was converted to target 7
using the six-step sequence shown in Scheme 5. Final product 7
was obtained in an overall yield of 13% from the last eight steps.
antiproliferative activities (IC50 0.01e0.03 mM) of analogue 6 were
observed in the cultures of the following malignant cells: K562
(over 90-fold more active then lead 1), Jurkat (a similar activity as
1), Raji (over 500-fold more active then 1), and HT-29 (13-fold more
active then 1). The remaining five analogues (2e5 and 7) showed
selective cytotoxicities only against individual cancer cell lines.
Thus, analogue 2 exhibited the remarkable antiproliferative activ-
ities against K562 (IC50 0.31
mM, 6-fold more potent than 1) and
MCF-7 cells (IC50 4 nM, 445-fold more potent than 1). Against HeLa
Table 1
In vitro cytotoxicity of tiazofurin (1) and analogues 2e7.
Compounds
IC50
K562
(m
M)a
HL-60
0.19
>100
12.17
0.02
>100
Jurkat
0.04
>100
>100
>100
>100
Raji
HT-29
0.26
>100
>100
>100
>100
MCF-7
1.78
0.004
>100
>100
3.92
HeLa
3.82
4.08
0.15
>100
>100
>100
>100
MRC-5
0.36
>100
>100
>100
5.26
0.05
>100
1
2
3
4
5
6
7
1.89
0.31
0.51
5.28
>100
2.59
9.28
14.74
0.01
>100
>100
0.02
>100
20.43
2.69
0.03
23.58
0.02
>100
19.86
9.54
6.29
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 experiments.
Coefficients of variation were less than 10%.

118
M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
Table 2
with synthesized compounds for 24 and 72 h was analyzed by flow
cytometry in cells stained with propidium iodide. Untreated cells
served as a control. The results are presented in Table 2 (for graphic
presentation see Fig. S1 in the Supplementary data). As the data in
Table 2 indicate, a 24-h treatment of K562 cells with analogues 2e7
slightly change the percentage of cells in G0/G1 and S phase of cell
cycle compared to control. As it is further seen from Table 2, ana-
logues induce various changes in G2/M phase of cell cycle
compared to both tiazofurin and control: analogue 3 significantly
increases the percentage of G2M phase cells, while tiazofurin and
analogues 5e7 decrease it. After the exposure of K562 cells to an-
alogues 2e7 during 72 h the increased percentage of cells in G0/G1
phase was detected when compared to tiazofurin, while this per-
centage is almost the same with respect to control. The percentage
of cells in S phase was similar to the values obtained for both tia-
zofurin and control. All analogues induced a similar percentage of
cells in G2/M phase, with the values similar to those obtained after
24-h treatment. However, each of these values was approximately
2-fold higher than the value detected after a 72-h treatment with
tiazofurin.
After 72-h cells treatment, the percentage of cells in sub-G1
phase (which is suggestive of apoptosis) was significantly higher
than the values recorded after 24-h treatment of cells. After pro-
longed treatment, each of the synthesized compounds introduced
3e4 times more cells in sub-G1 phase relative to control. Tiazofurin
caused significant apoptosis only after 72 h, as observed in our
previous study [26].
Based upon these results it may be concluded that synthesized
tiazofurin analogues affect the distribution of K562 cells in the cell
cycle phases. These changes depend on the nature of the analogue
as well as the duration of cells treatment with the tested
compounds.
Distribution of K562 cells in cell cycle phases (%) after 24- and 72-h cell treatment.
Compound
Sub-G1
G0/G1
24 h
S
G2/M
24 h
24 h 72 h
72 h
24 h
72 h
72 h
control
1.05
3.04 36.28 45.92 48.38 35.73 14.29 15.31
7.55 5.74
1 (tiazofurin) 2.22 24.99 37.08 35.66 53.16 33.62
2
3
4
5
6
7
2.93 10.90 42.67 42.24 42.80 35.04 11.60 11.82
1.40 13.52 32.21 40.24 46.33 34.08 20.06 12.16
2.61 11.13 35.02 44.32 47.22 33.29 15.16 11.26
3.71 11.17 43.07 48.09 45.03 31.42
6.12 11.29 39.08 44.10 49.13 34.11
4.44 10.84 39.45 43.13 46.71 34.00
8.21
5.67 10.50
9.41 12.04
9.32
cells this molecule showed the similar potency as control com-
pound 1. At the same time, this analogue represents the most active
compound synthesized in this work. Analogue 3 was more potent
than lead 1 against the following malignant cell lines: K562 (IC50
0.51
m
M, almost 4-fold more active than 1), Raji (IC50 2.59
mM, 2-
fold more active than 1) and HeLa (IC50 0.15
more active than 1). The corresponding amine hydrochloride 4
however, was more potent than lead 1 only against HL-60 cell line
m
M, over 25-fold
(IC50 0.02 mM, 9.5-fold more active than 1). This molecule is also
active against Raji cells, but shows almost 2-fold weaker cytotox-
icity than tiazofurin (1). Analogues 5 and 7 gave the poorest results
in the biological assay, as neither of these two compounds was
more active than tiazofurin (1) toward any of the cell lines under
evaluation. In the preliminary communication [29] we have re-
ported the results of in vitro antitumour activity of analogues 2 and
6 recorded after 24-h cells treatment. As expected, compound 2
exhibited significantly stronger activities after 72-h cells treatment
(Table 1), including a submicromolar activity against K562 cell line
(IC50 0.31
mM). However, this molecule was completely inactive
after treatment of this cell line for 24 h [29]. Analogue 6 behaves
similarly but with one exception: the treatment of HL-60 cells for
2.4. Detection of apoptosis
72 h (Table 1) results in a lower potency (IC50 20.43
the submicromolar activity recorded after 24-h cells treatment
(IC50 0.12 M) [29]. The synthesized tiazofurin isosteres 2e4 are
mM) relative to
As data in Table 2 revealed, all tested compounds increased
percentage of K562 cells in sub-G1 phase compared with untreated
control. These findings suggested that synthesized analogues may
induce apoptosis. Percentage of apoptotic, necrotic and live cells
was determined with flow cytometry after double staining of
treated cells with Annexin V-FITC/PI and the results are shown in
Table 3 (for a graphic presentation see Fig. S2 in the Supplementary
data).
Apoptotic response, which was presented as a percentage of
specific apoptosis, shows that all analogues increase the percentage
of Annexin V-positive cells compared to tiazofurin. Percentage of
specific apoptosis increases with prolonging treatment time and
after 72 h is 2- to 4-fold higher than the values recorded after 24 h.
Compound 6 causes the greatest specific apoptosis in both exam-
ined periods (29% after 24 h and 50.41% after 72 h).
m
fully non-toxic towards normal human MRC-5 cells while lead
compound 1, as well as analogues 5 and 6 exhibited sub-
micromolar toxicity to the normal cells. The large variations in
the results for each cell line suggest that compounds 2e7 are not
acting at the same biological target as tiazofurin (IMPDH) and
points to a different mechanism of action, probably through in-
duction of apoptosis. Despite its strong toxicity toward normal
cells, we believe that analogue 6 represents the best candidate for
further optimization because of its strong antiproliferative activity
against the majority of tumour cell lines under evaluation.
2.3. Cell cycle analysis
The percentage of specific necrosis also increases with
increasing incubation time. The highest percentage of specific ne-
crosis causes analogue 5 (24.55% after 72 h), while the other ana-
logues showed slightly lower values (5e10%).
The cell cycle profile of exponentially growing K562 cells treated
Table 3
Percentage of specific apoptosis and necrosis induced with synthesized compounds
in the K562 cell culture (24- and 72-h cell treatment).
To resolve the mechanisms underlying the apoptosis induced
with synthesized tiazofurin analogues (2e7) we have studied the
ability of these compounds to modulate expression of selected
apoptosis markers, such as Bcl-2, Bax, caspase 3 and PARP. The
results are presented in Fig. 2 (for more details see pages S10 and
S11 in the Supplementary data).
Western blot analysis indicates that analogues 3 and 4 after a
24-h treatment decrease the expression of Bcl-2 in comparison to
the control and tiazofurin, while analogues 2, 5, 6 and 7 slightly
increase the total amount of the protein compared to the control.
However, this increase is much higher when compared to
Compound
Specific apoptosis (%)
Specific necrosis (%)
24 h
72 h
24 h
72 h
1 (tiazofurin)
ꢀ0.34
10.73
17.57
10.71
12.60
29.00
11.88
13.71
38.06
33.39
30.26
43.60
50.41
37.07
3.76
ꢀ0.82
4.97
3.60
5.88
10.71
5.33
7.11
6.22
24.55
6.86
2
3
4
5
6
7
11.71
3.68
10.33

M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
119
Fig. 2. Results of Western blot analysis after treatment of K562 cells with synthesized compounds for 24 h (A) and 72 h (B).
tiazofurin. After 72-h treatment analogue 3 reduces expression of
anti apoptotic Bcl-2 protein with respect to both control and tia-
zofurin, while analogues 2 and 4 slightly increased the total amount
of same protein compared to untreated control only. The greatest
decrease of the expression of Bcl-2 protein is caused by the com-
pounds 3 and 4 after both 24 and 72 h of cells treatment.
Some chemotherapeutics induce apoptosis that exerts a signif-
icant expression of Bax. However, in our experiments, tiazofurin
analogues did not effect the expression of Bax protein after 24 h.
Expression of Bax protein is increased after 72-h treatment with all
of the synthesized compounds, as well as tiazofurin. These results
are in agreement with increase of the specific apoptosis and also
with higher percent of cells in sub-G1 phase of cell cycle, after
treatment of cells for 72 h.
Expression levels of caspase 3 precursor and the corresponding
active subunit in cells treated with tiazofurin and analogues, were
determined in order to verify whether these compounds induce
apoptosis through a caspase-dependent way. It was confirmed that
treatment of K562 cells with all of the synthesized analogues in-
creases expression of the precursor of caspase 3, as well as the
corresponding active moiety. It was also found that the expression
level of active subunit of caspase 3 depends on the nature of the
analogue, as well as the time of treatment. After 24 h, most of the
analogues (except 3 and 4) including lead 1 enhance the expression
of the caspase 3, clearly indicating its participation in the apoptotic
process.
followed by subsequent treatment of the resulting thiazoline 17
with DBU and BrCCl3. Final exposure of protected C-nucleosides to
methanolic ammonia provided targets 2e7 in good yields.
Synthesized compounds were evaluated for their anti-
proliferative activity against a panel of human malignant cell lines.
20-Azido derivative 2 showed the highest potency toward MCF-
7 cells (IC50 0.004
mM) being the most active compound under
evaluation. Analogue 6 has shown potent to moderate anti-
proliferative effects against seven of eight investigated cell lines.
Remarkable antiproliferative activities of this analogue were
recorded in the following cell cultures: K562 (over 90-fold more
active then lead 1), Jurkat (a similar activity as 1), Raji (over 500-
fold more active then 1), and HT-29 (13-fold more active then 1).
It should be also emphasized that both analogues 2 and 6 exhibit
stronger antitumour potencies after cells treatment for 72 h unlike
the preliminary published results recorded after the cells treatment
for 24 h [29]. Analogues 3 and 4 also exhibited potent activities, but
only against individual cancer cell lines. The cell cycle analysis re-
veals that following treatment of cells with synthesized com-
pounds, there was an increase in the sub-G1 peak indicating that
the mechanism of action of these compounds probably involves
apoptosis. The flow-cytometry further confirmed a significant
percentage of specific apoptosis that was detected after treatment
with majority of analogues for both exposure times. Western blot
analysis of apoptosis markers (Bcl-2, Bax, caspase 3, PARP) sug-
gested that all analogues induced apoptosis in K562 cells in
caspase-dependent way.
Activation of caspase 3 leads to cleavage of different “down-
stream” proteins, including poly (ADP-ribose) polymerase (PARP),
which is specifically cleaved on a 24 kDa DNA-binding domain to
give a catalytic fragment of 89 kDa [32,33]. PARP participates in
repairing damaged DNA chains during the cell cycle and represent
one of the biochemical hallmarks of apoptosis. Western blot anal-
ysis confirmed the proteolytic cleavage of PARP upon treatment of
K562 cells with all synthesized analogues. In comparison to other
analogues, molecule 3 causes the weakest cleavage of PARP.
4. Experimental section
4.1. General experimental procedures
Melting points were determined on a Büchi 510 apparatus and
were not corrected. Optical rotations were measured on P 3002
(Krüss) 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) and band positions (nmax) are given in cmꢀ1. Low resolu-
tion mass spectra were recorded on Finnigan-MAT 8230 (CI), VG
AutoSpec (FAB) spectrometers and on an Agilent Technologies
HPLC/MS 3Q system, series 1200/6410 (ESI). High resolution mass
spectra (ESI) of synthesized compounds were acquired on an Agi-
lent technologies 6210 TOF LC/MS instrument (LC series 1200).
Column chromatography was performed on Kieselgel 60
(<0.063 mm, E. Merck). Flash column chromatography was per-
formed using Kieselgel 60 (0.040e0.063, E. Merck). Preparative TLC
3. Conclusions
Six tiazofurin mimics (2e7) bearing the nitrogen functionalities
at the C-20 position have been synthesized in multistep sequences
starting from monoacetone D-glucose. The synthetic strategy to
targets 2e7 assumed an initial preparation of the corresponding
ribofuranosyl thioamides as key intermediates followed by their
subsequent cyclo-condensation with ethyl bromopyruvate to form
the thiazole ring. An alternative and more efficient procedure for
the construction of the thiazole ring by avoiding the use of
hydrogen sulphide gas has also been developed. It comprises of
cysteine cyclocondensation with ribofuranosyl cyanide 16,

120
M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
was performed on hand-made plates, 20 ꢂ 20 cm size with ~1 mm
layer thickness. Kieselgel 60 G (E. Merck) with fluorescent indicator
F254 as additive was used as a stationary phase. The corresponding
bands were scraped and eluted with 1:1 EtOAc/iPrOH. 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.
4.6. 2,5-Anhydro-3-azido-6-O-benzoyl-4-O-benzyl-3-deoxy-D-
allononitrile (10)
Prepared from 9 by General procedure A. Purified by column
chromatography (4:1 light petroleum/Me2CO). Yield 40%. Colour-
less syrup, [
a
]
20 þ17.2 (c 1.0, CHCl3). IR (film): nmax 2280, 2100,1730.
D
1H NMR (250 MHz, CDCl3):
d
4.29 (dd, 1H, J2,3 ¼ 3.2, J3,4 ¼ 4.6 Hz, H-
3), 4.33e4.47 (m, 3H, H-4, H-5 and H-6a), 4.52 (dd, 1H, J5,6b ¼ 2.8,
J6a,6b ¼ 11.9 Hz, H-6b), 4.63 and 4.75 (2 ꢂ d, 2H, Jgem ¼ 11.6 Hz,
CH2Ph), 4.65 (d, 1H, J2,3 ¼ 3.2, H-2), 8.09e7.29 (m, 10H, 2 ꢂ Ph). 13
C
4.2. General procedure A: Synthesis of ribofuranosyl cyanides
NMR (62.5 MHz, CDCl3):
d 62.5 (C-6), 64.1 (C-3), 69.0 (C-2), 73.5
A solution of acetal 9, 15, 19 or 28 (1 equiv) in a 4:1 mixture of
TFA/6 M HCl (~0.25 M) was kept at þ4 ꢁC until the starting materials
were consumed (TLC, 70 h for 9, 140 h for 15, 240 h for 19 and 28).
The mixture was concentrated 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 extracts were washed successively with
saturated aq NaHCO3 and water, dried and evaporated. The
remaining crude aldehyde (9a, 15a, 19a, or 28a) was immediately
dissolved in a mixture of EtOH/CH2Cl2 (~0.25 M) and treated with
NH2OH$HCl (~2 equiv) and NaOAc (~3 equiv) while stirring at room
temperature until the starting materials were consumed (TLC, 2 h
for 9a, 15a and 19a; 2.5 h for 28a). The mixture was evaporated 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 (9b, 15b, 19b or 28b) as a
mixture of the corresponding E- and Z-isomers. To a cooled
(ꢀ15 ꢁC) and stirred solution of 9b, 15b, 19b or 28b in anhydrous
pyridine (~0.8 M) was added dropwise during 0.5 h a cold solution
of MsCl (~5 equiv) in dry pyridine (4.6 M). The mixture was first
stirred at 0 ꢁC for 0.5 h, then at room temperature for 2 h and then
poured into a 1:1 mixture of ice and concentrated HCl (pH ~2). 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 on a
column of silica gel.
(CH2Ph), 78.4 (C-4), 80.1 (C-5), 116.3 (C-1), 128.1, 128.4, 128.6, 128. 7,
129.2, 129.7, 133.3, 136.0 (2 ꢂ Ph), 166.0 (PhC ¼ O). LRMS (CI): m/z
379 (MþþH).
4.7. 2,5-Anhydro-3-azido-6-O-benzoyl-4-O-benzyl-3-deoxy-D-
allonothioamide (11)
Prepared from 10 by General procedure B. Purified by column
chromatography (17:1 toluene/EtOAc). Yield 34%. Colourless syrup,
20
[a
]
þ106.3 (c 1.1, CHCl3). IR (film): nmax 2110, 1720, 1536, 1315,
D
1112. 1H NMR (250 MHz, CDCl3):
d
4.02 (dd, 1H, J3,4 ¼ 3.8,
J4,5 ¼ 8.2 Hz, H-4), 4.42 (m, 1H, J5,6a ¼ 2.7, J5,6b ¼ 4.9 Hz, H-5),
4.48e4.59 (m, 3H, PhCHa, 2 ꢂ H-6), 4.67e4.74 (m, 2H, H-2 and H-3),
4.78 (d, 1H, Jgem ¼ 11.6 Hz, PhCHb), 7.22e7.93 (m, 10H, 2 ꢂ Ph), 8.12
and 8.53 (2 ꢂ bs, 2H, NH2). 13C NMR (62.5 MHz, CDCl3):
d 63.1 (C-6),
65.9 (C-3), 72.9 (CH2Ph), 76.6 (C-4), 79.4 (C-5), 87.0 (C-2), 127.9,
128.3, 128.46, 128.5, 129.0, 129.4, 133.4, 136.2 (2 ꢂ Ph), 166.4
(PhC ¼ O), 203.1 (C]S). LRMS (CI): m/z 413 (MþþH).
4.8. 2,5-Anhydro-3-azido-6-O-benzoyl-3-deoxy-D-allonothioamide
(12)
To a cooled (0 ꢁC) and stirred solution of 11 (0.038 g, 0.09 mmol)
in dry CH2Cl2 (2 mL) was added TiCl4 (0.02 mL, 0.18 mmol) in
anhydrous CH2Cl2 (0.3 mL). The mixture was first stirred at 0 ꢁC for
40 min, then poured into a mixture of ice and water and the
resulting suspension was extracted with CH2Cl2 (4 ꢂ 2 mL). The
combined extracts were washed with saturated aq NaHCO3 (1 mL)
and with water (1 mL), dried and evaporated. The residue was
4.3. General procedure B: Synthesis of ribofuranosyl thioamides
purified by column chromatography (8:1 / 4:1 toluene/EtOAc) to
20
give pure 12 (0.020 g, 67%) as a colourless oil, [
a
]
þ46.8 (c 1.0,
D
Through a solution of ribofuranosyl cyanides (1 mmol) in
anhydrous pyridine (0.1e0.2 M) was passed H2S gas at room tem-
perature until the starting materials were consumed (TLC, 2 h for
10, 8 h for 20, 23 and 29). The solvent was evaporated in vacuum
and the residue was purified on a column of silica gel, or by
crystallization.
CHCl3). IR (film): nmax 3350, 2110, 1500, 1310, 1100. 1H NMR
(200 MHz, CDCl3):
d
2.74 (bs, 1H, OH), 4.20 (dd, 1H, J4,5 ¼ 7.0,
J3,4 ¼ 4.9 Hz, H-4), 4.27 (td, 1H, J5,6a ¼ 2.4, J5,6b ¼ 4.9 Hz, H-5),
4.50e4.60 (m, 2H, H-3 and H-6a), 4.66 (dd, 1H, J5,6b ¼ 4.9,
J6a,6b ¼ 13.6 Hz, H-6b), 4.74 (d, 1H, J2,3 ¼ 3.4 Hz, H-2), 7.41e8.09 (m,
5H, Ph), 7.76 and 8.50 (2 ꢂ bs, 2H, NH2). 13C NMR (50 MHz, CDCl3):
d
63.6 (C-6), 68.7 (C-3), 71.1 (C-4), 81.7 (C-5), 86.8 (C-2), 128.7, 129.1,
129.6, 133.8 (Ph), 166.9 (PhC ¼ O), 203.5 (C]S). LRMS (CI): m/z 323
4.4. General procedure C: Hantzsch thiazole synthesis
(MþþH).
To a solution of thioamide 12, 21, 24, 26 or 30 (1 equiv) in ab-
solute EtOH (0.1 M) was added ethyl bromopyruvate (1.5e2 equiv)
and the mixture was stirred under reflux until the starting mate-
rials were consumed (TLC, 50 min for 12, 21, 24 and 30, 80 min for
26). The solvent was evaporated in vacuum and the residue was
purified on a column of silica gel.
4.9. Ethyl 2-(2-azido-5-O-benzoyl-2-deoxy-b-D-ribofuranosyl)
thiazole-4-carboxylate (13)
Prepared Prepared from 12 by General procedure C. Purified by
column chromatography (8:1 / 4:1 toluene/EtOAc). Yield 57%.
20
Colourless syrup, [
a
]
ꢀ2.0 (c 1.0, CHCl3). 1H NMR (250 MHz,
D
CDCl3):
d
1.39 (t, 3H, J ¼ 7.0 Hz, CO2CH2CH3), 3.08 (bs, 1H, OH),
4.5. General procedure D: Ester amonolysis
4.17e4.50 (m, 5H, CO2CH2CH3, H-20, H-30 and H-40), 4.54 (dd, 1H,
J4 ,5 a ¼ 3.9, J5 a,5 b ¼ 12.9 Hz, H-50a), 4.70 (dd, 1H, J5 a,5 b ¼ 12.9,
0
0
0
0
0
0
0
J4 ,5 b ¼ 2.9 Hz, H-50b), 5.29 (d,1H, J1 ,2 ¼ 3.6 Hz, H-1 ), 7.40e8.07 (m,
0
0
0
0
A solution of protected thiazole 13, 18, 22, 25, 27 or 31 (1 mmol)
in 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.
5H, Ph), 8.08 (s, 1H, H-5). 13C NMR (62.5 MHz, CDCl3):
d 14.2
(CO2CH2CH3), 61.6 (CO2CH2CH3), 63.6 (C-50), 67.9 (C-20), 72.3 (C-30),
80.8 (C-10), 81.7 (C-40), 128.1 (C-5), 128.4, 129.6, 129.7, 133.3 (Ph),

M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
121
147.5 (C-4), 161.2 (C-2), 166.4 (PhC ¼ O), 171.0 (CO2CH2CH3). HRMS
(ESIþ): m/e 419.1032 (MþþH). Calcd for C18H19N4O6S: 419.1025.
was added during 15 min. The reaction mixture was stirred for
5 h at 0 ꢁC and then stored for 17 h at þ4 ꢁC. The mixture was
evaporated and the residue was purified by flash column chroma-
4.10. 2,5-Anhydro-3-azido-4,6-di-O-benzoyl-3-deoxy-
ethylene acetal (15)
D
-allose
tography (4:1 cyclohexane/Me2CO) to give pure 18 (0.775 g, 84%) as
20
a colourless oil, [
CDCl3):
a
]
ꢀ65.6 (c 1.8, CHCl3). 1H NMR (250 MHz,
D
d
1.41 (t, 3H, J ¼ 7.0 Hz, CO2CH2CH3), 4.43 (q, 2H,
CO2CH2CH3), 4.58 (dd, 1H, J4 ,5 a ¼ 3.7, J5 a,5 b ¼ 12.0 Hz, H-50a),
0
0
0
0
To a solution of 14 [34] (4.66 g, 9.46 mmol) in DMSO (80 mL) was
added NaN3 (5.34 g, 82.22 mmol), and the resulting suspension was
stirred at 108e110 ꢁC for 24 h. The mixture was poured in water
(200 mL) and extracted with 1:1 benzene-hexane (4 ꢂ 80 mL). The
extract was washed with water (100 mL), dried and evaporated.
The remaining crude mixture was purified by flash column chro-
matography (17:3 cyclohexane/Me2CO) to afford pure 15 (1.95 g,
4.61e4.75 (m, 2H, H-20 and H-40), 4.78 (dd, 1H, J4 ,5 b ¼ 3.3,
0
0
J5 a,5 b ¼ 12.0 Hz, H-50b), 5.41 (d, 1H, J1 ,2 ¼ 5.1 Hz, H-10), 5.64 (t, 1H,
0
0
0
0
J ¼ 5.6 Hz, H-30), 7.38e8.10 (m, 10H, 2 ꢂ Ph), 8.08 (s, 1H, H-5). 13C
NMR (62.5 MHz, CDCl3):
d 14.2 (CO2CH2CH3), 61.5 (CO2CH2CH3),
63.3 (C-50), 66.2 (C-20), 73.6 (C-30), 80.3 (C-10), 81.1 (C-40), 128.1 (C-
5), 128.4, 128.6, 128.9, 129.3, 129.6, 129.9, 133.2, 133.8 (2 ꢂ Ph), 147.7
47%) as a colourless oil, [
a
]
20 ꢀ31.7 (c 1.0, CHCl3). Less pure fractions
(C-4), 161.0 (C-2), 165.5 and 165.9 (2
ꢂ
PhC
¼
O), 169.7
D
were additionally purified by preparative TLC (17:3 cyclohexane/
Me2CO) to give an additional amount of pure product 15 (0.570 g,
total yield 61%). IR (KBr): nmax 2100, 1730. 1H NMR (250 MHz,
(CO2CH2CH3). HRMS (ESIþ): m/e 523.1284 (MþþH). Calcd for
C25H23N4O7S: 523.1287.
CDCl3):
d
3.88e4.10 (m, 4H, 2 ꢂ CH2-dioxolane), 4.22 (dd, 1H,
4.13. 2,5-Anhydro-3-acetamido-4,6-di-O-benzoyl-3-deoxy-D-allose
ethylene acetal (19)
J1,2 ¼ 2.8, J2,3 ¼ 5.3 Hz, H-2), 4.34 (t, 1H, J ¼ 5.7 Hz, H-3), 4.46e4.55
(m, 2H, H-5 and H-6a), 4.62 (dd, 1H, J6a,6b ¼ 13.3, J5,6b ¼ 5.5 Hz, H-
6b), 5.08 (d, 1H, J1,2 ¼ 2.8 Hz, H-1), 5.55 (t, 1H, J ¼ 5.6 Hz, H-4),
7.35e8.13 (m, 10H, 2 ꢂ Ph). NOE contact: H-1 and H-3. 13C NMR
A solution of 15 (0.90 g, 2.05 mmol) in glacial AcOH (4.5 mL) and
Ac2O (4.5 mL), was hydrogenated over PtO2 (0.011 g) at normal
pressure of hydrogen, at rt for 48 h. The suspension was filtered and
the catalyst washed with MeOH (5 mL). The filtrate was concen-
trated and residual AcOH removed by co-distillation with a 1:1
mixture of toluene/MeOH (6 ꢂ 10 mL). Column chromatography on
silica gel (7:3 / 3:7 toluene/EtOAc) gave pure 19 (0.637 g, 68%) as a
(62.5 MHz, CDCl3):
d
60.8 (C-3), 63.8 (C-6), 65.4 and 65.6 (2 ꢂ CH2-
dioxolane), 74.3 (C-4), 79.5 (C-5), 82.2 (C-2), 102.2 (C-1), 128.3,
128.5, 128.7, 129.6, 129.8, 133.1, 133.6 (2 ꢂ Ph), 165.7 and 166.1
(2 ꢂ PhC ¼ O). LRMS (FAB): m/z 462 (MþþNa). Anal. Found: C, 60.10;
H, 4.85; N, 9.80. Calcd for C22H21N3O7: C, 60.14; H, 4.78; N, 9.57.
colourless solid. Recrystallization from a mixture of CH2Cl2/hexane
20
4.11. 2,5-Anhydro-3-azido-4,6-di-O-benzoyl-3-deoxy-
allononitrile (16)
D
-
gave colourless needles, mp 190.5e191.5 ꢁC, [
a]
ꢀ75.6 (c 1.2,
D
CHCl3). IR (KBr): nmax 3300, 1730, 1660, 1570, 1380, 1290. 1H NMR
(250 MHz, CDCl3):
d
1.97 (s, 3H, CH3CO), 3.81e4.05 (m, 4H, 2 ꢂ CH2-
Prepared from 15 (via 15a and 15b) by General procedure A.
dioxolane), 4.02 (dd,1H, J1,2 ¼ 2.9, J2,3 ¼ 9.0 Hz, H-2), 4.51 (m, 2H, H-
5 and H-6a), 4.60 (dd, 1H, J6a,6b ¼ 12.7, J5,6b 5.4 Hz, H-6b), 4.99 (td,
1H, J3,NH ¼ 8.4, J3,4 ¼ 5.8 Hz, H-3), 5.10 (d,1H, J1,2 ¼ 2.9 Hz, H-1), 5.62
(dd, 1H, J4,5 ¼ 1.7, J3,4 ¼ 5.8 Hz, H-4), 5.86 (d, 1H, J3,NH ¼ 8.4 Hz,
AcNH), 7.39e8.12 (m, 10H, 2 ꢂ Ph). 13C NMR (62.5 MHz, CDCl3):
Purified by column chromatography (19:1 toluene/EtOAc). Yield
20
59%. Colourless syrup, [
a]
ꢀ48.9 (c 1.0, CHCl3). IR (film): nmax
D
2280, 2100,1730. 1H NMR (250 MHz, CDCl3):
d 4.56e4.68 (m, 3H, H-
5 and 2 ꢂ H-6), 4.70 (d, 1H, J2,3 ¼ 4.9 Hz, H-2), 4.75 (t, 1H, J ¼ 4.9 Hz,
H-3), 5.74 (t, 1H, J ¼ 4.9 Hz, H-4), 7.40e8.14 (m, 10H, 2 ꢂ Ph). 13
C
d
23.1 (CH3CO), 50.9 (C-3), 64.2 (C-6), 65.4 and 65.7 (2 ꢂ CH2-
NMR (62.5 MHz, CDCl3):
d
62.9 (C-6), 64.7 (C-3), 69.2 (C-2), 73.2 (C-
dioxolane), 75.2 (C-4), 81.3 (C-2), 81.8 (C-5), 102.6 (C-1), 128.4,
128.6, 129.2, 129.55, 129.6, 129.8, 133.1, 133.6 (2 ꢂ Ph), 165.3 and
166.2 (2 ꢂ PhC ¼ O), 169.8 (CH3CO). LRMS (CI): m/z 456 (MþþH).
Anal. Found: C, 62.15; H, 5.64; N, 3.07. Calcd for C24H25NO8∙0.5H2O:
C, 62.06; H, 5.64; N, 3.01.
4), 80.7 (C-5), 116.0 (C-1), 128.1, 128.4, 128.9, 129.1, 129.6, 129.9,
133.3, 134.0 (2 ꢂ Ph), 165.3 and 165.91 (2 ꢂ PhC ¼ O). LRMS (FAB):
m/z 415 (MþþNa). HRMS (ESIþ): m/z 415.1028 (MþþNa). Calcd for
C
20H16N4O5Na: 415.1018.
4.12. Ethyl 2-(2-azido-3,5-di-O-benzoyl-2-deoxy-
b
-D-
4.14. 2,5-Anhydro-3-acetamido-4,6-di-O-benzoyl-3-deoxy-D-
ribofuranosyl)thiazole-4-carboxylate (18)
allononitrile (20)
To a stirred solution of 16 (0.904 g, 2.30 mmol) in dry MeOH
(10 mL) was added -cysteine ethyl ester hydrochloride (0.640 g,
Prepared from 19 (via 19a and 19b) by General procedure A.
Purified by column chromatography (3:2 toluene/EtOAc). Yield 39%.
L
3.45 mmol) followed by Et3N (0.45 mL, 3.22 mmol). The reaction
mixture was stirred for 2 h at room temperature and evaporated.
The residue was purified by flash column chromatography
(19:1 / 9:1 toluene/EtOAc) to afford thiazoline 17 (0.924 g, 76%) as
Colourless needles, mp 156e157 ꢁC (CH2Cl2/hexane), [
a]
20 ꢀ66.0 (c
D
1.4, CHCl3). IR (KBr): nmax 3300, 1730, 1660, 1540, 1275. 1H NMR
(250 MHz, CDCl3):
d 2.00 (s, 3H, CH3CO), 4.59 (bs, 3H, H-5 and
2 ꢂ H-6), 4.77 (d, 1H, J2,3 ¼ 8.1 Hz, H-2), 5.28 (dt, 1H, J3,NH ¼ 8.2,
J3,4 ¼ 6.1 Hz, H-3), 5.61 (dd, 1H, J3,4 ¼ 6.1, J4,5 2.0 Hz, H-4), 6.23 (d,
1H, J3,NH ¼ 8.2 Hz, AcNH), 7.38e8.18 (m, 10H, 2 ꢂ Ph). 13C NMR
a mixture of C-4 epimers. 1H NMR (250 MHz, CDCl3):
d 1.27 (t, 3H,
J ¼ 7.1 Hz, CO2CH2CH3), 3.27e3.61 (m, 2H, 2 ꢂ H-5), 4.22 (q, 2H,
CO2CH2CH3), 4.43e4.71 (m, 4H, H-20, H-40, 2 ꢂ H-50), 4.92 (d, 1H,
(62.5 MHz, CDCl3): d 22.9 (CH3CO), 54.9 (C-3), 63.6 (C-6), 68.9 (C-2),
J1 ,2 ¼ 6.2 Hz, H-10), 5.07e5.18 (2 ꢂ t, 0.51 and 0.37H, J4,5 ¼ 8.8 Hz,
H-4), 5.61 (t, 1H, J ¼ 5.0 Hz, H-30), 7.34e8.12 (m, 10H, 2 ꢂ Ph). 13C
73.5 (C-4), 82.7 (C-5), 116.6 (C-1), 128.4, 128.6, 128.8, 129.0, 129.7,
129.8, 133.5, 134.1 (2 ꢂ Ph), 165.1, 166.2 (2 ꢂ PhC ¼ O), 170.4
(CH3CO). LRMS (CI): m/z 407 (MþꢀH). Anal. Found: C, 64.77; H,
4.78; N, 6.72. Calcd for C22H20N2O6: C, 64.70; H, 4.94; N, 6.86.
0
0
NMR (62.5 MHz, CDCl3):
d
13.8 (CO2CH2CH3), 34.2 and 34.3 (2 ꢂ H-
5), 61.6 (CO2CH2CH3), 63.3 and 63.4 (C-50), 64.3 and 64.5 (C-20), 73.8
(C-30), 80.5 (C-4), 80.7 and 80.8 (C-10 and C-40), 128.1, 128.4, 128.7,
129.2, 129.4, 129.4, 129.6, 132.9, 133.4 (2 ꢂ Ph), 165.2, 165.7, 169.77,
169.8, 173.5, 174.1 (PhC ¼ O, CO2CH2CH3 and C-2).
4.15. 2,5-Anhydro-3-acetamido-4,6-di-O-benzoyl-3-deoxy-D-
allonothioamide (21)
To a stirred solution of 17 (0.924 g, 1.76 mmol) in anhydrous
CH2Cl2 (18 mL) was added DBU (0.53 mL, 3.50 mmol) while stirring.
The solution was cooled to 0 ꢁC and BrCCl3 (0.21 mL, 2.10 mmol)
Prepared from 20 by General procedure B. Purified by crystal-
lization. Yield 86%. Colourless needles, mp 190 ꢁC (Me2CO),

122
20
M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
[
a]
ꢀ111.5 (c 1.1, CHCl3). IR (KBr): nmax 1724, 1655, 1541, 1273. 1H
166.3 (2 ꢂ PhC ¼ O), 167.8 (CO2CH2CH3), 169.8 (PhCONH). LRMS
D
NMR (250 MHz, DMSO-d6):
d
1.78 (s, 3H, CH3CO), 4.45e4.60 (bs, 3H,
(CI): m/z 601 (MþþH). HRMS (ESIþ): m/e 601.1653 (MþþH). Calcd
H-5 and 2 ꢂ H-6), 4.65 (d, 1H, J2,3 ¼ 8.5 Hz, H-2), 4.74 (m, 1H, H-3),
5.40 (dd,1H, J3,4 ¼ 5.2, J4,5 ¼ 2.7 Hz, H-4), 7.41e8.11 (m,10H, 2 ꢂ Ph),
8.54 (d, 1H, J3,NH ¼ 7.9 Hz, AcNH), 9.00 and 9.86 (2 ꢂ bs, 2H, CSNH2).
for C32H29N2O8S: 601.1645.
4.19. 2,5-Anhydro-3-benzamido-4,6-di-O-benzoyl-3-deoxy-D-
allonothioamide (26)
13C NMR (62.5 MHz, DMSO-d6):
d 22.7 (CH3CO), 56.2 (C-3), 65.0 (C-
6), 74.2 (C-4), 81.2 (C-5), 85.1 (C-2), 129.1, 129.2, 129.4, 129.6, 129.8,
130.0, 134.0, 134.2 (2 ꢂ Ph), 165.6 and 166.3 (2 ꢂ PhC ¼ O), 170.3
(CH3CO), 202.5 (C]S). LRMS (FAB): m/z 443.1 (MþþH). HRMS
(ESIþ): m/z 465.1078 (MþþNa). Calcd for C22H22N2O6SNa:
465.1096.
Procedure A. Through a solution of 16 (0.065 g, 0.16 mmol) in
anhydrous pyridine (2 mL) was passed H2S gas at room tempera-
ture for 1.5 h. The mixture was evaporated and the residue was
purified on a column of silica gel (8:1 toluene/EtOAc). Eluted first
was the minor product 16a (0.018 g, 25%) isolated as a colourless oil,
4.16. Ethyl 2-(2-acetamido-3,5-di-O-benzoyl-2-deoxy-
b
-D
-
[a
]
20 ꢀ34.4 (c 1.2, CHCl3). IR (film): nmax 2116, 1716, 1522, 1315, 1111.
D
ribofuranosyl)thiazole-4-carboxylate (22)
1H NMR (250 MHz, DMSO-d6):
d
4.56e4.75 (m, 4H, H-3, H-5 and
2 ꢂ H-6), 4.84 (d, 1H, J2,3 ¼ 4.4 Hz, H-2), 5.46 (t, 1H, J ¼ 5.9 Hz, H-4),
Prepared from 21 by General procedure C. Purified by column
chromatography (7:3 / 7:4 CH2Cl2/EtOAc). Yield 73%. Colourless
7.41e8.15 (m, 10H, 2 ꢂ Ph), 7.76 and 8.50 (2 ꢂ bs, 2H, CSNH2). 13
C
NMR (62.5 MHz, CDCl3): d 63.6 (C-6), 66.5 (C-3), 72.7 (C-4), 80.3 (C-
20
syrup, [
a
]
ꢀ40.2 (c 1.0, CHCl3). IR (film): nmax 1732, 1539, 1372,
5), 86.8 (C-2), 128.5, 128.6, 128.7, 129.0, 129.6, 130.0, 133.7, 133.9
(2 ꢂ Ph),165.6 and 166.7 (2 ꢂ PhC ¼ O), 202.6 (C]S). LRMS (CI): m/z
427 (MþþH).
D
1268. 1H NMR (250 MHz, CDCl3):
d
1.35 (t, 3H, J ¼ 7.0 Hz,
CO2CH2CH3),1.95 (s, 3H, CH3CO), 4.56e4.69 (m, 2H, H-40 and H-50a),
4.82 (dd, 1H, J4 ,5 b ¼ 4.0, J5 a,5 b ¼ 12.5 Hz, H-50b), 4.94 (ddd, 1H,
Eluted second was the major product 26 (0.047 g, 71%) isolated
0
0
0
0
J1 ,2 ¼ 10.1, J2 ,NH ¼ 8.9, J2 ,3 ¼ 5.5 Hz, H-20), 5.45 (d, 1H,
as a colourless solid, mp 188 ꢁC (EtOH), [
a]
20 ꢀ91.9 (c 1.0, Me2CO).
0
0
0
0
0
D
J1 ,2 ¼ 10.1 Hz, H-10), 5.73 (d, 1H, J2 ,3 ¼ 5.5 Hz, H-30), 6.69 (d, 1H,
Procedure B: Through a solution of 16 (0.516 g, 1.32 mmol) and
DMAP (0.005 g, 0.04 mmol) in absolute EtOH (7 mL) was passed
H2S gas at room temperature for 8 h. The mixture was evaporated
and the residue was purified on a column of silica gel (5:1 / 7:3
toluene/EtOAc, 1:1 EtOAc/iPrOH), to afford pure 26 (0.434 g, 82%) as
a colourless solid. Recrystallization from EtOH gave colourless
0
0
0
0
0
J2 ,NH ¼ 8.9 Hz, AcNH), 7.35e8.21 (m, 10H, 2 ꢂ Ph), 8.07 (s, 1H, H-5).
13C NMR (62.5 MHz, CDCl3):
d 14.2 (CO2CH2CH3), 22.9 (CH3CO), 57.8
(C-20), 61.4 (CO2CH2CH3), 75.1 (C-30), 79.5 (C-10), 83.1 (C-40), 128.55,
128.6, 129.0, 129.3, 129.6, 129.8, 133.3, 133.7 (2 ꢂ Ph), 128.4 (C-5),
146.7 (C-4), 160.9 (C-2), 165.4 and 166.2 (2 ꢂ PhC ¼ O), 170.0 and
170.6 (CH3CO and CO2CH2CH3). LRMS (CI): m/z 539 (MþþH). HRMS
(ESIþ): m/e 539.1492 (MþþH). Calcd for C27H27N2O8S: 539.1488.
crystals, mp 188 ꢁC, [
a]
20 ꢀ91.9 (c 1.0, Me2CO). IR (KBr): nmax 3375,
D
1720, 1655, 1535, 1530, 1300, 1275, 1140. 1H NMR (250 MHz, DMSO-
d6):
d
4.17 (m, 1H, J3,4 ¼ 4.3, J4,5 ¼ 3.7 Hz, H-4), 4.27 (m, 1H, H-5),
4.50 (pseudo d, 2H, J5,6 ¼ 4.3 Hz, 2 ꢂ H-6), 4.64 (m,1H, H-3), 4.70 (d,
1H, J2,3 ¼ 7.9 Hz, H-2), 5.61 (d, 1H, J4,OH ¼ 4.9 Hz, OH), 7.42e8.10 (m,
10H, 2 ꢂ Ph), 8.40 (d, 1H, J3,NH ¼ 7.9 Hz, PhCONH), 9.01 and 9.87
4.17. 2,5-Anhydro-3-benzamido-4,6-di-O-benzoyl-3-deoxy-D-
allonothioamide (24)
(2 ꢂ bs, 2H, CSNH2). 13C NMR (62.5 MHz, DMSO-d6):
d 58.2 (C-3),
Prepared from 23 by General procedure B. Purified by crystal-
lization. Yield 74%. Colourless needles, mp 162e163 ꢁC (MeOH),
65.4 (C-6), 71.2 (C-4), 83.0 (C-5), 85.1 (C-2), 127.8, 128.3, 129.0,
129.5, 129.6, 131.4, 133.6, 134.7 (2 ꢂ Ph), 166.0 and 166.4 (PhC ¼ O
and PhCONH), 203.5 (C]S). LRMS (CI): m/z 401 (MþþH). Anal.
Found: C, 59.80; H, 5.23; N, 7.02; S, 8.01. Calcd for C20H20N2O5S: C,
59.99; H, 5.03; N, 7.00; S, 8.00.
20
[
a
]
ꢀ130.9 (c 1.2, CHCl3). IR (KBr): nmax 1722, 1654, 1528, 1316,
D
1273, 1110. 1H NMR (250 MHz, CDCl3):
d 4.44 (m, 1H, H-3), 4.61 (t,
1H, J ¼ 4.1 Hz, H-5), 4.80 (pseudo d, 2H, 2 ꢂ H-6), 5.00 (d, 1H,
J2,3 ¼ 10.4 Hz, H-2), 5.94 (bd, 1H, J3,4 ¼ 5.1 Hz, H-4), 7.21 (d, 1H,
J3,NH ¼ 4.9 Hz, PhCONH), 7.33e8.18 (m, 15H, 3 ꢂ Ph), 7.88 and 8.45
(2 ꢂ bs, 2H, CSNH2). 13C NMR (62.5 MHz, CDCl3):
d
57.4 (C-3), 64.3
4.20. Ethyl 2-(2-benzamido-5-O-benzoyl-2-deoxy-b-D-
ribofuranosyl)thiazole-4-carboxylate (27)
(C-6), 74.4 (C-4), 83.6 (C-5), 84.5 (C-2), 127.1, 128.3, 128.5, 128.52,
128.6, 129.0, 129.1, 129.6, 129.8, 131.8, 133.5, 133.6 (3 ꢂ Ph), 165.6,
167.1 and 167.6 (2 ꢂ PhC ¼ O and PhCONH), 202.4 (C]S). LRMS (CI):
m/z 505 (MþþH). Anal. Found: C, 61.69; H, 5.43; N, 5.05; S, 5.98.
Calcd for C27H24N2O6S$1.5 CH3OH: C, 61.94; H, 5.47; N, 5.07; S, 5.80.
Prepared from 26 by General procedure C. Purified by column
chromatography (7:3 / 3:2 / 1:1 toluene/EtOAc). Yield 55%.
20
Colourless syrup, [
a
]
ꢀ35.8 (c 1.0, CHCl3). IR (film): nmax 3450,
D
1735, 1670, 1530, 1290. 1H NMR (250 MHz, CDCl3):
d 1.26 (t, 3H,
J ¼ 7.0 Hz, CO2CH2CH3), 4.23 (q, 2H, CO2CH2CH3), 4.41e4.82 (m, 6H,
4.18. Ethyl 2-(2-benzamido-3,5-di-O-benzoyl-2-deoxy-b-D-
ribofuranosyl)thiazole-4-carboxylate (25)
H-20, H-30, H-40, 2 ꢂ H-50 and OH), 5.49 (d, 1H, J1 ,2 ¼ 8.2 Hz, H-10),
7.14e8.15 (m, 11H, 2 ꢂ Ph and PhCONH), 8.03 (s, 1H, H-5). 13C NMR
0
0
(62.5 MHz, CDCl3):
d
14.1 (CO2CH2CH3), 60.5 (C-20), 61.5
Prepared from 24 by General procedure C. Purified by column
(CO2CH2CH3), 64.6 (C-50), 72.5 (C-30), 79.5 (C-10), 84.4 (C-40), 127.2,
128.2, 128.4, 129.5, 129.7, 131.6, 133.2, 133.4 (2 ꢂ Ph), 128.2 (C-5),
146.5 (C-4), 161.2 (C-2), 166.4 (PhCO2), 168.6 and 171.7 (CO2CH2CH3
and PhCONH). LRMS (CI): m/z 496 (Mþ). HRMS (ESIþ): m/e 519.1208
(MþþNa). Calcd for C25H24N2NaO7S: 519.1202.
chromatography (4:1 toluene/EtOAc). Yield 78%. Colourless syrup,
[
a]
20 ꢀ102.6 (c 1.2, CHCl3). IR (KBr): nmax 1724, 1665, 1532, 1270. 1H
D
NMR (250 MHz, CDCl3):
d
1.34 (t, 3H, J ¼ 7.0 Hz, CO2CH2CH3), 4.36
(q, 2H, CO2CH2CH3), 4.65e4.75 (m, 2H, J4 ,5a ¼ 3.3 Hz, H-40 and H-
0
0
5a0), 4.90 (dd, 1H, J4 ,5 b ¼ 4.2, J5 a,5 b ¼ 13.0 Hz, H-50b), 5.06 (ddd, 1H,
0
0
0
0
J1 ,2 ¼ 10.1, J2 ,3 ¼ 5.3, J2 ,NH ¼ 8.4 Hz, H-20), 5.66 (d, 1H,
0
0
0
0
0
J1 ,2 ¼ 10.1 Hz, H-10), 5.87 (bd, 1H, J2 ,3 ¼ 5.3, J3 ,4 ¼ 0.6 Hz, H-30),
4.21. 2,5-Anhydro-6-O-benzoyl-3-deoxy-3-(3-
methoxybenzamido)-4-O-(3-methoxybenzoyl)-D-allose ethylene
acetal (28)
0
0
0
0
0
0
0
7.00 (d, 1H, J2 ,NH ¼ 8.4 Hz, PhCONH), 7.32e8.26 (m, 15H, 3 ꢂ Ph),
8.14 (s, 1H, H-5). 13C NMR (62.5 MHz, CDCl3):
d
14.3 (CO2CH2CH3),
58.6 (C-20), 61.4 (CO2CH2CH3), 64.2 (C-50), 75.2 (C-30), 79.4 (C-10),
83.3 (C-40), 128.3 (C-5), 128.5, 128.6, 128.7, 129.0, 129.4, 129.6, 129.8,
131.8, 133.4, 133.5, 133.8 (3 ꢂ Ph), 146.8 (C-4), 161.0 (C-2), 165.4 and
A solution of 9 [35] (2.05 g, 4.82 mmol) in a mixture of absolute
EtOH (70 mL) and CHCl3 (3.45 mL) was hydrogenated over 10% Pd/C

M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
123
(4.57 g) at 1 atm, for 168 h at room temperature. The mixture was
4.24. Ethyl 2-[5-O-benzoyl-2-deoxy-2-(3-methoxybenzamido)-3-
filtered and the catalyst washed with EtOH. The filtrate and
washings were combined and evaporated to give crude 9c as a
colourless solid. Crude 9c was dissolved in anhydrous pyridine
(38 mL) and 3-methoxybenzoyl chloride (2.85 mL, 20.92 mmol)
was added to the solution. The mixture was left at room temper-
ature for 72 h, then poured onto ice and acidified with 6 M HCl (pH
3e4), and the resulting suspension was extracted with CH2Cl2
(4 ꢂ 50 mL). The extract was washed with water (20 mL), saturated
aq NaHCO3 (20 mL) and again with water (20 mL), dried and
evaporated to afford a colourless solid. Recrystallization from
O-(3-methoxybenzoyl)-b-D-ribofuranosyl]thiazole-4-carboxylate
(31)
Prepared from 30 by General procedure C. Purified by column
chromatography (4:1 toluene/EtOAc). Yield 60%. Colourless syrup,
[
a
]
20 ꢀ99.9 (c 1.2, CHCl3). IR (film): nmax 1724, 1666, 1532, 1273. 1H
D
NMR (250 MHz, CDCl3):
d
1.33 (t, 3H, J ¼ 7.1 Hz, CO2CH2CH3), 3.75
and 3.82 (2 ꢂ s, 3H each, 2 ꢂ OCH3), 4.40 (q, 2H, CO2CH2CH3), 4.77
(m, 2H, J4 ,5 a ¼ 3.3 Hz, H-40 and H-50a), 4.96 (dd, 1H, J4 ,5 b ¼ 4.3,
0
0
0
0
J5 a,5 b ¼ 13.0 Hz, H-50b), 5.08 (ddd, 1H, J1 ,2 ¼ 10.1, J2 ,3 ¼ 5.4,
0
0
0
0
0
0
J2 ,NH ¼ 9.1 Hz, H-20), 5.65 (d, 1H, J1 ,2 ¼ 10.1 Hz, H-10), 5.91 (dd, 1H,
0
0
0
MeOH gave pure 28 (1.758 g, 63%) as white crystals, mp
20
159e160 ꢁC, [
a
]
ꢀ56.2 (c 1.0, CHCl3). IR (KBr): nmax 3330, 1730,
J2 ,3 ¼ 5.4, J3 ,4 ¼ 1 Hz, H-30), 7.12 (d, 1H, J2 ,NH ¼ 9.1 Hz, PhCONH),
0
0
0
0
0
D
1640, 1540, 1280. 1H NMR (250 MHz, CDCl3):
d
3.76 and 3.79 (2 ꢂ s,
7.01e8.30 (m, 13H, Ph and 2 ꢂ MeOC6H4), 8.19 (s, 1H, H-5). 13C NMR
3H each, 2 ꢂ OCH3), 3.83e4.10 (m, 4H, 2 ꢂ CH2-dioxolane), 4.21 (dd,
1H, J1,2 ¼ 2.7, J2,3 ¼ 8.7 Hz, H-2), 4.50e4.65 (m, 2H, H-5 and H-6a),
4.66 (dd, 1H, J5,6b ¼ 5.3, J6a,6b ¼ 12.7 Hz, H-6b), 5.12 (m, 1H, H-3),
5.19 (d, 1H, J1,2 ¼ 2.7 Hz, H-1), 5.73 (dd, 1H, J3,4 ¼ 6.4, J4,5 ¼ 1.0 Hz, H-
4), 6.43 (d, 1H, J3,NH ¼ 8.2 Hz, MeOC6H4CONH), 6.94e8.17 (m, 13H,
(62.5 MHz, CDCl3):
d
14.2 (CO2CH2CH3), 55.2 and 55.4 (2 ꢂ OCH3),
58.6 (C-20), 61.4 (CO2CH2CH3), 64.2 (C-50), 75.2 (C-30), 79.4 (C-10),
83.2 (C-40), 112.0, 112.1, 114.1, 118.5, 119.0, 120.2, 121.9, 128.4, 129.3,
129.5, 129.8, 130.2, 133.3, 134.8159.67, 159.7 (Ph and 2 ꢂ MeOC6H4),
128.0 (C-5), 146.8 (C-4), 160.9 (C-2), 165.3, 166.2 and 167.7 (3 ꢂ C]
O), 169.8 (CO2CH2CH3). LRMS (FAB): m/z 683 (MþþNa). HRMS
(ESIþ): m/e 683.1663 (MþþNa). Calcd for C34H32N2O10SNa:
683.1675.
Ph and 2 ꢂ MeOC6H4). 13C NMR (62.5 MHz, CDCl3):
d 51.4 (C-3), 55.3
and 55.4 (2 ꢂ OCH3), 64.2 (C-6), 65.5 and 65.8 (2 ꢂ CH2-dioxolane),
75.3 (C-4), 81.6 (C-2), 81.9 (C-5), 102.6 (C-1), 112.1, 114.2, 118.1, 118.6,
119.9, 121.8, 128.4, 129.6, 129.8, 130.4, 133.1, 135.4, 159.6, 159.8 (Ph
and 2 ꢂ MeOC6H4), 165.2, 166.2 and 167.0 (3 ꢂ C]O). LRMS (CI): m/
z 578 (MþþH). Anal. Found: C, 64.43; H, 5.35; N, 3.11. Calcd for
4.25. 2-(2-Azido-2-deoxy-b-D-ribofuranosyl)thiazole-4-
carboxamide (2)
C
31H31NO10: C, 64.46; H, 5.41; N, 2.42.
Prepared from 13 by General procedure D. Purified by prepar-
4.22. 2,5-Anhydro-6-O-benzoyl-3-deoxy-3-(3-
methoxybenzamido)-4-O-(3-methoxybenzoyl)-
ative TLC (5:1 CHCl3/MeOH). Yield 75% (from 13), 93% (from 18).
20
D
-allononitrile (29)
Colourless needles, mp 160 ꢁC (MeOH/iPr2O), [
CH3OH). For spectral data see ref. 29.
a
]
ꢀ48.2 (c 0.8,
D
Prepared from 28 (via 28a and 28b) by General procedure A.
Purified by column chromatography (17:3 toluene/EtOAc). Yield
4.26. 2-(2-Amino-2-deoxy-b-D-ribofuranosyl)thiazole-4-
carboxamide (3)
50%. Colourless needles, mp 146.5e147.5 ꢁC (MeOH), [
a
]
20 ꢀ83.2 (c
D
1.2, CHCl3). IR (KBr): nmax 3400, 1730, 1640, 1530, 1275. 1H NMR
(250 MHz, CDCl3):
d
3.76 and 3.80 (2 ꢂ s, 3H each, 2 ꢂ OCH3), 4.64
To a stirred solution of 2 (0.133 g, 0.47 mmol) in absolute EtOH
(14 mL) was added 10% Pd/C (0.043 g). The suspension was hy-
drogenated at room temperature and normal pressure of H2 for
24 h. The suspension was filtered through a celite pad, washed with
EtOH and evaporated to a colourless powder. Recrystallization from
(bs, 3H, H-5 and 2 ꢂ H-6), 4.90 (d,1H, J2,3 ¼ 7.6 Hz, H-2), 5.41 (m,1H,
J3,4 ¼ 6.0 Hz, H-3), 5.70 (dd, 1H, J3,4 ¼ 6.0, J4,5 ¼ 2.4 Hz, H-4), 6.76 (d,
1H, J3,NH ¼ 7.9 Hz, MeOC6H4CONH), 6.96e8.22 (m, 13H, Ph and
2 ꢂ MeOC6H4). 13C NMR (62.5 MHz, CDCl3):
d 55.3 and 55.4
(2 ꢂ OCH3), 55.5 (C-3), 63.6 (C-6), 69.3 (C-2), 73.7 (C-4), 82.6 (C-5),
116.6 (C-1), 112.2, 114.4, 116.6, 118.7, 120.3, 121.8, 128.6, 129.1, 129.6,
129.7, 129.8, 133.4, 134.2, 159.7, 159.8 (Ph and 2 ꢂ MeOC6H4), 165.0,
166.2 and 167.4 (3 ꢂ C]O). LRMS (CI): m/z 530 (MþþH). Anal.
Found: C, 65.86; H, 5.21; N, 5.45. Calcd for C29H26N2O8: C, 65.65; H,
4.94; N, 5.28.
a mixture MeOH/iPr2O afforded pure 3 (0.079 g, 65%), as colourless
20
needles, mp 164 ꢁC, [
a
]
ꢀ10.4 (c 0.5, H2O). IR (film): nmax 3433,
D
3365, 3298, 3200, 1688, 1598, 1241. 1H NMR (250 MHz, methanol-
0
0
0
d4):
d
3.36 (m, 1H, H-20), 3.71 (d, 2H, J4 ,5 ¼ 4.7 Hz, H-5 ), 4.08e4.21
(m, 2H, H-30 and H-40), 5.42 (d, 1H, J1 ,2 ¼ 8.5 Hz, H-10), 8.22 (s, 1H,
0
0
H-5). 13C NMR (62.5 MHz, methanol-d4):
d
61.9 (C-20), 63.3 (C-50),
74.1 (C-30), 83.0 (C-10), 88.3 (C-40), 126.3 (C-5), 150.1 (C-4), 166.1 (C-
2), 173.5 (CONH2). LRMS (CI): m/z 260 (MþþH). Anal. Found: C,
41.80; H, 5.46; N, 15.64; S, 11.95. Calcd for C9H13N3O4S: C, 41.69; H,
5.05; N, 16.21; S, 12.37.
4.23. 2,5-Anhydro-6-O-benzoyl-3-deoxy-3-(3-
methoxybenzamido)-4-O-(3-methoxybenzoyl)-D- allonothioamide
(30)
Prepared from 29 by General procedure B. Purified by crystal-
lization. Yield 93%. Colourless needles, mp 149e150 ꢁC (CH2Cl2/
4.27. 2-(2-Amino-2-deoxy-b-D-ribofuranosyl)thiazole-4-
carboxamide amine hydrochloride (4)
hexane), [
a
]
D
20 ꢀ128.0 (c 1.1, CHCl3). IR (KBr): nmax 1721, 1644, 1532,
1317, 1275, 1106. 1H NMR (250 MHz, CDCl3):
d
3.75 and 3.78 (2 ꢂ s,
A solution of 2 (0.076 g, 0.267 mmol) in a mixture of dry EtOH
(7 mL) and CHCl3 (0.7 mL) was hydrogenated over 10% Pd/C
(0.025 g) for 24 h at room temperature. The mixture was filtered
through a celite pad and the catalyst washed with EtOH. The filtrate
and washings were combined and evaporated to give crude 4
(0.070 g, 89%) as a colourless solid. Recrystallization from a mixture
3H each, 2 ꢂ OCH3), 4.38 (m, 1H, H-3), 4.60 (t, 1H, J ¼ 3.6 Hz, H-5),
4.79 (pseudo d, 2H, J5,6 ¼ 3.6 Hz, 2 ꢂ H-6), 4.96 (d, 1H, J2,3 ¼ 10.4 Hz,
H-2), 5.92 (d, 1H, J3,4 ¼ 5.2 Hz, H-4), 7.17 (d, 1H, J3,NH ¼ 4.9 Hz,
PhCONH), 7.76 and 8.46 (2 ꢂ bs, 2H, CSNH2), 6.98e8.20 (m, 13H, Ph
and 2 ꢂ MeOC6H4). 13C NMR (62.5 MHz, CDCl3):
d 55.6 (OCH3), 57.8
(C-3), 64.6 (C-6), 74.8 (C-4), 83.9 (C-5), 84.8 (C-2), 112.4, 114.5, 118.6,
119.3, 120.3,122.3, 129.0, 129.4,129.9, 130.1, 130.7, 134.0, 135.2159.9,
160.0 (Ph and 2 ꢂ MeOC6H4), 165.8, 167.4 and 167.7 (3 ꢂ C]O),
202.8 (C]S). LRMS (CI): m/z 565 (MþþH). Anal. Found: C, 61.47; H,
5.09; N, 5.10; S, 5.79. Calcd for C29H28N2O8S: C, 61.69; H, 5.00; N,
4.96; S, 5.68.
MeOH/iPr2O afforded pure 4, as colourless needles, mp 223 ꢁC,
20
[
a
]
ꢀ78.8 (c 0.9, H2O). IR (film): nmax 3440, 2924, 1670, 1460. 1H
D
0
0
0
0
NMR (250 MHz, D2O):
d
3.74 (dd,1H, J4 ,5 a ¼ 4.9, J5 a,5 b ¼ 12.5 Hz, H-
50a), 3.83 (dd, 1H, J5 a,5 b ¼ 12.5, J4 ,5 b ¼ 3.4 Hz, H-50b), 4.06 (dd, 1H,
0
0
0
0
0
0
0
0
0
0
0 0
J1 ,2 ¼ 7.4, J2 ,3 ¼ 5.8 Hz, H-2 ), 4.24 (m, 1H, J3 4 ¼ 3.8 Hz, H-4 ), 4.53
0
0
0
0
0
0
0
(dd, 1H, J2 ,3 ¼ 5.8, J3 4 ¼ 3.8 Hz, H-3 ), 5.42 (d, 1H, J1 ,2 ¼ 7.4 Hz, H-

124
M. Popsavin et al. / European Journal of Medicinal Chemistry 111 (2016) 114e125
10), 8.25 (s, 1H, H-5). 13C NMR (62.5 MHz, D2O):
d
60.0 (C-20), 64.0
procedure [27]. Results were presented as percent of Annexin V
positive gated cells. Percentage of specific apoptosis was calculated
according to Bender et al. [37].
(C-50), 72.6 (C-30), 80.9 (C-10), 89.5 (C-40), 129.6 (C-5), 151.0 (C-4),
168.0 (C-2), 171.5 (CONH2). LRMS (CI): m/z 296 (MþþH). Anal.
Found: C, 34.48; H, 4.77; N, 13.13; S, 10.35. Calcd for
C9H14ClN3O4S$H2O: C, 34.45; H, 5.14; N, 13.39; S, 10.22.
4.34. Western blot
4.28. 2-(2-Acetamido-2-deoxy-b-D-ribofuranosyl)thiazole-4-
carboxamide (5)
Western blot was carried out following the reported procedure
[27].
Prepared from 22 by General procedure D. Purified by column
Acknowledgement
chromatography (4:1 / 7:3 / 3:2 / 1:1 / 1:2 EtOAc/iPrOH).
20
Yield 62%. Colourless syrup, [
a]
ꢀ11.6 (c 1.1, CH3OH). IR (film):
D
Financial support from the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grant No
172006) is gratefully acknowledged.
nmax 3304,1667,1550, 1377,1295. 1H NMR (250 MHz, methanol-d4):
0
0
d
2.00 (s, 3H, CH3CO), 3.73 (pseudo d, 2H, J4 ,5 ¼ 4.5 Hz, 2 ꢂ H-50),
4.11 (m, 1H, J3 ,4 ¼ 2.8 Hz, H-40), 4.24 (dd, 1H, J2 ,3 ¼ 5.4,
0
0
0
0
0
0
0
0
0
0
0
0
J3 ,4 ¼ 2.8 Hz, H-3 ), 4.49 (dd,1H, J1 ,2 ¼ 8.4, J2 ,3 ¼ 5.4 Hz, H-2 ), 5.09
Appendix A. Supplementary data
(d, 1H, J1 ,2 ¼ 8.4 Hz, H-10), 8.21 (s, 1H, H-5). 13C NMR (62.5 MHz,
0
0
methanol-d4):
d
22.65 (CH3CO), 57.81 (C-20), 63.49 (C-50), 72.57 (C-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.01.037.
30). 80.93 (C-10), 88.62 (C-40), 125.96 (C-5), 150.59 (C-4), 165.60 (C-
2), 172.56 (CH3CO), 173.58 (CONH2). LRMS (CI): m/z 302 (MþþH).
HRMS (ESIþ): m/e 302.0816 (MþþH). Calcd for C11H16N3O5S:
302.0811.
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chromatography (9:1 / 7:3 EtOAc/iPrOH). Yield 72%. Colourless
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J1 ,2 ¼ 8.7 Hz, H, H-10), 7.06e7.51 (m, 4H, MeOC6H4), 8.22 (s, 1H, H-
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5). 13C NMR (62.5 MHz, methanol-d4):
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55.9 (OCH3), 60.7 (C-20),
63.5 (C-50), 72.8 (C-30), 80.6 (C-10), 88.9 (C-40), 113.8, 118.7, 120.6,
130.6, 136.8, 150.6 (MeOC6H4), 126.0 (C-5), 142.3 (C-4), 161.2 (C-2),
170.3 (MeOC6H4CO), 172.4 (CONH2). LRMS (CI): m/z 394 (MþþH).
Anal. Found: C, 51.61; H, 5.18; N, 10.31; S, 8.00. Calcd for
C
17H19N3O6S: C, 51.90; H, 4.87; N, 10.68; S, 8.15.
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