Synthesis and in vitro antitumour screening of 2-(β-d-xylofuranosyl) thiazole-4-carboxamide and two novel tiazofurin analogues with substituted tetrahydrofurodioxol moiety as a sugar mimic

Article abstract of DOI:10.1016/j.bmcl.2012.08.093

2-(β-d-xylofuranosyl)thiazole-4-carboxamide (2) and two new tiazofurin analogues with 5-hydroxymethyl-2-methyl-tetrahydro-furo[2,3-d][1,3]dioxol-6-ol moiety as a sugar mimic (27 and 28) have been synthesized and evaluated for their in vitro antitumour activity against a panel of human tumour cell lines (K562, HL 60, Jurkat, Raji and HeLa). In contrast to previous literature reports, a metabolic MTT assay revealed remarkable cytotoxicity of 2 against K562 (IC₅₀ = 0.15 μM) and HL-60 (IC₅₀ = 0.13 μM) cells. Flow cytometry data suggest that cytotoxic effects of analogue 2 in the culture of K562 cells might be mediated by apoptosis, in opposite to tiazofurin, which did not induce apoptosis of K562 cells after 24 h, thus suggesting a different mechanism of action. All three analogues 2, 27 and 28 were also active against Jurkat, Raji and HeLa cells, with IC₅₀ values in the range from 0.06 to 5.61 μM, but were completely inactive against the normal foetal lung fibroblasts (MRC-5).

Full text of DOI:10.1016/j.bmcl.2012.08.093

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6700–6704
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and in vitro antitumour screening of 2-(b-D-xylofuranosyl)thiazole-
4-carboxamide and two novel tiazofurin analogues with substituted
tetrahydrofurodioxol moiety as a sugar mimic
Mirjana Popsavin a, , Saša Spaic , Miloš Svircev , Vesna Kojic , Gordana Bogdanovic , Velimir Popsavin
⇑
a
a
b
b
a
´
ˇ
´
´
a
´
Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi Sad, Serbia
b Oncology Institute of Vojvodina, Institutski put 4, 21204 Sremska Kamenica, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
2-(b-D-xylofuranosyl)thiazole-4-carboxamide (2) and two new tiazofurin analogues with 5-hydroxy-
Received 27 July 2012
Revised 23 August 2012
Accepted 25 August 2012
Available online 3 September 2012
methyl-2-methyl-tetrahydro-furo[2,3-d][1,3]dioxol-6-ol moiety as a sugar mimic (27 and 28) have been
synthesized and evaluated for their in vitro antitumour activity against a panel of human tumour cell
lines (K562, HL 60, Jurkat, Raji and HeLa). In contrast to previous literature reports, a metabolic MTT assay
revealed remarkable cytotoxicity of 2 against K562 (IC50 = 0.15 lM) and HL-60 (IC50 = 0.13 lM) cells.
Flow cytometry data suggest that cytotoxic effects of analogue 2 in the culture of K562 cells might be
mediated by apoptosis, in opposite to tiazofurin, which did not induce apoptosis of K562 cells after
24 h, thus suggesting a different mechanism of action. All three analogues 2, 27 and 28 were also active
Keywords:
Tiazofurin
C-Nucleosides
Antitumour agent
Thiazole
against Jurkat, Raji and HeLa cells, with IC50 values in the range from 0.06 to 5.61
pletely inactive against the normal foetal lung fibroblasts (MRC-5).
lM, but were com-
Ó 2012 Elsevier Ltd. All rights reserved.
Analogue synthesis
Tiazofurin, 2-(b-
D-ribofuranosyl)thiazole-4-carboxamide (1,
number of malignant cells. The rationale underlying this study
Fig. 1) is a synthetic C-nucleoside that attracted considerable
attention because of its interesting anticancer activity.1 In phase
II clinical trials, it induced haematological responses in patients
with acute myelogenous leukaemia or chronic myeloid leukaemia
in blast crisis.1,2 The biological activity of tiazofurin derives from a
combination of cytotoxicity and maturation-inducing activities.3
Both effects are attributed to inhibition of the enzyme inosine 50-
monophosphate dehydrogenase (IMPDH) by the tiazofurin adenine
dinucleotide (an active metabolite of 1), which binds at the cofac-
tor-binding site and selectively inhibits IMPDH thus inducing the
shutdown of guanylate synthesis.4 Despite the remarkable efficacy
of tiazofurin, lack of specificity and a significant toxicity remains a
problem in its clinical use.1,5 In order to improve therapeutic prop-
erties, a number of structural modifications of the sugar moiety of
tiazofurin have been reported,6 including the synthesis of analogue
arises from our previous findings that tiazofurin analogues of D-
xylo geometry (3 and 4) exhibited more potent antitumour activi-
ties then tiazofurin itself against some human tumour cell
lines.10,11
Our strategy to synthesise the target C-nucleoside 2 was to syn-
thesize the aldononitrile 9 (Scheme 1) as a key intermediate and
then to cyclocondense it with cysteine ester hydrochloride accord-
ing to an adapted literature procedure.12 The commercially avail-
able 1,2:3,5-di-O-isopropylidene-a-D-xylofuranose (5) was used
as a convenient starting material for this part of the work.
Partial acid hydrolysis of 5 gave 1,2-O-isopropylidene-a-D-
xylofuranose (6), which was subsequently treated with benzoyl
chloride in a mixture of dichloromethane and pyridine to afford
1,2-O-isopropylidene-3,5-di-O-benzoyl-a-D-xylofuranose (7) in an
2 with b-
D
-xylofuranosyl moiety as a sugar segment.7 In an initial
-xylofuranosyl)thiazole-4-carboxamide
antitumour assay, 2-(b-
D
(2) was shown to be non-cytotoxic to murine lymphoma P388
cells.7 Subsequent studies revealed that this molecule devoid of
any significant cytotoxicity toward K562 and HL-60 cell lines.8,9
Despite to these discouraging results we decided to synthesize
analogue 2 and to evaluate its antitumour activity against a larger
⇑
Corresponding author. Tel.: +381 21 485 27 68; fax: +381 21 454 065.
E-mail address: mirjana.popsavin@dh.uns.ac.rs (M. Popsavin).
Figure 1. Structure of tiazofurin (1) and its D-xylo-analogues 2, 3 and 4.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.093

M. Popsavin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6700–6704
6701
Scheme 1. Reagents and conditions: (a) 30% AcOH, rt, 24 h, 68%; (b) BzCl, Py, CH2Cl2, rt, 44 h, 99%; (c) glac. AcOH, Ac2O, concd H2SO4, rt, 40 h, 100%; (d) 11 equiv Me3SiCN,
BF3ꢀOEt2, CH2Cl2, rt, 6 h, 21% of 9, 4% of 10, 1.5% of 11; (e) 4.5 equiv Me3SiCN, BF3ꢀOEt2, CH2Cl2, rt, 2 h, 56% of 10, 8% of 11; (f) cysteine ethyl ester hydrochloride, Et3N, MeOH, rt,
2 h; (g) BrCCl3, DBU, CH2Cl2, 0 °C, 5 h, then 4 °C, 68 h, 36% from 9; (h) NH3, MeOH, rt, 6 days, 89%.
almost quantitative yield. An acetolysis of 7 afforded a mixture of
anomeric glycosyl acetates 8. This procedure provided a signifi-
cantly higher yield (67% vs 28%) than previously reported.13 Treat-
ment of 8 with 11 molar equivalents of cyanotrimethylsilane in
dichloromethane, with 0.2 equiv of boron trifluoride etherate as
the catalyst, for 6 h at room temperature, provided a low yield of
xylofuranosyl cyanide 9 (21%), accompanied with traces of cyclic
acetals 10 (4%) and 11 (1.5%). Designation of the b-anomer 9 was
based upon observation of a NOE at H-2 and H-3 when H-4 was
irradiated, indicating that these protons are in close proximity on
the same side of the ring. All other physical and spectral data are
consistent with structures of 9, 10 and 11.14 Stereochemistry of
10 and 11 were also resolved by NOE differential 1H NMR spectros-
copy. It was found that when the methyl group hydrogens of 10
were irradiated, H-4 peak was enhanced, suggesting that the
methyl group and the H-4 have a syn orientation that means the
nitrile group is exo-oriented. This effect was not observed in 11
thus implying to the opposite stereochemistry at the quaternary
C-atom. However, when the methyl group hydrogens of 11 were
irradiated, the H-2 proton signal was enhanced, suggesting that
the methyl group and the H-2 are syn oriented. Such an arrange-
ment is only possible if the isomer 11 has the nitrile group endo-
positioned. Both cyclic acetals 10 and 11 represent convenient
intermediates for the preparation of novel tiazofurin surrogates
(27 and 28) with 5-hydroxymethyl-2-methyl-tetrahydro-
furo[2,3-d][1,3]dioxol-6-ol moiety as a sugar mimic (Scheme 3).
All our attempts to prepare 9 in higher yield failed, but we were
able to find reaction conditions suitable for more efficient prepara-
tion of cyanoethylidene derivatives 10 and 11. Namely, cyclic ace-
tals 10 and 11 are exclusively obtained upon treatment with
4.5 equiv of cyanotrimethylsilane and 0.1 equiv of boron trifluoride
etherate at room temperature for 2 h. Apart from 10 and 11 that
were obtained in 7:1 ratio and 64% combined yield, a minor
amount of starting compound 8 (26%) was also isolated as pure
treated with bromotrichloromethane and DBU to give the thiazole
13 in an overall yield of 36% over the two steps. Treatment of 13
with methanolic ammonia provided the target 2 (89%) as a result
of successive ester aminolysis, O-deacetylation and O-debenzoyla-
tion. This synthetic sequence produced the C-nucleoside 2 in 4.5%
overall yield in eight steps from 5. Spectral data of thus prepared
C-nucleoside 2 were in agreement with the reported data.16
An alternative route for the synthesis of 2 has been developed
starting from the epoxide 14 readily available from D
-glucose.10,17
In a previous Letter10 we have reported a reaction of 14 with
Me3SiN3/BF3ꢀOEt2/CH2Cl2 reagent system, which after treatment
with benzoyl chloride in pyridine, afforded the 6-azido derivative
15b (34%) as the major product, accompanied by a minor amount
(16%) of tri-O-benzoyl derivative 16 (Scheme 2). Both rearranged
products 15a and 16a were formed by the migration of the ben-
zoyloxy group via a regioselective cleavage of oxirane ring at the
C-4 position. It was assumed that performing the reaction without
Me3SiN3 would give tri-O-benzoyl derivative 16, a requisite inter-
mediate for the preparation of target 2. Hence, in the first step
we have focused on the ring opening of epoxide 14 by BF3ꢀOEt2.
The reaction was carried out in dry CH2Cl2, for 46 h at room tem-
perature, to afford 16a. Crude 16a was not purified further, but
was immediately treated with benzoyl chloride in pyridine to af-
ford the expected ester 16 (36% from two steps) accompanied with
a minor amount of 17 (29% from two steps). Nucleophilic displace-
ment of the primary chloride leaving group in 17 with benzoate
anion provided an additional amount of tri-O-benzoyl derivative
16 (52% overall yield from 14). The mechanism of the mentioned
regioselective chlorination at C-6 may be explained by the succes-
sive formation of orthoester derivatives 17a and 17b, followed by
subsequent regiospecific attack of chloride anion at C-6.
Having obtained the key intermediate 16, we next focused on
its further chemical transformations in order to elaborate the thia-
zole carboxamide aglycon at C-1. The sequence started with hydro-
lytic removal of the dioxolane protection in 16, which was
achieved with a 4:1 mixture of trifluoroacetic acid and 6 M hydro-
chloric acid at +4 °C. The resulting aldehyde 18 was not purified
but was treated with hydroxylamine hydrochloride and sodium
acetate immediately giving the oxime derivatives 19 as an insepa-
rable mixture of the corresponding E- and Z-isomers. The mixture
19 was used in the next step without purification and was reacted
with mesyl chloride in pyridine to give the corresponding nitrile 20
a-anomer. Application of the reaction to convert O-protected
b-D-ribofuranoses to b-D-ribofuranosyl cyanides was already
described in the literature.15 In some cases it was found necessary
to elevate the reaction temperature in order to destabilize the
cyanoethylidene by-products and hasten their conversion into
the desired b-nitrile. However, these conditions were ineffective
when applied to the D-xylofuranose derivative 8.
Compound 9 was allowed to react with cysteine ethyl ester
hydrochloride in the presence of triethylamine at room tempera-
ture to afford thiazoline 12 as an inseparable mixture of C-4 epi-
mers. The crude mixture was not separated but was immediately
(65% from 16). Finally, the b-D-xylofuranosyl cyanide 20 was con-
verted to the tiazofurin analogue 2 through the same three-step se-
quence as that already used for the conversion of nitrile 9 to target

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M. Popsavin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6700–6704
Scheme 2. Reagents and conditions: (a) see reference10; (b) BF3ꢀOEt2, CH2Cl2, rt, 46 h; (c) BzCl, Py, rt, 4 days, 36% of 16 (from 14), 29% of 17 (from 14); (d) 4:1 TFA/6 M HCl,
4 °C, 7 days; (e) NH2OHꢀHCl, NaOAc, EtOH, CH2Cl2, rt, 2 h; (f) MsCl, Py, ꢁ15 °C, 0.5 h, then rt, 2 h, 65% from 16; (g) cysteine ethyl ester hydrochloride, Et3N, MeOH, CH2Cl2, rt,
2 h; (h) BrCCl3, DBU, CH2Cl2, 0 °C, 5 h, then 4 °C, 66 h; (i) NH3, MeOH, rt, 7 days, 22% from 20.
Scheme 3. Reagents and conditions: (a) cysteine ethyl ester hydrochloride, Et3N, MeOH, rt, 2 h for 10, 3.5 h for 11; (b) BrCCl3, DBU, CH2Cl2, 0 °C, 5 h, than 4 °C, 4 days for 23,
54% of 25 (from 10), 18 h for 24, 50% of 26 (from 11); (c) NH3, MeOH, rt, 7 days for 25, 89% of 27, 6 days for 26, 90% of 28.
2. The same synthetic methodology was then applied for the prep-
aration of tiazofurin mimics starting from cyanoethylidene deriva-
tives 10 and 11 (Scheme 3). The corresponding tiazofurin mimics
2718 and 2819 were thus obtained ready for biological testing.
The biological activities of the synthesized derivatives 2, 27 and
28 were evaluated by an in vitro cytotoxicity test carried out with a
panel of five human tumour cell lines (myelogenous leukaemia
K562, promyelocytic leukaemia HL 60, Jurkat T cells leukaemia,
Raji Burkitt’s lymphoma, cervix carcinoma HeLa) and one normal
cell line (foetal lung fibroblasts MRC-5). Cell growth inhibition
was evaluated after 72-h cells treatment by using the MTT assay.
Tiazofurin (1) was selected as the positive control.
Table 1
Antiproliferative activities of 1, 2, 27 and 28
a
Compds
IC50
(lM)
K562
HL-60
Jurkat
Raji
HeLa
MRC-5
1
2
27
28
2.09
0.15
0.10
0.02
0.19
0.13
0.05
0.34
0.04
0.53
0.59
0.64
5.28
3.11
2.64
5.61
3.82
0.08
0.06
0.64
0.36
>100
>100
>100
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 done in quadruplicates. Coefficients of variation were <10%.
As shown in Table 1, analogues 2, 27 and 28 exhibited good to
excellent cytotoxic activity toward tumour cells with IC50 values
ranging from 0.02 to 5.61 lM, but were devoid of any cytotoxicity
more active than reference 1, respectively. The most potent anti-
proliferative activity of mimic 28 was recorded in K562 cells,
which was over 100-fold greater than that observed for lead 1.
As the data from Table 1 further reveal, analogue 2 showed a po-
tent antiproliferative activity against both K562 and HL-60 cells.
These findings are in disagreement with the previous biological
data,8,9 which suggested that analogue 2 is inactive against these
cell lines. Such different biological results can be mainly attributed
to differences in experimental conditions used for evaluation of
cytotoxic activity. Accordingly, we checked the antiproliferative
activity of 2 against K562 cells several times. Apart from MTT as-
say, its cytotoxic activity was evaluated by means of SRB, and
against the normal foetal lung fibroblasts (MRC-5). Remarkably,
the synthesized tiazofurin mimics 2, 27 and 28 demonstrated sub-
micromolar activities toward four tumour cell lines under evalua-
tion (K562, HL-60, Jurkat and HeLa). Against three of these cell
lines molecules 2 and 27 were more active than tiazofurin (1).
Compound 28 however, was more potent than lead 1 only against
two malignant cell lines (K562 and HeLa). Analogue 2 demon-
strated the most powerful activity toward HeLa cells being 48-fold
more potent than tiazofurin (1). Mimic 27 exhibited the most pow-
erful activity against HL-60 and HeLa cell lines being 4- and 64-fold

M. Popsavin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6700–6704
6703
DET assays (for details see Supplementary data). Consistent results
obtained by repeated testing in different assays confirmed the
remarkable growth inhibitory activity of 2 against K562 cells in
our experimental conditions.
Tiazofurin (1) and its analogue 2 were also evaluated against
the following human tumour cell lines: colon adenocarcinoma
HT-29, estrogen receptor positive breast adenocarcinoma MCF-7
and estrogen receptor negative breast adenocarcinoma MDA-MB-
231 (data not shown). It has been found that the parent molecule
1 shows a potent antiproliferative activity with IC50 values in the
two malignant cell lines (K562 and HeLa). Surprisingly, analogue 2
showed a potent antiproliferative activity against both K562 and
HL-60 cells. These results are in contrast with previous findings,8,9
which showed that analogue 2 is inactive against these cell lines. It
was concluded that disagreement of these biological data origi-
nates from differences in experimental conditions used for evalua-
tion of cytotoxic activities. Flow cytometry data suggest that
cytotoxic effects of analogue 2 in K562 might be mediated by apop-
tosis, in opposite to tiazofurin, which did not induce apoptosis of
K562 cells after 24 h, thus suggesting a different mechanism of ac-
tion, most probably through the inhibition of IMPDH.
rage from 0.26 to 1.78 lM. In contrast, the analogue 2 was com-
pletely inactive against these cell lines. The difference in cytotoxic
activity indicates that analogue 2 is not acting at the same biolog-
ical target as tiazofurin (IMPDH). It is possible that the observed
cytotoxicities of 2 originated from its ability to induce the apopto-
sis in target cells. In order to address this hypothesis, we examined
the apoptotic signalling induced by tiazofurin and analogue 2 in
the human K562 malignant cell line. Cells were treated with 1
and 2 for 24 and 72 h, at the IC50 concentrations, then stained with
the components of the Annexin-V-FLUOS Staining Kit and eventu-
ally analyzed by flow cytometry, according to the reported proce-
dure.20 Apoptotic response presented as a percentage of specific
apoptosis (Fig. 2) showed that the tiazofurin analogue 2 after 24
h induced several-fold more Annexin-V positive K562 cells com-
pared to the parent compound 1. As Figure 2 shows, analogue 2
after 72 h induced almost seven-fold more Annexin-V positive
K562 cells compared to tiazofurin (1). Flow cytometry data agreed
well with the results of MTT assay explaining the mechanism of
cytotoxic activity of 2 against the K562 cell line (induction of apop-
tosis). Such a different mechanism of action of 2 with respect to
lead 1 is not entirely unexpected, since it has rather different
molecular geometry with respect to 1, which acts as IMPDH inhib-
itor.21 Further work directed to the elucidation of the pathways
underlying the apoptosis with analogue 2 is ongoing and the re-
sults will be reported elsewhere.
Acknowledgment
Financial support from the Ministry of Education and Science of
the Republic of Serbia (Grant No. 172006) is gratefully
acknowledged.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
08.093. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
1. For a brief review on the biological and clinical impact of tiazofurin, see:
Grifantini, M. Curr. Opin. Investig. Drugs 2000, 1, 257.
2. Malek, K.; Boosalis, M.; Waraska, K.; Mitchell, B. S.; Wright, D. G. Leuk. Res.
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3. (a) Wright, D. G.; Boosalis, M. S.; Waraska, K.; Oshry, L. J.; Weintraub, L. R.;
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1991, 3, 61.
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L.; Petrelli, R.; Felczak, K.; Gao, G.; Bonnac, L.; Yu, J. S.; Bennett, E. M.;
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J.; Schinazi, R. F. Curr. Med. Chem. 2004, 11, 887.
In conclusion, the known7 tiazofurin stereoisomer 2-(b-
D-xylo-
furanosyl)thiazole-4-carboxamide (2) and two new tiazofurin
analogues bearing the 5-hydroxymethyl-2-methyl-tetrahydro-
furo[2,3-d][1,3]dioxol-6-ol moiety as a sugar mimic (27 and 28),
have been synthesized and evaluated for their in vitro antitumour
activity against a number of human neoplastic cell lines. Com-
pounds 2, 27 and 28 demonstrated submicromolar activities to-
ward four tumour cell lines (K562, HL-60, Jurkat and HeLa).
Molecules 2 and 27 were more active than tiazofurin (1) against
all of these cells. Compound 28 however, was more potent against
5. (a) Vranic´-Mandušic´, V.; Subota, V.; Savovski, K.; Medic´, L.; Dramic´anin, T.;
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+8.8 (c 1.2; CHCl3); 1H NMR (250 MHz,
Figure 2. Percentage of specific apoptosis of K562 cells induced by tiazofurin and
analogue 2 after 24 and 72 h-treatment. Cells were stained with Annexin-V-FLUOS
and propidium iodide and analyzed by flow cytometry. Percentage of specific
apoptosis of tiazofurin (1) and tiazofurin mimetic 2 was calculated according to
reference.22
14. Compound 9: colourless syrup, [a]
D
CDCl3): d 2.15 (s, 3H, MeCO), 4.63 (d, 2H, J5,6 = 5.8 Hz, H-6), 4.77 (m, 1H, H-5),
4.81 (s, 1H, H-2), 5.54 (s, 1H, H-3), 5.83 (d, 1H, J3,4 = 3.5 Hz, H-4), 7.35–8.16 (m,
10H, 2 ꢂ Ph); 13C NMR (62.9 MHz, CDCl3): d 20.4 (CH3CO), 61.7 (C-6), 70.5 (C-
2), 74.8 (C-4), 79.5 (C-3), 80.1 (C-5), 115.8 (CN), 127.9, 128.3, 128.6, 129.1,

6704
M. Popsavin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6700–6704
´
129.6, 129.9, 133.2, 133.9 (2 ꢂ Ph), 164.7 and 165.9 (2 ꢂ PhCO), 168.9 (CH3CO);
HRMS (ESI): m/z 410.1223 (M++H), Calcd for C22H20NO7: 410.1234.
Compound 10: colourless crystals, mp 127 °C (CH2Cl2/hexane), [
17. Popsavin, V.; Popsavin, M.; Radic´, Lj.; Beric´, O.; Cirin-Novta, V. Tetrahedron Lett.
1999, 40, 9305.
a
]
ꢁ46.2 (c
18. Compound 27: colourless needles, mp. 165 °C (iPr2O/MeOH), [
a
]D=ꢁ13.0 (c 0.3;
D
0.2; CHCl3); 1H NMR (250 MHz, CDCl3): d 1.85 (s, 3H, CH3), 4.59–4.72 (m, 3H,
2 ꢂ H-5 and H-4), 4.91 (d, 1H, J1,2 = 4.1 Hz, H-2), 5.64 (d, 1H, J3,4 = 2.6 Hz, H-3),
6.24 (d, 1H, J1,2 = 4.1 Hz, H-1), 7.33–8.07 (m, 10H, 2 ꢂ Ph); 13C NMR (62.9 MHz,
CDCl3): d 24.2 (CH3), 61.1 (C-5), 75.4 (C-3), 77.8 (C-4), 84.2 (C-2), 99.9 (Cq),
105.5 (C-1), 116.4 (CN), 128.2, 128.3, 128.4, 129.1, 129.5, 129.6, 133.1, 133.7
(2 ꢂ Ph), 164.8 and 165.8 (2 ꢂ PhCO); HRMS (ESI): m/z 432.1041 (M++Na),
Calcd for C22H19NO7Na: 432.1054.
MeOH); 1H NMR (250 MHz, methanol-d4): d 1.88 (s, 3H, CH3), 3.78 (dd, 1H,
J4 ,5a = 6.6, J5 a,5 b = 11.7 Hz, H-50a), 3.86 (dd, 1H, J4 ,5 b = 4.9, J5 a,5 b = 11.7 Hz, H-
0
0
0
0
0
0
0
0
50b), 4.23 (d, 1H, J3 ,4 = 2.8 Hz, H-30), 4.28 (m, 1H, H-40), 4.48 (d, 1H,
0
0
J1 ,2 = 3.8 Hz, H-20), 6.09 (d, 1H, J1 ,2 = 3.8 Hz, H-10), 8.21 (s, 1H, H-5); 13C
NMR (62.9 MHz, methanol-d4): d 26.23 (CH3), 60.8 (C-50), 75.4 (C-30), 83.2 (C-
40), 87.6 (C-20), 107.2 (C-10), 109.9 (Cq), 126.4 (C-5), 151.5 (C-4), 165.4 (C-2),
173.9 (CONH2); HRMS (ESI): m/z 303.0636 (M++H), Calcd for C11H15N2O6S:
303.0645.
0
0
0
0
Compound 11: colourless syrup, [
a
]D=ꢁ9.09 (c 0.4; CHCl3); 1H NMR (250 MHz,
CDCl3): d 1.82 (s, 3H, CH3), 4.57 (dd, 1H, J5a,5b = 11.7, J4,5a = 4.9 Hz, H-5a), 4.66
(dd, 1H, J5a,5b = 11.7, J4,5b = 6.4 Hz, H-5b), 4.86 (d, 1H, J1,2 = 3.8 Hz, H-2), 5.20 (m,
1H, H-4), 5.83 (d, 1H, J3,4 = 4.2 Hz, H-3), 6.20 (d, 1H, J1,2 = 3.8 Hz, H-1), 7.38–
8.07 (m, 10H, 2 ꢂ Ph); 13C NMR (62.9 MHz, CDCl3): d 26.9 (CH3), 62.2 (C-5),
75.6 (C-3), 79.4 (C-4), 86.1 (C-2), 101.6 (Cq), 106.1 (C-1), 117.7 (CN), 128.4,
128.6, 129.6, 129.7, 133.2, 133.3, 133.9 (2 ꢂ Ph), 165.0 and 165.9 (2 ꢂ PhCO);
HRMS (ESI): m/z 432.1041 (M++Na), Calcd for C22H19NO7Na: 432.1054.
15. Utimoto, K.; Wakabayashi, Y.; Horiie, T.; Inoue, M.; Shishiyama, Y.; Obayashi,
M.; Nozaki, H. Tetrahedron 1983, 39, 967.
19. Compound 28: colourless syrup, [
a
]D=ꢁ0.01 (c 0.4; MeOH); 1H NMR (250 MHz,
0
0
0
0
methanol-d4): d 1.78 (s, 3H, CH3), 3.70 (dd, 1H, J4 ,5 a = 6.5, J5 a,5 b = 11.7 Hz, H-
50a), 3.78 (dd, 1H, J4 ,5 b = 5.7, J5 a,5 b = 11.7 Hz, H-50b), 4.07 (m, 1H, H-40), 4.23
0
0
0
0
(d, 1H, J3 ,4 = 2.9 Hz, H-30), 4.71 (d, 1H, J1 ,2 = 3.7 Hz, H-20), 6.12 (d, 1H,
0
0
0
0
J1 ,2 = 3.7 Hz, H-10), 8.23 (s, 1H, H-5); 13C NMR (62.9 MHz, methanol-d4): d 26.0
(CH3), 60.9 (C-50), 75.3 (C-30), 83.5 (C-40), 88.1 (C-20), 107.1 (C-10), 110.6 (Cq),
126.7 (C-5), 151.0 (C-4), 165.4 (C-2), 172.9 (CONH2); HRMS (ESI): m/z 303.0648
(M++H), Calcd for C11H15N2O6S: 303.0645.
0
0
ˇ
´
´
´
20. Popsavin, M.; Svircev, M.; Torovic, Lj.; Bogdanovic, G.; Kojic, V.; Jakimov, D.;
a
]D=ꢁ5.3 (c 0.5; MeOH); 1H NMR (250 MHz,
Spaic, S.; Aleksic, L.; Popsavin, V. Tetrahedron 2011, 67, 6847.
´
´
16. Compound 2: colourless syrup, [
methanol-d4): d 3.90 (dd, 1H, J4 ,5a = 6.1, J5a ,5b = 11.7 Hz, H-5a0), 3.95 (dd, 1H,
21. (a) Franchetti, P.; Marchetti, S.; Cappellacci, L.; Jayaram, H. N.; Yalowitz, J. A.;
Goldstein, B. M.; Barascut, J.-L.; Dukhan, D.; Imbach, J.-L.; Grifantini, M. J. Med.
Chem. 2000, 43, 1264; (b) Makara, G. M.; Keserû, G. M. J. Med. Chem. 1997, 40,
4154; (c) Goldstein, B. M.; Bell, J. E.; Marquez, V. E. J. Med. Chem. 1990, 33, 1123.
22. Bender, A.; Opel, D.; Naumann, I.; Kappler, R.; Friedman, L.; von Scheinitz, D.;
Debatin, K.-M.; Fulda, S. Oncogene 2011, 30, 494.
0
0
0
0
J4 ,5b = 5.0, J5a ,5b = 11.8 Hz, H-5b0), 4.15 (dd, 1H, J2 ,3 = 1.7, J3 ,4 = 3.6 Hz, H-30),
0
0
0
0
0
0
0
0
4.30 (ddd, 1H, J3 ,4 = 3.7, J4 ,5a = 6.1, J4 ,5b = 5.0 Hz, H-40), 4.37 (t, 1H,
0
0
0
0
0
0
J1 ,2 = J2 ,3 = 1.9 Hz, H-20), 5.04 (d, 1H, J1 ,2 = 2.1 Hz, H-10), 8.13 (s, 1H, H-5);
13C NMR (62.9 MHz, methanol-d4): d 61.8 (C-50), 78.6 (C-30), 84.5 (overlapped
C-20 and C-40), 86.4 (C-10), 125.6 (C-5), 150.5 (C-4), 165.9 (C-2), 174.9 (CONH2);
HRMS (ESI): m/z 261.0536 (M++H), Calcd for C9H13N2O5S: 261.0540.
0
0
0
0
0
0