Design, synthesis and in vitro antitumour activity of new goniofufurone and 7-epi-goniofufurone mimics with halogen or azido groups at the C-7 position

Article abstract of DOI:10.1016/j.ejmech.2017.01.024

A series of new antitumour lactones containing the [3.3.0] bicyclic furano-lactone core and the halogen or azido group at the C-7 position have been designed, synthesized, and evaluated for their in vitro antitumour activity against a panel of human tumour cell lines. Some of the analogues displayed powerful antiproliferative effects to certain human tumour cells, but all of them were devoid of any cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). A SAR study reveals the structural features of these lactones that may affect their antiproliferative activity. These are: the nature of substituent present at the C-7 position, stereochemistry at the C-7 position, the absence of phenyl group at the C-7 position. Flow cytometry data indicate that the cytotoxic effects of the synthesized analogues in a culture of K562 cells are mediated by apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these cells. Results of Western blot analysis suggested that the most of synthesized compounds induce apoptosis in K562 cells in caspase-dependent way.

Full text of DOI:10.1016/j.ejmech.2017.01.024

Accepted Manuscript
Design, synthesis and in vitro antitumour activity of new goniofufurone and 7-epi-
goniofufurone mimics with halogen or azido groups at the C-7 position
Jovana Francuz, Ivana Kovačević, Mirjana Popsavin, Goran Benedeković, Bojana
Srećo Zelenović, Vesna Kojić, Dimitar Jakimov, Lidija Aleksić, Gordana Bogdanović,
Tatjana Srdić-Rajić, Eva Lončar, Marko V. Rodić, Vladimir Divjaković, Velimir
Popsavin
PII:
S0223-5234(17)30034-X
10.1016/j.ejmech.2017.01.024
EJMECH 9175
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 December 2016
Revised Date: 30 December 2016
Accepted Date: 16 January 2017
Please cite this article as: J. Francuz, I. Kovačević, M. Popsavin, G. Benedeković, B.S. Zelenović,
V. Kojić, D. Jakimov, L. Aleksić, G. Bogdanović, T. Srdić-Rajić, E. Lončar, M.V. Rodić, V. Divjaković,
V. Popsavin, Design, synthesis and in vitro antitumour activity of new goniofufurone and 7-epi-
goniofufurone mimics with halogen or azido groups at the C-7 position, European Journal of Medicinal
Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.024.
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ACCEPTED MANUSCRIPT
Graphycal Abstract
Design, synthesis and in vitro antitumour activity of new goniofufurone
and 7-epi-goniofufurone mimics with halogen or azido groups at the C-7
position
Jovana Francuz a, Ivana Kovačević a, Mirjana Popsavin a, Goran Benedeković a, Bojana Srećo
Zelenović a, Vesna Kojić c, Dimitar Jakimov c, Lidija Aleksić c, Gordana Bogdanović c, Tatjana
Srdić-Rajić d, Eva Lončar e, Marko V. Rodić a, Vladimir Divjaković b, Velimir Popsavin a
a Department of Chemistry, Biochemistry and Environmental Protection, b Department of Physics,
Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia
c Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put Dr Goldmana
4, 21204 Sremska Kamenica, Serbia
d Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
e Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia

ACCEPTED MANUSCRIPT
Design, synthesis and in vitro antitumour activity of new goniofufurone
and 7-epi-goniofufurone mimics with halogen or azido groups at the C-7
position
Jovana Francuz a, Ivana Kovačević a, Mirjana Popsavin a, Goran Benedeković a, Bojana Srećo
Zelenović a, Vesna Kojić c, Dimitar Jakimov c, Lidija Aleksić c, Gordana Bogdanović c, Tatjana
Srdić-Rajić d, Eva Lončar e, Marko V. Rodić a, Vladimir Divjaković b, Velimir Popsavin a,
a Department of Chemistry, Biochemistry and Environmental Protection, b Department of Physics,
Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad, Serbia
c Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put Dr Goldmana 4,
21204 Sremska Kamenica, Serbia
d Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
e Faculty of Technology, University of Novi Sad, Bulevar Cara Lazara 1, 21000 Novi Sad, Serbia
Abstract
A series of new antitumour lactones containing the [3.3.0] bicyclic furano-lactone core and the
halogen or azido group at the C-7 position have been designed, synthesized, and evaluated for their
in vitro antitumour activity against a panel of human tumour cell lines. Some of the analogues
displayed powerful antiproliferative effects to certain human tumour cells, but all of them were
devoid of any cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). A SAR study
reveals the structural features of these lactones that may affect their antiproliferative activity. These
are: the nature of substituent present at the C-7 position, stereochemistry at the C-7 position, the
absence of phenyl group at the C-7 position. Flow cytometry data indicate that the cytotoxic effects
of the synthesized analogues in a culture of K562 cells are mediated by apoptosis, additionally
revealing that these molecules induced changes in cell cycle distribution of these cells. Results of
Western blot analysis suggested that the most of synthesized compounds induce apoptosis in K562
cells in caspase-dependent way.
Corresponding author. Tel.: +381 21 485 27 68; fax: +381 21 454 065. E-mail address: velimir.popsavin@dh.uns.ac.rs
(V. Popsavin).
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Keywords: styryl lactones; halogen isosteres; analogues; detection of apoptosis; antitumour activity;
structure-activity relationships.
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1. Introduction
Nature is an inexhaustible source of biologically active compounds. Particularly interesting are
styryl lactones, which have been isolated from the Goniothalamus plant genus [1]. Special interest
of medicinal chemists is directed to the styryl lactones with furano-furone scaffold, such as (+)-
goniofufurone (1) and 7-epi-(+)-goniofufurone (2), which have been isolated from the stem bark of
Goniothalamus giganteus [2,3]. As both natural products (1 and 2) and a number of their
derivatives and analogues exhibited promising antitumour properties [1,4,5], they have been the
target of many synthetic efforts [6–8]. We have also been involved in the synthesis of natural styryl
lactones and their analogs [9–11]. Thus, we have recently reported a preliminary account [10] on a
new and facile route to (+)-goniofufurone and 7-epi-(+)-goniofufurone mimics bearing chloro
(analogues 3 and 4, Scheme 1), or bromo functions (analogues 5 and 6) at the C-7 position. The
results of preliminary in vitro antitumour assay [12] confirmed that the replacement of hydroxyl
group at the C-7 position with a halogen may increase the antitumour potency originally displayed
by leads 1 and 2 against several human tumour cell lines [10]. On the other hand, a study carried out
previously in our laboratory showed that 7-dephenyl analogue of 1 (compound 13), and in particular
the corresponding (3S,4R)-stereoisomer (not shown in the scheme) strongly inhibited the growth of
certain human neoplastic cell lines [13]. Based upon these observations, we designed a series of
bicyclic lactones, 3–12, as direct isosteres of 1 and 2. Finally, several new (+)-goniofufurone and 7-
epi-(+)-goniofufurone bioisosteres of type 14–18 have been designed by combining both isosterism
and ring removal methods. Detailed design strategy is outlined in Scheme 1.
<Insert Scheme 1>
2. Results and discussion
2.1. Chemistry
As both titanium(IV) chloride and bromide efficiently catalyzed rapid nucleophilic replacement of
benzoyloxy groups at C-7 with chloro or bromo nucleophiles to afford the corresponding isosteres
3–6 [10], we wanted to use titanium(IV) iodide and the similar methodology to prepare 7-iodo
analogues of (+)-goniofufurone and 7-epi-(+)-goniofufurone (compounds 7 and 8). However,
treatment of 19 with titanium(IV) iodide in dry dichloromethane (Scheme 2) did not afford the
expected 7-iodo derivatives, but resulted in 5-O-debenzylation with the formation of alcohol 20
(Scheme 2). Hence we were forced to look for an alternative procedure based on the Garegg-
Samuelsson reaction [14].
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<Insert Scheme 2>
The synthesis started from the known benzylic alcohols 21 and 22 (Scheme 2) that were previously
prepared in our laboratory starting from D-xylose [15]. Treatment of lactone 21 with iodine,
imidazole and triphenyl phosphine in dry toluene afforded 7-iodo derivate 23 (41%) as the main
reaction product, accompanied with a minor amounts of C-7 epimer 24 (11%) and olefin 25 (10%).
Under the similar reaction conditions, compound 22 gave the expected 7-iodo derivate 24 in a 44%
yield, along with traces of 23 (2%), and a minor amount of olefin 25 (11%). It should be underlined
that the major reaction products in both reactions have inverted configuration at the C-7 position.
Further treatment of compounds 23 or 24 with titanium(IV) iodide in dry dichloromethane gave a
mixture of 7 (17% from 23, 31% from 24,) and 8 (17% from 23, 11% from 24). Obviously,
titanium(IV) iodide apart from debenzylation partly causes the epimerization at the C-7 position.
Stereochemistry of iodo-lactones 7 and 24 were confirmed by single crystal X-ray diffraction
analysis (for the crystal structures of 7 and 24, see the Supplementary data).
Since our recent study showed that treatment of 19 (Scheme 2) with titanium(IV) fluoride does not
give the corresponding 7-fluoro derivatives [16], we decided to apply a different methodology for
the introduction of fluoro groups at the C-7 position. Again, the synthesis of target molecules 9 and
10 starts from known [15] styryl lactones 21 and 22 (Scheme 3).
<Insert Scheme 3>
Treatment of compound 21 with diethylaminosulfur trifluoride (DAST) in dichloromethane gave 7-
deoxy-7-fluoro derivative 26 (63%), as the major product, accompanied with a minor amount of the
C-7 epimer 27 (12%). Under the same reaction conditions, compound 22 gave the expected 7-
deoxy-7-fluoro derivative 27 as a major product (76%), along with a minor amount of C-7 epimer
26 (24%). It appears that the replacement of hydroxyl group at C-7 in both 21 and 22 preferentially
occurs with retention of configuration at the electrophilic position. 5-O-Benzyl protective groups
were removed under the standard reaction conditions (H2, Pd/C, cat. HCl, EtOAc) whereby the
desired 7-fluoro derivatives 9 and 10 were obtained in 70 and 79% yield, respectively.
Stereochemistry of fluoro-lactones 10 and 27 has been unambiguously confirmed by single crystal
X-ray diffraction analysis (for the crystal structures of 10 and 27, see the Supplementary data).
After obtaining the halogen isosteres of natural products 1 and 2, our further activities were focused
on the synthesis of the corresponding 7-azido derivatives. Apart from being an isostere, the azido
group has been extensively used in joining two entirely different building blocks (“click reaction”),
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thus enabling an easy access to highly complex molecular architectures of interest for both
medicinal chemistry and chemical biology [17,18]. In order to prepare azido isosteres 11 and 12 we
have adopted a literature procedure that was previously applied for the epoxide ring opening by
using trimethylsilyl azide/boron trifluoride diethyl etherate reagent system [19,20]. The synthesis of
azido derivatives 11 and 12 started from the known oxetane 28 (Scheme 4) that is readily available
from 7-epi-(+)-goniofufurone in one step [15].
<Insert Scheme 4>
Treatment of 28 with trimethylsilyl azide in dry dichloromethane, in presence of boron trifluoride
diethyl etherate gave the expected analogue 12 as a major product (24%) and the corresponding C-7
epimer 11 (10%) as a minor reaction product. Stereochemistries of these 7-azido-lactones were
unequivocally confirmed by single crystal X-ray diffraction analysis (for the crystal structures of 11
and 12 see the Supplementary data). An unexpected product 29 with unusual pyrano-furone
skeleton was also isolated in 12% yield. 1H NMR data of 29 (J5,6 = 12.4 Hz and H-6/H-3 NOE
contact) indicated that the six-member ring occupies a twist-boat conformation with all substituents
being pseudo-equatorially oriented. The single crystal X-ray analysis of 29, confirmed the twist-
boat geometry of the six-member ring (Fig. 1), as well as the anti (trans-diaxial) arrangement of H-
5 and H-6 (τ = 173.1°), which is in accordance with the observed vicinal coupling of 12.4 Hz.
Finally, the distance between H-3 and H-6 (2.87 Å) in the molecular structure of 29 is consistent
with NOE results.
<Insert Figure 1>
A possible mechanism of this reaction (Scheme 4) involves an initial formation of the stabilized
carbocation 28a. The attack of azide nucleophile from the less hindered re-face gave 12 as the
major product, while the minor stereoisomer 11 is formed as a result of attack to the electrophilic C-
7 position from the more hindered si-face. The formation of pyrano-furone 29 started with the
rearrangement of benzylic carbocation 28a to the intermediate 28b, followed by the carboxonium
ion capture preferentially from the less hindered β-side.
The synthesis of 7-dephenylated goniofufurone analogues bearing a halogen or azido group at the
C-7 position was carried out to examine the role of the phenyl group in the biological activities of
lactones of type 1. Preparation of these analogues (compounds 14–18) is presented in Scheme 5.
<Insert Scheme 5>
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The known furano-lactone 13 [13,21–23], which might be considered as a dephenylated analogue of
goniofufurone (1), was used as a convenient starting material for this part of the work. Apart from
being a starting material in the synthesis, compound 13 will be also used as a positive control in
biological testing of analogues 14–18. Lactone 13 readily reacted with carbon tetrachloride and
triphenyl phosphine in dry dichloromethane, in the presence of pyridine to afford 7-chloro
derivative 14 in 38% yield. The reaction of 13 with carbon tetrabromide and triphenyl phosphine
gave 7-bromo derivative 15 in 28% yield. Absolute value of optical rotation of our sample 15
{[α]20D +10.9 (c 1.8, CHCl3)} is somewhat smaller, but of an opposite sign, than the reported value
for the enantiomer ent-15 {[α]20D −22.0 (c 1.8, CHCl3)} [24]. However, the melting point {lit. [24]
mp = 88–90 °C, found 87–89 °C} and NMR data of synthesized sample 15 were in full agreement
with those reported for ent-15 [24]. Moreover, lactone 13 readily reacted with iodine, imidazole and
triphenyl phosphine, under the standard conditions of the Garegg-Samuelsson reaction [14], to
afford the known [25] 7-iodo derivative 16 in 63% yield. Physical constants (mp and optical
rotation) and NMR spectral data (1H and 13C) of thus obtained sample 16 are in reasonable
agreement with previously reported data [25]. Displacement of 7-iodo function in 16 with azido
group was achieved by treatment of compound 16 with sodium azide in DMF, in the presence of
ammonium chloride, to give the desired compound 18 in 66% yield. The molecular structure of 7-
azido-lactone 18 was confirmed by single crystal X-ray diffraction analysis (for the crystal structure
of 18, see the Supplementary data). The synthesis of dephenylated 7-fluoro derivative 17 was
completed by using the methodology similar to that already used for the preparation of 7-fluoro
derivatives 9 and 10 (Scheme 3). Thus, treatment of known lactone 30 [13] with DAST in
dichloromethane gave a moderate yield of the corresponding 7-fluoro derivative 31 (35%).
Catalytic hydrogenation of 31 over 10% Pd/C, in the presence of traces of concentrated
hydrochloric acid, gave the desired debenzylated derivative 17 in 57% yield.
2.2. In vitro antitumour activities and SAR
After completion of the synthesis, all synthesized compounds were evaluated for their in vitro
cytotoxic activity against a panel of seven human malignant cell lines, including human
myelogenous leukaemia (K562), human promyelocytic leukaemia (HL-60), T cell leukaemia
(Jurkat), Burkitt′s lymphoma (Raji), ER+ breast adenocarcinoma (MCF-7), cervix carcinoma
(HeLa), lung adenocarcinoma epithelial cells (A549) and a single normal cell line – foetal lung
fibroblasts (MRC-5). Cell growth inhibition was evaluated by using the standard MTT colorimetric
assay after exposure of cells to the test compounds for 72 h. (+)-Goniofufurone (1), 7-epi-(+)-
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goniofufurone (2), dephenyl goniofufurone (13) and the commercial antitumour agent doxorubicin
(DOX) were used as positive controls.
<Insert Table 1>
According to the resulting IC50 values of the cytotoxic assay (Table 1), the K562, HL-60, Jurkat,
Raji, HeLa and A549 cell lines were sensitive to all of the synthesized analogues. The highest
potency in the culture of K562 was recorded after treatment with 7-chloro derivative 4 (IC50 0.11
µM), which demonstrated about 2-fold higher potency than the commercial antitumour agent DOX.
A potent submicromolar antiproliferative activity was recorded after treatment of K562 cells with
bromo derivative 6 (IC50 0.98 µM). The highest potency in the HL-60 cell line was recorded after
treatment with 7-bromo isostere 5 (IC50 0.11 µM), which was approximately 8-fold more active
than DOX. All goniofufurone mimics (3, 5, 7, 9 and 11) demonstrated powerful to moderate
antiproliferative effects toward HL-60 cells, in contrast to lead 1, which was completely inactive
against this cell line. With the exception of analogue 8, the remaining four of 7-epi-(+)-
goniofufurone mimics (4, 6, 10 and 12) were more potent than lead 2. The most active compound in
the culture of Jurkat cells was analogue 4 (IC50 0.03 µM), which demonstrated the same potency as
the commercial antitumour drug DOX. The most active molecules against Raji cell line were the
following isosteres: 7-fluoro derivative 10 (IC50 0.03 µM), 7-chloro derivative 3 (IC50 0.04 µM) and
7-iodo derivative 7 (IC50 0.04 µM), which exhibited 75- and 99-fold higher potency than the
commercial antitumour agent DOX (IC50 2.98 µM), as well as 42- and 461-fold stronger activity
with respect to leads 2 and 1, respectively. Several compounds exhibited submicromolar activities
toward the HeLa cells. These are: 7-azido derivative 12 (IC50 0.12 µM), 7-fluoro derivative 17 (IC50
0.52 µM), 7-bromo derivative 5 (IC50 0.68 µM) and 7-chloro derivative 3 (IC50 0.81 µM). However,
the potencies of these analogues were significantly lower than that recorded for DOX in the same
cell line (IC50 0.07 µM). The highest potency in the culture of A549 was recorded after treatment
with dephenyl 7-chloro derivative 14 (IC50 4.02 µM), which demonstrated the similar potency as
DOX in the same cell line. The most potent compound against MCF-7 cells (IC50 0.85 µM) is
dephenyl azido derivative (molecule 18), but the potency of this compound was 4-fold lower than
that recorded for commercial drug doxorubicin. Remarkably, all of the synthesized styryl lactones
including natural products 1 and 2 were completely inactive toward normal MRC-5 cells, in
contrast to the commercial antitumour drug DOX, which exhibited a potent cytotoxic activity (IC50
0.10 µM) against this cell line. These results do suggest that the synthesized styryl lactones
represent selective antitumour agents, but this assumption should be verified by additional in vitro
experiments with additional normal cell lines.
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In order to correlate the structure of synthesized goniofufurone mimics with their cytotoxic
activities, we first considered the effect of halogen and azido groups at C-7 position. The natural
products 1, 2 and synthetic dephenyl goniofufurone 13 have been used as controls in this SAR
analysis. As the data in Table 1 reveal the introduction of halogen or azido group caused the
increased antitumour potency originally displayed by lead 1, but decreased the cytotoxicity
originally demonstrated by lead 2. However, almost each of dephenyl analogues (14–18) displayed
the increased antitumour potency with respect to that originally displayed by the control molecule
13, against the majority of tumour cell lines. However, several interesting exceptions deserve to be
commented: replacement of C-7 hydroxyl group by a bromine in the lead 1 (compound 5) has the
smallest impact on antitumour potency, but produces the most active analogue which exhibited
antitumour activity in a submicromolar range against HL-60 cell line (IC50 0.11 µM). The same
structural modification of lead 2 increased potency of resulting analogue 6 toward HL-60 and Jurkat
cells. Replacement of the hydroxyl group by a fluorine atom in the structure of lead 2 has a small
impact on antitumour potency of analogues against the majority of tumour cells. However it
produces the most active analogue (10) in this study which exhibited antitumour activity in a low
submicromolar range (IC50 0.03 µM) against Raji cell line (for more details see the Supplementary
data, Table S6 and Fig. S8 a–c).
The next round of modifications undertaken to improve the antiproliferative activities have been
performed by a formalremoval of phenyl group from the C-7 position. The relationships between
these structural changes and antiproliferative potencies were established by comparing the IC50
values of dephenyl derivatives (analogues 14–18) with those recorded for the corresponding
halogen and azido derivatives with phenyl group that are arbitrarily used as control molecules
(analogues 3–12). As the data in Table 1 indicate the removal of phenyl group decreased
antiproliferative activity of the chloro, bromo and iodo isosteres, against majority of tumour cell
lines under evaluation. Remarkably, the same structural modification of mimics bearing fluoro and
azido groups increased antitumour potency of resulting analogues (17 and 18), against almost the all
malignant cell lines under evaluation. We also compared the activity of dephenyl derivative 13 with
that of natural products 1 and 2, and found that compound 13 was more active against four cell lines
when compared to goniofufurone (1), but against three cell lines when compared to 7-epi-
goniofufurone (2). (For more details see the Supplementary data, Table S6 and Fig. S9).
Finally, we wanted to explore the effects of absolute stereochemistry at C-7 to antiproliferative
activity of analogues. Our previous studies [10,26] indicated that the styryl lactones having the (7S)-
stereochemistry represent more potent cytotoxic agents with respect to the corresponding (7R)-
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epimers. To further verify this assumption, we compared the IC50 values of six pairs of C-7 epimers
(1 and 2, 3 and 4, 5 and 6, 7 and 8, 9 and 10, 11 and 12), each of which contains exactly the same
substituents and differs only in their absolute stereochemistry at C-7. Epimers with (7R)-absolute
configuration (1, 3, 5, 7, 9 and 11) are considered as controls in this part of SAR analysis. Similarly
to natural products 1 and 2, the (7S)-stereoisomers bearing an azido group (analogue 12) showed a
more potent cytotoxicity than the corresponding (7R)-epimer 11. These results are consistent with
our previous findings [10,26]. In contrast to that, all stereoisomers of (7R)-series with chloro,
bromo, iodo and fluoro groups at the C-7 position (3, 5, 7 and 9) showed increased antitumour
potency then (7S)-epimers (4, 6, 8 and 10) against a majority of malignant cell lines under
evaluation (for details see the Supplementary data, Table S6 and Fig. S8 d). Unfortunately, these
findings disagree with the results of our previous studies [10,26].
2.3.
Cell cycle analysis
The cell passes through a series of events (cell cycle) leading to cell division and duplication. Cells
that actively pass through the cell cycle represent the targets in cancer chemotherapy. We studied
effects of (+)-goniofufurone (1) and analogues (3, 5, 7, 9 and 11), as well as 7-epi-(+)-
goniofufurone (2) and its analogues (4, 6, 8, 10 and 12), and finally dephenyl goniofufurone (13)
and analogues (14, 15, 16, 17 and 18) to the cell cycle perturbations. The cell cycle profile of
exponentially growing K562 cells treated with synthesized compounds for 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 2A (for graphic presentation see Figs. S13, S14 and S15 in the
Supplementary data). As the data in Table 2A indicate a 72-h treatment of K562 cells with
goniofufurone (1) and the corresponding analogues (3, 5, 7, 9 and 11) slightly change the
percentage of cells in G0/G1, S and G2/M phase of cell cycle compared to control. But all of these
compounds increased the percentage of cells in the sub-G1 phase (which is suggestive of apoptosis)
from cca. 2- to 10-fold compared to the control. 7-Azido mimetic of goniofufurone (compound 11)
induced the most prominent sub-G1 peak in K562 cells after 72-h treatment.
<Insert Table 2>
Table 2A further reveals that 7-epi-(+)-goniofufurone (2) and the corresponding analogues (4, 6, 8,
10 and 12), slightly change the percentage of cells in G0/G1, S and G2/M phase of cell cycle
compared to control. However, all analogues increased 2- to 10-fold the percentage of cells in the
sub-G1 phase when compared to untreated control. The exception is 7-epi-(+)-goniofufurone which
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decreased the percentage of cells in the sub-G1 phase with respect to control. 7-Chloro analogue of
7-epi-(+)-goniofufurone (compound 4) induced the most prominent sub-G1 peak in K562 cells after
72-h treatment.
The data in Table 2A also indicate that 72-h treatment of K562 cells with dephenyl goniofufurone
(13) and the corresponding analogues (14–18) slightly change the percentage of cells in G0/G1, S
and G2/M phase of cell cycle. But all of these compounds significantly increased the percentage of
cells in the sub-G1 phase (from 1.5- to 30-fold compared to the control). Bromo, fluoro and azido
derivatives of dephenyl goniofufurone (compound 15, 17 and 18) induced the most prominent sub-
G1 peaks in K562 cells after 72-h treatment.
The obtained results showed that synthesized halogen isosteres of 1 (compounds 3, 5, 7, 9, 11), 2
(compounds 4, 6, 8, 10, 12) and of 13 (compounds 14–18) induced changes in cell cycle distribution
of K562 cells.
2.4. Detection of apoptosis
Cell cycle analysis indicated the pro-apoptotic effect of synthesized analogues through the
formation of sub-G1 peak. Therefore, we further analyzed apoptotic cell death using double staining
with Annexin V-FITC and propidium iodide. Double staining enables detection of cells in the early
phase of apoptosis and clearly discriminates truly necrotic cells from the Annexin V positive cells.
The type of cell death induced by goniofufurone (1), 7-epi-goniofufurone (2), dephenyl
goniofufurone (13) and analogues (3–12, 14–18) in K562 human leukaemia cells, was determined
by flow cytometry after staining, and the results are shown in Table 2B (for a graphic presentation
see Figs. S10, S11 and S12 in the Supplementary data).
Apoptotic response, which was presented as a percentage of specific apoptosis, shows that all
analogues increased the percentage of Annexin V-positive cells compared to parent compound 1.
Compound 3 causes the greatest specific apoptosis (58.36%). As it can be seen, a few analogues
induced several-fold more Annexin V positive cells compared to parent compound 2. Exceptions
are compounds 6 and 12. However, the 7-chloro mimic of 7-epi-goniofufurone (compound 4)
causes the greatest specific apoptosis (38.77%). As it can be further seen, all 7-dephenyl analogues
induced several-fold more Annexin V positive cells when compared to the parent compound 13.
Compounds 17, 18 and 15 cause the greatest specific apoptosis (11.33, 18.10 and 19.59%,
respectively). These results confirmed that 7-chloro analogues 3 and 4, and dephenyl 7-fluoro, 7-
azido and 7-bromo isosteres 17, 18 and 15 increased the percentage of Annexin V positive cells 47-,
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8-, 226-, 362- and 391-fold respectively, compared to parent compounds 1, 2 and 13. These findings
agree well with the results of cell cycle analysis (sub-G1 peak). The highest percentage of specific
necrosis caused analogues bearing 7-bromo, 7-iodo and 7-azido groups, while the analogues with 7-
chloro and 7-fluoro groups showed slightly lower values. But all 7-dephenyl analogues increase the
percentage of specific necrosis to 13.4-fold.
To evaluate the mechanisms underlying the apoptosis induced with synthesized analogues (3–18)
we have studied the ability of these compounds to modulate expression of selected apoptosis
markers, such as Bcl-2, Bax, caspase-3 and Poly (ADP-ribose) polymerase (PARP). The results are
presented in Table 3 (for more details see the Supplementary data, Figs. S16, S17 and S18).
<Insert Table 3>
Semi-quantitative Western blot analysis revealed that the majority of examined compounds (1–5, 7–
12 with the exception of 6, 13–18) increased expression of anti-apoptotic Bcl-2 protein in K562
cells when compared to control. The majority of tested compounds (with the exception of 1, 15, 16
and 17) reduced expression of pro-apoptotic protein Bax (Bcl-2 associated protein X) which, when
activated, forms the oligomers that disrupt outer mitochondrial membrane. Cytochrome c released
from damaged mitochondria promotes caspase-9 activation on the scaffold protein APAF1 [27,28].
Caspases, a family of aspartate-specific cysteine proteases, play central role in the mechanism of
apoptosis as they both initiate and execute the apoptotic process. The expression level of the
caspase-3 precursor and active subunit of caspase-3 effector, as well as catalytic fragment of PARP
in the cells exposed to synthesized compounds were measured in order to determine whether
apoptosis was associated with the activation of caspases. Caspase-3 activation is followed by
cleavage of different downstream targets including poly ADP-ribose polymerase (PARP), which is
specifically cleaved on a DNA-binding domain and a catalytic fragment [29]. Western blot analysis
showed over-expression of both caspase-3 active subunit and cleaved catalytic PARP fragment (85
kD) in K562 cells after treatment with lactones 9, 11, 12 and 18 while all other synthesized
compounds (with exception of bromo derivative 6) influenced only PARP cleavage. During the
execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases and C-terminal
apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with
caspase-3 while N-terminal apoptotic fragment, retains DNA-binding activity and inhibits the
catalytic activity of uncleaved PARP-1 [29]. These results suggest that all the investigated
compounds (with exception of 6) induce apoptosis of K562 cells in caspase-dependent way.
11

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Biochemical changes in apoptosis, DNA fragmentation and caspase activation, may explain in part
some of the morphological changes in apoptosis. Therefore, it is important to note that apoptosis
may occur without oligonucleosomal DNA fragmentation and can be caspase-independent [30].
3. Conclusions
In conclusion, fifteen new analogues of goniofufurone (1) and 7-epi-goniofufurone (2), bearing a
halogen or azido functionality at the C-7 position, were designed and synthesized in this work. All
the synthesized compounds were tested in vitro toward a panel of human tumour cell lines, and the
structure–activity relationships are briefly discussed. The ability of synthesized compound to induce
apoptosis and to change the distribution of K562 cell cycle was also studied. Some of the
synthesized compounds showed potent antitumour activity, especially analogues 3, 7, 10 (0.03–0.04
µM against Raji), and 4 (0.03 µM against Jurkat), which displayed the highest activity of all
compounds under evaluation.
The preliminary SAR analysis suggested the following structural requirements for the
antiproliferative action of synthesized analogues: (A) the presence of halogen or azido group at C-7
increases the antitumour potency originally displayed by lead 1, but decreases the cytotoxicity
originally demonstrated by lead 2; (B) removal of the phenyl group may increase or decrease
antiproliferative activity depending on the nature of substituent at the C-7 position; (C) generally,
styryl lactones having the (7S)-stereochemistry represent more potent cytotoxic agents than the
corresponding (7R)-isomers, but the activity also depends on the nature of substituent present at the
C-7 position.
The cell cycle analysis reveals that treatment of cells with synthesized compounds increases
percentage of K562 cell in sub G1 phase indicating that the mechanism of action of these
compounds probably involves apoptosis. The flow-cytometry further confirmed a significant
percentage of specific apoptotic cells that was detected after treatment with majority of analogues.
Compound 3 causes the greatest specific apoptosis (58.36%). Western blot analysis of apoptosis
markers (Bcl-2, Bax, caspase-3, PARP) suggested that all of the synthesized compounds (with
exception of 6) induced apoptosis in K562 cells in caspase-dependent way.
4. Experimental section
4.1. Chemistry
4.1.1. General experimental procedures
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Melting points were determined on a Hot Stage Microscope Nagema PHMK 05 and were not
corrected. Optical rotations were measured on an Autopol IV (Rudolph Research) polarimeter at
room temperature. NMR spectra were recorded on a Bruker AC 250 E or a Bruker Avance III 400
MHz instrument and chemical shifts are expressed in ppm downfield from TMS. IR spectra were
recorded with an FTIR Nexus 670 spectrophotometer (Thermo-Nicolet). High resolution mass
spectra (ESI) of synthesized compounds were acquired on a Agilent Technologies 1200 series
instrument equipped with Zorbax Eclipse Plus C18 (100 mm × 2.1 mm i.d. 1.8 µm) column and
DAD detector (190–450 nm) in combination with a 6210 time-of-flight LC/MS instrument (ESI) in
the positive ion mode. Flash column chromatography was performed using Kieselgel 60 (0.040–
0.063, E. Merck). Preparative TLC was performed on hand-made plates, 20 × 20 cm size with ~1
mm layer thickness. Kieselgel 60 G (E. Merck) with fluorescent indicator F254 as additive was used
as stationary phase. 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 diminished pressure at a bath temperature below 35 °C. The purities of the tested
compounds were established by HPLC (compounds 3–13 and 15–18) or by combustion analysis
(compound 14) and were found to be >95% pure. HPLC analysis was carried out using an Agilent
1100 HPLC system (Agilent Technologies, Palo Alto, CA, USA) with DAD detector and Zorbax
SB-C 18 column (4.6 × 150 mm, 5 µm). Eluent: acetonitrile-water (HPLC grade); gradient elution:
60–100% acetonitrile; flow rate: 0.5 mL/min. Temperature: 25 °C. Detection: 254 nm (compounds
3–10, 12, 13 and 16) and 210 nm (compounds 11, 15, 17 and 18). For details see the Supplementary
data.
The procedures for preparation of 7-chloro (3 and 4) and 7-bromo derivatives (5 and 6) are
described in reference 10.
4.1.2. 3,6-Anhydro-7-O-benzoyl-2-deoxy-7-C-phenyl-D-glycero-D-ido-heptono-1,4-lactone (20)
To a cooled (0 °C) and stirred solution of 19 (0.103 g, 0.23 mmol) in anhydrous CH2Cl2 (6 mL) was
added TiI4 (0.419 g, 0.75 mmol). The mixture was stirred at 0 °C for 0.5 h and then at room
temperature for 3.5 h. The mixture was poured onto ice and water, and extracted with CH2Cl2. The
combined extracts were washed with 10% aq NaHCO3, dried and evaporated. The residue was first
purified by flash column chromatography (19:1 CH2Cl2/EtOAc) and then by preparative TLC (1:1
toluene/EtOAc) to give pure 20 (0.042 g, 55%) as a colourless syrup, [α]20D = +50.0 (c 1.0, CHCl3),
Rf = 0.25 (19:1 CH2Cl2/EtOAc). IR (film): νmax 3454 (OH), 1787 (C=O, lactone), 1722 (PhC=O).
1H NMR (250 MHz, CDCl3): δ 1.30 (br s, 1H, OH), 2.55 (d, 1H, J2a,2b = 18.8 Hz, H-2a), 2.67 (dd,
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1H, J2a,2b = 18.8, J2b,3 = 5.1 Hz, H-2b), 4.32 (dd, 1H, J5,6 = 2.1, J6,7 = 9.2 Hz, H-6), 4.45 (d, 1H, J5,6
= 2.1 Hz, H-5), 4.95–5.04 (m, 2H, H-3 and H-4), 6.12 (d, 1H, J6,7 = 9.3 Hz, H-7), 7.35–8.14 (m,
10H, Ph). 13C NMR (62.9 MHz, CDCl3): δ 35.8 (C-2), 73. 2 (C-5), 73.2 (C-7), 77.1 (C-3), 82.5 (C-
6), 87.0 (C-4), 127.6, 128.6, 128.7, 128.9, 129.9, 133.9, 136.8 (Ph), 166.9 (PhC=O), 175.40 (C-1).
HRMS (ESI): m/e 377.0984 (M+ + Na), calcd for C20H18NaO6: 377.0996; m/e 393.0723 (M+ + K),
calcd for C20H18KO6: 393.0735.
4.1.3. Preparation of 7-iodo derivatives 23 and 24
Procedure A. To a solution of 21 (0.081 g, 0.24 mmol) in dry toluene (5.5 mL) was added iodine
(0.118 g, 0.47 mmol), imidazole (0.043 g, 0.64 mmol) and Ph3P (0.162 g, 0.62 mmol). The mixture
was stirred at 70 °C for 3 h, then evaporated. The mixture of 23, 24 and 25 was first purified by
flash column chromatography (3:2 light petroleum/Et2O) and then by preparative TLC (3:2
CH2Cl2/light petroleum, 5 successive developments). Eluted first was the minor product 25 (0.008
g, 10%) that was isolated in the form of colourless needles, mp 165 °C, [α]20D = +7.5 (c 0.5,
CHCl3), Rf = 0.52 (CH2Cl2). Further elution gave pure compounds 23 (41%) and 24 (11%).
Procedure B. To a cooled (0 °C) and stirred solution of 22 (0.099 g, 0.29 mmol) in anhydrous
CH2Cl2 (1.5 mL) was added 2,6-lutidine (0.11 mL, 0.94 mmol), Ph3P (0.244 g, 0.93 mmol) and
iodine (0.246 g, 0.97 mmol). The mixture was stirred at 0 °C for 0.5 h and then at room temperature
for 28 h, under nitrogen atmosphere. The mixture was poured in 1 M HCl (pH 1), and extracted
with CH2Cl2. The combined extracts were washed with 10% aq NaCl (pH 7), dried and evaporated.
The mixture of 23, 24 and 25 was first purified by flash column chromatography (3:2 light
petroleum/Et2O) and then by preparative TLC (3:2 CH2Cl2/light petroleum, 5 successive
developments). Eluted first was the minor product 25 (0.011 g, 11%). Further elution gave pure
compounds 23 (2%) and 24 (44%).
3,6-Anhydro-5-O-benzyl-2,7-dideoxy-7-iodo-7-C-phenyl-L-glycero-D-ido-heptono-1,4-lactone (23)
Colourless needles, mp 136–138 °C, [α]20D = +35.0 (c 0.5, CHCl3), Rf = 0.30 (CH2Cl2). IR (film):
νmax 1789 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.76 (dd, 1H, J2a,2b = 18.9, J2a,3 = 5.8 Hz, H-2a),
2.88 (bd, 1H, J2a,2b = 18.6 Hz, H-2b), 3.78 (d, 1H, J5,6 = 3.2 Hz, H-5), 4.08 and 4.24 (2×d, 2H, Jgem
= 11.3 Hz, CH2Ph), 4.69 (dd, 1H, J5,6 = 3.2, J6,7 = 10.2 Hz, H-6), 4.92 (d, 1H, J3,4 = 4.5 Hz, H-4),
5.07 (bt, 1H, J3,4 = 4.6 Hz, H-3), 5.29 (d, 1H, J6,7 = 10.2 Hz, H-7), 7.08–7.48 (m, 10H, 2×Ph). 13C
NMR (62.9 MHz, CDCl3): δ 27.8 (C-7), 35.9 (C-2), 72.9 (CH2Ph), 77.7 (C-3), 79.3 (C-5), 85.1 (C-
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4), 85.5 (C-6), 127.5, 127.8, 128.2, 128.4, 128.5, 128.8, 136.4, 140.5 (2×Ph), 175.1 (C-1). HRMS
(ESI): m/e 468.0658 (M++NH4), calcd for C20H23INO4: 468.0666.
3,6-Anhydro-5-O-benzyl-2,7-dideoxy-7-iodo-7-C-phenyl-D-glycero-D-ido-heptono-1,4-lactone (24)
Colourless needles, mp 149–150 °C, [α]20D = −9.8 (c 0.5, CHCl3), Rf = 0.38 (CH2Cl2). IR (film):
νmax 1789 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.54 (d, 1H, J2a,2b = 18.9 Hz, H-2a), 2.64 (dd, 1H,
J2b,3 = 4.8, J2a,2b = 18.9 Hz, H-2b), 4.63 (d, 1H, J5,6 = 2.9 Hz, H-5), 4.75 (d, 1H, Jgem = 10.9 Hz,
CH2Ph), 4.81–5.03 (m, 4H, CH2Ph, H-3, H-4 and H-6), 5.31 (d, 1H, J6,7 = 10.7 Hz, H-7), 7.28–7.54
(m, 10H, 2×Ph). 13C NMR (62.9 MHz, CDCl3): δ 26.2 (C-7), 36.4 (C-2), 74.2 (CH2Ph), 78.8 (C-3),
82.7 (C-5), 84.1 and 84.2 (C-4 and C-6), 127.8, 128.3, 128.4, 128.43, 128.7, 128.8, 136.9, 140.8
(2×Ph), 175.1 (C-1). HRMS (ESI): m/e 468.0653 (M++NH4), calcd for C20H23INO4: 468.0666.
Olefin 25
Colourless needles, mp 165 °C, [α]20D = +7.5 (c 0.5, CHCl3), Rf = 0.52 (CH2Cl2). IR (film): νmax
1793 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.83 (dd, 1H, J2a,2b = 18.6, J2a,3 = 4.6 Hz, H-2a), 2.94
(d, 1H, J2a,2b = 18.6 Hz, H-2b), 4.60 (s, 1H, H-5), 4.61 and 4.79 (2×d, 2H, Jgem = 11.5 Hz, CH2Ph),
4.94 (d, 1H, J3,4 = 3.8 Hz, H-4), 5.34 (t, 1H, J2a,3 = 4.2, J3,4 = 4.0 Hz, H-3), 5.55 (s, 1H, H-7), 7.16–
7.62 (m, 10H, 2×Ph); NOE contact: H-5 and H-7. 13C NMR (62.9 MHz, CDCl3): δ 35.8 (C-2), 70.6
(CH2Ph), 81.0 (C-3), 81.5 (C-5), 83.5 (C-4), 105.7 (C-7), 126.5, 127.9, 128.2, 128.3, 128.6, 128.65,
134.6, 136.8 (2×Ph), 152.0 (C-6), 173.8 (C-1). HRMS (ESI): m/e 323.1270 (M++H), calcd for
C20H19O4: 323.1278; m/e 345.1089 (M++Na), calcd for C20H18NaO4: 345.1097; m/e 361.0838
(M++K), calcd for C20H18KO4: 361.0837.
4.1.4. General procedure for the preparation of 7-iodo analogues 7 and 8
To a cooled (0 °C) and stirred solution of 23 or 24 (1 equiv) in dry CH2Cl2 (0.04 M) was added TiI4
(2.5–2.7 equiv). The mixture was first stirred at 0 °C for 0.5 h and then at room temperature until
the starting materials were consumed (TLC, 3 h for 23, 2.5 h for 24). The mixture was poured onto
ice and water and the resulting suspension was extracted with CH2Cl2. The combined extracts were
washed with saturated aq NaHCO3, dried and evaporated. The residue was first purified by flash
column chromatography (19:1 CH2Cl2/EtOAc), and then by preparative TLC (9:1 CH2Cl2/EtOAc, 3
successive developments), to afford pure 7 and 8.
3,6-Anhydro-2,7-dideoxy-7-iodo-7-C-phenyl-D-glycero-D-ido-heptono-1,4-lactone (7). Yield: 31%
(from 24); 17% (from 23). Colourless needles, mp 123–125 °C, [α]20D = −71.2 (c 0.5, CHCl3), Rf =
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0.49 (4:1 CH2Cl2/EtOAc). IR (KBr): νmax 3396 (OH), 1752 (C=O). 1H NMR (250 MHz, CDCl3): δ
2.62 (d, 2H, J2a,2b = 17.4 Hz, H-2a and OH), 2.66 (dd, 1H, J2b,3 = 5.0, J2a,2b = 18.8 Hz, H-2b), 4.84–
4.91 (m, 2H, J5,6 = 2.7 Hz, H-5 and H-6), 4.96 (d, 1H, J3,4 = 4.2 Hz, H-4), 5.04 (m, 1H, H-3), 5.21
(d, 1H, J6,7 = 10.5 Hz, H-7), 7.28–7.54 (m, 5H, Ph). 13C NMR (62.9 MHz, CDCl3): δ 26.8 (C-7),
36.4 (C-2), 75.8 (C-5), 78.7 (C-3), 84.1 (C-6), 86.9 (C-4), 127.8, 128.5, 128.9, 140.5 (Ph), 175.2 (C-
1). HRMS (ESI): m/e 378.0196 (M++NH4), calcd for C13H17INO4: 378.0197.
3,6-Anhydro-2,7-dideoxy-7-iodo-7-C-phenyl-L-glycero-D-ido-heptono-1,4-lactone (8). Yield: 17%
(from 23); 11% (from 24). Colourless needles, mp 204–206 °C, [α]D = +38.6 (c 0.5, CHCl3), Rf =
0.44 (4:1 CH2Cl2/EtOAc). IR (CHCl3): νmax 3440 (OH), 1739 (C=O). 1H NMR (250 MHz, CDCl3):
δ 2.77 (dd, 1H, J2a,2b = 18.9, J2a,3 = 5.7 Hz, H-2a), 2.88 (d, 1H, J2a,2b = 18.8 Hz, H-2b), 4.09 (br s,
1H, H-5), 4.61 (d, 1H, J5,6 = 2.7, J6,7 = 10.1 Hz, H-6), 4.95 (d, 1H, J3,4 = 4.3 Hz, H-4), 5.10 (m, 1H,
H-3), 5.24 (d, 1H, J6,7 = 10.1 Hz, H-7), 7.28–7.51 (m, 5H, Ph). 13C NMR (62.9 MHz, CDCl3): δ
27.2 (C-7), 35.9 (C-2), 72.2 (C-5), 76.4 (C-3), 85.7 (C-6), 88.0 (C-4), 127.4, 128.5, 129.1. 140.2
(Ph), 175.1 (C-1). HRMS (ESI): m/e 378.0194 (M++NH4), calcd for C13H17INO4: 378.0197.
4.1.5. General procedure for the preparation of 7-fluoro derivatives 26 and 27
To a cooled (0 °C) and stirred solution of 21 or 22 (1 equiv) in dry CH2Cl2 (0.17 M) was added
DAST (3.0–3.1 equiv). The mixture was first stirred at 0 °C for 0.5 h and then at room temperature
until the starting materials were consumed (TLC, 3.5 h for 21, 2.5 h for 22). The mixture was
suspended in cold 10% aq NaHCO3, and the resulting suspension was extracted with CH2Cl2, dried
and evaporated. The residue was then purified by flash column chromatography (4:1 CH2Cl2/light
petroleum), to give pure products 26 and 27.
3,6-Anhydro-5-O-benzyl-2,7-dideoxy-7-fluoro-7-C-phenyl-D-glycero-D-ido-heptono-1,4-lactone
(26). Yield: 63% (from 21); 24% (from 22). Colourless needles, mp 89 °C, [α]D = +29.4 (c 0.5,
CHCl3), Rf = 0.30 (4:1 CH2Cl2/light petroleum). IR (KBr): νmax 1785 (C=O). 1H NMR (250 MHz,
CDCl3): δ 2.54 (d, 1H, J2a,2b = 18.5 Hz, H-2a), 2.66 (dd, 1H, J2a,2b = 18.5 Hz, H-2b), 4.34 (m, 1H,
J5,6 = 3.1, J6,7 = 8.8, JF,6 = 4.6 Hz, H-6), 4.46 (d, 1H, J5,6 = 3.1 Hz, H-5), 4.75 and 4.82 (2×d, 2H,
Jgem = 11.6 Hz, CH2Ph), 4.95 (m, 2H, H-3 and H-4), 5.70 (dd, 1H, J6,7 = 8.7, JF,7 = 44.8 Hz, H-7),
7.20–7.49 (m, 10H, 2×Ph). 13C NMR (62.9 MHz, CDCl3): δ 35.8 (C-2), 72.6 (CH2Ph), 77.5 (C-3),
80.8 (C-5), 81.8 (d, JF,6=36.2 Hz, C-6), 85.1 (C-4), 89.8 (d, JF,7=167.8 Hz, C-7), 126.7, 127.9,
128.3, 128.4, 128.6, 128.8, 137.0, 137.2 (2×Ph), 175.0 (C-1). HRMS (ESI): m/e 360.1608
(M++NH4), calcd for C20H23FNO4: 360.1606.
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3,6-Anhydro-5-O-benzyl-2,7-dideoxy-7-fluoro-7-C-phenyl-L-glycero-D-ido-heptono-1,4-lactone
(27). Yield: 12% (from 21); 76% (from 22). Colourless plates, mp 123–124 °C, [α]20D = +71.8 (c
0.5, CHCl3), Rf = 0.19 (4:1 CH2Cl2/light petroleum). IR (KBr): νmax 1793 (C=O). 1H NMR (250
MHz, CDCl3): δ 2.63–2.87 (m, 2H, 2×H-2), 3.85 (d, 1H, J5,6 = 3.9 Hz, H-5), 4.32 and 4.47 (2×d,
2H, Jgem = 11.5 Hz, CH2Ph), 4.49 (ddd, 1H, J5,6 = 4.1, J6,7 = 7.1, JF,6 = 12.0 Hz, H-6), 4.89 (t, 1H,
J3,4 = 3.1, J3,F = 3.6 Hz, H-4), 5.14 (m, 1H, H-3), 5.70 (dd, 1H, J6,7 = 7.1, JF,7 = 48.2 Hz, H-7), 7.23–
7.46 (m, 10H, 2×Ph). 13C NMR (62.9 MHz, CDCl3): δ 36.0 (C-2), 72.7 (CH2Ph), 77.8 (C-3), 82.0
(d, JF,5=6.7 Hz, C-5), 83.0 (d, JF,6=21.9 Hz, C-6), 84.6 (C-4), 93.3 (d, JF,7=172.6 Hz, C-7), 126.9,
127.2, 128.2, 128.55, 128.58, 128.9, 135.9, 136.6 (2×Ph), 175.0 (C-1). HRMS (ESI): m/e 360.1604
(M++NH4), calcd for C20H23FNO4: 360.1606.
4.1.6. General procedure for the preparation of fluoride analogues 9 and 10
A solution of 26 or 27 (1 equiv) in EtOAc (0.06 M + 2% conc. HCl) was hydrogenated over 10%
Pd/C (0.02 g; the catalyst contained 50% of water) for 30 h (for 26) or 21 h (for 27) at room
temperature. The mixture was filtered through a Celite pad and the catalyst washed with EtOAc.
The combined organic solutions were evaporated and the residues were purified by flash
chromatography (7:3 toluene/EtOAc for 9; 3:2 toluene/EtOAc for 10), to afford pure 9 or 10.
3,6-Anhydro-2,7-dideoxy-7-fluoro-7-C-phenyl-D-glycero-D-ido-heptono-1,4-lactone (9). Yield:
70%. Colourless needles, mp 124–125 °C, [α]20D = +18.2 (c 0.5, CHCl3), Rf = 0.32 (3:2
toluene/EtOAc). IR (KBr): νmax 3456 (OH), 1771 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.62 (d,
1H, J2a,2b = 18.4 Hz, H-2a), 2.74 (dd, 1H, J2a,2b = 18.8, J2b,3 = 5.4 Hz, H-2b), 4.26 (td, 1H, J5,6 = 2.9,
J6,7 = JF,6 = 7.5 Hz, H-6), 4.70 (br s, 1H, H-5), 4.95 (d, 1H, J3,4 = 4.2 Hz, H-4), 5.05 (t, 1H, J = 4.5
Hz, H-3), 5.66 (dd, 1H, J6,7 = 7.9, JF,7 = 45.5 Hz, H-7), 7.32–7.59 (m, 5H, Ph). 13C NMR (62.9
MHz, CDCl3): δ 35.9 (C-2), 74.0 (C-5), 77.5 (C-3), 82.1 (d, JF,6 = 31.9 Hz, C-6), 87.4 (C-4), 91.0
(d, JF,7 = 167.8 Hz, C-7), 126.5, 126.6, 129.0, 129.3, 136.3, 136.6 (Ph), 175.2 (C-1). HRMS (ESI):
m/e 270.1138 (M++NH4), calcd for C13H17FNO4: 270.1136.
3,6-Anhydro-2,7-dideoxy-7-fluoro-7-C-phenyl-L-glycero-D-ido-heptono-1,4-lactone (10). Yield:
79%. Colourless prisms, mp 152–154 °C, [α]20D = +132.9 (c 0.5, CHCl3), Rf = 0.24 (3:2
toluene/EtOAc). IR (KBr): νmax 3497 (OH), 1763 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.49 (d,
1H, J5,OH = 5.7 Hz, OH), 2.77 (m, 2H, 2×H-2), 4.12 (br s, 1H, H-5), 4.37 (ddd, 1H, J5,6 = 4.4, J6,7
=
7.1, JF,6 = 16.8 Hz, H-6), 4.85 (t, 1H, J3,4 = 3.2, J3,F = 3.3 Hz, H-4), 5.14 (m, 1H, H-3), 5.69 (dd, 1H,
J6,7 = 7.1, JF,7 = 48.1 Hz, H-7), 7.33–7.56 (m, 5H, Ph). 13C NMR (62.9 MHz, CDCl3): δ 36.0 (C-2),
17

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74.5 (JF,5 = 6.7 Hz, C-5), 77.0 (C-3), 83.1 (d, JF,6 = 21.0 Hz, C-6), 87.6 (C-4), 93.3 (d, JF,7 = 172.6
Hz, C-7), 126.7, 126.8, 129.0, 129.5, 135.5, 135.8 (Ph), 175.3 (C-1). HRMS (ESI): m/e 270.1135
(M++NH4), calcd for C13H17FNO4: 270.1136.
4.1.7. Preparation of azido derivatives 11, 12 and 29
To a cooled (0 °C) and stirred solution of 28 (0.042 g, 0.18 mmol) in dry CH2Cl2 (1 mL) was added
Me3SiN3 (0.12 mL, 0.90 mmol) and BF3·Et2O (0.03 mL, 0.24 mmol). The mixture was stirred at 0
°C for 4 h, then Et3N (0.05 mL, 0.33 mmol) was added, and the mixture was stirred at 0 °C for
additional 0.5 h. The reaction mixture was poured in water and extracted with CH2Cl2. The
combined organic solutions were dried and evaporated. The remaining crude residue was first
purified by flash column chromatography (19:1 CH2Cl2/EtOAc) and then by preparative TLC
(Et2O, 2 successive developments).
(3aR,5S,6S,7S,7aR)-5-Azido-7-hydroxy-6-phenyl-hexahydrofuro[3,2-b]piran-2-on (29). Yield:
12%. Colourless needles, mp 185−186 °C, [α]20D = +225.8 (c 0.4, CHCl3), Rf = 0.25 (9:1
CH2Cl2/EtOAc). IR (film): νmax 3439 (OH), 2105 (N3), 1774 (C=O). 1H NMR (250 MHz, CDCl3) δ
1.83 (br s, 1H, OH), 2.70 (dd, 1H, J5,6 = 12.4, J6,7 = 8.3 Hz, H-6), 2.73 (d, 1H, J2a,2b = 18.7 Hz, H-
2a), 2.98 (dd, 1H, J2a,2b = 18.7, J2b,3 = 6.9 Hz, H-2b), 4.23 (dd, 1H, J4,5 = 5.9, J5,6 = 12.4 Hz, H-5),
4.58 (dd, 1H, J3,4 = 4.9, J4,5 = 5.7 Hz, H-4), 4.93 (dd, 1H, J2b,3 = 6.4, J3,4 = 5.0, H-3), 5.37 (d, 1H,
J6,7 = 8.3 Hz, H-7), 7.20–7.48 (m, 5H, Ph). 13C NMR (62.9 MHz, CDCl3): δ 35.1 (C-2), 50.7 (C-6),
68.5 (C-3), 71.6 (C-5), 85.3 (C-4), 93.1 (C-7), 128.5, 128.6, 129.5, 135.7 (Ph), 174.3 (C-1). HRMS
(ESI): m/e 293.1243 (M++NH4), calcd for C13H17N4O4: 293.1244.
3,6-Anhydro-7-azido-2,7-dideoxy-7-C-phenyl-D-glycero-D-ido-heptono-1,4-lactone (11). Yield:
10%. Colourless needles, mp 137−138 °C, [α]20D = −75.2 (c 0.25, CHCl3), Rf = 0.66 (Et2O). IR
(film): νmax 3448 (OH), 2106 (N3), 1785 (C=O). 1H NMR (250 MHz, CDCl3) δ 2.43 (br s, 1H, OH),
2.61 (d, 1H, J2a,2b = 18.5 Hz, H-2a), 2.71 (dd, 1H, J2a,2b = 18.7, J2b,3 = 5.4 Hz, H-2b), 4.18 (dd, 1H,
J5,6 = 2.8, J6,7 = 8.0 Hz, H-6), 4.57 (d, 1H, J5,6 = 2.7 Hz, H-5), 4.85 (d, 1H, J6,7 = 8.0 Hz, H-7), 4.92
(d, 1H, J3,4 = 4.1 Hz, H-4), 5.04 (dd, 1H, J3,4 = 4.3, J2,3 = 5.2 Hz, H-3), 7.32–7.52 (m, 5H, Ph). 13C
NMR (62.9 MHz, CDCl3): δ 35.9 (C-2), 64.1 (C-7), 74.1 (C-5), 77.4 (C-3), 82.4 (C-6), 87.2 (C-4),
127.6, 129.07, 129.09, 135.59 (Ph), 175.1 (C-1). HRMS (ESI): m/e 298.0797 (M++Na), calcd for
C13H13N3NaO4: 298.0798.
3,6-Anhydro-7-azido-2,7-dideoxy-7-C-phenyl-L-glicero-D-ido-heptono-1,4-lactone (12). Yield:
24%. Colourless plates, mp 133−134 °C, [α]20D = +149.2 (c 0.25, CHCl3), Rf = 0.59 (Et2O). IR
18

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(film): νmax 3442 (OH), 2104 (N3), 1785 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.40 (br s, 1H,
OH), 2.79 (d, 2H, J2,3 = 3.2 Hz, 2×H-2), 4.04 (d, 1H, J5,6 = 2.6 Hz, H-5), 4.24 (dd, 1H, J5,6 = 2.9,
J6,7 = 8.3 Hz, H-6), 4.73–4.95 (m, 2H, H-4 and H-7), 5.13 (m, 1H, H-3), 7.32–7.55 (m, 5H, Ph). 13C
NMR (62.9 MHz, CDCl3): δ 35.6 (C-2), 64.8 (C-7), 74.0 (C-5), 77.4 (C-3), 83.6 (C-6), 87.3 (C-4),
127.7, 129.1, 135.7 (Ph), 175.2 (C-1). HRMS (ESI): m/e 298.0803 (M++Na), calcd for
C13H13N3NaO4: 298.0798.
4.1.8. 3,6-Anhydro-2-deoxy-D-ido-heptono-1,4-lactone (13)
Prepared according to the literature procedure [21]. Colourless crystals, mp 72–74 °C, [α]20
=
D
+26.8 (c 1.8, H2O), Rf = 0.72 (9:1 CH2Cl2/MeOH). lit. [22] mp 72–74 °C, [α]20D = +28.4 (c 1.97,
H2O), lit. [21] [α]20D = +23.0 (c 0.5, H2O). IR (film): νmax 3419 (OH), 1770 (C=O). 1H NMR (250
MHz, acetone-d6): δ 2.47 (d, 1H, J2a,2b = 18.8 Hz, H-2a), 2.85 (dd, 1H, J2b,3 = 6.1 Hz, H-2b), 3.66
(dd, 1H, J7a,7b = 11.6, J7a,6 = 5.2 Hz, H-7a), 3.75 (d, 1H, J6,7b = 5.1 Hz, H-7b), 3.96 (m, 1H, J5,6 = 1.8
Hz, H-6), 4.25 and 5.01 (br s, 2H, exchangeable with D2O, 2×OH), 4.34 (d, 1H, H-5), 4.88 (d, 1H,
J3,4 = 4.4 Hz, H-4), 4.94 (t, 1H, H-3). 13C NMR (62.9 MHz, acetone-d6): δ 36.5 (C-2), 60.9 (C-7),
75.1 (C-5), 77.5 (C-3), 82. 1 (C-6), 89.1 (C-4), 176.3 (C-1). HRMS (ESI): m/e 175.0597 (M++H),
calcd for C7H11O5: 175.0601.
4.1.9. 3,6-Anhydro-7-chloro-2,7-dideoxy-D-ido-heptono-1,4-lactone (14)
To a cooled (0 °C) and stirred solution of 13 (0.149 g, 0.86 mmol) in dry CH2Cl2 (15 mL) and Py
(0.74 mL) was added Ph3P (1.011 g, 3.85 mmol) and CCl4 (0.75 mL, 7.71 mmol). The mixture was
stirred at 0 °C for 0.5 h and then at room temperature for 51 h. The mixture was poured in 6 M HCl,
and extracted first with CH2Cl2 and then with EtOAc. The combined extracts were washed with
10% aq NaCl, dried and evaporated. The residue was purified by flash column chromatography (1:1
light petroleum/Et2O), to afford pure 14 (0.063 g, 38%) as colourless plates, mp 92–94 °C, [α]20
=
D
+21.0 (c 0.5, CHCl3), Rf = 0.49 (1:1 light petroleum/EtOAc). IR (film): νmax 1786 (C=O), 3391
(OH). 1H NMR (400 MHz, CDCl3): δ 2.61 (br s, 1 H, OH), 2.67 (br d, 1H, J2a,2b = 18.8 Hz, H-2a),
2.78 (dd, 1H, J2a,2b = 18.8, J2b,3 = 5.2 Hz, H-2b), 3.68 (dd, 1H, J6,7 = 6.3, J7a,7b = 10.8 Hz, H-7a),
3.72 (dd, 1H, J6,7 = 7.8, J7a,7b = 10.8 Hz, H-7b), 4.27 (m, 1H, H-6), 4.59 (br s, 1H, H-5), 4.93 (d, 1H,
J3,4 = 4.2 Hz, H-4), 5.01 (m, 1H, H-3). 13C NMR (100 MHz, CDCl3): δ 36.0 (C-2), 39.8 (C-7), 73.6
(C-5), 77.3 (C-3), 80.9 (C-6), 87.6 (C-4), 175.4 (C-1). HRMS (ESI): m/e 210.0526 (M++NH4),
calcd for C7H13ClNO4: 210.0528. Anal. Found: C, 43.44; H, 4.78. Calcd for C7H9ClO4: C, 43.65; H,
4.71.
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4.1.10. 3,6-Anhydro-7-bromo-2,7-dideoxy-D-ido-heptono-1,4-lactone (15)
To a cooled (0 °C) and stirred solution of 13 (0.306 g, 1.76 mmol) in dry CH2Cl2 (50 mL) and Py
(1.5 mL) was added Ph3P (1.152 g, 4.39 mmol) and CBr4 (1.457 g, 4.40 mmol). The mixture was
stirred at 0 °C for 0.5 h and then at room temperature for 72 h. The mixture was poured in 6 M HCl,
and extracted with EtOAc. The combined extracts were washed with 10% aq NaCl, dried and
evaporated. The residue was purified by three flash column chromatography (first Et2O; second and
third CHCl3), gave pure 15 (0.116 g, 28%) as colourless needles, mp 87–89 °C, [α]20D = +10.9 (c
1.8, CHCl3), Rf = 0.26 (1:1 light petroleum/EtOAc); lit. [24] mp (for enantiomer) 88–90 °C, [α]20
=
D
−22.0 (c 1.8, CHCl3) (for enantiomer). IR (film): νmax 1782 (C=O), 3448 (OH). 1H NMR (400 MHz,
CDCl3): δ 2.66 (d, 1H, , J2a,2b = 18.7 Hz, H-2a), 2.70 (dd, 1H, J2a,2b = 18.7, J2b,3 = 5.2 Hz , H-2b),
3.12 (br s, 1H, OH), 3.40–3.60 (m, 2H, J6,7 = 6.4 Hz, H-7), 4.30 (m, 1H, J5,6 = 3.0, J5,6 = 6.3 Hz, H-
6), 4.58 (d, 1H, J5,6 = 2.9 Hz, H-5), 4.93 (d, 1H, J3,4 = 4.2 Hz, H-4), 5.02 (m, 1H, H-3). 13C NMR
(100 MHz, D2O): δ 28.4 (C-7), 36.4 (C-2), 73.8 (C-5), 78.1 (C-3), 81.7 (C-6), 89.2 (C-4), 180.0 (C-
1). HRMS (ESI): m/e 254.0020 (M++NH4), calcd for C7H13NBrO4: 254.0022.
4.1.11. 3,6-Anhydro-2,7-dideoxy-7-iodo-D-ido-heptono-1,4-lactone (16)
To a solution of 13 (0.281 g, 1.61 mmol) in dry THF (2.5 mL) was added imidazole (0.219 g, 3.22
mmol) and Ph3P (0.845 g, 3.22 mmol). In the refluxing mixture was added dropwise a solution of
iodine (0.491 g, 1.93 mmol) in dry THF (1.4 mL). The mixture was stirred for 3 h under reflux, and
then evaporated. The residue was purified by flash column chromatography (2:1 light
petroleum/EtOAc), to afford pure 16 (0.290 g, 63%) as a colourless powder, mp 131 °C, [α]20
=
D
+10.7 (c 1, CHCl3), Rf = 0.32 (1:1 light petroleum/EtOAc). lit. [25] mp 131–133 °C, [α]20D = +17.2
(c 1.0, CHCl3). IR (film): νmax 1778 (C=O), 3436 (OH). 1H NMR (250 MHz, CDCl3): δ 2.64 (d, 1H,
J2a,2b = 18.7 Hz, H-2a), 2.76 (dd, 1H, J2a,2b = 18.7, J2b,3 = 5.3 Hz, H-2b), 3.10 (br s, 1H, OH), 3.18–
3.34 (m, 2H, H-7a and H-7b), 4.30 (m, 1H, J5,6 = 2.6, J6,7 = 7.2 Hz, H-6), 4.59 (br s, 1H, H-5), 4.93
(d, 1H, J3,4 = 4.2 Hz, H-4), 5.04 (m, 1H, H-3). 13C NMR (62.9 MHz, CDCl3): δ −1.1 (C-7), 36.2 (C-
2), 73.9 (C-5), 77.6 (C-3), 81.4 (C-6), 87.7 (C-4), 175.6 (C-1). HRMS (ESI): m/e 301.9887
(M++NH4), calcd for C7H13NIO4: 301.9884.
4.1.12. 3,6-Anhydro-7-azido-2,7-dideoxy-D-ido-heptono-1,4-lactone (18)
To a solution of 16 (0.208 g, 0.73 mmol) in anhydrous DMF (17 mL) was added NH4Cl (0.055 g,
1.03 mmol) and NaN3 (0.477 g, 7.33 mmol). The mixture was stirred for 7.5 h at 50 °C, then for 24
h at room temperature and evaporated. The residue was purified by flash column chromatography
20

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(1:1 light petroleum/EtOAc), to give pure 18 (0.096 g, 66%) as colourless plates, mp 85–87 °C,
[α]20D = +11.0 (c 0.2, CHCl3), Rf = 0.31 (1:1 light petroleum/EtOAc). IR (KBr): νmax 1755 (C=O),
2097 (N3), 3412 (OH). 1H NMR (250 MHz, CDCl3:) δ 2.68 (dd, 1H, J2a,2b = 18.7, J2a,3 = 1.2 Hz, H-
2a), 2.79 (dd, 1H, J2a,2b = 18.8, J2b,3 = 5.2 Hz, H-2b), 3.09 (br s, 1H, OH), 3.58 (d, 1H, J6,7 = 5.8 Hz,
H-7), 4.17 (td, 1H, J6,7 = 5.8, J5,6 = 3.4 Hz, H-6), 4.50 (br s, 1H, H-5), 4.90 (d, 1H, J3,4 = 4.2 Hz, H-
4), 5.02 (m, 1H, H-3). 13C NMR (62.9 MHz, CDCl3): δ 36.0 (C-2), 49.5 (C-7), 71.3 (C-5), 76.9 (C-
3), 79.2 (C-6), 88.0 (C-4), 175.6 (C-1). HRMS (ESI): m/e 217.0927 (M++NH4), calcd for
C7H13NO4: 217.0931.
4.1.13. 3,6-Anhydro-3-O-benzyl-2,7-dideoxy-7-fluoro-D-ido-heptono-1,4-lactone (31)
To a cooled (0 °C) and stirred solution of 30 (0.074 g, 0.28 mmol) in dry CH2Cl2 (1.5 mL) was
added DAST (0.11 mL g, 0.84 mmol). The mixture was stirred at 0 °C for 0.5 h and then at room
temperature for 3 h. The mixture was suspended in 10% aq NaHCO3, and the resulting suspension
was extracted first with CH2Cl2 and then with EtOAc, dried and evaporated. The residue was
purified by flash column chromatography (4:1 CH2Cl2/light petroleum), to give pure product 31
(0.026 g, 35%) as a colourless syrup, [α]20D = +16.2 (c 0.2, CHCl3), Rf = 0.80 (3:2 light
petroleum/acetone). IR (film): νmax 1782 (C=O). 1H NMR (250 MHz, CDCl3): δ 2.73 (m, 2H, H-2),
4.29 (d, 1H, J5,6 = 4.7 Hz, H-5), 4.31–4.45 (m, 1H, H-6), 4.48–4.79 (m, 3H, H-7 and CH2Ph), 4.95
(d, 1H, J3,4 = 4.7 Hz, H-4), 5.01 (m, 5H, H-3), 7.47–7.29 (m, 5H, Ph). 13C NMR (62.9 MHz,
CDCl3): δ 36.0 (C-2), 72.8 (CH2Ph), 77.2 (C-3), 78.9 and 79.2 (JF,6 = 21.4 Hz, C-6), 80.4 and 83.1
(JF,7 = 168.6 Hz,C-7), 81.4 and 81.5 (JF,5 = 5.0 Hz, C-5), 85.4 (C-4), 127.8, 128.3, 128.7, 136.7
(Ph), 174.9 (C-1). HRMS (ESI): m/e 284.1285 (M++NH4), calcd for C14H15FNO4: 284.1293.
4.1.14. 3,6-Anhydro-2,7-dideoxy-7-fluoro-D-ido-heptono-1,4-lactone (17)
A solution of 31 (0.047 g, 0.18 mmol) in EtOAc (3 mL + 1% conc. HCl) was hydrogenated over
10% Pd/C (0.047 g; the catalyst contained 50% of water) for 19 days at room temperature. The
mixture was filtered through a Celite pad and the catalyst washed with EtOAc. The combined
organic solutions were evaporated and the residue was purified by flash column chromatography
(7:3 light petroleum/acetone) to afford pure 17 (0.017 g, 57%) as a colourless syrup, [α]20D = +30.3
(c 0.3, CHCl3), Rf = 0.42 (7:3 light petroleum/acetone). IR (film): νmax 1783 (C=O), 3431 (OH). 1H
NMR (400 MHz, CDCl3): δ 2.20 (br s, 1H, OH), 2.70 (bd, 1H, J2a,2b = 18.8 Hz, H-2a), 2.80 (dd, 1H,
J2a,2b = 18.8, J2b,3 = 5.7 Hz, H-2b), 4.31 (m, 1H, J6,7 = 4.5, JF,7 = 13.6 Hz, H-6), 4.61 (d, 1H, J5,6
=
3.7 Hz, H-5), 4.70 (m, 2H, J6,7 = 4.5, J7a,7b = 10.1, JF,7 = 46.8 Hz, CH2-7), 4.91 (d, 1H, J3,4 = 4.2 Hz,
21

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H-4), 5.10 (m, 1H, H-3). 13C NMR (100 MHz, CDCl3): δ 36.0 (C-2), 74.6 and 74.7 (JF,5 = 4.9 Hz,
C-5), 77.0 (C-3), 79.2 and 81.0 (JF,6 = 20.9 Hz, C-6), 80.9 and 82.5 (JF,7 = 167.3 Hz, C-7), 88.2 (C-
4), 175.5 (C-1). HRMS (ESI): m/e 194.0821 (M++NH4), calcd for C7H13FNO4: 194.0821.
4.2. X-ray Crystal Structure Analysis
Diffraction experiments were performed on an Oxford Diffraction Gemini S diffractometer. Crystal
structures were solved by SHELXT [31] or SIR92 [32] and refined with SHELXL [33]. See the
Supplementary data for details. Crystallographic data have been deposited at the Cambridge
Crystallographic Data Centre. CCDC Numbers: 1514910 (7), 1514911 (10), 1514912 (11), 1514913
(12) 1514914 (18), 1514915 (24), 1514916 (27), and 1514917 (29). These data are available free of
charge via www.ccdc.cam.ac.uk/data_request/cif.
4.3. Biological materials
Rhodamine B, RPMI 1640 medium, 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide,
foetal calf serum, propidium iodide and RNase A, were purchased from Sigma (St. Louis, MO,
USA). Penicillin and streptomycin were purchased from ICN Galenika (Belgrade, Serbia). Annexin
V-FITC apoptosis detection kit was purchased from BD Biosciences Pharmingen (Belgium).
Proteins were detected by Western blotting using the following monoclonal antibodies against: Bax,
Bcl-2 and Caspase-3 (obtained from R&D Systems, Minneapolis, MN) and anti-Poly (ADP-ribose)
polymerase (PARP) (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.
4.4. Cell lines
Human chronic myelogenous leukaemia (K562), promyelocytic leukaemia (HL-60), human T cell
leukaemia (Jurkat) and Burkitt’s lymphoma (Raji) were grown in RPMI 1640 while ER+ breast
adenocarcinoma (MCF-7), cervix carcinoma (HeLa), lung adenocarcinoma epithelial (A549)
malignant cells, and normal foetal lung fibroblasts (MRC-5) were grown in DMEM medium. Both
media were supplemented with 10% of foetal calf serum (FTS, NIVNS) and antibiotics (100 IU/mL
of penicillin and 100 mg/mg of streptomycin). Cell lines were cultured in flasks (Costar, 25 mL) at
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37°C in the atmosphere of 100% humidity and 5% of CO2 (Heraeus). Exponentially growing viable
cells were used throughout the assays.
4.5. Cells treatment
The cells were seeded in six-well plates at a concentration of 5×105 cells/well. Cells were treated
for 72 h with goniofufurone (1), 7-epi-goniofufurone (2), dephenyl goniofufurone (13) and the
corresponding analogues (3–12, 14–18) at their IC50/72 h concentrations. Untreated cells were used
as control. Viable cells of treated and control samples were used for cell cycle and apoptosis
detection and Western blot analysis. Viability was determined using trypan blue dye-exclusion
assay.
4.6. MTT assay
Cells were harvested, counted by trypan blue and plated into 96-well microtitar plates (Costar) at
optimal seeding density of 5×103 cells per well to assure logarithmic growth rate throughout the
assay period. Viable cells were placed in a volume of 90 mL per well, and pre-incubated 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 incubated for 72 h. The wells containing cells
without tested substances were used as control. Three hours before the end of incubation period
MTT solution (10 mL) was added to each well. MTT was dissolved 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 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.
4.7. Cell cycle analysis
After treatment K562 cells were washed in cold PBS, fixed and incubated for 30 min in 70%
ethanol on ice, centrifuged and incubated with 500 µL RNase A (100 units/mL) and 500 µL
propidium iodide (400 µL/mL) for 30 min at 37 °C. Cell cycle was analyzed by FACS Calibur
E440 (Becton Dickinson) flow cytometer and the Cell Quest software. Results were presented as
percentage of cell cycle phases.
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4.8. Detection of apoptosis
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 1×106
cells/mL. The cell suspension (100 mL) was transferred to 5 mL culture tubes and mixed with
Annexin V (5 mL) and propidium iodide (5 mL). The cells were gently vortexed and incubated for
15 min at 25 °C. After incubation, 400 mL of binding buffer was added to each tube and 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 [34].
4.9. Western blot analysis
For the Western blot, 50 mg of proteins per sample were separated by electrophoresis and electro-
transferred to a PVDF membrane 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.
Acknowledgement
This work was supported by research grants from the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grant No. OI 172006 and OI 1612036), and
(in part) by a research project from the Serbian Academy of Sciences and Arts (Grant No. F-130).
Supplementary data
Contains the results of: X-ray crystal structure determination, SAR analysis, flow cytometry and
Western blot analysis. Copies of 1H, and 13C NMR spectra of final products, purity grade of final
compounds by HPLC.
References
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