Novel: O -methyl goniofufurone and 7- epi -goniofufurone derivatives: Synthesis, in vitro cytotoxicity and SAR analysis

Article abstract of DOI:10.1039/c8md00431e

Novel goniofufurone (1) and 7-epi-goniofufurone (2) derivatives bearing a methoxy group at the C-5 and/or C-7 positions were prepared and their in vitro antitumour activity against some human tumour cell lines was evaluated. Some of the analogues displayed powerful antiproliferative effects against the studied tumour cells, but almost all of them were non-cytotoxic toward the normal cells (MRC-5). A SAR study reveals that the introduction of a methoxy group at the C-7 position may increase the antiproliferative effects of the analogues. The most active compounds are 7-O-methyl derivatives of goniofufurone (3) and 7-epi-(+)-goniofufurone (6), which exhibited 1177- and 451-fold higher potencies than the leads 1 and 2 toward the MDA-MB 231 cell line. At the same time, compound 3 is almost 1.5-fold more active than the commercial drug doxorubicin (DOX) against the same cell line. Flow cytometry data confirmed that the cytotoxic effects of these analogues are mediated by apoptosis, additionally revealing that these molecules induced changes in the K562 cell cycle distribution.

Full text of DOI:10.1039/c8md00431e

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RESEARCH ARTICLE
Novel O-methyl goniofufurone and 7-epi-
goniofufurone derivatives: synthesis, in vitro
cytotoxicity and SAR analysis†
Cite this: Med. Chem. Commun.,
2018, 9, 2017
Jovana Francuz,a Mirjana Popsavin,a Sanja Djokić,a Vesna Kojić,b
a
Tatjana Srdić-Rajić,c Marko V. Rodić,
Dimitar Jakimovb and Velimir Popsavin
ad
*
Novel goniofufurone (1) and 7-epi-goniofufurone (2) derivatives bearing a methoxy group at the C-5 and/
or C-7 positions were prepared and their in vitro antitumour activity against some human tumour cell lines
was evaluated. Some of the analogues displayed powerful antiproliferative effects against the studied tu-
mour cells, but almost all of them were non-cytotoxic toward the normal cells (MRC-5). A SAR study re-
veals that the introduction of a methoxy group at the C-7 position may increase the antiproliferative effects
of the analogues. The most active compounds are 7-O-methyl derivatives of goniofufurone (3) and 7-epi-
(+)-goniofufurone (6), which exhibited 1177- and 451-fold higher potencies than the leads 1 and 2 toward
the MDA-MB 231 cell line. At the same time, compound 3 is almost 1.5-fold more active than the commer-
cial drug doxorubicin (DOX) against the same cell line. Flow cytometry data confirmed that the cytotoxic
effects of these analogues are mediated by apoptosis, additionally revealing that these molecules induced
changes in the K562 cell cycle distribution.
Received 29th August 2018,
Accepted 22nd October 2018
DOI: 10.1039/c8md00431e
rsc.li/medchemcomm
compound 1 (Fig. 1) has a restricted geometry of the C5–C7
Introduction
segment, due to an intramolecular H-bond formed between
[3.3.0]Furofuranone moieties are frequently found in natural
products that show a broad range of biological activities in-
cluding cytotoxic activity.1–4 As a consequence of that, the syn-
thesis of [3.3.0]furofuranone derivatives has received consider-
able attention from synthetic medicinal chemists in the last
twenty years.5 As a part of our continuing interest in the syn-
thesis and antiproliferative evaluation of natural products
and analogues with the [3.3.0]furofuranone scaffold,6–10 we
have planned the synthesis of new (+)-goniofufurone and
7-epi-goniofufurone derivatives with methoxy groups at the
C-5 and/or C-7 positions (3–10, Fig. 1). The rationale underly-
ing the preparation of 3 arises from the fact that the parent
the 5-OH and the 7-OH, as established by X-ray analysis.11 We
a Department of Chemistry, Biochemistry and Environmental Protection, Faculty of
Sciences, University of Novi Sad, Trg Dositeja Obradovića 3, 21000 Novi Sad,
Serbia. E-mail: velimir.popsavin@dh.uns.ac.rs
b Oncology Institute of Vojvodina, Faculty of Medicine, University of Novi Sad, Put
Dr Goldmana 4, 21204 Sremska Kamenica, Serbia
c Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade,
Serbia
d Serbian Academy of Sciences and Arts, Knez Mihailova 35, 11000 Belgrade,
Serbia
† Electronic supplementary information (ESI) available: Contains the results of:
X-ray crystal structure determination, SAR analysis and flow cytometry. Copies of
the 1H and 13C NMR spectra of the final products are also disclosed. CCDC
1843444–1843449. For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c8md00431e
Fig. 1 Structures of (+)-goniofufurone (1), 7-epi-(+)-goniofufurone (2)
and the targeted O-methyl derivatives 3–10.
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assumed that the replacement of the 7-OH group with a
7-OMe group will make the H-bond stronger which will fur-
ther strengthen the conformational constraint in the C5–C7
segment. On the other hand, we have recently demonstrated
that readily available (+)-goniofufurone oxetane deriva-
tives,12,13 which cause much greater structural rigidity,
strongly inhibit the growth of certain human neoplastic cell
lines.13 Herein, we report their synthesis starting from
D-glucose, as well as their effects on the proliferation of cer-
tain human tumour cell lines, including their impact on the
K562 cell cycle and their apoptosis inducing properties in the
mentioned cell culture.
biguously confirmed by single crystal X-ray diffraction analy-
sis (for the crystal structure of 3, see the ESI†).
The synthesis of analogues 4 and 7 started from commer-
cially available diacetone-D-glucose 11 (Scheme 2). Treatment
of 11 with MeI and NaH in dry DMF gave the known15 3-O-
methyl derivative 20 in 81% yield. When compound 20 was
treated with periodic acid in dry ethyl acetate and the crude
intermediate 20a was allowed to react with phenyl magne-
sium bromide in toluene, the benzyl alcohols 21 and 23 were
obtained, with the L-ido-derivative 23 as the major product
(23/21 ≈ 9 : 1). The stereochemistry of compounds 21 and 23
was confirmed by single crystal X-ray diffraction analysis (for
the crystal structures of 21 and 23, see the ESI†).
Hydrolytic removal of the isopropylidene protecting group
in 21 gave the expected pyranose 22 in an almost quantitative
yield (99%). Treatment of lactol 22 with Meldrum's acid in
the presence of triethylamine gave the desired 5-O-methyl-
goniofufurone (4) in 41% yield. Under similar reaction condi-
tions, compound 23 was converted to the expected lactol 24
(81%), which was finally converted to 7 (46%) by cyclo-
condensation with Meldrum's acid.
The synthesis of 5,7-di-O-methyl-goniofufurone (5) and 5,7-
di-O-methyl-7-epi-goniofufurone (8) is outlined in Scheme 3.
The preparation started from known16 benzyl alcohols 25 and
28 that were previously prepared in our laboratory according
to a modified procedure.10 Treatment of compounds 25 and
28 with MeI and NaH in dry DMF gave derivatives 26 and 29
(85% and 90%, respectively). Under the same reaction condi-
tions, compounds 21 and 23 gave the expected methoxy de-
rivatives 26 and 29 in 88% and 93% yields, respectively.
Treatment of 26 with aqueous trifluoroacetic acid gave 27
(89%). The reaction of 27 with Meldrum's acid in the pres-
ence of triethylamine gave the final product 5 in a 44% yield.
Under similar reaction conditions, compound 29 gave
lactol 30 (93%), the condensation of which with Meldrum's
acid gave analogue 8 in 51% yield. The stereochemistry of
Results and discussion
Chemistry
The preparation of 7-O-methyl-goniofufurone (3) and 7-O-
methyl-7-epi-goniofufurone (6) is outlined in Scheme 1. The
known14 benzyl alcohols 12 and 16, served as convenient
starting materials for this part of the work. These compounds
(12 and 16) have been synthesized earlier in our laboratory
starting from 11.8 Treatment of compound 12 with MeI and
NaH in dry DMF gave the 7-O-methyl derivative 13 (94%). Un-
der the same reaction conditions, compound 16 gave the
(7S)-isomer 17 in 94% yield. Hydrolytic removal of the iso-
propylidene protecting group in 13 gave the lactol 14 (85%),
which upon treatment with Meldrum's acid in the presence
of triethylamine gave the expected furanolactone 15 in 51%
yield. Under similar reaction conditions, compound 17 gave
lactol 18 (99%), which by subsequent cyclocondensation with
Meldrum's acid gave the protected lactone 19 (47%). 5-O-
Benzyl protective groups were removed under the standard re-
action conditions (H2, Pd/C, MeOH) whereby the desired
7-methoxy derivatives 3 and 6 were obtained in 82 and 68%
yield, respectively. The stereochemistry of 3 has been unam-
Scheme 1 Preparation of analogues 3 and 6: (a) MeI, NaH, DMF, 0 °C → rt, 2 h for 12, 94% of 13, 2 h for 16, 94% of 17; (b) 90% aq TFA, rt, 0.5 h
for 13, 85% of 14, 1 h for 17, 99% of 18; (c) Meldrum's acid, Et3N, DMF, 46 °C, 70 h for 14, 51% of 15, 68 h for 18, 47% of 19; (d) H2–Pd/C, MeOH, rt,
4 h for 15, 82% of 3, 24 h for 19, 68% of 6.
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Scheme 2 Preparation of analogues 4 and 7: (a) MeI, NaH, DMF, 0 °C → rt, 2 h, 81%; (b) H5IO6, EtOAc, rt, 1.5 h; (c) PhMgBr/THF, toluene, 0 °C, 5
h, 56% of 23, 6% of 21; (d) 90% aq TFA, rt, 0.5 h for 21, 99% of 22, 0.5 h for 23, 81% of 24; (e) Meldrum's acid, Et3N, DMF, 46 °C, 74 h for 22, 41%
of 4, 70 h for 24, 46% of 7.
compounds 5 and 8 has been unambiguously confirmed by
single crystal X-ray diffraction analysis (for crystal structures
of 5 and 8, see the ESI†).
In vitro antitumour activities and SAR
It is well known that many analogues of natural styryl lac-
tones of type 1 and 2 exhibited potent in vitro cytotoxic activ-
ity against certain human tumour cell lines.1–4 Hence, the
newly synthesized analogues were evaluated for their in vitro
cytotoxicity against a panel of human malignant cell lines
(myelogenous leukaemia, K562, promyelocytic leukaemia,
HL-60, T cell leukaemia, Jurkat, Burkitt's lymphoma, Raji, es-
trogen receptor positive breast adenocarcinoma, MCF-7, es-
trogen receptor negative breast adenocarcinoma, MDA-MB
231, cervix carcinoma, HeLa, lung adenocarcinoma, A549),
and against a single normal cell line, foetal lung fibroblasts
(MRC-5). The purpose of the evaluation of cytotoxicity against
the normal cells is to check the eventual selectivity of ana-
logues against the malignant cell lines. Cell growth inhibition
was evaluated by MTT colorimetric assay,17 after exposure of
cells to the tested compounds for 72 h. (+)-Goniofufurone (1),
An attempted preparation of O-methyl goniofufurone de-
rivatives by direct alkylation of parent compounds (MeI,
NaH, and DMF) gave a complex reaction mixture from which
the desired products could not be isolated in pure form.
Finally, a simple two step synthesis sequence was also ac-
complished by oxidation of compounds 4 or 7 with PCC
whereby target 9 was obtained in 99% and 91% yields, re-
spectively, (Scheme 4). The Grignard addition of PhMgBr to 9
gave target 10 in 35% yield. The stereochemistry of com-
pound 9 was confirmed by single crystal X-ray diffraction
analysis (for the crystal structure of 9, see the ESI†).
Scheme 3 Synthesis of 5,7-di-O-methyl derivatives 5 and 8: (a) MeI,
NaH, DMF, 0 °C → rt, 2 h for 25, 85% of 26, 2 h for 21, 88% of 26, 2 h
for 28, 90% of 29, 2 h for 23, 93% of 29; (b) 90% aq TFA, rt, 1 h for 26,
89% of 27, 1 h for 29, 93% of 30; (c) Meldrum's acid, Et3N, DMF, 46 °C,
71 h for 27, 44% of 5, 71 h for 30, 51% of 8.
Scheme 4 Preparation of analogues 9 and 10: (a) PCC, CH2Cl2, reflux,
2.5 h for 4, 99% of 9, 2 h for 7, 91% of 9; (b) PhMgBr, THF, 0 °C, 1.5 h, 35%.
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7-epi-(+)-goniofufurone (2) and the commercial antitumour
agent doxorubicin (DOX) were used as positive controls. The
results are presented in Table 1.
other analogues including natural products 1 and 2 were
completely inactive toward the normal cell line, MRC-5. In
contrast, the commercial drug doxorubicin exhibited potent
cytotoxicity (IC50 0.10 μM) against these cells. These results
do suggest that these molecules represent selective anti-
tumour agents, but this should be verified by additional
in vitro experiments with different normal cell lines.
In an attempt to correlate the structures of synthesized
goniofufurone analogues with their cytotoxic activities, we
first considered the effects of methoxy groups at the C-7 and/
or C-5 positions. The natural products 1 and 2 are used as
the controls in this SAR analysis. According to the data in
Table 1, the introduction of a methoxy group at the C-7 or
C-5 position caused the increased cytotoxicity originally
displayed by lead 1, but decreased the antiproliferative poten-
cies originally demonstrated by lead 2. Also, we wanted to ex-
plore the effects of methoxy groups at both C-7 and C-5 posi-
tions, and obtained very similar results as in the previous
examples. The analogue with two methoxy groups (5) was a
more potent cytotoxic agent than lead 1, but the correspond-
ing (7S)-epimer (8) was a less active compound with respect
to that originally shown by the control molecule 2 (for more
details see ESI,† Table S2 and Fig. S7).
The next round of modifications undertaken to improve
the antiproliferative activities have been performed by intro-
ducing a keto group at the C-7 position (compound 9), as well
as a methoxy group at C-5 and the second phenyl group at
the C-7 position (compound 10). The relationships between
these structural changes and cytotoxic activities were
established by comparing the IC50 values of the methoxy ana-
logues 9 and 10 with those recorded for the natural products
1 and 2 that are arbitrarily used as control molecules. As the
results shown in Table 1 indicate, in the great majority of the
cases, the analogue 9 showed a more potent antiproliferative
activity than the control molecules 1 and 2. Furthermore, in-
sertion of a phenyl group at the C-7 position of the 5-O-
methoxy derivative (10) resulted in the increased activity of
the lead 1 against five of the eight studied tumour cell lines.
However, compound 10 was a more potent cytotoxic agent
According to the results of the cytotoxicity assay, all the
malignant cell lines under evaluation were sensitive to all of
the synthesized analogues. The normal MCF-7 cell line was
not sensitive to compounds 3, 5, 6 and 8. The highest po-
tency in the culture of K562 was recorded after treatment
with 5-O-methyl-goniofufurone 4 (IC50 5.36 μM). All five
goniofufurone analogues (3–5, 9, and 10) demonstrated di-
verse antiproliferative activities toward HL-60 cells, in con-
trast to the lead 1, which was completely inactive against
this cell line. All 7-epi-goniofufurone derivatives (6–8) were
more potent than the lead 2 against the same cell line. The
highest potency in the culture of HL-60 (IC50 1.63 μM) cells
was recorded after treatment with 7-O-methyl-7-epi-
(+)-goniofufurone (6). The most active compound in the cul-
ture of Jurkat cells is the derivative 8 (IC50 1.69 μM). The
most active molecule against the Raji cells was the
goniofufurone derivative 4 (IC50 8.69 μM), which exhibited a
2-fold higher potency than lead 1. Analogue 9 was the most
potent compound against the MCF-7 cells (IC50 3.44 μM),
compared to the natural products 1 and 2, this analogue
exhibited 4.5- and 2.5-fold higher activity against these cells.
Two analogues demonstrated submicromolar activities to-
ward MDA-MB 231 cells. These are: 7-O-methyl-
(+)-goniofufurone 3 (IC50 0.064 μM) and 7-O-methyl-7-epi-
(+)-goniofufurone 6 (IC50 0.13 μM), which exhibited 1177-
and 451-fold higher potency than leads 1 and 2, respectively.
Compound 3 was almost 1.5-fold more active than the com-
mercial antitumour agent DOX. The highest potency in the
culture of HeLa was recorded after treatment with 7-O-
methyl-(+)-goniofufurone 3 (IC50 5.64 μM). Compounds 3 and
9 were the most potent against the A549 cell line (IC50 2.64
and 2.58 μM, respectively). These analogues were 13-fold
more active than lead 1, 8-fold more active than lead 2, and
almost 2-fold more active than doxorubicin. Interestingly,
only one of eight derivatives (molecule 8) showed negligible
activity against normal MRC-5 cells (IC50 87.61 μM). The
Table 1 In vitro cytotoxicities of natural products 1 and 2, their O-methylated derivatives 3–10 and DOX
IC50 (μM),a 72 hours
Compounds
K562
HL-60
Jurkat
Raji
MCF-7
MDA-MB 231
HeLa
A549
MRC-5
1
2
3
4
5
6
7
8
0.41
0.028
7.95
>100
22.02
2.96
25.01
2.21
1.63
18.95
4.36
32.45
18.64
15.64
24.85
2.36
12.64
15.46
1.69
18.45
1.25
15.64
8.69
12.36
21.08
12.22
10.47
25.61
18.95
2.98
16.59
9.24
>100
4.04
>100
>100
12.65
>100
3.44
75.34
58.70
0.064
28.64
1.36
0.13
35.22
2.65
8.32
0.89
5.64
8.63
56.37
8.46
11.01
89.34
6.39
12.64
0.07
35.21
21.02
2.64
23.55
26.34
5.31
38.25
25.05
2.58
15.08
4.91
>100
>100
>100
>100
>100
>100
>100
87.61
>100
>100
0.10
5.36
14.65
11.54
8.45
9.24
9
10
DOX
22.47
28.69
0.25
11.21
2.36
0.92
8.97
9.79
0.03
41.02
51.02
0.09
11.36
0.20
a
IC50 is the concentration of the compound required to inhibit the cell growth by 50% compared to an untreated control. The values are
means of three independent experiments done in quadruplicates. Coefficients of variation were <10%.
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than lead 2 against only four of the eight malignant cell lines
(for more details see ESI,† Table S2 and Fig. S7).
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) and an-
alogues 3–10 in K562 human leukaemia cells was determined
by flow cytometry after staining, and the results are shown in
Table 2B (for the graphical presentation see Fig. S8 and S9 in
the ESI†).
The apoptotic response, which was presented as the per-
centage of specific apoptosis, shows that all analogues in-
crease the percentage of Annexin V positive cells compared to
the parent compound 1. Compound 10 causes the greatest
specific apoptosis (33.41% after 72 h). Also, we can see that
all analogues induced several-fold more Annexin V positive
cells compared to the parent compound 2. However, ana-
logue 7 causes the greatest specific apoptosis (55.48% after
72 h). Compound 6 also causes notable specific apoptosis
(11.17% after 72 h). These results suggested that the
diphenylated analogue 10 and (7S) isomers 6 and 7 increased
the percentage of Annexin V positive cells which is in accor-
dance with results obtained during the cell cycle analysis
(sub-G1 peak, 10.13, 6.03 and 28.67% respectively). The
highest percentage of specific necrosis caused analogues 10
and 7 (8.73 and 2.57%, respectively).
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 are the targets in cancer therapy. Ef-
fects of (+)-goniofufurone (1), 7-epi-(+)-goniofufurone (2) and
analogues 3–10 on the cell cycle changes were further stud-
ied. The cell cycle profile of exponentially growing K562 cells
treated with the synthesized compounds for 72 h was ana-
lyzed by flow cytometry using cells stained with propidium io-
dide. Untreated cells served as the control. The results are
presented in Table 2A (for the graphical presentation see Fig.
S10 and S11 in the ESI†).
As the data in Table 2A indicate, treatment of K562 cells
with goniofufurone (1) and the corresponding analogues
(3–5, 9 and 10) slightly change the percentage of cells in the
S, G2/M and G0/G1 phases of the cell cycle compared to the
control. But only compounds 5 and 10 increased the percent-
age of cells in the sub-G1 phase (which is suggestive of apo-
ptosis), about 3.5- and 5.5-fold, compared to the control. Ana-
logue 10 induced the most prominent sub-G1 peak in K562
cells after 72-h of cell treatment.
Table 2A further reveals that 7-epi-(+)-goniofufurone (2)
and the corresponding analogues (6–8) slightly change the
percentage of cells in the G0/G1, S and G2/M phases of the
cell cycle compared to the control. However, analogues 6 and
7 increased about 3- and 15.5-fold the percentage of cells in
the sub-G1 phase when compared to the untreated control.
Analogue 7 induced the most prominent sub-G1 peak in the
K562 cells after 72-h of cell treatment.
A study on the ability of these compounds to modulate
the expression of selected apoptosis markers (Bcl-2, Bax,
caspase 3 and poly (ADP-ribose) polymerase-PARP) gives us
the opportunity to assume the mechanisms of the actions of
the synthesized analogues 3–10. The results are presented in
Fig. 2 (for more details see ESI,† Table S3).
The semi-quantitative western blot analysis indicates that
the compounds 5, 8, 9 and 10 and natural products 1 and 2
increased the expression of the anti-apoptotic Bcl-2 protein
when compared to the control, while the compounds 3, 4, 6
and 7 reduced the expression of the same protein. On the
other hand, analogues 4, 5, 8, 9, and 10 and the lead com-
pound 1 increased expression of the pro-apoptotic Bax
The obtained results showed that some of the synthesized
O-methyl derivatives (compounds 5–7 and 10) induced
changes in the cell cycle distribution of the K562 cells.
Detection of apoptosis
The cell cycle analysis indicated the pro-apoptotic effect of
the synthesized analogues through the formation of a sub-G1
Table 2 (A) Influence of the synthesized compound on the K562 cell cycle; (B) percentage of specific apoptosis and necrosis induced after treatment
with the synthesized compounds
(A) Distribution of K562 cells in the
cell cycle phases (%)
(B) Type of cell death
Specific apoptosis (%)
Compd
Sub G1
G0/G1
S
G2/M
Specific necrosis (%)
Control
1
3
4
5
9
10
Control
2
6
7
8
1.84
2.16
2.40
1.82
6.36
1.33
10.13
1.84
2.73
6.03
28.67
2.40
47.51
52.47
52.09
47.34
44.86
45.46
43.35
47.51
51.46
53.39
35.04
48.59
26.52
21.03
20.61
26.52
20.48
23.68
17.77
26.52
22.11
19.68
15.08
22.66
24.13
24.34
24.89
24.32
28.30
29.53
28.75
24.13
23.71
20.90
21.21
26.35
—
—
−0.36
−0.27
2.69
4.32
2.10
33.41
—
−0.22
11.17
55.48
1.70
−0.31
−0.50
−0.54
0.11
−0.59
8.73
—
−0.33
0.55
2.57
−0.44
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Fig. 2 Results of the western blot analysis after treatment of K562 cells with lead compounds 1 and 2 and synthesized compounds (3–10) for 72 h.
protein, while analogues 3, 6 and 7 and the lead compound 2
decreased expression of this protein. Since the equilibrium of
Bax and Bcl-2 can lead to the induction of apoptosis, we can
assume that the synthesized analogues do not cause apopto-
sis based on these groups of proteins.
given in cm−1. High resolution mass spectra (ESI) of the syn-
thesized compounds were acquired on an Agilent Technolo-
gies 1200 series instrument equipped with a Zorbax Eclipse
Plus C18 (100 mm × 2.1 mm, i.d. 1.8 μm) column and a 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 in size with an ≈1 mm layer
thickness. Kieselgel 60 G (E. Merck) with a fluorescent indica-
tor F254 as an additive was used as the stationary phase. The
corresponding bands were scraped and eluted with EtOAc.
All organic extracts were dried with anhydrous Na2SO4. The
organic solutions were concentrated in a rotary evaporator
under diminished pressure at a bath temperature below 35
°C. The purities of the final products were established by
HRMS and were found to be >95% pure.
However, the expression levels of caspase 3 (precursor and
active subunit) and the catalytic fragment of PARP in the
K562 cells exposed to compounds 1–10 were measured in or-
der to determine whether apoptosis was associated with the
activation of caspase. The western blot analysis revealed over-
expression of both caspase-3 units and the cleaved catalytic
PARP fragment (85 kD) in K562 cells after treatment with an-
alogues 5 and 10 while all other synthesized analogues
influenced only the PARP cleavage. With the exception of
compounds 8 and 9, the analogues increased the expression
of the active subunit of caspase 3 (p18) and PARP cleavage.
The most expressed level of the cleavage of the PARP unit (85
kD) was recorded in K562 cells after treatment with analogue
7, whereby this compound caused the greatest specific apo-
ptosis (55.48% after 72 h). These results allowed us to hy-
pothesize that all of the synthesized analogues and natural
products 1 and 2 caused apoptosis in the K562 cells in a
caspase-dependent manner.
General procedure for the preparation of O-methyl derivatives
13, 17, 20, 26 and 29
To a cooled (0 °C) and stirred solution of 12, 16, 11, 21, 23,
25 or 28 (1 equiv.) in dry DMF (0.05 M) was added NaH (3.0–
5.0 equiv.) and MeI (3.0–3.3 equiv.). The mixture was stirred
at 0 °C for 0.5 h and then at room temperature for 1.5 h. The
mixture was evaporated and extracted with EtOAc. The com-
bined extracts were dried and evaporated. The residues were
purified by flash column chromatography (9 : 1 light petro-
leum/Et2O for 13, 4 : 1 light petroleum/Et2O for 17, 7 : 3 light
petroleum/Et2O for 20, 17 : 3 light petroleum/Et2O for 26, and
3 : 1 light petroleum/Et2O for 29) to afford pure products 13,
17, 20, 26 or 29.
All these biological results can be helpful for the further
development of new antitumour agents derived from (+)-
goniofufurone and 7-epi-(+)-goniofufurone and from the re-
lated styryl lactones.
Experimental
General experimental procedures
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) polar-
imeter at room temperature. NMR spectra were recorded on a
Bruker AC 250 E or a Bruker Avance III 400 MHz instrument
and the chemical shifts are expressed in ppm downfield from
TMS. IR spectra were recorded with an FTIR Nexus 670
spectrophotometer (Thermo-Nicolet). The band positions are
3-O-Benzyl-1,2-O-isopropylidene-5-O-methyl-5-C-phenyl-α-D-
gluco-pentofuranose (13). Yield 94%. Colourless syrup, [α]D
=
−65.6 (c 0.5, CHCl3); Rf = 0.45 (4 : 1 light petroleum/Et2O). IR
1
(film): νmax 3018 and 2936 (CH), 1216 (C–O–C). H NMR (400
MHz, CDCl3): δ 1.32 and 1.46 (2 × s, 3 H each, Me2C), 3.21 (s,
3 H, OMe), 4.23 (d, 1 H, J3,4 = 2.0 Hz, H-3), 4.31 (dd, 1 H, J3,4
= 2.0, J4,5 = 9.2 Hz, H-4), 4.50 (d, 1 H, J4,5 = 9.2 Hz, H-5), 4.66
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(d, 1 H, J1,2 = 3.5 Hz, H-2), 4.71 and 4.80 (2 × d, 2 H, Jgem
=
H, J3,4 = 2.9, J4,5 = 8.6 Hz, H-4), 4.45–4.50 (m, 2 H, J1,2 = 4.0,
J4,5 = 8.8 Hz, H-2 and H-5), 6.01 (d, 1 H, J1,2 = 3.8 Hz, H-1),
7.30–7.45 (m, 5 H, Ph). 13C NMR (100 MHz, CDCl3): δ 26.2
and 26.7 (2 × Me2C), 56.6 (C5–OMe), 57.1 (C3–OMe), 80.7 (C-
2), 82.1 (C-5), 83.6 (C-4), 83.8 (C-3), 105.5 (C-1), 111.7 (Me2C),
127.8, 128.2, 128.3 and 138.1 (Ph). HRMS (ESI): m/z 312.1793
(M+ + NH4), calcd. for C16H26NO5: 312.1806; m/z 333.1096 (M+
+ K), calcd. for C16H22KO5: 333.1099.
11.7 Hz, CH2Ph), 5.91 (d, 1 H, J1,2 = 3.4 Hz, H-1), 7.31–7.59
(m, 10 H, Ph). 13C NMR (100 MHz, CDCl3): δ 26.3 and 26.7
(Me2C), 56.1 (OMe), 72.4 (CH2Ph), 79.8 (C-5), 81.7 (C-3), 82.3
(C-2), 82.8 (C-4), 105.0 (C-1), 111.5 (Me2C), 127.7, 127.8,
127.84, 128.0, 128.3, 128.4, 137.8 and 139.1 (Ph). HRMS (ESI):
m/z 409.1407 (M+ + K), calcd. for C22H26KO5: 409.1412.
3-O-Benzyl-1,2-O-isopropylidene-5-O-methyl-5-C-phenyl-β-L-
ido-pentofuranose (17). Yield 94%. Colourless syrup, [α]D
=
1,2-O-Isopropylidene-3-O-methyl-5-C-phenyl-α-D-gluco- (21)
and β-L-ido-pentofuranose (23)
−12.8 (c 0.5, CHCl3); Rf = 0.28 (7 : 3 light petroleum/Et2O). IR
1
(film): νmax 3031 (CH) and 2987, 1075 and 1036 (C–O–C). H
NMR (400 MHz, CDCl3): δ 1.31 and 1.54 (2 × s, 3 H each,
Me2C), 3.24 (s, 3 H, OMe), 3.37 (d, 1 H, J3,4 = 3.0 Hz, H-3), 4.09
and 4.41 (2 × d, 2 H, Jgem = 11.3 Hz, CH2Ph), 4.47 (dd, 1 H, J3,4
= 3.0, J4,5 = 8.8 Hz, H-4), 4.53 (d, 1 H, J1,2 = 3.8 Hz, H-2), 4.55
(d, 1 H, J4,5 = 8.8 Hz, H-5), 6.06 (d, 1 H, J1,2 = 3.7 Hz, H-1),
7.29–7.50 (m, 10 H, Ph). 13C NMR (100 MHz, CDCl3): δ 26.1
and 26.6 (Me2C), 56.3 (OMe), 71.4 (CH2Ph), 81.1 (C-2), 81.9 (C-
5), 82.0 (C-3), 83.6 (C-4), 105.3 (C-1), 111.5 (Me2C), 127.3,
127.6, 127.8, 128.1, 128.2, 137.1 and 137.8 (Ph). HRMS (ESI):
m/z 393.1658 (M+ + Na), calcd. for C22H26NaO5: 393.1673; m/z
409.1399 (M+ + K), calcd. for C22H26KO5: 409.1412.
1,2 : 5,6-Di-O-isopropylidene-3-O-methyl-α-D-glucofuranose
(20). Yield 81%. Colourless syrup, [α]D = −37.0 (c 0.5, EtOH),
lit15 [α]D = −33.0 (c 0.4, EtOH); Rf = 0.63 (1 : 1 light petroleum/
Et2O). IR (film): νmax 2987 and 2937 (CH), 1083 and 1020 (C–
O–C). 1H NMR (250 MHz, CDCl3): δ 1.21, 1.25, 1.32 and 1.38 (4
To a solution of compound 20 (3.40 g, 12.39 mmol) in dry
EtOAc (340 mL) was added H5IO6 (3.54 g, 15.55 mmol). The
mixture was stirred at room temperature for 1.5 h, then filtered
and evaporated to afford crude aldehyde 20a. To a stirred and
cooled (0 °C) solution of 20a (2.38 g) in dry toluene (80 mL)
was added a solution of PhMgBr (16 mL, 32.00 mmol) in dry
THF. The mixture was stirred for 5 h at 0 °C, in an atmosphere
of nitrogen, then poured into 10% aq NH4Cl (150 mL) and
extracted with EtOAc (2 × 100 mL). The combined extracts were
washed with 10% aq NaCl (100 mL), the organic phase was
dried and evaporated and the residue purified by flash column
chromatography (1: 1 light petroleum/Et2O). Eluted first was
the pure minor product 21 (0.20 g, 6%) isolated as a colourless
oil. Crystallization from CH2Cl2/hexane gave colourless prisms,
mp 71–73 °C, [α]D = −43.7 (c 0.5, CHCl3), Rf = 0.33 (1 : 1 light pe-
troleum/Et2O). IR (KBr): νmax 3508 (OH). 1H NMR (250 MHz,
CDCl3): δ 1.31 and 1.46 (2 × s, 3 H each, Me2C), 3.41 (s, 3 H,
OMe), 3.62 (bs, 1 H, OH), 3.71 (d, 1 H, J3,4 = 3.2 Hz, H-3), 4.28
(dd, 1 H, J3,4 = 3.2, J4,5 = 5.7 Hz, H-4), 4.56 (d, 1 H, J1,2 = 3.8 Hz,
H-2), 5.09 (d, 1 H, J4,5 = 5.6 Hz, H-5), 6.01 (d, 1 H, J1,2 = 3.8 Hz,
H-1), 7.20–7.50 (m, 5 H, Ph). 13C NMR (62.9 MHz, CDCl3): δ
26.1 and 26.7 (Me2C), 57.5 (OMe), 72.3 (C-5), 81.0 (C-2), 82.3 (C-
4), 85.1 (C-3), 105.1 (C-1), 111.5 (Me2C), 126.0, 127.6, 128.4 and
141.4 (Ph). HRMS (ESI): m/z 298.1646 (M+ + NH4), calcd. for
C15H24NO5: 298.1649; m/z 561.2679 (2 M+ + H), calcd. for
C30H41O10: 561.2694.
Eluted second was the pure major product 23 (1.95 g, 56%)
isolated as a colourless oil. Crystallization from CH2Cl2/
hexane gave colourless prisms, mp 97–98 °C, [α]D = −70.6 (c
0.5, CHCl3), Rf = 0.25 (1 : 1 light petroleum/Et2O). IR (KBr):
νmax 3528 (OH). 1H NMR (250 MHz, CDCl3): δ 1.29 and 1.46 (2
× s, 3 H each, Me2C), 2.88 (bs, 1 H, OH), 3.25 (s, 3 H, OMe),
3.25 (d, 1 H, H-3), 4.23 (dd, 1 H, J3,4 = 3.2, J4,5 = 7.9 Hz, H-4),
4.53 (d, 1 H, J1,2 = 3.8 Hz, H-2), 5.00 (d, 1 H, J4,5 = 7.9 Hz, H-5),
5.97 (d, 1 H, J1,2 = 3.8 Hz, H-1), 7.20–7.55 (m, 5 H, Ph). 13C
NMR (62.9 MHz, CDCl3): δ 26.1 and 26.6 (Me2C), 57.2 (OMe),
72.2 (C-5), 81.4 (C-2), 83.7 (C-3), 84.6 (C-4), 105.0 (C-1), 111.7
(Me2C), 126.9, 128.0, 128.2 and 139.7 (Ph). HRMS (ESI): m/z
298.1648 (M+ + NH4), calcd. for C15H24NO5: 298.1649.
× s, 3 H each, 2 × Me2C), 3.34 (s, 3 H, OMe), 3.66 (d, 1 H, J3,4
=
2.9 Hz, H-3), 3.82–4.03 (m, 3 H, 2 × H-6 and H-4), 4.18 (m, 1 H,
H-5), 4.45 (d, 1 H, J1,2 = 3.7 Hz, H-2), 5.75 (d, 1 H, J1,2 = 3.7 Hz,
H-1). 13C NMR (62.9 MHz, CDCl3): δ 25.1, 26.0 and 26.6 (2 ×
Me2C), 57.9 (OMe), 66.9 (C-6), 72.2 (C-5), 80.8 (C-4), 81.7 (C-2),
83.5 (C-3), 104.9 (C-1), 108.7 and 111.4 (2 × Me2C). HRMS
(ESI): m/z 275.1487 (M+ + H), calcd. for C13H23O6: 275.1489; m/
z 292.1755 (M+ + NH4), calcd. for C13H26NO6: 292.1755.
1,2-O-Isopropylidene-3,5-di-O-methyl-5-C-phenyl-α-D-gluco-
pentofuranose (26). Yield 85% (from 25), 88% (from 21).
Colourless prisms, mp 54–55 °C (CH2Cl2/hexane), [α]D = −98.0
(c 0.5, CHCl3); Rf = 0.33 (4 : 1 light petroleum/Et2O). IR (KBr):
1
νmax 2933 and 2918 (CH), 1089 (C–O–C). H NMR (400 MHz,
CDCl3): δ 1.30 and 1.44 (2 × s, 3 H each, Me2C), 3.25 and 3.54
(2 × s, 3 H each, 2 × OMe), 3.93 (d, 1 H, J3,4 = 2.9 Hz, H-3),
4.24 (dd, 1 H, J3,4 = 2.9, J4,5 = 9.2 Hz, H-4), 4.40 (d, 1 H, J4,5
=
9.2 Hz, H-5), 4.59 (d, 1 H, J1,2 = 3.7 Hz, H-2), 5.86 (d, 1 H, J1,2
= 3.7 Hz, H-1), 7.29–7.43 (m, 5 H, Ph). 13C NMR (100 MHz,
CDCl3): δ 26.2 and 26.7 (Me2C), 56.2 and 58.2 (2 × OMe), 79.7
(C-5), 81.7 (C-2), 82.6 (C-4), 83.7 (C-3), 104.9 (C-1), 111.4
(Me2C), 127.6, 127.9, 128.2 and 139.1 (Ph). HRMS (ESI): m/z
333.1090 (M+ + K), calcd. for C16H22KO5: 333.1099.
1,2-O-Isopropylidene-3,5-di-O-methyl-5-C-phenyl-β-L-ido-
pentofuranose (29). Yield 90% (from 28), 93% (from 23).
Colourless syrup, [α]D = +66.6 (c 0.5, CHCl3); Rf = 0.29 (7 : 3
light petroleum/Et2O). IR (film): νmax 3018 and 2935 (CH),
1217 and 1081 (C–O–C). 1H NMR (400 MHz, CDCl3): δ 1.31
and 1.50 (2 × s, 3 H each, Me2C), 3.14 (s, 3 H, C3–OMe), 3.25
(s, 3 H, C5–OMe), 3.93 (d, 1 H, J3,4 = 2.9 Hz, H-3), 4.34 (dd, 1
General procedure for the cyclocondensation with Meldrum's
acid (preparation of 15, 19, 4, 7, 5 and 8)
The starting compound 13, 17, 21, 23, 26 or 29 (1 equiv.) was
dissolved in 90% TFA (0.1 M) and the resulting solution was
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stirred at room temperature until the starting material was
consumed (TLC, 0.5 h for 13, 21 and 23, 1 h for 17, 26 and
29). The mixture was concentrated by co-distillation with tol-
uene, and the residue was purified by flash column chroma-
tography (7 : 3 light petroleum/Et2O for 14, 4 : 1 Et2O/light pe-
troleum for 18 and 27, 4 : 1 EtOAc/toluene for 22, 3 : 2
toluene/EtOAc for 24, and Et2O for 30). To a stirred solution
of compound 14, 18, 22, 24, 27 or 30 (1 equiv.) in dry DMF
(0.07 M) was added Meldrum's acid (3.0–3.6 equiv.) and dry
Et3N (3.0–3.1 equiv.). The mixture was stirred at 44–46 °C un-
til the starting material was consumed (TLC, 70 h for 14 and
24, 68 h for 18, 74 h for 22, 71 h for 27 and 30). The residue
was evaporated and purified by flash column chromatogra-
phy (3 : 2 light petroleum/Et2O for 15, 1 : 1 light petroleum/
Et2O for 5, 7 : 3 toluene/EtOAc for 4, 4 : 1 Et2O/light petroleum
for 7, and 7 : 3 Et2O/light petroleum for 19 and 8).
3,6-Anhydro-5-O-benzyl-2-deoxy-7-O-methyl-7-C-phenyl-D-
glycero-D-ido-heptono-1,4-lactone (15). Yield 51%. Colourless
needles, mp 172–173 °C (CH2Cl2/hexane), [α]D = −10.0 (c 0.5,
CHCl3); Rf = 0.39 (3 : 2 Et2O/light petroleum). IR (KBr): νmax
1780 (CO). 1H NMR (400 MHz, CDCl3): δ 2.51 (bd, 1 H, J2a,2b
= 18.8 Hz, H-2a), 2.61 (dd, 1 H, J2b,3 = 5.7, J2a,2b = 18.8 Hz, H-
2b), 3.19 (s, 3 H, OMe), 4.14 (dd, 1 H, J5,6 = 3.2, J6,7 = 8.9 Hz,
H-6), 4.44 (d, 1 H, J5,6 = 3.2 Hz, H-5), 4.46 (d, 1 H, J6,7 = 8.9 Hz,
H-7), 4.79 (s, 2 H, CH2Ph), 4.91–4.94 (m, 2 H, H-3 and H-4),
7.31–7.59 (m, 10 H, Ph). 13C NMR (100 MHz, CDCl3): δ 35.9
(C-2), 56.15 (OMe), 73.3 (CH2Ph), 77.1 (C-3), 78.0 (C-7), 81.0
(C-5), 83.4 (C-6), 85.2 (C-4), 127.8, 128.1, 128.2, 128.3, 128.5,
128.6, 137.4, 139.0 (Ph) and 175.50 (C-1). HRMS (ESI): m/z
377.1349 (M+ + Na), calcd. for C21H22NaO5: 377.1359; m/z
393.1088 (M+ + K), calcd. for C21H22KO5: 393.1099.
CDCl3): δ 35.7 (C-2), 58.3 (OMe), 71.8 (C-7), 77.0 (C-3), 83.0
(C-6), 83.8 (C-5), 84.0 (C-4), 126.1, 127.8, 128.3, 141.0 (Ph)
and 175.3 (C-1). HRMS (ESI): m/z 282.1334 (M+ + NH4), calcd.
for C14H20NO5: 282.1336.
3,6-Anhydro-2-deoxy-5-O-methyl-7-C-phenyl-L-glycero-D-ido-
heptono-1,4-lactone (7). Yield 46%. Colourless needles, mp
144–146 °C (CH2Cl2/hexane), [α]D = +122.4 (c 0.5, CHCl3); Rf =
0.36 (Et2O). IR (KBr): νmax 3450 (OH), 1783 (CO). 1H NMR
(250 MHz, CDCl3): δ 2.63 (d, 1 H, J2a,2b = 18.8 Hz, H-2a), 2.72
(dd, 1 H, J2a,2b = 18.9, J2b,3 = 4.9 Hz, H-2b), 2.99 (bs, 1 H, OH),
3.29 (s, 3 H, OMe), 3.52 (d, 1 H, J5,6 = 3.7 Hz, H-5), 4.61 (d, 1
H, J5,6 = 3.8, J6,7 = 7.1 Hz, H-6), 4.87 (d, 1 H, J3,4 = 4.6 Hz, H-4),
4.94 (d, 1 H, J6,7 = 7.1 Hz, H-7), 5.00 (m, 1 H, H-3), 7.20–7.44
(m, 5 H, Ph). 13C NMR (62.9 MHz, CDCl3): δ 35.8 (C-2), 57.9
(OMe), 72.5 (C-7), 77.2 (C-3), 83.3 (C-5), 84.3 (C-4), 84.7 (C-6),
126.7, 128.0, 128.2, 139.7 (Ph) and 175.3 (C-1). HRMS (ESI): m/
z 282.1343 (M+ + NH4), calcd. for C14H20NO5: 282.1336.
3,6-Anhydro-2-deoxy-5,7-di-O-methyl-7-C-phenyl-D-glycero-
D-ido-heptono-1,4-lactone (5). Yield 44%. Colourless needles,
mp 96–97 °C (CH2Cl2/hexane), [α]D = −57.0 (c 0.5, CHCl3); Rf =
1
0.23 (CH2Cl2). IR (KBr): νmax 1784 (CO). H NMR (400 MHz,
CDCl3): δ 2.47 (bd, 1 H, J2a,2b = 18.9 Hz, H-2a), 2.61 (dd, 1 H,
J2a,2b = 18.9, J2b,3 = 6.4 Hz, H-2b), 3.21 (s, 3 H, C7–OMe), 3.58
(s, 3 H, C5–OMe), 4.07 (dd, 1 H, J5,6 = 3.2, J6,7 = 8.9 Hz, H-6),
4.14 (d, 1 H, J5,6 = 3.0 Hz, H-5), 4.35 (d, 1 H, J6,7 = 8.9 Hz, H-
7), 4.87 (bt, 1 H, J = 5.6, J = 5.1 Hz, H-3), 4.93 (d, 1 H, J3,4
=
4.5 Hz, H-4), 7.28–7.41 (m, 5 H, Ph). 13C NMR (100 MHz,
CDCl3): δ 36.0 (C-2), 56.1 (C7–OMe), 58.8 (C5–OMe), 76.8 (C-
3), 79.8 (C-7), 82.8 (C-5), 83.1 (C-6), 84.5 (C-4), 127.6, 128.1,
128.3 and 139.0 (Ph) and 175.4 (C-1). HRMS (ESI): m/z
317.0777 (M+ + K), calcd. for C15H18KO5: 317.0786.
3,6-Anhydro-5-O-benzyl-2-deoxy-7-O-methyl-7-C-phenyl-L-
glycero-D-ido-heptono-1,4-lactone (19). Yield 47%. Colourless
syrup, [α]D = +92.0 (c 0.5, CHCl3); Rf = 0.24 (49 : 1 CH2Cl2/
EtOAc). IR (film): νmax 1789 (CO). 1H NMR (400 MHz, CDCl3):
δ 2.73 (dd, 1 H, J2a,3 = 6.3, J2a,2b = 18.9 Hz, H-2a), 2.84 (d, 1 H,
J2a,2b = 18.8 Hz, H-2b), 3.23 (s, 3 H, OMe), 4.65 (d, 1 H, J5,6 =3.5
Hz, H-5), 4.29 and 4.40 (2 × d, 2 H, Jgem = 11.5 Hz, CH2Ph), 4.32
(dd, 1 H, J5,6 = 3.6, J6,7 = 7.9 Hz, H-6), 4.46 (d, 1 H, J6,7 = 7.9 Hz,
H-7), 4.84 (d, 1 H, J3,4 = 4.6 Hz, H-4), 5.12 (t, 1 H, J2a,3 =5.5, J3,4
= 5.2 Hz, H-3), 7.23–7.50 (m, 10 H, Ph). 13C NMR (100 MHz,
CDCl3): δ 36.0 (C-2), 56.5 (OMe), 72.6 (CH2Ph), 77.6 (C-3), 81.8
(C-5), 82.2 (C-7), 84.2 (C-6), 84.4 (C-4), 127.6, 127.8, 128.1,
128.4, 128.5, 128.6, 136.9, 137.6 (Ph) and 175.3 (C-1). HRMS
(ESI): m/z 372.1813 (M+ + NH4), calcd. for C21H26NO5: 372.1806;
m/z 377.1361 (M+ + Na), calcd. for C21H22NaO5: 377.1359; m/z
393.1102 (M+ + K), calcd. for C21H22KO5: 393.1099.
3,6-Anhydro-2-deoxy-5,7-di-O-methyl-7-C-phenyl-L-glycero-D-
ido-heptono-1,4-lactone (8). Yield 51%. Colourless prisms,
mp 119–120 °C (CH2Cl2/hexane), [α]D = +206.8 (c 0.5, CHCl3);
1
Rf = 0.14 (24 : 1 CH2Cl2/EtOAc). IR (KBr): νmax 1787 (CO). H
NMR (400 MHz, CDCl3): δ 2.74 (dd, 1 H, J2a,3 = 6.1, J2a,2b
=
18.9 Hz, H-2a), 2.82 (d, 1 H, J2a,2b = 18.8 Hz, H-2b), 3.22 and
3.23 (2 × s, 6 H, 2 × OMe), 3.31 (d, 1 H, J5,6 = 2.9 Hz, H-5),
4.23 (dd, 1 H, J5,6 = 3.2, J6,7 = 8.2 Hz, H-6), 4.46 (d, 1 H, J6,7
=
8.2 Hz, H-7), 4.86 (d, 1 H, J3,4 = 4.6 Hz, H-4), 5.08 (t, 1 H, J =
5.3 Hz, H-3), 7.31–7.44 (m, 5 H, Ph). 13C NMR (100 MHz,
CDCl3): δ 36.0 (C-2), 56.6 and 57.8 (2 × OMe), 77.6 (C-3), 82.2
(C-7), 83.1 (C-5), 83.7 (C-4), 84.2 (C-6), 127.8, 128.5, 128.55,
137.7 (Ph) and 175.39 (C-1). HRMS (ESI): m/z 301.1038 (M+ +
Na), calcd. for C15H18NaO5: 301.1046; m/z 317.0774 (M+ + K),
calcd. for C15H18KO5: 317.0786.
General procedure for benzyl deprotection (compounds 3 and 6)
3,6-Anhydro-2-deoxy-5-O-methyl-7-C-phenyl-D-glycero-D-ido-
heptono-1,4-lactone (4). Yield 41%. Colourless syrup, [α]D
=
A solution of 15 or 19 (1 equiv.) in MeOH (0.01 M) was hydro-
genated over 10% Pd/C (0.01 or 0.02 g; the catalyst contained
50% of water) for 4 h (for 15) or 24 h (for 19) at room temper-
ature. The mixture was filtered through a Celite pad and the
catalyst was washed with MeOH. The combined organic solu-
tions were evaporated and the residues were purified by flash
chromatography (4 : 1 Et2O/light petroleum for 3; 9 : 1 Et2O/
light petroleum for 6) to afford pure 3 and 6.
+5.5 (c 0.5, CHCl3); Rf = 0.23 (7 : 3 Et2O/light petroleum). IR
(film): νmax 3482 (OH), 1786 (CO). 1H NMR (250 MHz,
CDCl3): δ 2.63 (bd, 1 H, J2a,2b = 18.3 Hz, H-2a), 2.69 (dd, 1 H,
J2a,2b = 18.9, J2b,3 = 5.6 Hz, H-2b), 3.20 (bs, 1 H, OH), 3.48 (s,
3 H, OMe), 4.01 (d, 1 H, J5,6 = 3.6 Hz, H-5), 4.61 (d, 1 H, J5,6
3.6, J6,7 = 7.0 Hz, H-6), 4.88–5.01 (m, 3 H, J3,4 = 4.6 Hz, H-4,
H-7 and H-3), 7.26–7.49 (m, 5 H, Ph). 13C NMR (62.9 MHz,
=
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3,6-Anhydro-2-deoxy-7-O-methyl-7-C-phenyl-D-glycero-D-ido-
heptono-1,4-lactone (3). Yield 82%. Colourless needles, mp
123–124 °C (CH2Cl2/hexane), [α]D = +6.4 (c 0.5, CHCl3); Rf =
0.32 (4 : 1 Et2O/light petroleum). IR (KBr): νmax 3421 (OH),
in an atmosphere of nitrogen, then poured into 10% aq NH4-
Cl (15 mL) and extracted with CH2Cl2 (2 × 10 mL). The com-
bined extracts were washed with 10% aq NaCl (15 mL), the
organic phase was dried and evaporated and the residue puri-
fied by two flash column chromatography (first 7 : 3 Et2O/
light petroleum; second 19 : 1 CH2Cl2/EtOAc). Eluted first was
the pure product 10 (0.05 g, 35%) isolated as a colourless
solid. Crystallization from Et2O/hexane gave colourless cloudy
crystals, mp 122–125 °C, [α]D = −18.6 (c 1.0, CHCl3), Rf = 0.35
(24 : 1 CH2Cl2/EtOAc). IR (KBr): νmax 3424 (OH), 1738 (CO).
1H NMR (250 MHz, CDCl3): δ 2.78 (d, 2 H, J2,3 = 3.4 Hz, H-2),
3.13 (s, 3 H, OMe), 3.80 (d, 1 H, J5,6 = 3.5 Hz, H-5), 4.70 (s, 1
H, exchangeable with D2O, OH), 4.84 (d, 1 H, J3,4 = 4.4 Hz, H-
4), 5.04 (d, 1 H, J5,6 = 3.5 Hz, H-6), 5.12 (m, 1 H, H-3), 7.14–
7.56 (m, 10 H, Ph). NOE contact: H-4 and H-5. 13C NMR (62.9
MHz, CDCl3): δ 36.0 (C-2), 58.4 (OMe), 77.8 (C-3), 78.2 (C-7),
82.7 (C-6), 83.6 (C-4), 85.1 (C-5), 125.0, 126.0, 126.9, 126.96,
128.0, 128.2, 144.3, 145.5 (Ph) and 175.2 (C-1). HRMS (ESI):
m/z 323.1274 (MH+ − H2O), calcd. for C20H18O4: 323.1278; m/z
363.1200 (M+ + Na), calcd. for C20H20NaO5: 363.1203. Further
elution gave the pure unreacted starting compound 9 (0.04 g,
35%).
1
1784 (CO). H NMR (400 MHz, CDCl3): δ 2.65 (d, 1 H, J2a,2b
= 18.7 Hz, H-2a), 2.74 (dd, 1 H, J2a,2b = 18.8, J2b,3 = 5.9 Hz, H-
2b), 3.35 (s, 3 H, OMe), 3.98 (dd, 1 H, J5,6 = 2.7, J6,7 = 4.3 Hz,
H-6), 4.42 (d, 1 H, J5,6 = 2.4 Hz, H-5), 4.71 (d, 1 H, J6,7 = 4.3
Hz, H-7), 4.88 (d, 1 H, J3,4 = 4.2 Hz, H-4), 5.10 (m, 1 H, H-3),
7.34–7.46 (m, 5 H, Ph). 13C NMR (100 MHz, CDCl3): δ 36.0 (C-
2), 57.9 (OMe), 74.1 (C-5), 77.0 (C-3), 83.0 (C-7), 83.5 (C-6),
87.4 (C-4), 126.9, 128.7, 128.73, 128.9, 129.0, 137.0 (Ph) and
175.4 (C-1). HRMS (ESI): m/z 282.1332 (M+ + NH4), calcd. for
C14H20NO5: 282.1336; m/z 287.0899 (M+ + Na), calcd. for
C14H16NaO5: 287.0890.
3,6-Anhydro-2-deoxy-7-O-methyl-7-C-phenyl-L-glycero-D-ido-
heptono-1,4-lactone (6). Yield 68%. Colourless powder, mp
118 °C (CH2Cl2/hexane), [α]D = +187.0 (c 0.5, CHCl3); Rf = 0.17
(Et2O). IR (KBr): νmax 3413 (OH), 1782 (CO). 1H NMR (400
MHz, CDCl3): δ 2.65 (dd, 1 H, J2a,3 = 2.9, J2a,2b = 18.8 Hz, H-
2a), 2.76 (dd, 1 H, J2a,2b = 18.8, J2b,3 = 4.5 Hz, H-2b), 3.28 (s, 3
H, OMe), 3.98 (dd, 1 H, J6,7 = 5.2 Hz, H-6), 4.22 (bs, 1 H, H-5),
4.57 (d, 1 H, J6,7 = 5.1 Hz, H-7), 4.86 (d, 1 H, J3,4 = 4.6 Hz, H-
4), 5.11 (m, 1 H, H-3), 7.34–7.43 (m, 5 H, Ph). 13C NMR (100
MHz, CDCl3): δ 35.9 (C-2), 56.6 (OMe), 75.1 (C-5), 77.0 (C-3),
82.4 (C-7), 83.0 (C-6), 87.5 (C-4), 127.6, 128.7, 137.2 (Ph), and
175.4 (C-1). HRMS (ESI): m/z 282.1329 (M+ + NH4), calcd. for
C14H20NO5: 282.1336; m/z 287.0893 (M+ + Na), calcd. for
C14H16NaO5: 287.0890; m/z 303.0630 (M+ + K), calcd. for
C14H16KO5: 303.0629.
X-ray crystal structure analysis
Diffraction experiments were performed on an Oxford Dif-
fraction Gemini S diffractometer. The crystal structures were
solved using SHELXT,18 or SIR92,19 and refined with
SHELXL.20 The structures are shown in the ESI.† The crystal-
lographic data have been deposited at the Cambridge Crystal-
lographic Data Centre. CCDC numbers: 1843444 (5), 1843445
(23), 1843446 (3), 1843447 (21), 1843448 (9) and 1843449 (8).
3,6-Anhydro-2-deoxy-5-O-methyl-7-C-phenyl-D-ido-hept-7-
ulosono-1,4-lactone (9). To a solution of compound 4 or 7 (1
equiv.) in dry CH2Cl2 (0.01 M) was added PCC (2.6 equiv.).
The mixture was stirred at reflux for 2.5 h (for 4) or 2 h (for
7) then concentrated, and the residues were purified by flash
column chromatography 4 : 1 Et2O/light petroleum. Pure 9
(yields 99% from 4, 91% from 7) was isolated as a colourless
solid. Recrystallization from CH2Cl2/hexane gave colourless
needles, mp 135–136 °C, [α]D = +27.4 (c 0.5, CHCl3); Rf = 0.30
(4 : 1 Et2O/light petroleum). IR (KBr): νmax 1779 (C1O, lac-
Biological evaluations
Chemicals and cell lines. Rhodamine B, RPMI 1640 me-
dium, foetal calf serum, propidium iodide 3-(4,5-
dimethylthiazol)-2,5-diphenyltetrazolium bromide and RNase
A were purchased from Sigma (St. Louis, MO, USA). Penicillin
and streptomycin were purchased from Galenika AD (Bel-
grade, Serbia). The Annexin V FLUOS apoptosis detection kit
was purchased from BD Biosciences Pharmingen (Belgium).
All other chemicals used in the experiments were commercial
products of reagent grade. The stock solution (10 mM) was
prepared in DMSO and diluted to various concentrations with
serum-free culture medium. Human chronic myelogenous
leukaemia (K562, ATCC CCL 243), promyelocytic leukaemia
(HL-60, ATCC CCL 240), human T cell leukaemia (Jurkat,
ATCC TIB 152) and Burkitt's lymphoma (Raji, ATCC CCL 86)
were grown in RPMI 1640, while ER+ breast adenocarcinoma
(MCF-7, ATCC HTB 22), ER− breast adenocarcinoma (MDA-
MB 231, ATCC HTB 26), cervix carcinoma (HeLa, ATCC CCL
2), lung adenocarcinoma epithelial (A549, ATCC CCL-185) ma-
lignant cells, and normal foetal lung fibroblasts (MRC-5,
ATCC CCL 171) were grown in DMEM medium. Both media
were supplemented with 10% of foetal calf serum (FTS,
NIVNS) and antibiotics (100 IU mL−1 of penicillin and 100 mg
1
tone), 1698 (C7O). H NMR (250 MHz, CDCl3): δ 2.63 (d, 1
H, J2a,2b = 18.4 Hz, H-2a), 2.72 (dd, 1 H, J2a,2b = 18.6, J2b,3
=
4.5 Hz, H-2b), 3.12 (s, 3 H, OMe), 4.43 (d, 1 H, J5,6 = 5.1 Hz,
H-5), 4.94 (d, 1 H, J3,4 = 4.0 Hz, H-4), 5.19 (m, 1 H, J = 4.3 Hz,
H-3), 5.56 (d, 1 H, J5,6 = 5.1 Hz, H-6), 7.32–7.92 (m, 5 H, Ph).
13C NMR (62.9 MHz, CDCl3): δ 35.9 (C-2), 58.5 (OMe), 78.2 (C-
3), 83.2 (C-6), 84.6 (C-4), 85.3 (C-5), 127.6, 128.5, 133.1, 135.6
(Ph), 174.7 (C-1) and 194.4 (C-7). HRMS (ESI): m/z 263.0909
(M+ + H), calcd. for C14H15O5: 263.0914; m/z 280.1175 (M+
NH4), calcd. for C14H18NO5: 280.1180; m/z 285.0729 (M+
+
+
Na), calcd. for C14H14NaO5: 285.0733; m/z 301.0476 (M+ + K),
calcd. for C14H14KO5: 301.0473.
5-O-Methyl-7-C-phenyl-goniofufurone (10). To a stirred and
cooled (0 °C) solution of 9 (0.11 g, 0.42 mmol) in dry THF
(1.9 mL) was added a solution of PhMgBr (0.46 mL, 0.46
mmol) in dry THF. The mixture was stirred for 1.5 h at 0 °C,
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mg−1 of streptomycin). The cell lines were cultured in flasks
(Costar, 25 mL) at 37 °C in an atmosphere of 100% humidity
and 5% of CO2 (Heraeus). Exponentially growing viable cells
were used throughout the assays. All of the cell lines were
obtained from the ATCC (American Type Culture Collection).
Cell treatment. The cells were seeded in six-well plates at
a concentration of 5 × 105 cells per well. The cells were
treated for 72 h with goniofufurone (1), 7-epi-goniofufurone
(2) and the analogues (3–10) at their IC50 concentrations.
Untreated cells were used as the positive control. Viable cells
of the treated and control samples were used for apoptosis
detection. Viability was determined using the trypan blue
dye-exclusion assay.
age of specific apoptosis was detected after treatment with
some of the analogues. Compounds 6, 7 and 10 caused the
greatest specific apoptosis (11.17, 55.48, and 33.41%,
respectively).
X-ray analysis of the methylated goniofufurone analogues
did not confirm the existence of intramolecular H-bonds in
their molecules. This suggests that the formation of a quasi-
ring is unlikely (at least in solid state). However, there are nu-
merous other reasons that could render the mono- or
dialkylated derivatives more or less active such as a change
in lipophilicity, increased or decreased uptake, etc.
Conflicts of interest
MTT assay. The colorimetric MTT assay was carried out
following the literature procedure.17
There are no conflicts to declare.
Cell cycle analysis. The cell cycle analysis was carried out
following a previously reported procedure.21
Acknowledgements
Detection of apoptosis. Apoptosis of K562 cells was evalu-
ated according to a previously reported procedure.21
Western blot. Western blot was carried out as reported
previously.21
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 (in part) by a re-
search project from the Serbian Academy of Sciences and Arts
(Grant No. F-130).
Conclusions
Notes and references
In conclusion, eight new analogues of (+)-goniofufurone and
7-epi-(+)-goniofufurone bearing methoxy groups at C-7 and/or
C-5 were synthesized and evaluated for their antiproliferative
activity against a panel of human malignant cell lines. All syn-
thesized analogues showed a stronger activity than lead 1
against at least two tumour cell lines, while seven analogues
are more active than lead 2 toward at least two malignant cell
lines. Two analogues were more active than DOX, while a single
analogue shows similar activity to DOX against at least two tu-
mour cell lines. The 7-O-methoxy derivative 3 showed the
highest potency toward MDA-MB 231 cells (IC50 0.064 μM) be-
ing the most active compound under evaluation. None of the
analogues showed any significant cytotoxicity toward normal
MRC-5 cells. In contrast, the commercial drug DOX showed a
potent cytotoxic activity with an IC50 value of 0.10 μM.
The preliminary SAR analysis suggested the following
structural requirements for the cytotoxic effects of the synthe-
sized compounds: (A) introduction of methoxy groups at the
C-7 and/or C-5 positions caused the increased cytotoxicity
originally displayed by lead 1, but decreased the anti-
proliferative potency originally demonstrated by lead 2; (B)
the keto derivative 9 with a methoxy group at C-5, showed a
more potent antiproliferative activity than the control mole-
cules 1 and 2; (C) insertion of an additional phenyl group at
the C-7 position of the 5-O-methoxy derivative (compound 10)
resulted in an increase in the cytotoxic activity with respect to
lead 1, but decreased the activity with respect to 2.
1 C. Wiart, J. Evidence-Based Complementary Altern. Med.,
2007, 4, 299–311.
2 A. de Fatima, L. V. Modolo, L. S. Conegero, R. A. Pilli, C. V.
Ferreira, L. K. Kohn and J. E. de Carvalho, Curr. Med. Chem.,
2006, 13, 3371–3384.
3 H. B. Mereyala and M. Joe, Curr. Med. Chem.: Anti-Cancer
Agents, 2001, 1, 293–300.
4 M. A. Blazquez, A. Bermejo, M. C. Zafra-Polo and D. Cortes,
Phytochem. Anal., 1999, 10, 161–170.
5 X.-S. Peng, R. M. P. Ylagan, Y. M. Siu and H. N. C. Wong,
Chem. – Asian J., 2015, 10, 2070–2083.
6 J. Francuz, I. Kovačević, M. Popsavin, G. Benedeković, B. Srećo
Zelenović, V. Kojić, D. Jakimov, L. Aleksić, G. Bogdanović, T.
Srdić-Rajić, E. Lončar, M. V. Rodić, V. Divjaković and V.
Popsavin, Eur. J. Med. Chem., 2017, 128, 13–24.
7 V. Popsavin, B. Srećo, G. Benedeković, J. Francuz, M.
Popsavin, V. Kojić and G. Bogdanović, Eur. J. Med. Chem.,
2010, 45, 2876–2883.
8 V. Popsavin, J. Francuz, B. Srećo Zelenović, G. Benedeković,
M. Popsavin, V. Kojić and G. Bogdanović, Bioorg. Med. Chem.
Lett., 2013, 23, 5507–5510.
9 G. Benedeković, J. Francuz, I. Kovačević, M. Popsavin, B.
Srećo Zelenović, V. Kojić, G. Bogdanović, V. Divjaković and
V. Popsavin, Eur. J. Med. Chem., 2014, 82, 449–458.
10 G. Benedeković, I. Kovačević, M. Popsavin, J. Francuz, V.
Kojić, G. Bogdanović and V. Popsavin, Tetrahedron, 2015, 71,
4581–4589.
11 X. P. Fang, J. E. Anderson, C. J. Chang, P. E. Fanwick and J. L.
McLaughlin, J. Chem. Soc., Perkin Trans. 1, 1990, 1655–1661.
12 M. Ralph, S. Ng and K. I. Booker-Milburn, Org. Lett.,
2016, 18, 968–971.
The cell cycle analysis reveals that the treatment of cells
with some of the synthesized analogues increases the inten-
sity of the sub-G1 peak of K562 cells indicating that the
mechanism of action of these compounds involves apoptosis.
Flow cytometry further confirmed that a significant percent-
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13 V. Popsavin, G. Benedeković, B. Srećo, J. Francuz, M.
Popsavin, V. Kojić, G. Bogdanović and V. Divjaković,
Tetrahedron, 2009, 65, 10596–10607.
14 T. D. Inch, Carbohydr. Res., 1967, 5, 45–52.
15 J. Kovar and H. H. Baer, Can. J. Chem., 1973, 51, 1801–1811.
16 R. Bruns, A. Wernicke and P. Köll, Tetrahedron, 1999, 55,
9793–9800.
17 D. A. Scudiero, R. H. Shoemaker, K. D. Paull, A. Monks, S.
Tierney, T. H. Nofziger, M. J. Currens, D. Seniff and M. R.
Boyd, Cancer Res., 1988, 48, 4827–4833.
18 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Adv.,
2015, 71, 3–8.
19 A. Altomare, G. Cascarano, C. Giacovazzo and A. Gualardi,
J. Appl. Crystallogr., 1993, 26, 343–350.
20 G. M. Sheldrick, Acta Crystallogr., Sect. C: Struct. Chem.,
2015, 71, 3–8.
21 I. Kovačević, M. Popsavin, G. Benedeković, J. Kesić, V.
Kojić, D. Jakimov, T. Srdić-Rajić, G. Bogdanović, V.
Divjaković and V. Popsavin, Eur. J. Med. Chem., 2017, 134,
293–303.
This journal is © The Royal Society of Chemistry 2018
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