Synthesis and antiproliferative activity of goniobutenolides A and B, 5-halogenated crassalactone D derivatives and the corresponding 7-epimers

Article abstract of DOI:10.1016/j.ejmech.2015.12.011

A new synthesis of goniobutenolides A (1) and B (2) and the corresponding 7-epimers has been achieved starting from diacetone d-glucose. The key step of the synthesis is a new one-pot sequence that commenced with Z-selective Wittig (or Horner-Wadsworth-Emmons) olefination, followed by successive γ-lactonisation and β-elimination. The above-mentioned unsaturated lactones were then converted to the corresponding 5-halogenated crassalactone D derivatives by using the appropriate haloetherification protocol. The most of synthesized compounds exhibited potent cytotoxic activities against a panel of tumour cell lines. The main structural features responsible for their antitumour potency have been revealed by means of SAR analysis. Flow cytometry data suggested that cytotoxic effects of these compounds in the culture of K562 cells might be mediated by apoptosis, additionally revealing that these molecules induced changes in cell cycle distribution of these cells. Results of semi-quantitative Western blot analysis indicate that the most of synthesized compounds induce apoptosis in a caspase-dependent manner.

Full text of DOI:10.1016/j.ejmech.2015.12.011

European Journal of Medicinal Chemistry 108 (2016) 594e604
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and antiproliferative activity of goniobutenolides A and B, 5-
halogenated crassalactone D derivatives and the corresponding 7-
epimers
a
ꢀ
ꢁ a
ꢁ a
ꢁ c
ꢁ b
Ivana Kovacevic , Mirjana Popsavin , Goran Benedekovic , Vesna Kojic ,
b
ꢁ a
ꢁ
ꢁ b
Dimitar Jakimov , Marko V. Rodic , Tatjana Srdic-Rajic , Gordana Bogdanovic ,
Vladimir Divjakovic , Velimir Popsavin
a, *
ꢁ d
a
ꢁ
Department of Chemistry, Biochemistry and Environmental Protection, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 3, 21000 Novi
Sad, Serbia
b Oncology Institute of Vojvodina, Put Doktora Goldmana 4, 21204, Sremska Kamenica, Serbia
c Institute for Oncology and Radiology of Serbia, Pasterova 14, 11000 Belgrade, Serbia
d
ꢁ
Department of Physics, Faculty of Sciences, University of Novi Sad, Trg Dositeja Obradovica 4, 21000 Novi Sad, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
A new synthesis of goniobutenolides A (1) and B (2) and the corresponding 7-epimers has been achieved
Received 30 July 2015
Received in revised form
4 December 2015
Accepted 9 December 2015
Available online 12 December 2015
starting from diacetone
commenced with Z-selective Wittig (or Horner-Wadsworth-Emmons) olefination, followed by successive
-lactonisation and -elimination. The above-mentioned unsaturated lactones were then converted to
D-glucose. The key step of the synthesis is a new one-pot sequence that
g
b
the corresponding 5-halogenated crassalactone D derivatives by using the appropriate haloetherification
protocol. The most of synthesized compounds exhibited potent cytotoxic activities against a panel of
tumour cell lines. The main structural features responsible for their antitumour potency have been
revealed by means of SAR analysis. Flow cytometry data suggested that cytotoxic effects of these com-
pounds in the culture of K562 cells might be mediated by apoptosis, additionally revealing that these
molecules induced changes in cell cycle distribution of these cells. Results of semi-quantitative Western
blot analysis indicate that the most of synthesized compounds induce apoptosis in a caspase-dependent
manner.
Keywords:
Goniobutenolide A
Goniobutenolide B
Crassalactone D
Haloetherification
Antitumour activity
Structure-activity relationships
Apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
cell lines [10]. In 2009 two research groups reported independently
total synthesis of 3 [11,12]. Recent report disclosed by Bermejo et al.
Goniobutenolide A (1), goniobutenolide B (2) and crassalactone
D (3) (Fig. 1) are products of secondary metabolism of Annonaceae
plant family, the largest family in Magnoliales. Goniobutenolides A
and B have been isolated from the stem bark of Gonithalamus
giganteus by brine shrimp lethality-directed fractionation of the
ethanolic extract and have been found to exhibit cytotoxicity to-
ward few cancer cell lines [1]. Although these compounds were the
targets of synthetic efforts by several groups [2e9], their biological
activities were not investigated in details. Tuchinda and co-workers
isolated crassalactone D (3) from the tropical plant Polyalthia Crassa
and evaluated its cytotoxicity against several mammalian cancer
[13] describes a bromoetherification of 6-epi-goniobutenolides A
and B that afforded a mixture of isomeric bromo-spirolactones. This
finding prompted us to extend this reaction to the natural gonio-
butenolides A (1) and B (2), as well as to the corresponding 7-
epimers, in order to prepare novel crassalactone D and 7-epi-
crassalactone D 5-halogen-derivatives for biological testing. The
additional reason for carrying out of such a research is based on
recent findings, which showed that some halogenated natural
products exhibit a variety of biological activities [14,15].
Herein, we disclose full details on the synthesis of 1 and 2 [16],
as well as their conversion to novel halogenated spiro-lactones 3a,
3b, 4aed including a number of selected 7-epimers (5e8, 7a, 7b,
8aec). The effects of these compounds to the proliferation of
certain human tumour cell lines, including their effects on the
K562 cell cycle and their apoptosis inducing properties in the same
* Corresponding author.
E-mail address: velimir.popsavin@dh.uns.ac.rs (V. Popsavin).
http://dx.doi.org/10.1016/j.ejmech.2015.12.011
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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595
Fig. 1. Chemical structures of goniobutenolide A (1), goniobutenolide B (2), crassalactone D (3), (4R)-crassalactone D (4), the corresponding halogen derivatives (3a, 3b, 4aed) and
(7S)-stereoisomers (5e8, 7a, 7b, 8aec).
cell culture, have also been studied in this work.
depend on the structure of the starting lactol, but also on the re-
action temperature (for details, see Table S1 in the Supplementary
data). The best combined yield of 1 and 2 (44%, Z/E ~2:1) was ob-
tained when lactol 14 was allowed to react with Still's reagent
(reagent B) under the HWE conditions (NaH, THF, Ar, 22 h, ꢀ10 ꢁC).
For possible mechanism of this process see Scheme S1 in the
Supplementary data. Our optical rotation values were different
from the values reported for synthetic [2e9] goniobutenolides A (1)
and B (2) but were in reasonable agreement with the reported data
of isolated 1 and 2 [1]. However, the NMR data of synthesized
samples 1 and 2 were in full agreement with those reported in the
literature for both isolated [1] and synthetic [2e9] samples 1 and 2.
The most convenient procedure for the preparation of lactones 5
and 6 appeared to be a Z-selective Wittig reaction of 15 with
(methoxycarbonylmethylene)triphenylphosphorane (reagent A), in
anhydrous MeOH (Et3N, 4 h, ꢀ10 ꢁC). The desired unsaturated
lactones 5 and 6 were thus obtained in 73% combined yield and in
the approximate Z/E isomer ratio of 2:1.
2. Results and discussion
2.1. Chemistry
Synthesis of goniobutenolides A (1) and B (2) that started from
the commercially available 1,2:5,6-di-O-isopropylidene-a-D-gluco-
furanose (9) is shown in Scheme 1. Compound 9 was transformed
into benzylic alcohols 10 and 11 by well established chemical re-
actions [17,18]. Cyclic carbonate can act as a protecting group [19]
for the diol functionalities in 10 and 11, but also it can undergo
ring-opening reactions due to its good leaving group properties
[20]. Treatment of 10 and 11 either with 1,10-carbonyldiimidazole in
toluene or with triphosgene and pyridine in dichloromethane
afforded crystalline compounds 12 and 13 in high yields. Hydrolytic
removal of the isopropylidene protective groups in both 12 and 13
gave the corresponding lactols 14 and 15 in almost quantitative
yields. Both products are rather hygroscopic so they should be used
in the next synthetic step immediately after their brief isolation.
In order to convert lactols 14 and 15 to the corresponding un-
saturated lactones 1 and 2 we have studied two different olefina-
tion procedures, such as Z-selective Wittig reaction [5,21] with
(methoxycarbonylmethylene)triphenylphosphorane (reagent A)
and an alternative Z-selective olefination process in the presence of
methyl P,P-bis(2,2,2-trifluoroethyl)phosphonoacetate (reagent B),
under the standard Horner-Wadsworth-Emmons (HWE) condi-
tions [22]. The stereoselectivity of both processes seems to strongly
Stereochemistry of unsaturated lactones
2 and 5 were
confirmed by single crystal X-ray diffraction analysis (for the crystal
structures of 2 and 5, see the Supplementary data).
Having obtained unsaturated lactones 1, 2, 5 and 6, we next
focused on the synthesis of 5-halogeno crassalactone D derivatives
using different haloetherification protocols [23,24]. Iodoether-
ification of 2 with I2 (NaHCO3, CH3CN) gave 5-iodo derivatives 3a
(14%), 4a (27%) and 4b (10%) (entry 2, Table 1). The same products
were synthesized in slightly higher overall yield (3a 28%, 4a 21%, 4b
8%) after exposure of natural product 1 to the same reaction
Scheme 1. Reagents and conditions: (a) Imd2CO, toluene, for 10: reflux, 1.5 h, 95% of 12, for 11: 1.5 h, 80e85 ꢁC, 76% of 13; (b) triphosgene, Py, CH2Cl2, for 10: 0 ꢁC, 1 h, then rt, 1 h,
81% of 12, for 11: 0 ꢁC, 1 h, then rt, 2 h, 83% of 13; (c) 9:1 TFA/H2O, 0 ꢁC/rt, for 12: 1.5 h, 100% of 14, for 13: 2 h, 96% of 15; (d) reagent B, NaH, THF, Ar, 22 h, ꢀ10 ꢁC, 29% of 1, 15% of 2;
(e) reagent A, Et3N, MeOH, N2, 4 h, ꢀ10 ꢁC, 49% of 5, 24% of 6.

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Table 1
Haloetherification of goniobutenolides A (1) and B (2).
Entry
1
Starting compound
Reagents and conditions
Products (% yield)a
Goniobutenolide A (1)
I2, NaHCO3, CH3CN, 6 days, rt/35 ꢁC
3a R1 ¼ I, R2 ¼ H (28)
4a R1 ¼ H, R2 ¼ I (21)
4b R1 ¼ I, R2 ¼ H (8)
3a R1 ¼ I, R2 ¼ H (14)
4a R1 ¼ H, R2 ¼ I (27)
4b R1 ¼ I, R2 ¼ H (10)
4c R1 ¼ Cl, R2 ¼ H (40)
3b R1 ¼ Cl, R2 ¼ H (26)
4d R1 ¼ H, R2 ¼ Cl (17)
2
Goniobutenolide B (2)
I2, NaHCO3, CH3CN, 7 days, rt/35 ꢁC
3
4
Goniobutenolide A (1)
Goniobutenolide B (2)
NCS, DMF, 53 h, 35 ꢁC
NCS, DMF, 70 h, 36 ꢁC
a
Isolated yields.
conditions (entry 1, Table 1).
Treatment of 1 with NCS in DMF gave 5-chloro derivative 4c in
40% (Table 1, entry 3). Under the similar conditions styryl-lactone 2
was transformed into the mixture of 3b (26%) and 4d (17%) (Table 1,
entry 4).
Scheme 2. Reagents and conditions: (a) 9:1 TFA/H2O, CHCl3, 5 days, 22 ꢁC, 42% of 7a,
42% of 8a.
Iodoetherification of
5 and 6 (I2, NaHCO3) afforded 5-
iodolactones 7b and 8a (9% and 40%, Table 2 entry 1; 3% and 51%,
entry 2). Changing the source of iodine from I2 to NIS gave the same
products 7b and 8a but in lower yields (4% 7b and 15% 8a from 5,
entry 3; 12% 7b, 19% 8a from 6, entry 4).
Chloroetherification of 7-epimers 5 and 6 afforded the same
molecule 8b in very low yields (13% from 5, entry 5; 19% from 6,
entry 6).
Similarly, bromocyclisation of 5 (Br2, NaHCO3, CH2Cl2) gave
compound 8c in a yield of only 12% (entry 7). Bromoetherifaction of
5 was also attempted by using NBS in DMF. Under these reaction
conditions product 8c was obtained in a slightly better yield (29%,
entry 8).
Stereoisomer 7a could not be prepared by iodocyclisation of 5 or
6. However treatment of 8a with TFA in mixture of CHCl3/H2O
provided an equimolar mixture of 7a and 8a, thus providing a 42%
yield of 7a for biological testing (Scheme 2).
3a, 4a and 4b did not give the crystals of X-ray quality. In order to
confirm the stereochemistry at C-4 of iodo derivative 3a, it was
dehalogenated with tributyltin hydride, in dry toluene and in the
presence of AIBN, whereupon crassalactone D (3) was obtained in
81% yield (Scheme 3). This finding is consistent with (4R)-stereo-
chemistry in 3a. NOE interaction between H-5 and H-7 in 3a
confirmed its absolute configuration at C-5. Dehalogenation of both
4a and 4b under the same reaction conditions gave (4R)-crassa-
lactone D (4) in 94% and 95% respective yields. These results are
consistent with (4S)-stereochemistry of both 4a and 4b. NOE in-
teractions between H-5 and Ph-H in 4a and between H-5 and H-7 in
4b confirmed their absolute configuration at C-5.
Dehalogenation of 5-iodo derivatives 7a and 8a under the re-
action conditions similar to that described above provided the
corresponding dehalogenated derivatives 7 (93%) and 8 (71%) as the
only reaction products (Scheme 3).
It appears, that the multi-step sequence comprised of the con-
version of 10 to goniobutenolides 1 and 2, followed by their
Stereochemistry of 5-halo-derivatives 3b, 4c,d, 7a,b and 8aec
were unequivocally confirmed by single crystal X-ray diffraction
analysis (see Supplementary data for details). However, compounds
Table 2
Haloetherification of (7S)-goniobutenolides A (5) and B (6).
Entry
1
Starting compound
Reagents and conditions
Products (% yield)a
(7S)-goniobutenolide A (5)
I2, NaHCO3, CH3CN, 9 days, rt/35 ꢁC
7b (9)
8a R1 ¼ H, R2 ¼ I (40)
7b (3)
2
3
4
(7S)-goniobutenolide B (6)
(7S)-goniobutenolide A (5)
(7S)-goniobutenolide B (6)
I2, NaHCO3, CH3CN, 10 days, rt/35 ꢁC
NIS, CH3CN, 48 h, rt
8a R1 ¼ H, R2 ¼ I (51)
7b (4)
8a R1 ¼ H, R2 ¼ I (15)
7b (12)
NIS, DMF, 71 h, rt/50 ꢁC
8a R1 ¼ H, R2 ¼ I (19)
8b R1 ¼ Cl, R2 ¼ H (13)
8b R1 ¼ Cl, R2 ¼ H (19)
8c R1 ¼ Br, R2 ¼ H (12)
8c R1 ¼ Br, R2 ¼ H (29)
5
6
7
8
(7S)-goniobutenolide A (5)
(7S)-goniobutenolide B (6)
(7S)-goniobutenolide A (5)
(7S)-goniobutenolide B (6)
NCS, DMF, 69.5 h, 40 ꢁC
NCS, DMF, 69 h, 37 ꢁC
Br2, NaHCO3, CH2Cl2, 2.5 h, rt
NBS, DMF, atm. N2, 20 days, rt/45ꢁ
a
Isolated yields.

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597
Scheme 3. Reagents and conditions: (a) Bu3SnH, AIBN, toluene, 1 h, 100 ꢁC, 81% of 3 (from 3a), 94% of 4 (from 4a), 95% of 4 (from 4b), 93% of 7 (from 7a), 71% of 8 (from 8a).
subsequent iodoetherification (to 3a, 3b, 4aed), and their final
free-radical dehalogenation (3a/3, 4a,b/4) represent a new
synthesis of (þ)-crassalactone D (3) and (4R)-crassalactone D (4)
in the same cell lines. (7S)-Goniobutenolide A (5) exhibited the
most powerful activity against K562 cells being 1.7-fold more active
than DOX and exhibited over 2-fold higher potency with respect to
natural product 1. Also, compound 5 showed submicromolar ac-
tivity toward HeLa cells with a IC50 value 3-fold higher than that
recorded for DOX against the same cell line. The highest potency in
the culture of K562 cells between halogenated compounds was
from diacetone -glucose (9) as a chiral precursor. By a similar re-
D
action sequence compound 11 was converted to spiro-lactones 7
and 8 (via goniobutenolides 5 or 6 and iodo-lactones 7a,b or 8aec
as the key intermediates).
recorded after treatment with 4d (IC50 0.18 mM), which was 2-fold
2.2. In vitro antitumour activities and SAR
more potent than natural product 1, 161-fold more potent than 2
and 1.4-fold more active than DOX. Other compounds showed
Antiproliferative activities of synthesized natural products, their
stereoisomers and analogues were evaluated against eight tumour
cell lines (myelogenous leukaemia K562, promyelocytic leukaemia
HL-60, Jurkat T cells leukaemia, Raji Burkitt's lymphoma, estrogen
receptor positive breast adenocarcinoma MCF-7, estrogen receptor
negative breast adenocarcinoma MDA-MB-231, cervix carcinoma
HeLa, alveolar basal adenocarcinoma A549) and a single human
normal cell line (MRC-5). The use of foetal lung fibroblasts (MRC-5)
serves to demonstrate the toxicity of synthesized compounds to-
ward normal cells. Cytotoxicity was evaluated by using the stan-
dard MTT assay, after exposure of cells to the tested compounds for
72 h. The commercial antitumour agent doxorubicin (DOX) was
used as a reference compound in this assay.
micromolar activities (IC50 1.21e11.68 mM). In the cultures of HL-60
and A549 cells the most active was 5-bromo derivative 8c while
other halogenated molecules showed moderate activities. A potent
submicromolar activity was recorded after treatment of Jurkat cells
with analogue 7a, which was 2-fold more active than 1 and over
46-fold more potent than 2. Iodolactone 7b was 3-fold more active
with respect to DOX, 106-fold more potent than 1 and approxi-
mately 2100-fold more active than 2. Bromolactone 8c showed the
same antiproliferative activity as DOX (IC50 0.03 mM) against Jurkat
cells. However its activity was significantly stronger than that
exhibited by both natural products 1 and 2 (35- and 700-fold
respectively) in the same cell line. Several compounds exhibited
very strong activities toward Raji cells. These are 4c (IC50 0.95
mM),
As shown in Table 3 the natural product 1 showed sub-
micromolar activity toward promyelocytic leukaemia (HL-60), be-
ing notably more active than commercial cytostatic doxorubicin
(DOX). Although somewhat less active than DOX, compound 1
acted as a very potent growth inhibitor of K562, MCF-7, HeLa, Jurkat
7a (IC50 0.07 M), 7b (IC50 1.01 M), 8c (IC50 2.37 M) and their
m
m
m
potencies are higher than that recorded for 1, 2 and DOX. De-
rivatives 7a, 7b and 8c also showed notable antiproliferative effects
in the cultures of MCF-7 and MDA-MB-231 cells with IC50 values in
the low submicromolar or micromolar range. Comparing to DOX,
these analogues were more potent against MCF-7 cells but they
showed the same or lower potency in the culture of MDA-MB
231 cells. All halogenated compound were moderately active to-
ward HeLa cells. Remarkably, neither the natural products 1 and 2,
and Raji cells (IC50 0.35e1.99 mM). Unsaturated lactone 2 was found
less active with respect to 1. It demonstrated micromolar activities
against the following cell lines: MCF-7, MDA-MB 231 and Hela, but
these activities are significantly lower then those recorded for DOX
Table 3
In vitro cytotoxicity of natural lactones 1 and 2, their 7-epimers 5 and 6 halogenated analogues of crassalactone D (3a,b, 4aec, 7a,b, 8aec) and DOX.
Compound
IC50
K562
(m
M)a
HL-60
Jurkat
Raji
3.55
32.74
12.22
9.88
8.76
21.66
3.54
11.78
0.95
18.64
0.07
MCF-7
MDA-MB 231
HeLa
A549
18.14
>100
74.23
MRC-5
1
2
5
6
0.35
28.94
0.15
21.21
5.48
4.69
9.78
11.68
5.69
0.18
1.21
6.78
8.58
2.79
5.51
0.25
0.25
45.78
33.54
18.26
14.23
15.66
13.74
8.69
10.24
37.79
5.63
24.79
21.54
41.02
4.22
1.06
21.01
32.32
2.78
12.88
14.23
24.36
15.46
17.65
39.21
0.45
1.08
4.74
13.53
3.55
14.67
7.01
3.48
5.69
7.55
25.32
0.18
0.01
>100
8.11
1.99
5.34
0.21
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
89.63
91.21
>100
>100
88.78
0.10
2.89
29.91
47.63
29.64
18.97
23.36
48.36
39.62
0.12
1.32
5.69
42.15
0.09
0.09
6.91
68.98
23.15
22.63
18.08
15.54
14.56
19.85
26.54
21.85
16.78
14.24
13.24
4.91
3a
3b
4a
4b
4c
4d
7a
7b
8a
8b
8c
DOX
14.36
29.08
8.06
10.98
25.66
21.04
16.48
6.69
0.01
1.01
21.36
24.11
0.03
11.32
21.35
2.37
18.64
28.26
0.05
4.36
15.33
28.31
0.07
0.92
0.03
2.98
0.20
a
IC50 is the concentration of compound required to inhibit 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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Table 4
(A) Influence of synthesized compound on the K562 cell cycle; (B) percentage of specific apoptosis and necrosis induced with synthesized compounds in the K562 cell culture.
Compound
(A) distribution of K562 cells in cell cycle phases (%)
(B) type of cell death
Sub G1
G0/G1
S
G2/M
Specific apoptosis (%)
Specific necrosis (%)
control
1
2
5
6
3a
3b
4a
4b
4c
4d
7a
7b
8a
8b
8c
1.48
3.87
3.01
2.57
1.57
44.61
20.05
2.11
24.02
37.14
51.01
5.15
8.25
16.14
2.58
38.48
35.54
32.11
40.34
44.03
32.97
18.21
46.16
24.51
20.01
24.49
35.28
45.27
30.49
44.01
38.64
41.03
40.22
44.23
39.02
36.25
18.83
49.2
28.16
40.37
34.72
21.45
32.16
29.13
31.64
31.20
35.44
19.01
20.37
20.65
18.07
18.15
3.59
12.54
23.57
11.1
e
e
62.42
3.99
4.10
3.93
3.87
3.46
3.87
1.56
3.66
2.75
3.49
3.04
3.50
2.82
2.16
4.05
2.02
3.19
0.75
71.87
78.38
1.43
84.50
75.96
69.61
7.30
9.45
86.29
0.76
8.13
3.06
27.41
17.35
21.73
22.21
20.12
5.80
59.19
as well as their 7-epimers 5 and 6, nor halogenated spiro-lactones
were toxic toward normal human cells (MRC-5), while the com-
mercial antitumour agent DOX exhibited sub-micromolar toxicity
against this cell line (Table 4).
In order to determine influence of synthesized compounds on cell
distribution in cell cycle phases, K562 cells were treated with
synthesized compounds in their IC50 concentration. Staining of
apoptotic cells with propidium iodide, resulted in broad hypodip-
loid picks after flow cytometry analysis. Percentage of apoptotic,
necrotic and live cells were determined with flow cytometry after
double staining of treated cells with Annexin V-FITC/PI.
As data in Table 4 revealed all tested compounds increased
percentage of K562 cells in sub G1 phase compared with untreated
control. These findings suggested that synthesized lactones may
induce apoptosis. Generally, both unsaturated and halogenated
lactones with 7R configuration increased the cell percentage in sub
G1 phase compared to stereoisomers of 7S configuration. Chloro
derivatives 4d and 4c increased the cell percentage 34.5-fold and
25-fold respectively, while the iodo derivative 3a induced 30-fold
percentage of K562 cells in sub G1 phase compared with control.
Analogues 3b, 4b and 8a also significantly increase the percentages
of K562 cells in sub G1 phase.
Although natural product 1 increases only 2.6-fold percentage of
K562 cells in sub G1 phase with respect to control, it induced
apoptosis in 62.42% of cells. Compounds 8a was the most potent in
inducing apoptosis, but generally compounds with 7R configura-
tion induced apoptosis in larger number of cells compared with 7S
isomeres. This is in agreement with the results obtained for the
distribution of cells in the cell cycle phases. High percentage of
specific apoptosis induced after treatment of K562 cells with lac-
tones 4b (84.50%), 3b (78.38%), 3a (71.87%), 4d (69.61%) and 8c
(59.19%) is noteworthy. All tested compounds induced low per-
centage of specific necrosis in K562 cells (1.56e4.05%).
To resolve the mechanisms underlying the apoptosis induced
with synthesized styryl lactones we have investigated the ability of
these compounds (1, 2, 5, 6, 3a, 3b, 4aed, 7a, 7b, 8aec) to modulate
expression of some apoptosis proteins, such as Bcl-2, Bax, caspase 3
and PARP. Semi-quantitative Western blot analysis revealed that
the most of examined compounds (1, 2, 3a, 3b, 4a, 4c, 5, 7a, 8a)
reduced expression of anti-apoptotic Bcl-2 protein (cell guardians)
in K562 cells compared with control (Fig. 2a). The majority of tested
compounds (with the exception of 3a, 3b, 4a, 6) induced over-
expression of pro-apoptotic effector protein Bax (Bcl-2 associated
protein X, Fig. 2b) 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 [26,27].
In order to establish possible structure-activity relationships we
first considered the influence of double bond configuration to
antitumour activity of unsaturated styryl lactones (see Fig. S11 in
the Supplementary data for details). The importance of this struc-
tural feature for the cytotoxic activities was accessed by comparing
the IC50 values of Z-isomers (1, 5) and E-isomers (2, 6). The results
have shown that the geometry of double bond has different influ-
ence on antiproliferative activity of these molecules. Thus the
presence of the Z-configured C4]C5 double bond in 1 significantly
decreases the IC50 values, that is, increases the cytotoxicity of
goniobutenolide A (1) with respect to goniobutenolide B (2) against
seven of eight investigated cell lines. However, the same structural
change in molecule 5 increases the IC50 values, namely decreases
the cytotoxic activity of (7S)-goniobutenolide A (5) with respect to
(7S)-goniobutenolide B (6) against five cell lines. It appears that the
presence of Z-configured C4]C5 double bond increases the potency
of the corresponding isomers against at least three malignant cell
lines (K562, MDA-MB 231 and HeLa). Next, we studied the influ-
ence of absolute configuration at C-7 on growth-inhibitory activity
of isomeric goniobutenolides. The data in Table 3 indicate that, in
most cases, change of configuration from the 7R to the 7S increases
antitumour activity. The same trend was observed when activities
of 7R and 7S stereoisomeric halo-lactones were compared. Com-
parison of the IC50 values of 5R (4a, 4c, 5a) and 5S (4b, 4d, 5b)
halogenated compounds revealed that molecules with 5R config-
uration were more cytotoxic. Finally, we considered influence of
halogen atom type on antiproliferative activity of 5-halo-lactones.
The results revealed that the analogues bearing iodine or bromine
atom are more active than the corresponding 5-chloro derivatives.
This suggests that the presence of a bulkier halogen atom increases
antitumour activity of analogues.
2.3. Detection of apoptosis
Induction of apoptosis (a form of programmed cell death) in
tumour cells is desirable mode of action for chemotherapeutic
agents. Encouraged with findings that some of natural styryl-
lactones from Goniothalamus genus induced apoptosis in various
cell lines [25], we set the goal to examine apoptotic signalling
induced by natural products 1 and 2, their 7-epimers 5 and 6, as
well as by the halogenated spiro-lactones (3a,b, 4aed, 7a,b, 8aec).
Caspases, a family of aspartate-specific cysteine proteases, play
central role in the mechanism of apoptosis as they both initiate and

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Fig. 2. Results of Western blot analysis after treatment of K562 cells with synthesized compounds.
execute the apoptotic process. Downstream, effector caspases (3, 6,
7) contribute to the biochemical and morphological changes that
occur during the execution phase of apoptosis [28]. Three major
pathways to apoptosis-associated caspase activation have been
identified the extrinsic (death receptor), the intrinsic (mitochon-
drial) and the intrinsic endoplasmic reticulum pathway [29].
The expression level of the precursor and active subunit of
effector caspase 3 and catalytic fragment of PARP in the cells
exposed to synthesized compounds were measured in order to
determine induced apoptosis 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 24 kDa DNA-binding domain and
catalytic fragment (89 kDa) [30]. Western blot analysis showed
cleavage of procaspase 3 (Fig. 2c) and PARP (Fig. 2d) in K562 cells
after treatment with 1, 2, 4c, 5, 6, 8a, 8c. Expression of activated
caspase 3 higher than in control, but without cleavage of PARP, was
also induced with 4a, 4d and 8b. Induction of PARP cleavage
without expression of activated caspase 3 was detected after
treatment with halo-lactones 3b and 7b (see Fig. S18 in the Sup-
plementary data).
olefination at ꢀ10 ꢁC (73%, Z/E ~ 2:1). Finally, unsaturated lactones
1, 2, 5 and 6 were converted to eleven novel halogenated crassa-
lactone D derivatives (3a,b, 4aed, 7a,b, 8aec) by using different
haloetherification protocols.
Synthesized compounds were evaluated for their anti-
proliferative activity against a panel of malignant cell lines as well
as against a single normal cell line by using the MTT test. Natural
product 1 showed highest potency toward HL-60 cells (IC50
0.25
antitumour activity, especially analogues 8c (IC50 0.03
Jurkat cells) and 7a (IC50 0.07 M toward Raji cells). 5-Iodo deriv-
ative 7b was shown to be the most potent of all molecules under
mM). Some of the halogenated derivatives showed very potent
mM against
m
evaluation with IC50 0.01
against MCF-7 cells.
mM against Jurkat, and IC50 0.01 mM
The preliminary SAR analysis suggested that: (a) the presence of
the Z-configured double bond significantly decreases the IC50
values, that is, increases the cytotoxic activity of the corresponding
goniobutenolides against at least three malignant cell lines; (b)
changes in stereochemistry from 7R to 7S increases the antitumour
activity both of goniobutenolides and of halo-lactones; (c) halo-
lactones of 5R stereochemistry are more cytotoxic than the 5S
isomers toward the majority of cells investigated; (d) the presence
of 5-bromo and 5-iodo functionalities enhances antiproliferative
activity compared to the activity of corresponding 5-chloro
isosteres.
The flow-cytometry revealed that all synthesized compounds
increase percentage of K562 cells in sub G1 phase compared with
control, with analogue 4d being the most potent (51.01% cells). High
percentage (>50%) of specific apoptosis was detected for eight
compounds (1, 3a, 3b, 4bed, 8a and 8c). Western blot analysis of
apoptosis markers (Bcl-3, Bax, caspase 3, PARP) suggested that the
majority of investigated compounds induced apoptosis in
K562 cells in caspase-dependent way.
Based on all this results we hypothesized that the most of
investigated compounds induced apoptosis in K562 cells in
caspase-dependent way. 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 [31].
3. Conclusions
A new divergent synthesis of goniobutenolides A (1), B (2) and
the corresponding 7-epimers has been achieved starting from
diacetone D-glucose. The key step of the synthesis represents a
novel cascade reaction comprised of a Z-selective Wittig (or HWE)
olefination, followed by a concomitant lactonisation and the final E2
elimination. HWE olefination afforded a higher yield of 1 and 2
(44%, Z/E ~ 2:1) compared with Wittig olefination. The highest yield
and selectivity for 5 and 6 were achieved employing Wittig
4. Experimental section
4.1. General experimental procedures
Melting points were determined on a Hot Stage Microscope

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600
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 com-
pounds were acquired on an Agilent technologies 6210 TOF LC/MS
instrument (LC series 1200). Flash column chromatography was
performed using Kieselgel 60 (0.040e0.063, E. Merck). Preparative
TLC was performed on hand-made plates, 20 ꢂ 20 cm size with
~1 mm layer thickness. Kieselgel 60 G (E. Merck) with fluorescent
indicator F254 as additive was used as stationary phase. All organic
extracts were dried with anhydrous Na2SO4. Organic solutions were
concentrated in a rotary evaporator under reduced pressure at a
bath temperature below 30 ꢁC. Purity of the tested compounds was
determined by HRMS (errors were less than 5 ppm).
and then with Et2O (3 ꢂ 7 mL). The combined organic solutions
were washed with brine, dried and evaporated. The residue was
purified by flash column chromatography (24:1 CH2Cl2/Me2CO) to
afford pure 13 (0.432 g, 83%). Recrystallization from CH2Cl2/hexane
gave short white needles, mp 187 ꢁC, [
a
]
¼ þ23.8 (c 0.5, CHCl3),
D
Rf ¼ 0.45 (49:1 CH2Cl2/Me2CO). IR (film): nmax 1761 (C¼O). 1H NMR
(250 MHz, CDCl3):
d
1.32 and 1.46 (2 ꢂ s, 3H each, CMe2), 4.65 (dd,
1H, J3,4 ¼ 3.0, J4,5 ¼ 1.2 Hz, H-4), 4.78 (d, 1H, J1,2 ¼ 3.7 Hz, H-2), 5.01
(d, 1H, J3,4 ¼ 3.1 Hz, H-3), 5.55 (bs, 1H, H-5), 6.01 (d, 1H, J1,2 ¼ 3.7 Hz,
H-1), 7.35e7.51 (m, 5H, Ph). NOE contact H-1 and Ph-H, H-2 and Ph-
H. 13C NMR (62.5 MHz, CDCl3):
d 26.1 and 26.5 (CMe2), 72.6 (C-4),
77.7 (C-5), 82.8 (C-3), 83.1 (C-2), 105.1 (C-1), 112.9 (CMe2), 126.9,
128.5, 129.1, 134.0 (Ph), 146.8 (C¼O). HRMS (ESI): m/e 293.1005
(MþþH), calcd for C15H17O6: 293.1020; m/e 331.0564 (MþþK), calcd
for C15H16KO6: 331.0578.
4.1.3. (þ)-goniobutenolide A (1) and (ꢀ)-goniobutenolide B (2)
Solution of 12 (0.364 g, 1.24 mmol) in 90% TFA (5 mL) was stirred
for 30 min minutes at 0 ꢁC and then for additional 1 h at room
temperature. The mixture was concentrated by co-distillation with
toluene and the residue was purified by flash column chromatog-
raphy (24:1 CH2Cl2/MeOH) to afford pure hygroscopic syrup 14
4.1.1. 3,5-O-Carbonyl-1,2-O-isopropyildene-5-C-phenyl-a-D-gluco-
pentofuranose (12)
Procedure A: To a solution of 10 (0.962 g, 3.61 mmol) in dry
toluene (40 mL) was added 1,10-carbonyldiimidazole (1.464 g,
9.03 mmol) and the mixture was stirred at boiling temperature for
1.5 h. After the mixture was cooled down to room temperature it
was concentrated and the residue purified by flesh chromatog-
raphy (4:1 light petroleum/EtOAc) to afford pure 12 (0.998 g, 95%).
Procedure B: To a cooled (0 ꢁC) solution of 10 (0.323 g,
1.21 mmol) in mixture of dry pyridine (0.71 mL, 8.83 mmol) and dry
CH2Cl2 (9.2 mL) solution of triphosgene (0.215 g, 0.73 mmol) in dry
CH2Cl2 (1 mL) was added dropwise. The mixture was stirred at 0 ꢁC
for 1 h and then at room temperature for 1 h. The mixture was
poured into saturated solution of NH4Cl and extracted first with
CH2Cl2 (2 ꢂ 15 mL) and then with Et2O (3 ꢂ 15 mL). The combined
organic solutions were washed with brine, dried and evaporated.
The residue was purified by flash column chromatography (9:1
Et2O/light petroleum) to afford pure 12 (0.285 g, 81%). Recrystalli-
zation from CH2Cl2/hexane gave white needles, mp 138 ꢁC,
(0.347 g, 100%), [
a
]
¼ ꢀ73.8 (c 1.0, EtOH), Rf ¼ 0.26 (24:1 CH2Cl2/
D
MeOH). IR (KBr): nmax 3411 (OH), 1786 (C ¼ O). HRMS (ESI): m/e
297.0602 (M þ HCOOꢀ), calcd for C13H13O8: 297.0616. To a cooled
(ꢀ10 ꢁC) and stirred suspension of 95% NaH (0.036 g, 1.51 mmol) in
dry THF (4 mL) was added dropwise [bis-(2,2,2-trifluoro-ethoxy)-
phosphoryl]-acetic acid methyl ester (reagent B; 0.3 mL,
1.45 mmol). The resulting mixture was stirred in argon atmosphere
for 0.5 h and a solution of 14 (0.190 g, 0.75 mmol) in dry THF (4 mL)
was added. After being stirred at ꢀ10 ꢁC in argon atmosphere for
22 h the mixture was poured into 5% aq HCl (20 mL) and the
resulting suspension was extracted first with Et2O (3 ꢂ 10 mL) and
then with EtOAc (3 ꢂ 10 mL). Combined organic solutions were
washed with brine, dried and concentrated. Purification by flash
column chromatography (4:1 Et2O/light petroleum) followed by
preparative TLC (19:1 iPr2O/MeOH, three successive developments
for 1 and 2) afforded pure 1 (0.052 g, 29%) and 2 (0.026 g, 15%).
[
a
]
¼ þ33.2 (c 1, CHCl3), Rf ¼ 0.25 (4:1 light petroleum/EtOAc). IR
D
(film): nmax 1764 (C¼O). 1H NMR (250 MHz, CDCl3):
d 1.29 and 1.45
(2 ꢂ s, 3H each, CMe2), 4.50 (dd, 1H, J3,4 ¼ 2.4, J4,5 ¼ 2.2 Hz, H-4),
4.68 (d, 1H, J3,4 ¼ 2.7 Hz, H-3), 4.72 (d, 1H, J1,2 ¼ 3.6 Hz, H-2), 5.73
4.1.3.1. (þ)-goniobutenolide A (1). Colourless syrup, [
a
]
¼ þ86.8 (c
D
0.4, CHCl3), Rf ¼ 0.28 (19:1 iPr2O/MeOH), lit [1]. [
a
]
¼ þ82.0 (c 0.25,
D
(bs,1H, H-5), 6.02 (d,1H, J1,2 ¼ 3.6 Hz, H-1), 7.23e7.5 (m, 5H, Ph). 13
C
CHCl3). Both 1H and 13C NMR data of compound 1 are consistent
with the naturally occurring goniobutenolide A [1], and are in good
agreement with the data previously reported by us [16].
NMR (62.5 MHz, CDCl3): d 25.8 and 26.3 (CMe2), 73.8 (C-4), 79.4 (C-
5), 79.8 (C-3), 83.1 (C-2), 104.8 (C-1), 112.8 (CMe2), 124.8, 129.0,
129.1, 134.9 (Ph), 146.0 (C¼O). HRMS (ESI): m/e 293.1009 (MþþH),
calcd for C15H17O6: 293.1020; m/e 331.0566 (MþþK), calcd for
4.1.3.2. (ꢀ)-goniobutenolide
142e145 ꢁC (CH2Cl2), [
DIPE/MeOH), lit [1]. mp 148e149 ꢁC, [
B
(2). Colourless
sticks,
mp
C
15H16KO6: 331.0578.
a
]
¼ ꢀ39.0 (c 0.1, CHCl3), Rf ¼ 0.33 (19:1
D
a]
¼ ꢀ36.5 (c 0.2, CHCl3). 1H
D
4.1.2. 3,5-O-Carbonyl-1,2-O-isopropyildene-5-C-phenyl-
b-L
-ido-
and 13C NMR data of compound 2 are consistent with the naturally
occurring goniobutenolide B [1], and are in good agreement with
the data previously reported by us [16].
pentofuranose (13)
Procedure A: To a solution of 11 (0.210 g, 0.79 mmol) in dry
toluene (4 mL) was added 1,10-carbonyldiimidazole (0.192 g,
1.18 mmol) and the mixture was stirred at 80e85 ꢁC for 1.5 h. After
the mixture was cooled down to room temperature it was
concentrated and the residue purified by flesh column chroma-
tography (24:1 CH2Cl2/Me2CO) to afford pure 13 (0.174 g, 76%).
Procedure B: To a cooled (0 ꢁC) solution of 11 (0.394 g,1.47 mmol)
in a mixture of dry pyridine (0.12 mL) and CH2Cl2 (7 mL) a solution
of triphosgene (0.144 g, 0.48 mmol) in dry CH2Cl2 (0.3 mL) was
added dropwise. The mixture was stirred at 0 ꢁC for 1 h and then at
room temperature for 1 h. A new portion of dry pyridine (0.05 mL)
and solution of triphosgene (0.087 g, 0.16 mmol) in dry CH2Cl2
(0.2 mL) was added and stirring at room temperature was
continued for additional 1 h. The mixture was poured into satu-
rated solution of NH4Cl and extracted first with CH2Cl2 (2 ꢂ 7 mL)
4.1.4. (7S)-goniobutenolide A (5) and (7S)-goniobutenolide B (6)
Solution of 13 (0.367 g, 1.24 mmol) in 90% TFA (10.5 mL) was
stirred for 30 minutes at 0 ꢁC and then for additional 1.5 h at room
temperature. The mixture was concentrated by co-distillation with
toluene and the resulting residue was purified by flash column
chromatography (47:3 / 23:2 CH2Cl2/MeOH) to afford pure lactol
15 (0.302 g, 96%) as a colourless syrup. [
a
]
¼ þ29.0 (c 1.0, EtOH),
D
Rf ¼ 0.53 (23:2 CH2Cl2/MeOH). IR (CH2Cl2): nmax 3434 (OH),
1786e1746 (C¼O). HRMS (ESI): m/e 253.0715 (MþþH), calcd for
C
12H13O6: 253.0707; m/e 270.0984 (MþþNH4), calcd for C12H16NO6:
270.0972; m/e 291.0280 (MþþK), calcd for C12H12KO6: 291.0266. To
a cooled (ꢀ10 ꢁC) and stirred solution of 15 (0.186 g, 0.74 mmol) in
dry MeOH (8 mL) were added three equal portions on every 40 min

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of Et3N (0.06 mL, 0.44 mmol) and 2-(triphenylphosphoranylidene)-
acetic acid methyl ester (reagent A, 0.148 g, 0.44 mmol). The
mixture was stirred under nitrogen atmosphere at ꢀ10 ꢁC for 4 h
and then evaporated. Multiple purification by flesh column chro-
matography (9:1 Et2O/light petroleum) followed by preparative TLC
(4:1 CH2Cl2/EtOAc, three successive developments for 5 and 6)
afford pure 5 (0.084 g, 49%) and 6 (0.041 g, 24%).
NMR (62.5 MHz, CDCl3): d 37.7 (C-5), 76.8 (C-6), 86.7 (C-7), 113.7 (C-
4),124.9 (C-2), 126.3,128.7, 128.8, 137.1 (Ph), 154.3 (C-3), 169.1 (C-1).
HRMS (ESI): m/e 376.0041 (MþþNH4), calcd for C13H15NIO4:
376.0040.
4.1.5.3. (4S,5S,7R)-5-iodo-crassalactone D (4b). Colourless syrup,
[
a
]
¼ ꢀ32.5 (c 0.2, CHCl3), Rf ¼ 0.70 (iPr2O). IR (film): nmax 3445
D
(OH), 1770 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.82 (bs, 1H, OH),
4.1.4.1. (7S)-goniobutenolide
A
(5). Colourless needles, mp
4.49 (d, 1H, J5,6 ¼ 10.1 Hz, H-5), 4.57 (dd, 1H, J5,6 ¼ 10.2, J6,7 ¼ 7.4 Hz,
H-6), 4.95 (d, 1H, J6,7 ¼ 7.4 Hz, H-7), 6.37 (d, 1H, J2,3 ¼ 5.6 Hz, H-2),
7.09 (d, 1H, J2,3 ¼ 5.6 Hz, H-3), 7.30e7.54 (m, 5H, Ph). NOE contact:
94e95 ꢁC (EtOAc/hexane, at 0 ꢁC), [
a]
¼ þ110.7 (c 0.3, CHCl3),
D
Rf ¼ 0.29 (19:1 iPr2O/MeOH), lit [4]. mp 94e96 ꢁC, [
a
]
¼ þ144.0 (c
D
0.3, CHCl3). 1H and 13C NMR data for 5 matched those previously
H-5 and H-7, H-3 and H-5. 13C NMR (62.5 MHz, CDCl3):
d 31.3 (C-5),
reported in the literature [4,16].
83.7 (C-6), 86.4 (C-7),111.1 (C-4),126.4,127.2,128.77,128.8,137.8 (C-
2 and Ph), 149.1 (C-3), 168.6 (C-1). HRMS (ESI): m/e 376.0041
(MþþNH4), calcd for C13H15NIO4: 376.0040.
4.1.4.2. (7S)-Goniotebutenolide
115e118 ꢁC (CH2Cl2), [
B
(6). Colourless needles, mp
a
]
¼ ꢀ134.3 (c 0.3, CHCl3), Rf ¼ 0.39 (19:1
D
iPr2O/MeOH), lit [4]. mp 117e119 ꢁC, [
a
]
¼ ꢀ196.0 (c 0.3, CHCl3). 1H
4.1.6. (4R,5S,7S)-5-iodo-crassalactone D (7b) and (4S,5R,7S)-5-
iodo-crassalactone D (8a)
D
and 13C NMR data for 5 matched those previously reported in the
literature [4,16].
Procedure A: Solution of 5 (0.109 g, 0.47 mmol), I2 (0.412 g,
1.64 mmol) and NaHCO3 (0.138 g, 1.64 mmol) in dry CH3CN (6.5 mL)
was stirred at room temperature for 0.5 h and then at 35 ꢁC for 9
days. The mixture was poured in H2O (20 mL) and resulting sus-
pension was extracted with CH2Cl2 (4 ꢂ 10 mL). Combined organic
solutions were washed with 10% sodium sulphite (20 mL) and brine
(20 mL), dried and concentrated. The residue was purified by pre-
parative TLC (1:1 Et2O/light petroleum, two successive de-
velopments) to afford pure 8a (0.068 g, 40%) and impure 7b which
was additionally purified by preparative TLC (iPr2O, two successive
developments). Pure 7b was isolated (0.0144 g, 9%) as a colourless
solid.
Procedure B: Solution of 5 (0.044 g, 0.19 mmol) and NIS (0.064 g,
0.28 mmol) in dry CH3CN (1.2 mL) was stirred at room temperature
for 48 h. The mixture was poured into 10% sodium sulphite (20 mL)
and resulting suspension was extracted with CH2Cl2 (3 ꢂ 10 mL).
Combined organic solutions were washed with brine (20 mL), dried
and concentrated. The residue was purified by preparative TLC
(iPr2O) to afford pure 8a (0.010 g, 15%) and 7b (0.003 g, 4%).
Procedure C: Solution of 6 (0.022 g, 0.09 mmol), I2 (0.078 g,
0.31 mmol) and NaHCO3 (0.026 g, 0.31 mmol) in dry CH3CN (1.9 mL)
was stirred at room temperature for 0.5 h and then at 35 ꢁC for ten
days. Mixture was poured in H2O (20 mL) and the resulting sus-
pension was extracted with CH2Cl2 (4 ꢂ 10 mL). Combined organic
solutions were washed with 10% sodium sulphite (20 mL) and brine
(20 mL), dried and concentrated. The residue was purified by pre-
parative TLC (1:1 Et2O/light petroleum, two successive de-
velopments) to afford pure 8a (0.017 g, 51%) and impure 7b which
was additionally purified by preparative TLC (iPr2O, two successive
developments). Pure 7b was isolated (0.001 g, 3%) as a colourless
solid.
4.1.5. (4R,5S,7R)-5-iodo-crassalactone D (3a), (4S,5R,7R)-5-iodo-
crassalactone D (4a) and (4S,5S,7R)-5-iodo-crassalactone D (4b)
Procedure A: Solution of 1 (0.091 g, 0.39 mmol), I2 (0.329 g,
1.3 mmol) and NaHCO3 (0.109 g, 1.3 mmol) in dry CH3CN (5.5 mL)
was stirred at room temperature for 0.5 h and then at 35 ꢁC for 6
days. Mixture was poured in H2O (25 mL) and resulting suspension
was extracted with CH2Cl2 (3 ꢂ 15 mL). Combined organic solutions
were washed with 10% sodium sulphite (25 mL) and brine (25 mL),
dried and concentrated. The residue was purified by preparative
TLC (1:1 Et2O/light petroleum, two successive developments) to
afford impure 4b and a mixture of 3a and 4a which was separated
by preparative TLC (49:1 CH2Cl2/Me2CO, two successive de-
velopments). Pure 3a (0.04 g, 28%) and 4a (0.029 g, 21%) were
isolated. Purification of impure 4b by preparative TLC (1:1 Et2O/
light petroleum, two successive developments) afforded pure 4b
(0.012 g, 8%).
Procedure B: Suspension of 2 (0.036 g, 0.16 mmol), I2 (0.135 g,
0.53 mmol) and NaHCO3 (0.043 g, 0.53 mmol) in dry CH3CN
(2.5 mL) was stirred at room temperature for 0.5 h and then at 35 ꢁC
for seven days. The mixture was poured in H2O (20 mL) and
resulting suspension was extracted with CH2Cl2 (3 ꢂ 10 mL).
Combined organic solutions were washed with 10% sodium sul-
phite (20 mL) and brine (20 mL), dried and concentrated. The res-
idue was purified by preparative TLC (1:1 Et2O/light petroleum) to
afford pure 4b (0.006 g, 10%) and a mixture of 3a and 4a which was
separated by preparative TLC (49:1 CH2Cl2/Me2CO, two successive
developments). Pure 3a (0.008 g, 14%) and 4a (0.015 g, 27%) were
isolated.
4.1.5.1. (4R,5S,7R)-5-iodo-crassalactone D (3a). Colourless syrup,
Procedure D: Solution of 6 (0.031 g, 0.14 mmol) and NIS (0.061 g,
0.27 mmol) in dry DMF (2 mL) was stirred at room temperature for
0.5 h and then at 40 ꢁC additional 46.5 h. A new portion of NIS
(0.031 g, 0.14 mmol) was added and stirring continued for 24 h at
50 ꢁC. The mixture was diluted with EtOAc (5 mL). Organic solution
was washed with 10% sodium sulphite (20 mL) and brine (20 mL),
dried and concentrated. The residue was purified by preparative
TLC (7:3 Et2O/light petroleum) to give pure 8a (0.009 g, 19%) and 7b
(0.006 g, 12%).
[
a
]
¼ þ26.0 (c 0.2, CHCl3), Rf ¼ 0.49 (49:1 CH2Cl2/Me2CO), IR
D
(film): nmax 3445 (OH), 1779 (C¼O). 1H NMR (250 MHz, CDCl3):
d
3.20 (bs, 1H, OH), 4.45e4.63 (m, 2H, H-5 and H-6), 5.10 (d, 1H,
J6,7 ¼ 5.5 Hz, H-7), 6.20 (d, 1H, J2,3 ¼ 5.5 Hz, H-2), 7.30e7.44 (m, 6H,
H-3 and Ph). NOE contact: H-5 and. H-7. 13C NMR (62.5 MHz,
CDCl3):
d 28.6 (C-5), 86.4 (C-6), 87.6 (C-7), 113.1 (C-4), 124.0 (C-2).
125.9, 128.6, 128.7, 136.7 (Ph), 153.6 (C-3), 169.4 (C-1). HRMS (ESI):
m/e 376.0040 (MþþNH4), calcd for C13H15NIO4: 376.0040.
4.1.5.2. (4S,5R,7R)-5-iodo-crassalactone D (4a). Colourless syrup,
4.1.6.1. (4R,5S,7S)-5-iodo-crassalactone D (7b). Colourless needles,
[
a
]
¼ ꢀ5.5 (c 0.2, CHCl3), Rf ¼ 0.61 (49:1 CH2Cl2/Me2CO). IR (film):
mp 108e112 ꢁC (Et2O/hexane), [
a
]
¼ þ12.0 (c 0.2, CHCl3), Rf ¼ 0.56
D
D
nmax 3459 (OH), 1770 (C¼O). 1H NMR (250 MHz, CDCl3):
d
2.74 (bs,
(iPr2O). IR (KBr): nmax 3464 (OH), 1777 (C¼O). 1H NMR (250 MHz,
1H, OH), 3.91 (m,1H, H-6), 4.67 (d,1H, J5,6 ¼ 3.6 Hz, H-5), 5.10 (d,1H,
J6,7 ¼ 7.1 Hz, H-7), 6.22 (d, 1H, J2,3 ¼ 5.5 Hz, H-2), 7.30e7.56 (m, 5H,
CDCl3):
d
2.47 (d, 1H, J6,OH ¼ 9.9 Hz, OH), 4.54 (s, 1H, H-5), 4.72 (dd,
1H, J6,7 ¼4.2, J6,OH ¼ 9.6 Hz, H-6), 5.99 (d,1H, J6,7 ¼4.3 Hz, H-7), 6.28
Ph), 7.53 (d, 1H, J2,3 ¼ 5.6 Hz, H-3). NOE contact: H-5 and Ph-H. 13
C
(d, 1H, J2,3 ¼ 5.6 Hz, H-2), 7.31e7.50 (m, 5H, Ph), 7.55 (d, 1H,

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I. Kovacevic et al. / European Journal of Medicinal Chemistry 108 (2016) 594e604
602
J2,3 ¼ 5.7 Hz, H-3). 13C NMR (62.5 MHz, CDCl3):
d
29.8 (C-5), 81.3 (C-
6), 5.15 (d, 1H, J6,7 ¼ 4.8 Hz, H-7), 6.35 (d, 1H, J2,3 ¼ 5.7 Hz, H-2),
7.32e7.50 (m, 6H, H-3, Ph). 13C NMR (62.5 MHz, CDCl3):
66.2 (C-5),
85.2 (C-6), 88.9 (C-7), 113.6 (C-4), 125.4 (C-2), 126.0, 128.7, 128.8,
137.0 (Ph), 149.8 (C-3), 168.5 (C-1). HRMS (ESI): m/e 284.0687
(MþþNH4), calcd for C13H15NClO4: 284.0684.
6), 86.1 (C-7), 114.8 (C-4), 125.5 (C-2), 126.5, 128.6, 128.8, 134.4 (Ph),
154.0 (C-3), 168.4 (C-1). HRMS (ESI): m/e 376.0041 (MþþNH4), calcd
for C13H15NIO4: 376.0040.
d
4.1.6.2. (4S,5R,7S)-5-iodo-crassalactone D (8a). Colourless needles,
mp 107e110 ꢁC (CH2Cl2/hexane), [
a]
¼ ꢀ9.0 (c 0.2, CHCl3),
4.1.9.2. (4S,5R,7R)-5-Chloro-crassalactone D (4d). Colourless nee-
D
Rf ¼ 0.42 (1:1 Et2O/light petroleum). IR (film): nmax 3404 (OH), 1760
dles, mp 110e111 ꢁC (CH2Cl2/hexane), [
a
]
D
¼ ꢀ1.0 (c 0.2, CHCl3),
(C¼O). 1H NMR (250 MHz, CDCl3):
d
1.92 (bs, 1H, OH), 4.41 (t, 1H,
Rf ¼ 0.46 (iPr2O). IR (KBr): nmax 3446 (OH), 1766 (C¼O). 1H NMR
J5,6 ¼ 3.5 Hz, J6,7 ¼ 3.2 Hz, H-6), 4.94 (d, 1H, J5,6 ¼ 3.9 Hz, H-5), 5.56
(d, 1H, J6,7 ¼ 2.8 Hz, H-7), 6.12 (d, 1H, J2,3 ¼ 5.5 Hz, H-2), 7.33e7.59
(m, 6H, Ph and H-3). NOE contact: H-5 and H-7. 13C NMR (62.5 MHz,
(250 MHz, CDCl3):
d
2.53 (d, 1H, J6,OH ¼ 8.4 Hz, OH), 4.52 (d, 1H,
J5,6 ¼ 4.4 Hz, H-5), 4.70 (m, 1H, H-6), 5.09 (d, 1H, J6,7 ¼ 7.8 Hz, H-7),
6.34 (d, 1H, J2,3 ¼ 5.6 Hz, H-2), 7.33e7.51 (m, 5H, Ph), 7.48 (d, 1H,
CDCl3):
d
30.2 (C-5), 74.9 (C-6), 83.8 (C-7), 114.2 (C-4), 122.2 (C-2),
J2,3 ¼ 5.6 Hz, H-3). 13C NMR (62.5 MHz, CDCl3):
d 66.1 (C-5), 77.2 (C-
126.6, 128.7, 128.9, 134.2 (Ph), 154.8 (C-3), 169.8 (C-1). HRMS (ESI):
6), 85.7 (C-7), 112.9 (C-4), 125.5 (C-2), 126.3, 126.4, 128.8, 137.2 (Ph),
150.6 (C-3), 168.5 (C-1). HRMS (ESI): m/e 284.0684 (MþþNH4),
calcd for C13H15NClO4: 284.0684.
m/e 376.0029 (MþþNH4), calcd for C13H15NIO4: 376.0040.
4.1.7. (4R,5R,7S)-5-iodo-crassalactone D (7a)
To a stirred solution of 8a (0.031 g, 0.09 mmol) in a mixture of
CHCl3 (1.5 mL) and H2O (0.01 mL) was added TFA (0.06 mL,
0.86 mmol). The resulting mixture was stirred at 22 ꢁC for 5 days
and then concentrated by co-distillation with toluene. The resulting
residue was purified by preparative TLC (7:3 Et2O/light petroleum)
to give pure 7a (0.013 g, 42%) and 8a (0.013 g, 42%). Recrystalliza-
tion from CH2Cl2/hexane gave pure 7a as long colourless prisms,
4.1.10. (4S,5S,7S)-5-Chloro-crassalactone D (8b)
Procedure A: Solution of 5 (0.037 g, 0.16 mmol) and NCS (0.042 g,
0.32 mmol) in dry DMF (1.1 mL) was stirred at 40 ꢁC for 69.5 h. The
mixture was diluted with EtOAc (7 mL) and poured in 10% sodium
sulphite (20 mL). Organic layer was separated and water solution
extracted with EtOAc (3 ꢂ 9 mL). Combined organic solution were
washed with brine (20 mL), dried and concentrated. Double puri-
fication by TLC (iPr2O, two successive developments) afforded pure
8b (0.006 g, 13%).
Procedure B: Solution of 6 (0.035 g, 0.15 mmol) and NCS (0.040 g,
0.30 mmol) in dry DMF (0.85 mL) was stirred at 37 ꢁC for 69 h. The
mixture was diluted with EtOAc (5 mL) and poured in 10% sodium
sulphite (20 mL). Organic layer was separated and water solution
extracted with EtOAc (3 ꢂ 9 mL). Combined organic solution were
washed with brine (20 mL), dried and concentrated. Purification by
preparative TLC (iPr2O) afforded pure 8b (0.008 g, 19%), as colour-
less irregular shaped crystals, mp 145e148 ꢁC (CH2Cl2/hexane),
mp 163e167 ꢁC (CH2Cl2/hexane), [
a
]
¼ þ19.0 (c 0.1, CHCl3),
D
Rf ¼ 0.58 (4:1 Et2O/light petroleum). IR (KBr): nmax 3461 (OH),
1779e1739 (C¼O). 1H NMR (250 MHz, CDCl3):
d
2.20 (bs, 1H, OH),
4.45 (bs, 1H, H-6), 4.67 (d, 1H, J5,6 ¼ 4.4 Hz, H-5), 5.50 (d, 1H,
J6,7 ¼ 4.1 Hz, H-7), 6.38 (d, 1H, J2,3 ¼ 5.6 Hz, H-2), 7.09 (d, 1H,
J2,3 ¼ 5.6 Hz, H-3), 7.31e7.57 (m, 5H, Ph). 13C NMR (62.5 MHz,
CDCl3):
d 30.9 (C-5), 73.7 (C-6), 86.3 (C-7), 112.5 (C-4), 126.7, 127.0,
128.55, 128.6, 134.8 (C-2 and Ph), 149.2 (C-3), 168.4 (C-1). HRMS
(ESI): m/e 376.0040 (MþþNH4), calcd for C13H15NIO4: 376.0040.
4.1.8. (4S,5S,7R)-5-Chloro-crassalactone D (4c)
[
a
]
¼ ꢀ33.0 (c 0.2, CHCl3), Rf ¼ 0.43 (iPr2O). IR (KBr): nmax 3456
D
Solution of 1 (0.050 g, 0.21 mmol) and NCS (0.043 g, 0.32 mmol)
in dry DMF (1.3 mL) was stirred at 35 ꢁC for 53 h. The mixture was
diluted with EtOAc (7 mL). Organic solution was washed with 10%
sodium sulphite (20 mL) and brine (20 mL), dried and concentrated.
The residue purification by preparative TLC (iPr2O) afforded pure 4c
(0.023 g, 40%). Recrystallization from CH2Cl2/hexane gave pure 4c
(OH), 1772 (C¼O). 1H NMR (400 MHz, CDCl3):
d 1.78 (br s, 1H, OH),
4.32 (d, 1H, J5,6 ¼ 6.6 Hz, H-5), 4.83 (t, 1H, J5,6zJ6,7 ¼ 6.8 Hz, H-6),
5.62 (d, 1H, J6,7 ¼ 6.9 Hz, H-7), 6.38 (d, 1H, J2,3 ¼ 5.6 Hz, H-2), 7.30 (d,
1H, J2,3 ¼ 5.6 Hz, H-3), 7.31e7.59 (m, 5H, Ph). NOE contact: H-3 and
H-5. 13C NMR (62.5 MHz, CDCl3):
d 63.9 (C-5), 77.8 (C-6), 83.0 (C-7),
110.0 (C-4), 126.5 (C-2), 127.1, 128.8, 129.1, 134.0 (Ph), 148.8 (C-3),
as colourless plates, mp 130e134 ꢁC (CH2Cl2/hexane), [
a
]
D
¼ þ32.3
173.5 (C-1). HRMS (ESI): m/e 284.0683 (MþþNH4), calcd for
(c 0.3, CHCl3), Rf ¼ 0.45 (iPr2O). IR (KBr): nmax 3423 (OH), 1745
C13H15NClO4: 284.0684.
(C¼O). 1H NMR (250 MHz, CDCl3):
d
3.10 (d, 1H, J6,OH ¼ 3.4 Hz, OH),
4.37 (d, 1H, J5,6 ¼ 9.5 Hz, H-5), 4.47 (m, 1H, J6,7 ¼ 7.7, J5,6 ¼ 9.2,
J6,OH ¼ 3.4 Hz, H-6), 4.95 (d, 1H, J6,7 ¼ 7.6 Hz, H-7), 6.35 (d, 1H,
J2,3 ¼ 5.6 Hz, H-2), 7.14 (d, 1H, J2,3 ¼ 5.6 Hz, H-3), 7.29e7.53 (m, 5H,
4.1.11. (4S,5S,7S)-5-bromo-crassalctone D (8c)
Procedure A: Suspension of 5 (0.042 g, 0.18 mmol), Br2 (0.2 mL,
0.36 mmol) and NaHCO3 (0.02 g, 0.36 mmol) in dry CH2Cl2 (2 mL)
was stirred at room temperature for 2.5 h. The mixture was poured
in 10% sodium sulphite and extracted with CH2Cl2 (4 ꢂ 10 mL).
Organic solution was washed with water (20 mL) and brine (20 mL),
dried and concentrated. The residue was purified by preparative
TLC (7:3 Et2O/light petroleum, two successive developments) to
afford pure 8c (0.007 g, 12%) as a colourless solid.
Procedure B: To a stirred solution of 5 (0.046 g, 0.20 mmol) in dry
DMF (0.6 mL) in an argon atmosphere was added a solution of NBS
(0.035 g, 0.20 mmol) in dry DMF (0.6 mL). The resulting mixture
was stirred at 20e25 ꢁC for 149 h and then temperature was raised
to 30 ꢁC. After 10 days the temperature was raised at 35 ꢁC and
stirring was continued for 77 h. A new portion of NBS (0.018 g,
0.10 mmol) in dry DMF (0.3 mL) was added. After 15 days tem-
perature was raised at 45 ꢁC and stirring continued for 2 days. After
stirring for two additional days at room temperature, mixture was
diluted with EtOAc (15 mL). Organic solution was washed with 5%
NaHCO3 (20 mL), water and brine, dried and concentrated. Residue
was purified by preparative TLC (3:2 Et2O/light petroleum) to give
Ph). NOE contact: H-3 and H-5. 13C NMR (62.5 MHz, CDCl3):
d 63.5
(C-5), 81.6 (C-6), 84.8 (C-7), 110.4 (C-4), 126.9 (C-2), 126.4, 128.8,
128.9, 137.7 (Ph), 148.8 (C-3), 168.7 (C-1). HRMS (ESI): m/e 284.0696
(MþþNH4), calcd for C13H15NClO4: 284.0684.
4.1.9. (4R,5S,7R)-5-Chloro-crassalactone D (3b) and (4S,5R,7R)-5-
chloro-crassalactone D (4d)
Solution of 2 (0.010 g, 0.43 mmol) and NCS (0.086 g, 0.64 mmol)
in dry DMF (2 mL) was stirred at 36 ꢁC for 70 h. The mixture was
diluted with EtOAc (5 mL). Organic solution was washed with 10%
sodium sulphite (20 mL) and brine (20 mL), dried and concentrated.
Multiple purification by flesh chromatography (1:1 Et2O/light pe-
troleum) afforded pure 3b (0.029 g, 26%) and 4d (0.019 g, 17%).
4.1.9.1. (4R,5S,7R)-5-Chloro-crassalactone D (3b). Colourless nee-
dles, mp 106e109 ꢁC (CH2Cl2/hexane), [
a
]
D
¼ þ11.3 (c 0.3, CHCl3),
Rf ¼ 0.49 (iPr2O). IR (KBr): nmax 3430 (OH), 1765 (C¼O). 1H NMR
(250 MHz, CDCl3): d 2.88 (bs,1H, OH), 4.39e4.52 (m, 2H, H-5 and H-

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603
impure 8c that was additionally purified by preparative TLC (iPr2O,
4.4. Detection of apoptosis
two successive developments). Pure compound 8c (0.018 g, 29%)
was obtained as a colourless solid.
Apoptosis of K562 cells was evaluated with an Annexin V-FITC
detection kit. Cells from each sample were collected (800 rpm/
5 min, Megafuge 1.0R, 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 ꢂ 106 cells/mL. The
Colourless prisms, mp 147e151 ꢁC (Et2O/hexane), [
a]
¼ ꢀ21.0
D
(c 0.2, CHCl3), Rf ¼ 0.60 (iPr2O). IR (film): nmax 3449 (OH),
1783e1767 (C¼O). 1H NMR (250 MHz, CDCl3):
d 1.86 (bs, 1H, OH),
4.31 (d, 1H, J5,6 ¼ 6.9 Hz, H-5), 4.91 (t, 1H, J ¼ 6.5 Hz, H-6), 5.59 (d,
1H, J6,7 ¼ 6.9 Hz, H-7), 6.38 (d, 1H, J2,3 ¼ 5.6 Hz, H-2), 7.29 (d, 1H,
J2,3 ¼ 5.6 Hz, H-3) 7.31e7.52 (m, 5H, Ph). NOE contact: H-3 and H-5.
cell suspension (100
m
L) was transferred to 5 mL culture tubes and
13C NMR (62.5 MHz, CDCl3):
d
53.52 (C-5), 78.21 (C-6), 83.11 (C-7),
mixed with Annexin V (5
m
L) and propidium iodide (5 L). The cells
m
110.03 (C-4), 122.18 (C-2), 126.64, 127.05, 128.78, 129.40, 134.04
(Ph), 148.83 (C-3), 168.66 (C-1). HRMS (ESI): m/e 328.0178
(MþþNH4), calcd for C13H15NBrO4: 328.0179.
were gently vortexed and incubated for 15 min at 25 ꢁC. After in-
cubation, 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. [33].
4.1.12. General procedure for dehalogenation of iodo derivatives 3a,
4a, 4b, 7a and 8a
Solution of 5-iodo derivatives 3a, 4a, 4b, 7a and 8a (1 equiv),
AIBN (0.2 equiv) and Bu3SnH (2 equiv) in dry toluene
(0.05e0.06 mmol) was stirred in argon atmosphere at 100 ꢁC for
1 h. The mixtures were evaporated and the residue purified by flash
column chromatography (4:1 CH2Cl2/EtOAc; products 3, 4 and 7),
or by preparative TLC (17:3 CH2Cl2/EtOAc; product 8).
4.5. Western blot
For the Western blot, 50 mg of proteins per sample were sepa-
rated by electrophoresis and electro-transferred to a PVDF mem-
brane Hybond-P and then blotted with primary antibodies against
Bcl-2, Bax, caspase 3 and PARP. b-Actin was used as internal control.
4.1.12.1. (þ)-crassalactone D (3). Yield 81%. Colourless needles, mp
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. Images of protein expression were analyzed in ImageJ
computer program (NIH Image, http://imagej.nih.gov) after minor
levels adjustments. Expression of investigated proteins was
measured by densitometry and compared with control sample.
140e142 ꢁC, (EtOAc/hexane), [
a
]
D
¼ þ20.5 (c 0.2, EtOH),1Rf ¼ 0.63
(5:2 EtOAc/hexane), Ref. [12] mp 138e140 ꢁC, [
a
]
D
¼ þ13.6 (c 0.2,
EtOH). Both 1H and 13C NMR data of compound 3 are consistent
with the naturally occurring (þ)-crassalactone D [10] and its
physical properties are in agreement with those reported in the
literature [12].
4.1.12.2. (4R)-crassalactone D (4). Yields: 94% from 4a, 95% from 4b.
Colourless needles, mp 137e142 ꢁC, (EtOAc/hexane), [
a
]
¼ þ28.0
Acknowledgement
D
(c 0.5, EtOH), Rf ¼ 0.61 (5:2 EtOAc/hexane). 1H and 13C NMR data for
4 matched those previously reported by us [16].
Financial support from the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grant No
172006) is gratefully acknowledged.
4.1.12.3. (7S)-(þ)-crassalactone D (7). Yield 93%. Colourless nee-
dles, mp 170e175 ꢁC (CH2Cl2/hexane), [
a
]
¼ þ32.0 (c 0.2, EtOH),
D
Rf ¼ 0.24 (Et2O). 1H and 13C NMR data for 4 matched those previ-
Appendix A. Supplementary data
ously reported by us [16].
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.12.011.
4.1.12.4. (4R,7S)-(þ)-crassalactone
D (8). Yield 71%. Colourless
needles, mp 139e141 ꢁC (EtOAc/hexane), [
a
]
¼ þ73.0 (c 0.2, EtOH),
D
These data include additional characterization and biological
data, as well as copies of 1H, and 13C NMR spectra of all final
compounds. Crystallographic results are also included in this sec-
tion. Supplementary data associated with this article can be found
in online version.
Rf ¼ 0.54 (Et2O). 1H and 13C NMR data for 4 matched those previ-
ously reported by us [16].
4.2. MTT assay
The colorimetric MTT assay was carried out following the re-
ported procedure [32].
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