Synthesis and in?vitro antitumour activity of crassalactone D, its stereoisomers and novel cinnamic ester derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.03.088

Naturally occurring styryl lactone, crassalactone D (1), unnatural 4-epi-crassalactone D (2), and the corresponding 7-epimers (3 and 4) have been synthesized starting from D-glucose. The key step of the synthesis is a new one-pot sequence that commenced with a Z-selective Wittig olefination of suitably functionalized sugar lactols with a stabilized ylide, (methoxycarbonylmethylene)-triphenylphosphorane, in dry methanol, to afford 1 or 3, in the mixtures with the corresponding 4-epimers (2 or 4, respectively). A number of 6-O-cinnamoyl derivatives of styryl lactones 1–4 have been prepared, bearing electron donating or electron withdrawing functionalities in the C-4 position of cinnamic acid residue. The synthesized products were evaluated for their in?vitro antiproliferative activity against selected human tumour cell lines, whereupon very potent cytotoxicities have been recorded in many cases. SAR analysis indicated some important structural features responsible for biological activity, such as stereochemistry at the C-4 and C-7 positions, as well as the nature of a substituent at the C-4 position in the aromatic ring of cinnamoate moiety. Flow cytometry and Western blot analysis data gave insight in the mechanism underlying antiproliferative effects of the synthesized compounds.

Full text of DOI:10.1016/j.ejmech.2017.03.088

European Journal of Medicinal Chemistry 134 (2017) 293e303
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis and in vitro antitumour activity of crassalactone D, its
stereoisomers and novel cinnamic ester derivatives
a
ꢀ
ꢁ a
ꢁ a
ꢁ a
ꢁ b
Ivana Kovacevic , Mirjana Popsavin , Goran Benedekovic , Jelena Kesic , Vesna Kojic ,
b
ꢁ
ꢁ c
ꢁ b
ꢁ d
Dimitar Jakimov , Tatjana Srdic-Rajic , Gordana Bogdanovic , Vladimir Divjakovic ,
Velimir Popsavin a
,
*
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, 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
ꢁ
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:
Naturally occurring styryl lactone, crassalactone D (1), unnatural 4-epi-crassalactone D (2), and the
corresponding 7-epimers (3 and 4) have been synthesized starting from D-glucose. The key step of the
Received 9 February 2017
Received in revised form
20 March 2017
Accepted 21 March 2017
Available online 13 April 2017
synthesis is a new one-pot sequence that commenced with a Z-selective Wittig olefination of suitably
functionalized sugar lactols with a stabilized ylide, (methoxycarbonylmethylene)-triphenylphosphorane,
in dry methanol, to afford 1 or 3, in the mixtures with the corresponding 4-epimers (2 or 4, respectively).
A number of 6-O-cinnamoyl derivatives of styryl lactones 1e4 have been prepared, bearing electron
donating or electron withdrawing functionalities in the C-4 position of cinnamic acid residue. The
synthesized products were evaluated for their in vitro antiproliferative activity against selected human
tumour cell lines, whereupon very potent cytotoxicities have been recorded in many cases. SAR analysis
indicated some important structural features responsible for biological activity, such as stereochemistry
at the C-4 and C-7 positions, as well as the nature of a substituent at the C-4 position in the aromatic ring
of cinnamoate moiety. Flow cytometry and Western blot analysis data gave insight in the mechanism
underlying antiproliferative effects of the synthesized compounds.
Keywords:
Crassalactone D
Cinnamoates
Apoptosis
Antitumour lactones
SAR analysis
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
proliferation of some human malignant cell lines have also been
preliminary reported [5]. Herein, we wish to disclose full details of
Crassalactone D (1, Fig. 1) is a naturally occurring styryl lactone
that shows notable cytotoxic effects against certain mammalian
tumour cell lines. It was isolated from the tropical plant Polyalthia
crassa by Tuchinda and co-workers in 2006 [1]. The relative
configuration of 1 was established by X-ray diffraction analysis and
its absolute stereochemistry was determined by NMR studies of its
(R)-and (S)-MTPA esters. Two asymmetric total syntheses of cras-
salactone D (1) reported in 2009 [2,3] have been followed by a
formal synthesis published in 2012 [4]. In the same year, we pub-
lished a preliminary account describing a new chiral pool approach
to the total synthesis of the natural product 1 and several new
epimers [5]. The effects of the synthesized compounds on the
the synthesis and biological evaluation of styryl lactones 1e4, as
well as some new 6-O-cinnamoyl derivatives (1aed, 2aed, 3aed
and 4aed) along with their effects on the proliferation of certain
malignant cell lines. Rationale for the preparation of cinnamic ester
derivatives of crassalactone D and epimers arises from the fact that
cinnamoates represent a promising class of anti-cancer agents [6].
Such concept of drug design and development is known as ‘mo-
lecular hybridization’. It is based on the combination of pharma-
cophoric moieties of different bioactive substances to produce a
new hybrid compound with improved affinity and efficacy. Addi-
tionally, this strategy can result in compounds presenting modified
selectivity profile, different and/or dual modes of action and
reduced undesired side effects [7]. Another reason for studying the
cinnamic ester derivatives of type 1bed, 2bed, 3bed, 4bed is
derived from our earlier findings that the introduction of either
* Corresponding author.
E-mail address: velimir.popsavin@dh.uns.ac.rs (V. Popsavin).
http://dx.doi.org/10.1016/j.ejmech.2017.03.088
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

ꢀ ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
294
Scheme 1. Reagents and conditions: (a) 90% aq TFA, 0 ꢀC for 0.5 h, then rt, 1 h for 5,
1.5 h for 7; (b) Ph3P ¼ CHCO2Me, dry MeOH, 0 ꢀC for 0.5, then rt, 21 h for 6, 47 h for 8,
51% (2:1 mixture of 2 and 1), 29% of 3, 38% of 4; (c) Ph3P ¼ CHCO2Et, dry MeOH, 0 ꢀC for
1.5 h, then rt, 25 h for 8, 10% of 3, 37% of 4; (d) TFA, CHCl3, rt, 63 h for 1:2 mixture of 1
and 2, 99% of 2:1 mixture of 1 and 2, 23 h for 4, 1:1 mixture of 3 and 4.
In an attempt to generalize this methodology by using a similar
substrate, but with the opposite stereochemistry at the C-5 posi-
tion, we treated lactol 8 under the same Wittig olefination condi-
tions. Gratifyingly, reaction of 8 with MCMP gave unnatural spiro-
lactones 3 and 4 in 67% combined yield. Unfortunately, a drop in
selectivity was observed, whereupon an equimolar mixture of 3
and 4 was obtained. However, when the Wittig olefination of 8 was
performed with ECMP, products 3 and 4 were obtained in respec-
tive isomeric ratio of 1:4, in slightly lower total yield (46%). The
products 1 and 2, as well as 3 and 4, were separated with diffi-
culties. In order to obtain pure products several flash column
chromatographic separations were required, followed by prepara-
tive TLC. This significantly decreased the yields of all products, and
in particular the yields of minor stereoisomers. Fortunately, it was
found that the less stable isomer 2 could be converted to the more
stable 1, after treatment of their mixture (2:1 in favour of 2) with a
solution of trifluoroacetic acid in chloroform, to give a 2:1 mixture
of 1 and 2 (by 1H NMR analysis). The similar results were obtained
when the reaction was carried out in the presence of Lewis acids
(for more details, see the Supplementary data). Greater stability of
naturally occurring 1 compared to 2, could be a result of stabili-
zation by anomeric effect and the related stereoelectronic effects
[15,16]. In contrast, when pure 4 was treated under similar reaction
conditions, a 1:1 mixture of 3 and 4 was obtained indicating a
similarity in stability of both stereoisomers.
Fig. 1. Chemical structures of crassalactone D (1), its epimers 2e4 and 6-O-cinnamoyl
derivatives 1aed, 2aed, 3aed and 4aed.
electron withdrawing or electron donating groups in the C-4 po-
sition of the cinnamic acid aromatic ring may increase antitumour
activity of the resulting (þ)-crassalactone B and C analogues [8,9].
Apoptotic signalling induced by the synthesized compounds was
also studied.
2. Results and discussion
2.1. Chemistry
Synthesis of crassalactone D (1) and the corresponding 7-
epimer 3 is shown in Scheme 1. The starting compounds 5 and 7
were prepared from commercially available diacetone-D-glucose in
three steps as we reported earlier [10]. Hydrolytic removal of the
isopropylidene protective groups in both 5 and 7 afforded the
corresponding lactols 6 and 8 in almost quantitative yields. Both
products, and particularly stereoisomer 6, are rather hygroscopic.
They were therefore used in the next synthetic step immediately
after their brief isolation. Accordingly, lactol 6 was submitted to the
reaction with a stabilized ylide, (methoxycarbonylmethylene)tri-
phenylphosphorane (MCMP) in dry methanol, to afford (þ)-cras-
salactone D (1) along with the corresponding 4-epimer 2 in 1:2
respective ratio and in 51% combined yield. This one-pot sequence
is comprised of an initial Z-selective Wittig olefination [11,12], fol-
Both 1H and 13C NMR data and physical properties of com-
pounds 1 and 2 were in agreement with those reported previously
[2,3]. Stereochemistry of lactones 3 and 4 were confirmed by single
crystal X-ray diffraction analysis (for the crystal structures of 3 and
4, see the Supplementary data).
Two independent procedures were used for the conversion of
spiro-lactones 1 and 2 to the cinnamic esters 1aed and 2aed and
the results are presented in Table 1.
lowed by successive g-lactonization, b-elimination and the final 5-
In the first experiments, a mixture of stereoisomeric lactones 1
and 2 (in 1:2 respective ratio) was treated with cinnamoyl chloride
(entry 1) or with 4-nitrocinnamoyl chloride (entry 2) in the pres-
ence of DMAP in dry dichloromethane, to afford mixtures of 1a and
2a or 1b and 2b in 72 and 85% combined yields, respectively.
endo-trig spirocyclization [5,10]. In order to increase the yield and/
or epimer ratio we carried out the olefination step with (ethox-
ycarbonylmethylene)triphenylphosphorane (ECMP) [13,14]. How-
ever, the isomers 1 and 2 were isolated in the similar total yield
(48%) and in the same isomer ratio (1/2 ¼ 1:2).

ꢀ
ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
295
Table 1
Preparation of cinnamic ester derivatives 1aed and 2aed.
Entry
Starting compound
Cinnamic acid
derivative
Reagents and conditions
Products
(isolated yield, %)
1
2
3
4
1 þ 2 (1:2 mixture)
1 þ 2 (1:2 mixture)
1 þ 2 (1:2 mixture)
1 þ 2 (1:2 mixture)
R ¼ H, X ¼ Cl
DMAP, CH2Cl2, 3 h, 0 ꢀC, 20 h, rt
DMAP, CH2Cl2, 3 h, 0 ꢀC
1a (34), 2a (38),
R ¼ H
R ¼ NO2, X ¼ Cl
R ¼ OMe, X ¼ OH
R ¼ F, X ¼ OH
1b (29), 2b (56),
R ¼ NO2
DMAP, DCC, CH2Cl2, 5 h, rt
DMAP, DCC, CH2Cl2, 5 h, rt
1c (32), 2c (60),
R ¼ OMe
1d (36), 2d (50),
R ¼ F
Next, the esterification step was carried out by using the Steglich
esterification protocol [17]. We were pleased to find that treatment
Accordingly, a treatment of 3 with 4-methoxycinnamic or with 4-
fluorocinnamic acid in anhydrous dichloromethane, in the pres-
ence of DCC and DMAP, gave the expected 6-O-(4-
methoxycinnamoyl) derivative 3c (entry 3) and 6-O-(4-
fluorocinnamoyl) derivative 3d (entry 4) in 86 and 73% yields
respectively. Finally, compound 4 reacted with 4-methoxy- or 4-
fluorocinnamic acid, under the same reaction conditions, to afford
the corresponding 4-substituted cinnamoates 4c and 4d in 77 and
88% respective yields (entries 7 and 8).
It should be noted that all C-6 signals of (7S)-isomers (3, 4, 3aed,
4aed) appeared downfield compared to the corresponding signals
of (7R)-isomers (1, 2, 1aed, 2aed), presumably due to the steric
compressions caused by the OH and Ph groups.
of mixture of
1 and 2 (in 1:2 respective ratio) with 4-
methoxycinnamic acid, in the presence of DCC and DMAP in
anhydrous dichloromethane, gave the corresponding esters 1c and
2c in 32 and 60% respective yields (entry 3). Finally when the
mixture of 1 and 2 was treated with 4-fluorocinnamic acid, under
the same reaction conditions as those described above, the ex-
pected products 1d and 2d were obtained in 36 and 50% yields,
respectively (entry 4).
The results related to the synthesis of 6-O-cinnamoyl derivatives
3aed and 4aed are presented in Table 2.
Treatment of
3
with cinnamoyl chloride or with 4-
nitrocinnamoyl chloride in the presence of DMAP in dry dichloro-
methane, gave the corresponding 6-O-cinnamoyl derivatives 3a
and 3b in 68 and 74% respective yields (entries 1 and 2). The same
synthetic protocol was then used for the conversion of 4 in 4a and
4b in 62 and 89% yields, respectively (entries 5 and 6). Next, the
Steglich esterification protocol was used for the synthesis of the
Structure and stereochemistry of esters 1bed, 2a and 4a were
confirmed by single crystal X-ray diffraction analysis (for the crystal
structures see the Supplementary data).
2.2. In vitro antitumour activities and SAR
corresponding
6-O-(4-methoxycinnamoyl)
or
6-O-(4-
Crassalactone D (1), its stereoisomers (2e4) and 6-O-cinnamoyl
derivatives (1aed, 2aed, 3aed, 4aed) were evaluated for their
fluorocinnamoyl) derivatives (Table 2, entries 3, 4, 7 and 8).
Table 2
Preparation of cinnamic ester derivatives 3aed and 4aed.
Entry
Starting compound
Cinnamic acid
derivative
Reagents and conditions
Products
(isolated yield, %)
1
2
3
4
R ¼ H, X ¼ Cl
DMAP, CH2Cl2, 3 h, 0 ꢀC, 45 min, rt
DMAP, CH2Cl2, 3.5 h, 0 ꢀC
DMAP, DCC, CH2Cl2, 3.5 h, rt
DMAP, DCC, CH2Cl2, 5.5 h, rt
3a (68), R ¼ H
3b (74), R ¼ NO2
3c (86), R ¼ OMe
3d (73), R ¼ F
R ¼ NO2, X ¼ Cl
R ¼ OMe, X ¼ OH
R ¼ F, X ¼ OH
5
6
7
8
R ¼ H, X ¼ Cl
DMAP, CH2Cl2, 3 h, 0 ꢀC, 22.5 h, rt
DMAP, CH2Cl2, 3.5 h, 0 ꢀC
DMAP, DCC, CH2Cl2, 3 h, rt
DMAP, DCC, CH2Cl2, 4 h, rt
4a (62), R ¼ H
4b (89), R ¼ NO2
4c (77), R ¼ OMe
4d (88), R ¼ F
R ¼ NO2, X ¼ Cl
R ¼ OMe, X ¼ OH
R ¼ F, X ¼ OH

ꢀ ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
296
in vitro antitumour activity against eight human malignant cell
lines including myelogenous leukaemia (K562), promyelocytic
leukaemia (HL-60), T-cells leukaemia (Jurkat), Burkitt's lymphoma
(Raji), oestrogen receptor positive breast adenocarcinoma (MCF-7),
oestrogen receptor negative breast adenocarcinoma (MDA-MB-
231), cervix carcinoma (HeLa), alveolar basal adenocarcinoma
(A549) and a single human normal cell line (MRC-5). Cytotoxic
activity was evaluated by using the standard MTT assay [18], 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.
compounds were more potent then leads 3 and 4 and control
compound DOX against human adenocarcinoma cells (A549).
Moreover, none of the synthesized styryl lactones exhibited any
toxicity toward normal MRC-5 cells, in contrast to the commercial
antitumour agent doxorubicin that exhibited a strong cytotoxicity
in submicromolar range (IC50 0.10 mM).
In an attempt to determine influence of newly introduced cin-
namoyl ester groups on antitumour activity, we compared IC50
values of cinnamoates 1aed, 2aed, 3aed, 4aed with those recor-
ded for leads 1e4 (see the Supplementary data for details). The
results have shown that presence of cinnamoyl ester group in
molecule 1 increased cytotoxicity of analogue 1a against MCF-7 and
A549 cells, while the introduction of 4-substituted cinnamoyl
groups increased cytotoxicity only against MCF-7 cell line. The
presence of non-substituted cinnamoyl moiety in the structure of
lead 2 increased the potency of resulting analogue 2a against four
malignant cell lines (Raji, MCF-7, MDA-MB231, HeLa), while intro-
duction of electron withdrawing group in the position C-4 of cin-
namoyl moiety (NO2 and F) decreased activity of analogues 2b and
2d toward the all tumour cell lines under evaluation. However,
introduction of electron donating group (OMe) at C-4 of cinnamoyl
moiety increased the potency of resulting analogue 2c only against
MCF-7 cell line. Presence of cinnamoyloxy groups at the C-6 posi-
tion of lead 3 has more favourable effects. Introduction of non-
substituted cinnamoyl moiety as well as 4-nitrocinnamoyl res-
idue in the structure of lead 3 increased the potency of resulting
analogues 3a and 3b against majority of tumour cell lines under
evaluation, while introduction of 4-methoxy- and 4-
fluorocinnamoyl moieties increased activity of analogues 3c and
3d against three investigated cell lines (MDA-MB231, HeLa, A549).
Cinnamoates 4a and 4b were more active against three cell lines
(MDA-MB231, A549, Raji), while analogues 4c and 4d were more
active against two cell lines (MDA-MB231, A549), compared to
parent compound 4. These results implied that introduction of
cinnamoyloxy groups at the C-6 positions of leads 3 and 4 may
increase antitumour activities of 6-O-cinnamoyl derivatives.
Crassalactone D (1) and stereoisomers 2 and 4 exhibited sub-
micromolar activity against K562 cell line (Table 3). Natural lactone
1 was 2-fold more active than commercial antitumour agent DOX,
5-fold more active with respect to 2 and 4 and 10-fold more potent
than 3. Compound 1 (IC50 0.34 mM) was 6-, 14- and 104-fold more
potent compared to 2, 4 and 3, respectively in the culture of HeLa
cells. Although spiro- lactones 1e4 were very potent inhibitors of
Jurkat cells growth, all of them were less active than DOX. In the
culture of Raji cells the most active was natural product 1 being as
active as DOX, but more active than stereoisomers 2e4. Crassa-
lactone D (1) showed the highest potency against MDA-MB231 cells
and the least potency against MCF-7 cells, when compared to the
remaining styryl lactones 2e4.
Twelve 6-O-cinnamoyl derivatives (1bec, 2bec, 3a,d, and 4aed)
exhibited activities in the micromolar range toward K562 cells,
while thirteen of them (1b,c, 2aed, 3aec, and 4aed) showed
micromolar activity against HL-60 cells. However none of the an-
alogues were more active than DOX in the culture of these cell lines.
Cinnamoate 1a demonstrated a submicromolar cytotoxicity (IC50
0.21 mM) against Jurkat cells, while compounds 3a and 4aed were
found to be active in micromolar range toward the same cell line. In
the culture of Raji cells compounds 2a, 4a and 4b exhibited a
greater activity than DOX. Cinnamoyl esters 1aec were more active
than lead 1 toward MCF-7 cells. Compound 2a was very potent
growth inhibitor of MCF-7, MDA-MB 231 and HeLa cells being more
active than (4R)-crassalactone D (2). Derivatives 3aed and 4aed
were more active than lead compounds 3 and 4 against human
breast adenocarcinoma cells (ERꢁ, MDA-MB 231). These
Next we considered the influence of electron withdrawing (NO2,
F) and electron donating (OMe) groups attached in the C-4 position
of cinnamate aromatic ring. Non-substituted cinnamoyl derivatives
Table 3
In vitro cytotoxicity of natural product 1, its analogues 2e4, 6-O-cinnamoyil derivatives 1aed, 2aed, 3aed, 4aed and DOX.
Compd
IC50
K562
(m
M)a
HL-60
Jurkat
Raji
MCF-7
MDA-MB 231
HeLa
A549
MRC-5
1
0.12 0.01
20.67 1.21
5.64 0.11
2.12 0.25
8.45 0.99
0.54 0.11
20.53 2.25
7.45 1.25
1.11 0.98
1.05 0.79
1.21 0.25
4.62 0.69
14.32 2.58
12.78 1.64
9.05 2.54
0.54 0.04
4.65 0.94
2.22 0.54
1.02 0.56
5.36 0.87
0.25 0.02
1.06 0.69
10.02 2.79
2.24 0.69
8.67 0.79
24.13 3.54
1.02 0.87
5.61 0.67
1.32 0.54
6.37 0.69
4.48 0.77
6.11 0.54
3.41 0.02
4.32 0.32
8.67 0.78
12.02 0.65
0.19 0.05
9.65 0.55
4.36 0.05
2.25 0.09
1.02 0.12
0.92 0.09
0.21 0.04
0.21 0.01
31.25 2.54
58.14 3.35
15.64 1.54
0.34 0.02
87.63 2.99
86.54 3.54
21.14 2.07
22.17 3.52
1.01 0.11
5.63 0.66
10.22 0.98
14.22 1.32
15.36 1.58
0.96 0.05
1.02 0.07
4.03 0.36
2.54 0.77
4.63 0.87
0.03 0.005
2.34 0.58
6.68 1.25
>100
89.68 4.56
34.12 3.41
5.97 1.12
2.38 0.56
>100
99.78 5.84
55.36 3.12
8.84 0.88
9.68 0.87
11.84 2.02
8.69 1.21
17.33 1.32
2.69 0.56
2.28 0.24
1.64 0.12
3.05 0.01
5.37 0.21
2.98 0.98
11.64 0.53
1.65 0.21
8.58 0.31
3.14 0.11
9.65 0.05
3.57 0.18
0.58 0.02
12.46 1.21
2.22 0.21
21.48 2.54
11.02 0.45
5.34 0.88
7.55 0.65
11.25 1.08
15.63 2.51
1.97 0.89
11.44 1.23
4.31 0.16
3.25 0.14
2.31 0.12
0.20 0.02
1.89 0.22
5.45 1.21
>100
>100
>100
>100
3.45 0.89
>100
>100
>100
45.13 4.32
5.23 0.89
4.48 0.15
2.64 0.11
1.02 0.02
>100
3.64 0.38
1.25 0.15
2.69 0.22
7.54 0.65
0.09 0.002
0.34 0.01
1.15 0.15
2.22 0.08
3.34 0.03
6.12 0.65
2.12 0.32
0.58 0.01
9.45 1.52
3.64 0.38
2.21 0.12
35.41 2.55
14.46 0.84
11.48 0.54
12.78 1.32
11.98 0.12
4.61 0.41
58.45 4.22
24.77 3.52
9.94 1.52
8.45 0.87
0.07 0.01
28.99 1.25
14.58 1.52
45.36 3.41
33.56 1.58
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
0.10 0.01
1a
1b
1c
1d
2
2a
2b
2c
2d
3
3a
3b
3c
3d
4
4a
4b
4c
4d
DOX
21.33 2.54
28.07 2.84
56.21 3.41
23.14 1.42
25.47 0.52
35.21 2.45
1.02 0.11
3.12 0.12
1.58 0.05
1.21 0.09
31.37 2.54
0.25 0.01
0.85 0.03
0.45 0.01
1.17 0.21
4.91 0.31
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%.

ꢀ
ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
297
1a, 2a, 3a and 4a were used as controls in this SAR analysis.
Insertion of electron donating methoxy substituent in the C-4 po-
sition of cinnamic acid moiety in derivatives 1a, 2a and 3a influ-
enced increase in activity toward two (1c), or three (2c and 3c) cell
lines. The most significant influence methoxy group was recorded
in 4c that exhibited higher potencies against five cell lines with
respect to non-substituted cinnamate 4a. The presence of electron
withdrawing fluoro group at the C-4 position of cinnamoyl ester
group of 1a lead to increase of potency only against K562 cells
(compound 1d). The presence of nitro group however, influenced
increase of activity against both K562 and HL-60 cells (1b and 2b),
while insertion of fluoro group in 2a lead to favourable antitumour
properties compared to nitro group (four vs. two cell lines). Pres-
ence of both fluoro and nitro group in the C-4 position of cinnamic
acid moiety of 3a exhibited the same effects, that is increase of
cytotoxicity against HeLa and MDA-MB231 cells. Analogues with
fluoro (4d) and nitro (4b) groups showed higher potency toward
three i.e. six tumour cell lines, respectively compared to control 4a.
These results indicated that introduction of either electron with-
drawing (NO2 and F) or electron donating (OMe) groups in the C-4
position of cinnamoyloxy moiety decreases antitumour potencies
against majority tumour cell lines under evaluation.
In the next phase of SAR analysis we compared IC50 values of
derivatives with electron donating (1c, 2c, 3c, 4c) and electron
withdrawing (1b,d, 2b,d, 3b,d, 4b,d) groups in the position C-4 of
cinnamate aromatic ring. The results showed that compounds
bearing a methoxy group at the position C-4 in cinnamoyloxy
moiety were more active then 4-fluoro and 4-nitro derivatives,
with one exception (2c vs. 2d, three vs. five cell lines).
Finally, we considered influence of absolute stereochemistry at
C-4 on antitumour activity. The data in Table 3 indicate that (4R)-
stereoisomers showed higher potency toward majority of investi-
gated cell lines compared to (4S)-isomers with one exception.
Namely, 4-nitrocinnamoyl-crasslactone D (1b) exhibited higher
cytotoxicity against five of eight cell lines under investigation
compared to (4R)-analogue (2b).
in correlation with the amount of DNA they contain, hence staining
of apoptotic cells resulted in broad hypodiploid picks after flow
cytometry analysis.
As data in Table 4A reveal crassalactone D (1) slightly increased,
while its epimers 2e4 decreased percentage of K562 cells in sub G1
phase compared with untreated control. All cinnamoyl derivatives
1aed, 2aed, 3aed and 4aed increased the percent of cells in sub
G1 phase compared both with leads 1e4 and control. Significant
accumulation of cells in the sub G1 phase was detected after
treatment of cells with compounds 1a (19-fold), 2a (38-fold), 3b
(14-fold), 3d (10-fold), 4a (8-fold) and 4b (27-fold) along with the
decrease of cells in G0/G1 and S phase of cell cycle. Cinnamoates 1c,
1d, 3a, 3c and 4d significantly increased the cell percentage in G2/
M phase of cell cycle.
The observed apoptosis-inducing effect of investigated com-
pounds (Table 4A) was also confirmed by flow cytometric evalua-
tion of Annexin V binding, which measures phosphatidyl serine on
the external leaflet of the plasma membrane, an event character-
istic of early apoptosis.
Double staining (PI and Annexin V) of K562 cells treated with
the synthesized compounds followed by fluorescence activated cell
storing (FACS) enabled differentiation of normal, living cells (low
Annexin and low PI staining), apoptotic cells (high Annexin and low
PI staining) and necrotic cells (low Annexin and high PI staining).
Results are presented in Table 4B as the percentage of specific
apoptosis and necrosis [22].
Although natural product 1 just slightly increases percentage of
K562 cells in sub G1 phase with respect to control, it induced
apoptosis in 22.6% of cells. Similarly, 6-O-cinnamoyl derivative 3a
increased only 3.8-fold percentage of K562 cells in sub G1 phase,
but induced apoptosis in 81.79% cells. Very high percentage of
specific apoptosis after treatment of K562 cells with cinnamoates
1a (83.35%), 2a (86.01%), 3b (81.97%), 3d (85.69%) and 4b (70.03%) is
in agreement with detection of apoptotic DNA fragmentation
(increased sub G1 cell fraction), an event which follows the acti-
vation of executioner caspases. All tested compounds induced low
percentage of specific necrosis in K562 cells (0.64e4.06%), except
compound 4a (28.29%).
Next, we investigated how the synthesized compounds modu-
late expression of some apoptosis proteins (Bcl-2, Bax, caspase 3
and PARP). Diverse cytotoxic stimuli, including oncogenic stress
and chemotherapeutic agents could engage the mitochondrial
apoptosis pathway, which is regulated by Bcl-2 family members.
These stimuli activate BH3-only family members (initiators), which
inhibit the pro-survival Bcl-2-like proteins (guardians), thereby
enabling activation of the pro-apoptotic effectors Bax and Bak,
which then disrupt the mitochondrial outer membrane. Released
cytochrome c promotes caspase activation [23].
Caspase 3 is the key executioner caspase, and it exists as an
inactive zymogen that is activated by upstream signals. Caspase 3
could be activated in the apoptotic cell both by extrinsic (death
ligand) and intrinsic (mitochondrial) pathways [24]. The conver-
sion of procaspase 3 to caspase 3 results in the generation of the
active ‘executioner’ caspase that subsequently catalyzes the hy-
drolysis of many protein substrates [24] and other downstream
targets including PARP which primary function is to detect and
repair DNA damage. Cleavage of PARP by caspases is considered to
be a hallmark of apoptosis [25].
Our previous studies [8e10] indicated that the styryl lactones
having the (7S)-stereochemistry represent more potent cytotoxic
agents with respect to the corresponding (7R)-epimers. The results
obtained from comparison of the IC50 values of (7R)- and (7S)-
crassalactone D derivatives, which showed that (7S)-stereoisomers
were are more active then (7R)-analogues toward the majority of
investigated cell lines, agrees well with our previous findings
[8e10].
2.3. Detection of apoptosis and apoptotic pathways
Apoptosis is a cell suicide program that plays a critical role in
development and tissue homeostasis. The ability of cancer cells to
evade this programmed cell death is one of the major characteris-
tics that enables their uncontrolled growth [19]. The efficiency of
chemotherapy in killing such cells depends on the successful in-
duction of apoptosis, since defects in apoptosis signalling repre-
sents one of the causes of drug resistance [20].
Recent report disclosed by Choo et al. [21] showed that some of
natural styryl-lactones isolated from Goniothalamus genus induced
apoptosis in various tumour cell lines. This finding prompted us to
examine apoptotic signalling induced by (þ)-crassalactone D (1), its
epimers 2e4 and cinnamoyl derivatives 1aed, 2aed, 3aed, 4aed.
Influence of the synthesized compounds on cell distribution in
cell cycle phases was investigated first. After treatment of
K562 cells with the synthesized compounds at their IC50 concen-
trations, the cells were permeabilised and treated with a fluores-
cent dye (propidium iodide, PI) that stains DNA quantitatively. The
fluorescence intensity of the stained cells at certain wavelengths is
Western blot analysis revealed that five (1, 4aed) of 20 evalu-
ated compounds decreased expression of both anti-apoptotic Bcl-2
protein and pro-apoptotic Bax protein in K562 cells compared with
control (Fig. 2). Compounds 1, 4aed increased expression of acti-
vated caspase 3 or 85 kDa fragment of cleaved PARP or both which
allowed us to hypothesize that these compounds induce apoptosis
in caspase-dependent way.

ꢀ ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
298
Table 4
(A) Influence of synthesized compound on the K562 cell cycle; (B) percentage of specific apoptosis and necrosis induced with the 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
1.48
1.60
28.63
2.13
3.39
2.02
1.26
56.93
2.93
1.57
2.61
1.09
5.69
20.43
3.41
14.87
1.10
12.49
40.08
3.23
3.99
38.48
32.52
16.04
48.17
21.08
40.27
39.26
23.10
33.85
30.72
41.18
15.18
21.01
30.42
41.60
23.20
28.98
27.11
30.15
47.26
37.01
41.03
59.02
31.55
28.04
41.08
28.31
35.20
14.63
38.08
42.64
33.71
17.17
38.09
29.17
27.14
37.11
52.34
42.16
22.70
29.40
30.34
19.01
6.86
e
e
22.60
83.35
0.75
37.78
8.05
11.74
86.01
10.54
15.35
4.02
1.08
4.06
3.40
3.86
3.28
4.80
4.12
2.18
3.15
3.58
2.84
1.97
0.64
3.76
3.78
0.67
28.29
1.76
1.60
1.22
1a
1b
1c
1d
2
2a
2b
2c
2d
3
3a
3b
3c
3d
4
4a
4b
4c
4d
23.78
21.66
34.45
29.40
24.28
5.34
25.14
25.07
22.50
66.56
35.21
19.98
27.85
24.82
17.58
18.24
7.07
7.78
81.79
81.97
2.28
85.69
10.05
6.76
70.03
2.36
18.71
20.11
28.66
Fig. 2. Results of Western blot analysis after treatment of K562 cells with the synthesized compounds.
Lactones 1a, 2a, 2d, 3, 3a, 3b, 3d, 4 decreased expression of anti-
apoptotic Bcl-2 protein and increased of pro-apoptotic Bax protein
while 1bed, 2, 2b, 2c and 3c increased expression of both Bcl-2 and
Bax. Majority of these compounds induced over-expression of
activated caspase 3 subunit or 85 kDa fragment of cleaved PARP or
both (with the exception of 1b and 3a which decreased both).
Hence, these results have suggested that the triggered caspase-
dependent apoptotic cell death was influenced with the Bcl-2
protein family.
selective Wittig reaction of lactol 6 with MCMP or ECMP as a key
step, followed by acid-promoted equilibration of dominant isomer
2. Spiro-lactones 3 and 4 were obtained in a similar manner from
monoacetonide 7. Compounds 1e4 were treated with cinnamoyl
chloride, p-nitrocinnamoyl chloride, p-methoxycinnamic acid and
p-fluorocinnamic acid to afford the corresponding ester derivatives
1aed, 2aed, 3aed and 4aed.
Evaluation of antitumour activity in vitro showed that all of the
synthesized compounds, with the exception of 2b, showed higher
potency then natural product 1 against at least one tumour cell line.
Eleven of twenty synthesized compounds were more active then
DOX against at least one cell line, while none of them was active
against the normal MRC-5 cells. The preliminary SAR study reveals
that an electron donating group attached in the para position of
cinnamoate aromatic ring is beneficial for antiproliferative activity,
as well as (4R) i.e. (7S) stereochemistry. The flow-cytometry and
3. Conclusions
A novel synthesis of crassalactone D (1) and its epimers 2e4 has
been achieved starting from the commercially available chiral
template, diacetone-
D
-glucose. Synthesis of natural 1 has been
achieved employing a new one-pot process that started with a Z-

ꢀ
ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
299
Western blot analysis showed that apoptotic cell death induced by
majority of the synthesized compounds was caspase-dependent.
4.1.2.2. (4R)-(þ)-crassalactone
137e142 ꢀC, (EtOAc/hexane), [
D
(2). Colourless needles, mp
a
]D ¼ þ28.0 (c 0.5, EtOH), Rf ¼ 0.61
(5:2 EtOAc/hexane). 1H and 13C NMR data for 4 matched those
previously recorded by us [5].
4. Experimental section
4.1. Chemistry
4.1.3. (7S)-(þ)-crassalactone D (3) and (4R,7S)-(þ)-crassalactone D
(4)
4.1.1. General experimental procedures
Procedure A. Solution of 7 (0.117 g, 0.40 mmol) in 90% TFA
(3.4 mL) was stirred for 30 min at 0 ꢀC and then for additional
1.5 h at room temperature. The mixture was concentrated by co-
distillation with toluene and the residue was purified by flash
column chromatography (47:3 / 23:2 CH2Cl2/MeOH) to afford
pure hygroscopic syrup 8 (0.097 g, 96%). To a cooled (0 ꢀC) solution
of lactol 8 (0.097 g, 0.38 mmol) in dry MeOH (10 mL) was added
MCMP (0.193 g, 0.58 mmol). After 30 min cooling bath was
removed and the mixture was stirred at room temperature for 3 h.
Another portion of MCMP (0.129 g, 0.38 mmol) was added
following previous procedure but stirring was continued at room
temperature for another 44 h. The mixture was concentrated.
Multiple purification by flash column chromatography (9:1 Et2O/
light petroleum, 17:3 CH2Cl2/EtOAc) afforded pure 4 (0.034 g, 38%)
and 3 (0.026g, 29%).
Procedure B. Solution of 7 (0.070 g, 0.24 mmol) in 90% TFA (2 mL)
was stirred for 30 min at 0 ꢀC and then for additional 1.5 h at room
temperature. The mixture was concentrated by co-distillation with
toluene and the residue was purified by flash column chromatog-
raphy (47:3 / 23:2 CH2Cl2/MeOH) to afford pure hygroscopic
syrup 8 (0.059 g, 96%). To a cooled (0 ꢀC) solution of lactol 8
(0.059 g, 0.23 mmol) in dry MeOH (6 mL) was added ECMP (0.122 g,
0.35 mmol). After 30 min cooling bath was removed and the
mixture was stirred at room temperature for 3 h. Another portion of
ECMP (0.081 g, 0.23 mmol) was added following previous pro-
cedure but stirring was continued at room temperature for another
22 h. The mixture was concentrated. Multiple purification by flsh
column chromatography (9:1 Et2O/light petroleum, 17:3 CH2Cl2/
EtOAc) afforded pure 4 (0.020 g, 37%) and 3 (0.0054g, 10%).
Procedure C: A solution of 4 (0.035 g, 0.15 mmol) in CHCl3
(0.8 mL) and TFA (0.11 mL, 1.51 mmol) was stirred for 23 h at room
temperature. The mixture was concentrated by co-distillation with
toluene and residue purified by flash column chromatography (7:3
Et2O/light petroleum). An inseparable mixture of 3 and 4 in the
respective 1:2 ratio (1H NMR) was obtained in an almost quanti-
tative yield. Repeated flash column chromatography gave pure 3
(0.014 g, 41%) and 4 (0.018 g, 51%).
Melting points were determined on a Hot Stage Microscope
Nagema PHMK 05 and were not corrected. Optical rotations were
measured on an Autopol IV (Rudolph Research) polarimeter at
room temperature. NMR spectra were recorded on a Bruker AC
250 E or a Bruker Avance III 400 MHz instrument and chemical
shifts are expressed in ppm downfield from TMS. IR spectra were
recorded with an FTIR Nexus 670 spectrophotometer (Thermo-
Nicolet). High resolution mass spectra (ESI) were acquired on a
Agilent Technologies 1200 series instrument equipped with Zorbax
Eclipse Plus C18 (100 mm ꢂ 2.1 mm i.d. 1.8
mm) column and DAD
detector (190e450 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 (0.040e0.063,
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. The corresponding bands were scraped and
eluted with EtOAc. All organic extracts were dried with anhydrous
Na2SO4. Organic solutions were concentrated in a rotary evaporator
under diminished pressure at a bath temperature below 35 ꢀC.
4.1.2. (þ)-Crassalactone D (1) and (4R)-(þ)-crassalactone D (2)
Procedure A: A solution of 5 (0.443 g, 1.51 mmol) in 90% TFA
(6.1 mL) was stirred for 30 min 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 chro-
matography (24:1 CH2Cl2/MeOH) to afford pure hygroscopic syrup
6 (0.380 g, 100%). To a cooled (0 ꢀC) solution of lactol 6 (0.380 g,
1.51 mmol) in dry MeOH (38 mL) was added MCMP (0.753 g,
2.32 mmol). After 30 min cooling bath was removed and the
mixture was stirred at room temperature for 3 h. Another portion of
MCMP (0.540 g, 1.51 mmol) was added following previous pro-
cedure but stirring was continued at room temperature for another
18 h. The mixture was concentrated. Multiple purification by flash
column chromatography (4:1 Et2O/light petroleum, 17:3 CH2Cl2/
EtOAc) afforded pure mixture of 1 and 2 (0.179 g, 51%), in the
respective ratio of 1:2 (1H NMR).
Procedure B: To a solution of 1 and 2 (1:2 mixture, 0.174 g,
0.75 mmol) in dry CH2Cl2 (3.6 mL) was added TFA (0.56 mL,
7.59 mmol). Reaction mixture was stirred for 16 h at room tem-
perature and then concentrated by co-distillation with toluene. The
mixture of 1 and 2 (99%) was obtained in the respective ratio of 2:1
(1H NMR). Separation by preparative TLC (15:1 / 1:3 hexane/
EtOAc, two successive developments) gave pure 1 (0.059 g,17%) and
2 (0.031 g, 9%).
4.1.3.1. (7S)-(þ)-crassalactone
170e175 ꢀC (CH2Cl2/hexane), [
D
(3). Colourless needles, mp
a
]D ¼ þ32.0 (c 0.2, EtOH), Rf ¼ 0.24
(Et2O). 1H and 13C NMR data for 4 matched those previously
recorded by us [5].
4.1.3.2. (4R,7S)-(þ)-crassalactone D (4). Colourless needles, mp
139e141 ꢀC (EtOAc/hexane), [
a
]
¼ þ73.0 (c 0.2, EtOH), Rf ¼ 0.54
D
(Et2O). 1H and 13C NMR data for 4 matched those previously
recorded by us [5].
4.1.2.1. (þ)-Crassalactone
D
(1). Colourless
needles,
mp
140e142 ꢀC, (EtOAc/hexane), [
a
]D ¼ þ20.5 (c 0.2, EtOH),1 Rf ¼ 0.63
4.1.4. General procedure for the preparation of cinnamoates 1ae4a
To a cooled (0 ꢀC) and stirred solution of cinnamoyl-chloride
(1.28 equiv) and DMAP (1.56 equiv) in dry CH2Cl2 (0.12e0.16 M)
was added a solution of 1e4 (1 equiv)2 in dry CH2Cl2 (0.04 M). After
stirring at 0 ꢀC for 2 h additional portions of cinnamoyl-chloride
(0.064 equiv) and DMAP (0.77 equiv) were added. The mixture
(5:2 EtOAc/hexane), Ref. [3] mp 138e140 ꢀC, [
a
]
¼ þ13.6 (c 0.2,
D
EtOH). Both 1H and 13C NMR data of compound 1 are consistent with
the naturally occurring (þ)-crassalactone D [1] and its physical
properties are in agreement with those reported in the literature [3].
1
Our optical rotation value is slightly greater than the data for [
]D¼ þ7 (c 0.2, EtOH)}, but were in reasonable agreement with the reported
data for synthetic (þ)-crassalactone D reported in ref [3].
a]D reported in ref
[1] {[
a
2
Compounds 1 and 2 were used in the reaction as a mixture of 1:2, respectively.

ꢀ ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
300
was stirred at 0 ꢀC for 1 h and then at room temperature for the
next 20 h (for 1 and 2), 45 min (for 3) or 22.5 h (for 4). The mixture
was poured into 5e10% aq HCl and extracted with CH2Cl2. The
combined extracts were washed with 10% aq NaCl (to pH 7), dried
and evaporated. The residue was purified by preparative TLC (1:1
Et2O/light petroleum, two successive developments for 1a, 2a and
4a; 4:1 Et2O/light petroleum for 3a).
124.0 (C-2), 126.9, 128.0, 128.1, 128.2, 128.8, 130.5, 133.9, 134.2
(2 ꢂ Ph), 145.6 (C-30), 151.6 (C-3), 165.4 (C-10), 169.5 (C-1). HRMS
(ESI): m/z 363.1220 (MþþH), calcd for C22H19O5: 363.1227; m/z
380.1486 (MþþNH4), calcd for C22H22NO5: 380.1492; m/z 385.1036
(MþþNa), calcd for C22H18NaO5: 385.1046.
4.1.5. General procedure for the preparation of 4-nitrocinnamoates
1be4b
4.1.4.1. 6-O-cinnamoyl-(þ)-crassalactone D (1a). Yield 34%. White
To a cooled (0 ꢀC) solution of 1e4 (1 equiv)2 in dry CH2Cl2
(0.06e0.07 M) were added 4-nitrocinnamoyl chloride (1.3e1.7
equiv) and DMAP (1.6e2.1 equiv). After stirring at 0 ꢀC for 1.5e2 h
additional portions of 4-nitrocinnamoyl chloride (0.6e0.8 equiv)
and DMAP (0.8e1 equiv) were added. The mixture was stirred at
0 ꢀC for another 1e2 h. The mixture was poured into 5e10% aq NaCl
and extracted with CH2Cl2. The combined extracts were dried and
evaporated. The residue was purified by preparative TLC (3:2 light
petroleum/EtOAc, three successive developments for 1b and 2b;
7:3 light petroleum/EtOAc, two successive developments for 4b) or
by flash column chromatography (3:2 light petroleum/EtOAc for
3b).
powder, mp 123e126 ꢀC (CH2Cl2/hexane), [
a
]
¼ þ118.0 (c 0.1,
D
CHCl3), Rf ¼ 0.31 (1:1 Et2O/light petroleum). IR (film): nmax 1775 and
1713 (C¼O). 1H NMR (250 MHz, CDCl3):
2.47 (dd, 1 H, J5a,5b ¼ 14.8,
d
J5a,6 ¼ 1.3 Hz, H-5a), 2.73 (dd, 1 H, J5a,5b ¼ 14.8, J5b,6 ¼ 6.9 Hz, H-5b),
5.45 (m, 1 H, J5a,6 ¼ 1.3, J6,7 ¼ 2.6, J5b,6 ¼ 6.9 Hz, H-6), 5.57 (d, 1 H,
J6,7 ¼ 2.6 Hz, H-7), 6.30 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.57 (d, 1 H,
J2 ,3 ¼ 16.0 Hz, H-20), 7.31 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3), 7.34e7.65 (m,
0
0
10 H, 2 ꢂ Ph), 7.82 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-30). NOE contact: H-3
0
0
and ortho H-atoms from Ph group at C-7. 13C NMR (62.5 MHz,
CDCl3):
d
40.2 (C-5), 78.9 (C-6), 88.0 (C-7), 113.7 (C-4), 117.2 (C-20),
125.0 (C-2), 125.4, 128.3, 128.7, 128.9, 129.3, 130.6, 134.0, 137.8
(2 ꢂ Ph), 146.2 (C-30), 151.1 (C-3), 166.6 (C-10), 169.6 (C-1). HRMS
(ESI): m/z 363.1228 (MþþH), calcd for C22H19O5: 363.1227; m/z
380.1491 (MþþNH4), calcd for C22H22NO5: 380.1492; m/z 385.1045
(MþþNa), calcd for C22H18NaO5: 385.1046.
4.1.5.1. 6-O-(4-nitrocinnamoyl)-(þ)-crassalactone D (1b). Yield 29%.
Colourless plates, mp 189e191 ꢀC (EtOH), [
a
]
D
¼ þ52.0 (c 0.1,
CHCl3), Rf ¼ 0.43 (3:2 light petroleum/EtOAc). IR (film): nmax 1774
and 1717 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.47 (bd, 1 H,
4.1.4.2. (4R)-6-O-cinnamoyl-(þ)-crassalactone D (2a). Yield 38%.
J5a,5b ¼ 14.7, H-5a), 2.73 (dd, 1 H, J5a,5b ¼ 14.7, J5b,6 ¼ 6.7 Hz, H-5b),
5.47 (m, 1 H, H-6), 5.57 (d, 1 H, J6,7 ¼ 2.0 Hz, H-7), 6.32 (d, 1 H,
Colourless needles, mp 112e115 ꢀC (CH2Cl2/hexane), [
a
]D ¼ þ39.0 (c
J2,3 ¼ 5.5 Hz, H-2), 6.69 (d, 1 H, J2 ,3 ¼ 15.9 Hz, H-20), 7.34 (d, 1 H,
J2,3 ¼ 5.5 Hz, H-3), 7.34e7.49 (m, 5 H, Ph), 7.74 and 8.29 (2 ꢂ d, 4 H,
0
0
0.1, CHCl3), Rf ¼ 0.46 (1:1 Et2O/light petroleum). IR (film): nmax 1777
and 1716 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.48 (dd, 1 H,
J ¼ 8.6 Hz, p-NO2Ph), 7.84 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR
0
0
J5a,5b ¼ 14.3, J5a,6 ¼ 4.3 Hz, H-5a), 2.90 (dd, 1 H, J5a,5b ¼ 14.3,
J5b,6 ¼ 5.6 Hz, H-5b), 5.42 (d, 1 H, J6,7 ¼ 3.2 Hz, H-7), 5.54 (m, 1 H, H-
(62.5 MHz, CDCl3):
d 40.1 (C-5), 79.5 (C-6), 88.1 (C-7), 113.7 (C-4),
0
6), 6.21 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.51 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-2 ),
7.35 (d, 1 H, J2,3 ¼ 5.6 Hz, H-3), 7.38e7.65 (m, 10 H, 2 ꢂ Ph), 7.78 (d,
121.6 (C-20), 125.1 (C-2), 124.2, 128.4, 128.5, 128.8, 128.9, 137.7, 140.1,
148.8 (2 ꢂ Ph), 143.2 (C-30), 151.0 (C-3), 165.7 (C-10), 169.6 (C-1).
HRMS (ESI): m/z 408.1069 (MþþH), calcd for C22H18NO7: 408.1078;
m/z 425.1333 (MþþNH4), calcd for C22H21N2O7: 425.1343; m/z
430.0893 (MþþNa), calcd for C22H17NaO7: 430.0897.
0
0
1 H, J2 ,3 ¼ 16.0 Hz, H-30). NOE contact: H-7 and H-3. 13C NMR
0
0
(62.5 MHz, CDCl3):
d 39.1 (C-5), 79.8 (C-6), 87.6 (C-7), 114.2 (C-4),
116.8 (C-20), 122.9 (C-2), 125.8, 128.2, 128.3, 128.7, 129.0, 130.8,
133.9, 138.0 (2 ꢂ Ph), 146.4 (C-30), 152.7 (C-3), 166.0 (C-10), 170.2 (C-
1). HRMS (ESI): m/z 363.1232 (MþþH), calcd for C22H19O5:
363.1227; m/z 380.1493 (MþþNH4), calcd for C22H22NO5: 380.1492;
m/z 385.1048 (MþþNa), calcd for C22H18NaO5: 385.1046.
4.1.5.2. (4R)-6-O-(4-nitrocinnamoyl)-(þ)-crassalactone
D
(2b).
Yield 56%. White powder, mp 162e165 ꢀC (EtOH), [
a
]D ¼ þ24.0 (c
0.1, CHCl3), Rf ¼ 0.51 (3:2 light petroleum/EtOAc). IR (film): nmax
1774 and 1720 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.49 (dd, 1 H,
4.1.4.3. (7S)-6-O-cinnamoyl-(þ)-crassalactone
D
(3a). Yield 68%.
]D ¼ þ36.5
J5a,5b ¼ 14.2, J5a,6 ¼ 4.7 Hz, H-5a), 2.92 (dd, 1 H, J5a,5b ¼ 14.3,
J5b,6 ¼ 5.8 Hz, H-5b), 5.40 (d, 1 H, J6,7 ¼ 3.5 Hz, H-7), 5.56 (m, 1 H, H-
Colourless crystals, mp 168e172 ꢀC (CH2Cl2/hexane), [
a
0
0
0
(c 0.2, CHCl3), Rf ¼ 0.42 (4:1 Et2O/light petroleum). IR (film): nmax
6), 6.23 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.63 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-2 ),
7.31 (d, 1 H, J2,3 ¼ 5.4 Hz, H-3), 7.38e7.63 (m, 5 H, Ph), 7.72 and 8.29
1776 and 1708 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.63 (d, 1 H,
(2 ꢂ d, 4 H, J ¼ 8.7 Hz, p-NO2Ph), 7.80 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-30).
0
0
J5a,5b ¼ 14.6, H-5a), 2.69 (dd, 1 H, J5a,5b ¼ 14.7, J5b,6 ¼ 5.3 Hz, H-5b),
5.52 (d, 1 H, J6,7 ¼ 4.5 Hz, H-7), 5.81 (t, 1 H, J ¼ 4.8 Hz, H-6), 6.20 (d,
NOE contact: H-7 and H-3. 13C NMR (62.5 MHz, CDCl3):
d 39.2 (C-5),
0
1 H, J2 ,3 ¼ 16.0 Hz, H-2 ), 6.26 (d,1 H, J2,3 ¼ 5.5 Hz, H-2), 7.23 (d,1 H,
80.2 (C-6), 87.5 (C-7), 114.0 (C-4), 121.1 (C-20), 123.2 (C-2), 124.2,
125.9, 128.5, 128.8, 128.83, 137.7, 139.9, 148.8 (2 ꢂ Ph), 143.4 (C-30),
152.4 (C-3), 165.2 (C-10), 169.4 (C-1). HRMS (ESI): m/z 408.1070
(MþþH), calcd for C22H18NO7: 408.1078; m/z 425.1338 (MþþNH4),
calcd for C22H21N2O7: 425.1343.
0
0
J2,3 ¼ 5.5 Hz, H-3), 7.25e7.55 (m, 11 H, 2 ꢂ Ph and H-30). 13C NMR
(62.5 MHz, CDCl3):
d 42.3 (C-5), 73.0 (C-6), 87.4 (C-7), 113.5 (C-4),
117.0 (C-20), 124.5 (C-2), 127.4, 128.0, 128.2, 128.3, 128.8, 130.4, 134.1,
134.8 (2 ꢂ Ph), 145.6 (C-30), 151.8 (C-3), 165.6 (C-10), 169.9 (C-1).
HRMS (ESI): m/z 363.1223 (MþþH), calcd for C22H19O5: 363.1227;
m/z 380.1486 (MþþNH4), calcd for C22H22NO5: 380.1492; m/z
385.1038 (MþþNa), calcd for C22H18NaO5: 385.1046.
4.1.5.3. (7S)-6-O-(4-nitrocinnamoyl)-(þ)-crassalactone
D
(3b).
Yield 74%. White powder, mp 105e125 ꢀC (Et2O/hexane),
[
a
]D ¼ þ21.0 (c 0.2, CHCl3), Rf ¼ 0.37 (1:1 light petroleum/EtOAc). IR
4.1.4.4. (4R,7S)-6-O-cinnamoyl-(þ)-crassalactone D (4a). Yield 62%.
(film): nmax 1773 and 1721 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.64
Colourless needles, mp 163e166 ꢀC (CH2Cl2/hexane), [
a]D ¼ þ51.0 (c
(d, 1 H, J5a,5b ¼ 14.6 Hz, H-5a), 2.82 (dd, 1 H, J5a,5b ¼ 14.7,
J5b,6 ¼ 5.3 Hz, H-5b), 5.55 (d, 1 H, J6,7 ¼ 4.4 Hz, H-7), 5.83 (t, 1 H,
J ¼ 4.8 Hz, H-6), 6.27 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.31 (d, 1 H,
0.2, CHCl3), Rf ¼ 0.58 (1:1 Et2O/light petroleum). IR (film): nmax 1775
and 1713 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.63 (dd, 1 H,
J5a,5b ¼ 15.1, J5a,6 ¼ 2.6 Hz, H-5a), 3.00 (dd, 1 H, J5a,5b ¼ 15.1,
J5b,6 ¼ 6.4 Hz, H-5b), 5.59 (d, 1 H, J6,7 ¼ 4.4 Hz, H-7), 5.54 (m, 1 H, H-
J2 ,3 ¼ 16.0 Hz, H-20), 7.23 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3), 7.15e8.30 (m,
0
0
10 H, 2 ꢂ Ph and H-30). 13C NMR (62.5 MHz, CDCl3):
d 42.3 (C-5),
0
6), 6.14 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-2 ), 6.25 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2),
73.5 (C-6), 87.4 (C-7), 113.5 (C-4), 121.3 (C-20), 124.1 (C-2), 124.6,
127.1, 128.0, 128.4, 128.8, 134.8, 140.2, 148.5 (2 ꢂ Ph), 142.5 (C-30),
151.7 (C-3), 164.6 (C-10), 169.9 (C-1). HRMS (ESI): m/z 425.1340
0
0
7.26e7.65 (m, 12 H, 2 ꢂ Ph, H-3 and H-30). 13C NMR (62.5 MHz,
CDCl3):
d
42.1 (C-5), 74.4 (C-6), 84.9 (C-7), 112.6 (C-4), 116.8 (C-20),

ꢀ
ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
301
(MþþNH4), calcd for C22H21NO7: 425.1343.
4.1.6.3. (7S)-6-O-(4-methoxycinnamoyl)-(ꢁ)-crassalactone
D
(3c).
Yield 86%. Colourless syrup, [
a]
¼ ꢁ14.0 (c 0.1, CHCl3), Rf ¼ 0.49
D
(3:2 EtOAc/light petroleum). IR (film): nmax 1771 and 1717 (C¼O). 1H
4.1.5.4. (4R,7S)-6-O-(4-nitrocinnamoyl)-(þ)-crassalactone
D (4b).
]D ¼ þ18.0 (c 0.1,
NMR (250 MHz, CDCl3):
d
2.64 (d, 1 H, J5a,5b ¼ 14.6, H-5a), 2.79 (dd,
Yield 89%. White powder, mp 85e90 ꢀC (EtOH), [
a
1 H, J5a,5b ¼ 14.6, J5b,6 ¼ 5.3 Hz, H-5b), 3.83 (s, 3 H, OMe), 5.52 (d,1 H,
J6,7 ¼ 4.5 Hz, H-7), 5.81 (t, 1 H, J ¼ 4.8 Hz, H-6), 6.06 (d, 1 H,
CHCl3), Rf ¼ 0.46 (13:7 light petroleum/EtOAc). IR (film): nmax 1775
and 1720 (C¼O). 1H NMR (400 MHz, CDCl3):
d 2.63 (dd, 1 H,
J2 ,3 ¼ 16.0 Hz, H-20), 6.26 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.88 (d, 2 H,
J ¼ 8.8 Hz, p-MeOPh). 7.23 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3), 7.25e7.52 (m,
0
0
J5a,5b ¼ 15.2, J5a,6 ¼ 2.8 Hz, H-5a), 3.04 (dd, 1 H, J5a,5b ¼ 15.2,
J5b,6 ¼ 6.5 Hz, H-5b), 5.62 (d, 1 H, J6,7 ¼ 4.5 Hz, H-7), 5.89 (ddd, 1 H,
8 H, Ph and H-30). 13C NMR (62.5 MHz, CDCl3):
d 42.3 (C-5), 55.3
0
0
J5a,6 ¼ 2.8, J6,7 ¼ 4.5, J5b,6 ¼ 6.5 Hz, H-6), 6.26 (d, 1 H, J2 ,3 ¼ 16.0 Hz,
(OMe), 72.8 (C-6), 87.6 (C-7), 113.6 (C-4), 114.5 (C-20), 124.5 (C-2),
114.2, 126.9, 127.4, 127.7, 128.3, 128.4, 129.9, 130.4, 134.9, 161.5
(2 ꢂ Ph), 145.3 (C-30), 151.9 (C-3), 166.0 (C-10), 170.0 (C-1). HRMS
(ESI): m/z 393.1327 (MþþH), calcd for C23H21O6: 393.1333; m/z
410.1588 (MþþNH4), calcd for C23H24NO6: 410.1598; m/z 415.1139
(MþþNa), calcd for C23H20NaO6: 415.1152.
H-20), 6.28 (d, J2,3 ¼ 5.5 Hz, H-2), 7.21e8.30 (m,11 H, 2 ꢂ Ph, H-3 and
H-30). 13C NMR (100 MHz, CDCl3):
d 42.1 (C-5), 74.9 (C-6), 84.9 (C-7),
112.5 (C-4), 121.0 (C-20), 124.2 (C-2), 124.3, 127.0, 128.2, 128.4, 128.7,
134.2, 140.0, 148.6 (2 ꢂ Ph), 142.6 (C-30), 151.4 (C-3), 164.6 (C-10),
169.4 (C-1). HRMS (ESI): m/z 425.1341 (MþþNH4), calcd for
C
22H21NO7: 425.1343.
4.1.6.4. (4R,7S)-6-O-(4-methoxycinnamoyl)-(þ)-crassalactone
D
4.1.6. General procedure for the preparation of 4-
methoxycinnamoates 1ce4c
(4c). Yield 77%. Colourless syrup, [
a]
¼ þ17.5 (c 0.2, CHCl3),
D
Rf ¼ 0.68 (3:2 light petroleum/EtOAc). IR (film): nmax 1775 and 1710
To a solution of 1e4 (1 equiv)2 in dry CH2Cl2 (0.03e0.04 M) were
added 4-methoxycinnamic acid (2.1 equiv), DMAP (4.2 equiv) and
DCC (2.5 equiv). The mixture was stirred at room temperature for
3e5 h, then poured into water and extracted with CH2Cl2. The
combined extracts were dried and evaporated. The residue was
purified by flash column chromatography (7:3 light petroleum/
EtOAc for 1c and 2c; 3:2 light petroleum/EtOAc for 3c; 3:2 light
petroleum/EtOAc for 4c) and then by preparative TLC (7:3 light
petroleum/EtOAc for 1c and 2c, two successive developments; 3:2
light petroleum/EtOAc for 3c; 4:1 light petroleum/EtOAc three
successive developments for 4c).
(C¼O). 1H NMR (250 MHz, CDCl3):
d
2.61 (dd, 1 H, J5a,5b ¼ 15.2,
J5a,6 ¼ 2.6 Hz, H-5a), 3.01 (dd, 1 H, J5a,5b ¼ 15.2, J5b,6 ¼ 6.3 Hz, H-5b),
3.84 (s, 3 H, OMe), 5.58 (d, 1 H, J6,7 ¼ 4.4 Hz, H-7), 5.86 (ddd, 1 H,
0
0
J5a,6 ¼ 2.6, J6,7 ¼ 4.4, J5b,6 ¼ 6.5 Hz, H-6), 6.01 (d, 1 H, J2 ,3 ¼ 15.9 Hz,
H-20), 6.24 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.88 (d, 2 H, J ¼ 8.8 Hz, p-
MeOPh), 7.27e8.42 (m, 9 H, 2 ꢂ Ph, H-3 and H-30). 13C NMR
(62.5 MHz, CDCl3):
d 42.1 (C-5), 55.3 (OMe), 74.3 (C-6), 85.0 (C-7),
112.7 (C-4), 114.2 (C-20), 123.9 (C-2), 114.3, 126.7, 126.9, 128.1, 128.3,
129.8, 134.3, 161.6 (2 ꢂ Ph), 145.3 (C-30), 151.7 (C-3), 165.8 (C-10),
169.6 (C-1). HRMS (ESI): m/z 393.1324 (MþþH), calcd for C23H21O6:
393.1333; m/z 410.1594 (MþþNH4), calcd for C23H24NO6: 410.1598;
m/z 415.1142 (MþþNa), calcd for C23H20NaO6: 415.1152.
4.1.6.1. 6-O-(4-methoxycinnamoyl)-(þ)-crassalactone
D
(1c).
Yield 32%. Colourless needles, mp 153e156 ꢀC (CH2Cl2/hexane),
4.1.7. General procedure for the preparation of 4-fluorocinnamoates
1de4d
[
a
]D ¼ þ77.0 (c 0.1, CHCl3), Rf ¼ 0.26 (7:3 light petroleum/EtOAc). IR
To a solution 1e4 (1 equiv)2 in dry CH2Cl2 (0.04e0.05 M) were
added 4-fluorocinnamic acid (2.3 equiv), DMAP (4.6 equiv) and DCC
(2.4 equiv). The mixture was stirred at room temperature for
4e5 h,3 then poured into water and extracted with CH2Cl2. The
combined extracts were washed with 10% aq NaCl, dried and
evaporated. The residue was purified by flash column chromatog-
raphy (3:2 light petroleum/EtOAc for 3d, 4:1 light petroleum/EtOAc
for 4d) or by flash chromatography (7:3 light petroleum/EtOAc)
followed by preparative TLC (7:3 light petroleum/EtOAc for 1d and
2d).
(film): nmax 1773 and 1709 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.46
(dd, 1 H, J5a,5b ¼ 14.8, J5a,6 ¼ 1.3 Hz, H-5a), 2.73 (dd, 1 H, J5a,5b ¼ 14.8,
J5b,6 ¼ 6.9 Hz, H-5b), 3.86 (s, 3 H, OMe), 5.44 (m, 1 H, J5a,6 ¼ 1.6,
J6,7 ¼ 2.5, J5b,6 ¼ 6.9 Hz, H-6), 5.56 (d, 1 H, J6,7 ¼ 2.6 Hz, H-7), 6.30 (d,
1 H, J2,3 ¼ 5.5 Hz, H-2), 6.44 (d, 1 H, J2 ,3 ¼ 15.9 Hz, H-20), 6.94 and
7.54 (2d, 4 H, J ¼ 8.7 Hz, p-MeOPh), 7.31 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3)
0
0
7.33e7.49 (m, 5 H, Ph), 7.77 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-30). NOE
0
0
contact: H-3 and ortho-H-atoms from Ph. 13C NMR (62.5 MHz,
CDCl3):
d 40.2 (C-5), 55.4 (OMe), 78.8 (C-6), 88.1 (C-7), 113.8 (C-4),
114.6 (C-20),125.0 (C-2),114.4,125.4,125.5,126.8,128.4,128.7,130.0,
137.9, 161.6 (2 ꢂ Ph), 145.9 (C-30), 151.1 (C-3), 166.6 (C-10), 169.6 (C-
1). HRMS (ESI): m/z 393.1328 (MþþH), calcd for C23H21O6:
393.1333; m/z 410.1589 (MþþNH4), calcd for C23H24NO6: 410.1598;
m/z 415.1147 (MþþNa), calcd for C23H20NaO6: 415.1152.
4.1.7.1. 6-O-(4-fluorocinnamoyl)-(þ)-crassalactone
D
(1d).
Yield 36%. Colourless needles, mp 152e155 ꢀC (CH2Cl2/hexane),
[
a
]D ¼ þ62.0 (c 0.1, CHCl3), Rf ¼ 0.34 (7:3 light petroleum/EtOAc). IR
(film): nmax 1775 and 1715 (C¼O). 1H NMR (250 MHz, CDCl3):
d 2.46
(bd, 1 H, J5a,5b ¼ 14.8 Hz, H-5a), 2.73 (dd, 1 H, J5a,5b ¼ 14.8,
J5b,6 ¼ 6.8 Hz, H-5b), 5.45 (m, 1 H, H-6), 5.57 (d, 1 H, J6,7 ¼ 2.4 Hz, H-
4.1.6.2. (4R)-6-O-(4-methoxycinnamoyl)-(þ)-crassalactone
D (2c).
Yield 60%. Colourless syrup, [
a
]
¼ þ29.0 (c 0.1, CHCl3), Rf ¼ 0.38
D
0
0
0
7), 6.31 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 6.51 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-2 ),
7.31 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3), 7.03e7.64 (m, 9 H, 2 ꢂ Ph), 7.78 (d,
(7:3 light petroleum/EtOAc). IR (film): nmax 1775 and 1712 (C¼O). 1H
NMR (250 MHz, CDCl3):
d
2.47 (dd, 1 H, J5a,5b ¼ 14.3, J5a,6 ¼ 4.2 Hz,
1 H, J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (62.5 MHz, CDCl3):
d
40.2 (C-5),
0
0
H-5a), 2.89 (dd, 1 H, J5a,5b ¼ 14.3, J5b,6 ¼ 5.7 Hz, H-5b), 3.87 (s, 3 H,
79.0 (C-6), 88.1 (C-7), 113.7 (C-4), 116.97 and 117.0 (C-20,
OMe), 5.41 (d, 1 H, J6,7 ¼ 3.1 Hz, H-7), 5.52 (m, 1 H, H-6), 6.21 (d, 1 H,
J2 ,F ¼ 1.8 Hz), 125.0 (C-2), 116.0 and 116.3 (C-300, J3 ,F ¼ 18.8 Hz),
125.4, 128.4, 128.8, 130.2, 130.3, 137.8 (2 ꢂ Ph), 162.1 and 166.5 (C-
0
00
J2,3 ¼ 5.5 Hz, H-2), 6.37 (d, 1 H, J2 ,3 ¼ 15.9 Hz, H-20), 6.94 and 7.52
(2 ꢂ d, 4 H, J ¼ 8.7 Hz, p-MeOPh), 7.30e7.65 (m, 6 H, H-3 and Ph),
0
0
400, J4 ,F ¼ 275.0 Hz),145.0 (C-30),151.1 (C-3),166.1 (C-10),169.6 (C-1).
00
7.72 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-3 ). 13C NMR (62.5 MHz, CDCl3):
d
39.0
0
0
0
HRMS (ESI): m/z 381.1122 (MþþH), calcd for C22H18FO5: 381.1133;
(C-5), 55.4 (OMe), 79.7 (C-6), 87.7 (C-7), 114.2 (C-20), 114.3 (C-4),
122.8 (C-2), 114.4, 125.7, 125.8, 126.6, 128.2, 130.0, 138.0, 161.8
(2 ꢂ Ph), 146.0 (C-30), 152.8 (C-3), 166.4 (C-10), 169.6 (C-1). HRMS
(ESI): m/z 393.1319 (MþþH), calcd for C23H21O6: 393.1333; m/z
410.1581 (MþþNH4), calcd for C23H24NO6: 410.1598; m/z 415.1139
(MþþNa), calcd for C23H20NaO6: 415.1152.
m/z 398.1388 (MþþNH4), calcd for C22H21NFO5: 398.1398; m/z
3
For 3 additional portions of reagents have been added: 4-fluorocinnamic acid
(1.2 equiv), DMAP (2.3 equiv) and DCC (2.3 equiv) and stirring continued for
additional 1 h at room temperature.

ꢀ ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
302
403.0943 (MþþNa), calcd for C22H17NaFO5: 403.0952.
4.3. MTT assay
The colorimetric MTT assay was carried out following reported
procedure [18].
4.1.7.2. (4R)-6-O-(4-fluorocinnamoyl)-(þ)-crassalactone
D
(2d).
Yield 50%. Colourless syrup, [
a
]
D
¼ þ24.0 (c 0.1, CHCl3), Rf ¼ 0.54
(7:3 light petroleum/EtOAc). IR (film): nmax 1776 and 1717 (C¼O). 1H
4.4. Cell cycle analysis
NMR (250 MHz, CDCl3):
d
2.48 (dd, 1 H, J5a,5b ¼ 14.3, J5a,6 ¼ 4.3 Hz,
H-5a), 2.90 (dd, 1 H, J5a,5b ¼ 14.3, J5b,6 ¼ 5.7 Hz, H-5b), 5.42 (d, 1 H,
K562 cells were treated with tested compounds for 72 h at their
IC50 concentrations. After treatment, K562 cells were washed in
cold PBS and incubated for 30 min in 70% ethanol on ice, centri-
J6,7 ¼ 3.2 Hz, H-7), 5.54 (m, 1 H, H-6), 6.21 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2),
6.43 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-20), 7.35 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3),
0
0
7.08e7.64 (m, 9 H, 2 ꢂ Ph), 7.73 (d, 1 H, J2 ,3 ¼ 16.0 Hz, H-30). 13C
0
0
fuged and incubated with 500
mL Rnase A (100 units/mL) and
NMR (62.5 MHz, CDCl3):
d
39.1 (C-5), 79.8 (C-6), 87.6 (C-7), 114.2 (C-
0
500 L propidium iodide (400
m
m
L/mL) for 30 min at 37 ꢀC. Cell cycle
4), 116.5 and 116.53 (C-20, J2 ,F ¼ 1.8 Hz), 123.0 (C-2), 116.0 and 116.3
was analyzed by FACS Calibur E440 (Becton Dickinson) flow cy-
tometer and the Cell Quest software. Results were presented as
percentage of cell cycle phases.
(C-300, J3 ,F ¼ 18.8 Hz), 125.8, 128.3, 128.7, 130.1, 130.2, 137.9 (2 ꢂ Ph),
00
162.1 and 166.2 (C-400, J4 ,F ¼ 256.3 Hz), 145.0 (C-30), 152.7 (C-3),
00
165.9 (C-10), 169.6 (C-1). HRMS (ESI): m/z 381.1122 (MþþH), calcd
for
C
22H18FO5: 381.1133; m/z 398.1388 (MþþNH4), calcd for
4.5. Detection of apoptosis
C
C
22H21NFO5: 398.1398; m/z 403.0945 (MþþNa), calcd for
22H17NaFO5: 403.0952.
Apoptosis of K562 cells was evaluated with an Annexin V-FITC
detection kit. Treated 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
4.1.7.3. (7S)-6-O-(4-fluorocinnamoyl)-(þ)-crassalactone
D
(3d).
Yield 73%. Colourless syrup, [
a
]
¼ þ60.0 (c 0.1, CHCl3), Rf ¼ 0.58
D
(1:1 EtOAc/light petroleum). IR (film): nmax 1774 and 1713 (C¼O). 1H
NMR (250 MHz, CDCl3):
d
2.64 (d, 1 H, J5a,5b ¼ 14.6 Hz, H-5a), 2.79
1 ꢂ 106 cells/mL. The cell suspension (100
m
L) was transferred to
(dd, 1 H, J5a,5b ¼ 14.6, J5b,6 ¼ 5.3 Hz, H-5b), 5.53 (d, 1 H, J6,7 ¼ 4.4,
5 mL culture tubes and mixed with Annexin V (5
mL) and propidium
J5b,6 ¼ 5.2 Hz, H-70), 5.81 (dd, 1 H, J6,7 ¼ 4.6 Hz, H-6), 6.11 (d, 1 H,
iodide (5
mL). The cells were gently vortexed and incubated for
0
0
J2 ,3 ¼ 16.0 Hz, H-2 ), 6.27 (d, 1 H, J2,3 ¼ 5.5 Hz, H-2), 7.06 (t, 2 H, p-F-
15 min at 25 ꢀC. After incubation, 400 mL of binding buffer was
added to each tube and suspension was analyzed after 1 h on FACS
Calibur E440 (Becton Dickinson) flow cytometer. Results were
presented as percent of Annexin V positive gated cells. Percentage
of specific apoptosis was calculated according to Bender et al. [22].
cinnamate), 7.24 (d, 1 H, J2,3 ¼ 5.5 Hz, H-3), 7.26e7.51 (m, 8 H, Ph
and H-30). 13C NMR (62.5 MHz, CDCl3):
d
42.3 (C-5), 73.0 (C-6), 87.4
(C-7), 113.5 (C-4), 116.8 and 116.83 (C-20, J2 ,F ¼ 1.8 Hz), 124.6 (C-2),
0
115.8 and 116.1 (C-300, J3 ,F ¼ 18.8 Hz), 127.4, 128.0, 128.3, 130.0,
00
130.36, 130.4,134.8 (2 ꢂ Ph),161.9 and 165.9 (C-400, J4 ,F ¼ 250.0 Hz),
00
144.3 (C-30), 151.8 (C-3), 165.5 (C-10), 170.0 (C-1). HRMS (ESI): m/z
398.1411 (MþþNH4), calcd for C22H21NFO5: 398.1398; m/z 381.1138
(MþþH), calcd for C122H18FO5: 381.1133.
4.6. 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.
4.1.7.4. (4R,7S)-6-O-(4-fluorocinnamoyl)-(þ)-crassalactone D (4d).
Yield 88%. Colourless syrup, [
a
]D ¼ þ34.5 (c 0.2, CHCl3), Rf ¼ 0.56
(7:3 light petroleum/EtOAc). IR (film): nmax 1778 and 1716 (C¼O). 1H
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 proteins was measured by
densitometry and compared with control sample.
NMR (250 MHz, CDCl3):
d
2.61 (dd, 1 H, J5a,5b ¼ 15.2, J5a,6 ¼ 2.7 Hz,
H-5a), 3.01 (dd, 1 H, J5a,5b ¼ 15.2, J5b,6 ¼ 6.4 Hz, H-5b), 5.58 (d, 1 H,
J6,7 ¼4.4 Hz, H-7), 5.87 (ddd, 1 H, J5a,5b ¼ 2.7, J6,7 ¼4.4, J5b,6 ¼ 6.5 Hz,
0
0
0
H-6), 6.06 (d, 1 H, J2 ,3 ¼ 15.9 Hz, H-2 ), 6.26 (d, 1 H, J2,3 ¼ 5.5 Hz, H-
2), 7.00e7.47 (m, 11 H, 2 ꢂ Ph, H-3 and H-30). 13C NMR (62.5 MHz,
CDCl3):
d
42.1 (C-5), 74.5 (C-6), 84.9 (C-7), 112.6 (C-4), 116.46 and
116.49 (C-20, J2 ,F ¼ 1.9 Hz), 124.0 (C-2), 115.9 and 116.2 (C-300,
0
00
J3 ,F ¼ 18.8 Hz), 126.9, 128.1, 128.3, 129.9, 130.0, 134.2 (2 ꢂ Ph), 162.0
and 165.99 (C-400, J4 ,F ¼ 249.4 Hz),144.2 (C-30), 151.5 (C-3),166.0 (C-
00
Acknowledgement
10), 169.5 (C-1). HRMS (ESI): m/z 381.1128 (MþþH), calcd for
C
C
C
22H18FO5: 381.1133; m/z 398.1392 (MþþNH4), calcd for
22H21FNO5: 398.1398; m/z 403.0947 (MþþNa), calcd for
22H17FNaO5: 403.0952.
This work was supported by research grants from the Ministry
of Education, Science and Technological Development of the Re-
public of Serbia (Grant No. OI 172006), and (in part) by a research
project from the Serbian Academy of Sciences and Arts (Grant No. F-
130).
4.2. X-ray crystal structure analysis
Appendix A. Supplementary data
Diffraction experiments were performed on an Oxford Diffrac-
tion Gemini S diffractometer. Crystal structures were solved by
SHELXT [26] or SIR92 [27] and refined with SHELXL [28]. See the
Supplementary data for details. Crystallographic data have been
deposited at the Cambridge Crystallographic Data Centre. CCDC
Numbers: 1530428 (4), 1530429 (3), 1530430 (2a), 1530431 (1b),
1530432 (1c), 1530433 (1d) and 1530434 (4a). These data are
available free of charge via www.ccdc.cam.ac.uk/data_request/cif.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.03.088.
References
[1] P. Tuchinda, B. Munyoo, M. Pohmakotr, P. Thinapong, S. Sophasan, T. Santisuk,
V. Reutrakul, Cytotoxic styryl-lactones from the leaves and twigs of Polyalthia

ꢀ
ꢁ
I. Kovacevic et al. / European Journal of Medicinal Chemistry 134 (2017) 293e303
303
crassa, J. Nat. Prod. 69 (2006) 1728e1733.
[2] E. Pavlakos, T. Georgiou, M. Tofi, T. Montagnon, G. Vassilikogiannakis,
roketal -lactones from 2-( -hydrokylalkyl)furans: syntheses of epi-
pyrenolides D and crassalactone D, Org. Lett. 11 (2009) 4556e4559.
efficient synthesis of (ꢁ)-epigoniofufuron, Synlett 2 (1993) 123e124.
g-Spi-
[15] J.E. Aho, P.M. Pihko, T.K. Rissa, Nonanomeric spiroketals in natural products:
structures, sources, and synthetic strategies, Chem. Rev. 105 (2005)
4406e4440.
g
g
[3] Z. Yang, P. Tang, J.F. Gauuan, B.F. Molino, Asymmetric total synthesis of
[16] A.J. Kirby, The anomeric effect and related stereoelectronic effects at oxygen,
in: K. Hafner, C.W. Rees, B.M. Trost, J.-M. Lehn, P. von Rague Schleyer,
R. Zahradnik (Eds.), Reactivity and Structure Concepts in Organic Chemistry,
vol. 15, Springer-Verlag, Berlin, Heidelberg, New York, 1983, pp. 1e147.
[17] B. Neises, W. Steglich, Esterification of carboxylic acids with
dicylohexylcarbodiimide/4-dimethylaminopyridine: tert-butyl ethyl fuma-
rate, Org. Synth. 63 (1985) 183e186.
[18] D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger,
M.J. Currens, D. Seniff, M.R. Boyd, Evaluation of a soluble tetrazolium/for-
mazan assay for cell growth and drug sensitivity in culture using human and
other tumor cell lines, Cancer. Res. 48 (1988) 4827e4833.
(þ)-crassalactone D, J. Org. Chem. 74 (2009) 9546e9549.
ꢁ
ꢁ
[4] R. Galan-Fernadez, D. Clemente-Tejeda, F.A. Bermejo, Oxidative cyclization of
g-alkylidene butenolides. Stereoselective preparation of spirolactones, Ark. ix
(2012) 171e184.
[5] A part of this work appeared as a preliminary communication V. Popsavin,
ꢀ
ꢁ
ꢁ
ꢁ
ꢁ
I. Kovacevic, G. Benedekovic, M. Popsavin, V. Kojic, G. Bogdanovic, Divergent
synthesis of cytotoxic styryl lactones related to goniobutenolides A and B, and
to crassalactone D, Org. Lett. 14 (2012) 5956e5959.
[6] P. De, M. Baltas, F. Bedos-Belval, Cinnamic acid derivatives as anticancer
agents e a review, Curr. Med. Chem. 18 (2011) 1672e1703.
[7] C. Viegas-Junior, A. Danuello, V. da Silva Bolzani, E.J. Barreiro, C.A.M. Fraga,
Molecular hybridization: a useful tool in the design of new drug prototypes,
Curr. Med. Chem. 14 (2007) 1829e1852.
[19] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144
(2011) 646e674.
[20] G. Housman, S. Byler, S. Heerboth, K. Lapinska, M. Longrace, N. Snyder,
ꢁ
ꢀ
ꢁ
ꢁ
ꢁ
[8] G. Benedekovic, J. Francuz, I. Kovacevic, M. Popsavin, B. Sreco Zelenovic,
S. Sarkar, Drug resistance in cancer: an overview, Cancers 6 (2014)
1769e1792.
ꢁ
ꢁ
ꢁ
V. Kojic, G. Bogdanovic, V. Divjakovic, V. Popsavin, Conformationally con-
strained goniofufurone mimics as inhibitors of tumour cells growth: design,
synthesis and SAR study, Eur. J. Med. Chem. 82 (2014) 449e458.
[21] C.-Y. Choo, N. Abdullah, M. Diederich, Cytotoxic activity and mechanism of
action of metabolites from the Goniothalamus genus, Phytochem. Rev. 13
(2014) 835e851.
[22] A. Bender, D. Opel, I. Naumann, R. Kappler, L. Friedman, D. Von Schwinitz, K.-
M. Debatin, S. Fulda, PI3K inhibitors prime neuroblastoma cells for chemo-
therapy by shifting the balance towards pro-apoptotic Bcl-2 proteins and
enhanced mitochondrial apoptosis, Oncogene 30 (2011) 494e503.
[23] P.E. Czabotar, G. Lessene, A. Strasser, J.M. Adams, Control of apoptosis by the
BCL-2 protein family: implications for physiology and therapy, Nat. Rev. Mol.
Cell Biol. 15 (2014) 49e63.
ꢁ
ꢀ
ꢁ
ꢁ
ꢁ
[9] G. Benedekovic, M. Popsavin, J. Francuz, I. Kovacevic, V. Kojic, G. Bogdanovic,
ꢁ
V. Divjakovic, V. Popsavin, Design, synthesis and SAR analysis of antitumour
styryl lactones related to (þ)-crassalactones B and C, Eur. J. Med. Chem. 87
(2014) 237e247.
ꢀ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
[10] I. Kovacevic, M. Popsavin, G. Benedekovic, V. Kojic, D. Jakimov, M.V. Rodic,
ꢁ
ꢁ
ꢁ
T. Srdic-Rajic, G. Bogdanovic, V. Divjakovic, V. Popsavin, Synthesis and anti-
proliferative activity of goniobutenolides and B, 5-halogenated crassa-
A
lactone D derivatives and the corresponding 7-epimers, Eur. J. Med. Chem.
108 (2016) 594e604.
[11] J.E. Harvey, S.A. Raw, R.J.K. Taylor, A versatile and stereocontrolled route to
pyranose and furanose C-glycosides, Org. Lett. 6 (2004) 2611e2614.
[12] S. Valverde, M. Martin-Lomas, B. Herradon, S. Garcia-Ochoa, The reaction of
carbohydrate-derived alkoxyaldehydes with methoxycarbonylmethylene-
[24] G.S. Salvesen, Caspases: opening the boxes and interpreting the arrows, Cell
Death Differ. 9 (2002) 3e5.
ꢂ
[25] C. Soldani, M.C. Lazze, M.G. Bottone, G. Tognon, M. Biggiogera, C.E. Pellicciari,
A.I. Scovassi, Poly(ADP-ribose) polymerase cleavage during apoptosis: when
and where? Exp. Cell. Res. 269 (2001) 193e201.
triphenylphosphorane: stereoselective synthesis of
b
-unsaturated esters,
[26] G.M. Sheldrick, SHELXT e integrated space-group and crystal structure
Tetrahedron 43 (1987) 1895e1901.
determination, Acta Crystallogr. Sect. A 71 (2015) 3e8.
[27] A. Altomare, G. Cascarano, C. Giacovazzo, A. Gualardi, Completion and
refinement of crystal structures with SIR92, J. Appl. Crystallogr. 26 (1993)
343e350.
[28] G.M. Sheldrick, Crystal structure refinement with SHELXL, Acta Crystallogr.
Sect. C 71 (2015) 3e8.
[13] K.R.C.
Prakash,
S.
Prahlada
Rao,
Ethoxycarbonylmethylene-
triphenylphosphorane in carbohydrate chemistry: an easy access to func-
tionalized 2-deoxy-3,6-anhydrohexono-1,4-lactones, Tetrahedron Lett. 32
(1991) 7473e7476.
[14] K.R.C.
Prakash,
S.
Prahlada
Rao,
Ethoxycarbonylmethylene-
triphenylphosphorane in carbohydrate chemistry, part III: a short and highly