Design, synthesis and SAR analysis of antitumour styryl lactones related to (+)-crassalactones B and C

Article abstract of DOI:10.1016/j.ejmech.2014.09.064

A series of styryl lactones containing the cinnamic acid ester groups such as (+)-crassalactones B (3a) and C (4a), 5,7-di-O-cinamoyl derivative 6, the corresponding 7-epimers and 7-deoxy derivatives have been synthesized, characterized and evaluated for their in vitro antitumour activity against a panel of several human tumour cell lines. Twelve new analogues such as 5-O-or 7-O-(4-methoxycinnamoyl), 5-O-or 7-O-(4-nitrocinnamoyl) and 5-O-or 7-O-(4-fluorocinnamoyl) esters of (+)-goniofufurone (3b-d), 7-epi-(+)-goniofufurone (epi-3b-d), as well as 7-deoxy derivatives 5b-d have been prepared to correlate all compounds in a SAR study. Some of the analogues displayed powerful antiproliferative effects on selected human tumour cell lines, but none of them demonstrated cytotoxicity towards the normal foetal lung fibroblasts (MRC-5). Thus, for the 7-epi-crassalactone B (epi-3a) was found to be a potent inhibitor of HL-60 cells growth, with an IC₅₀value that is approximately 46-fold lower than that observed for the commercial antitumour drug doxorubicin in the culture of the same cells. A SAR analysis performed on these lactones reveals the main structural features that affect their antiproliferative activity, such as nature of the substituents at the C-4 in the aromatic rings of cinnamoyl moieties, the absolute stereochemistry, as well as the presence of a deoxy function at the C-7 position.

Full text of DOI:10.1016/j.ejmech.2014.09.064

European Journal of Medicinal Chemistry 87 (2014) 237e247
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and SAR analysis of antitumour styryl lactones
related to (þ)-crassalactones B and C
a
a
ꢀ a
ꢁ
ꢀ a
Goran Benedekovic , Mirjana Popsavin , Jovana Francuz , Ivana Kovacevic ,
ꢀ b
ꢀ b
ꢀ c
a, *
Vesna Kojic , Gordana Bogdanovic , Vladimir Divjakovic , Velimir Popsavin
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
ꢀ
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 series of styryl lactones containing the cinnamic acid ester groups such as (þ)-crassalactones B (3a) and
C (4a), 5,7-di-O-cinamoyl derivative 6, the corresponding 7-epimers and 7-deoxy derivatives have been
synthesized, characterized and evaluated for their in vitro antitumour activity against a panel of several
human tumour cell lines. Twelve new analogues such as 5-O- or 7-O-(4-methoxycinnamoyl), 5-O- or 7-
O-(4-nitrocinnamoyl) and 5-O- or 7-O-(4-fluorocinnamoyl) esters of (þ)-goniofufurone (3bed), 7-epi-
(þ)-goniofufurone (epi-3bed), as well as 7-deoxy derivatives 5bed have been prepared to correlate all
compounds in a SAR study. Some of the analogues displayed powerful antiproliferative effects on
selected human tumour cell lines, but none of them demonstrated cytotoxicity towards the normal foetal
lung fibroblasts (MRC-5). Thus, for the 7-epi-crassalactone B (epi-3a) was found to be a potent inhibitor of
HL-60 cells growth, with an IC50 value that is approximately 46-fold lower than that observed for the
commercial antitumour drug doxorubicin in the culture of the same cells. A SAR analysis performed on
these lactones reveals the main structural features that affect their antiproliferative activity, such as
nature of the substituents at the C-4 in the aromatic rings of cinnamoyl moieties, the absolute stereo-
chemistry, as well as the presence of a deoxy function at the C-7 position.
Received 20 August 2014
Received in revised form
10 September 2014
Accepted 19 September 2014
Available online 22 September 2014
Keywords:
Cinnamoates
Styryl lactone
Crassalactone B
Crassalactone C
Antitumour activity
Structureeactivity relationships
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
of Goniothalamus giganteus (Annonaceae) by McLaughlin and co-
workers [3,4], and found to exhibit notable antiproliferative ef-
Cinnamic acid, its natural analogues and derivatives are known
for the treatment of cancer for over centuries [1]. Among its de-
rivatives, the cinnamoates represent a well known class of anti-
cancer agents. The variation in esters arises from both natural
resources as well as synthesized compounds. A lot of naturally
occurring and biologically active alcohols have been linked with
cinnamoyl residues through the ester linkage to amend their
anticancer efficacy. A number of styryl lactones have been recently
isolated from the tropical plant Polyalthia crassa [2]. The bioassay-
directed fractionation of the ethyl acetate extract of its leaves and
twigs led to the isolation of the known antitumour styryl lactone
(þ)-goniofufurone (1, Fig. 1) that was, along with its stereoisomer
7-epi-(þ)-goniofufurone (2), previously isolated from the stem bark
fects toward a number of human tumour cell lines [3e8]. Several
new cytotoxic compounds, including (þ)-crassalactones B (3a) and
C (4a), which represent the 5-O- or the 7-O-cinnamoyl esters of 1,
have also been isolated and fully characterized by spectroscopic
methods [2]. We have recently reported several synthetic routes to
(þ)-crassalactone C (4a) starting from
preliminary account related the first total synthesis of (þ)-crassa-
lactone B (3a) starting from -glucose [12]. Immediately thereafter
a new total synthesis of (þ)-crassalactones B and C starting from
diacetone -glucose [13], as well as a novel enantiospecific total
D-xylose [9e11], as well as a
D
D
synthesis of (ꢀ)-crassalactone C from tartaric acid [14] has been
reported by other groups.
In this work, we report efficient semi-syntheses of (þ)-crassa-
lactones B (3a) and C (4a), as well as the corresponding 5,7-
dicinamoates 6 and epi-6 (the structures not shown in Fig. 1),
starting
from
(þ)-goniofufurone
(1)
or
from 7-epi-
* Corresponding author.
E-mail address: velimir.popsavin@dh.uns.ac.rs (V. Popsavin).
http://dx.doi.org/10.1016/j.ejmech.2014.09.064
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
238
1H and 13C NMR spectroscopic data [16] of synthesized natural
product 3a were in full agreement with those reported in the
literature for both isolated [2] and synthetic [13] sample 3a.
The same three-step sequence was then applied for the prepa-
ration of 7-epi-(þ)-crassalactone B (epi-3a) starting from natural
product 2. Thus, treatment of 2 with tert-butyldimethylsilyl chlo-
ride gave the expected 7-O-silyl ether epi-7 (92%) which was sub-
sequently converted to the corresponding cinnamoate epi-8a in
97% yield. Final treatment of epi-8a with aqueous TFA afforded 7-
epi-(þ)-crassalactone B (epi-3a) in 85% yield. The target epi-3a was
thus obtained in an overall yield of 76% over the last three synthetic
steps.
Treatment of 7 and epi-7 with 4-methoxy-, 4-nitro-, or 4-
fluorocinnamic acid, by using the Steglich esterification protocol
[17] gave the expected 4-substituted cinnamic ester derivatives in
good to excellent yields. Generally, the cinnamic esters of (7R)-
absolute configuration (8bed) were formed in somewhat higher
yields (91, 97 and 96%, respectively) compared to those of (7S)-
absolute configuration (epi-8b, epi-8c and epi-8d) that were ob-
tained in 87, 80 and 75% yields respectively, presumably from the
steric reasons. Desilylation of 8bed with aqueous trifluoroacetic
acid in dichloromethane finally afforded the desired analogues 3b
(85%), 3c (74%) and 3d (98%), respectively. However, when the 7-
epimers (epi-8b, epi-8c and epi-8d) were treated under the same
reaction conditions (TFA, CH2Cl2/H2O), the corresponding 7-epi-
(þ)-crassalactone C derivatives (epi-4b, epi-4c and epi-4d) were
obtained as the major reaction products, accompanied by a minor
amounts of desired analogues epi-3b, epi-3c and epi-3d. The major
products epi-4b, epi-4c and epi-4d were presumably formed by a
competitive intramolecular cinnamate migration process, as
observed in our previous work when TBAF was used for removal of
the TBDPS protective group [12]. On the basis of the results ob-
tained for TBDMS removal by TFA, alternative acidic conditions
using ceric ammonium nitrate (CAN) were employed for this re-
action instead. These conditions previously prevented completely
the acetyl migration in certain furanoside derivatives [18]. We were
pleased to find that all three silyl ethers epi-8b, epi-8c and epi-8d,
upon the TBDMS deprotection with CAN in aqueous acetonitrile
afforded only the desired 5-O-cinnamoyl derivatives epi-3b, epi-3c
and epi-3d, respectively. No migrated product could be detected in
crude reaction mixtures by 1H NMR. The desilylated products epi-
3b and epi-3d were isolated in pure form after silica gel flash col-
umn chromatography in respective yields of 73 and 62%. However,
the attempted purification of 7-O-(4-nitrocinnamoyl) derivative
epi-3c on a column of flash silica predominantly gave the rear-
ranged product epi-4c (69%) accompanied with a minor amount of
desired epi-3c (19%). Therefore the chromatographic purification
was skipped, and pure epi-3c was isolated in 89% yield after crys-
tallization of crude reaction mixture from a mixture CH2Cl2/hexane.
Both intramolecular and intermolecular migrations of alkylox-
ycarbonyl groups during chromatographic purification on a silica
gel column were recently reported [19].
Fig. 1. Structures of (þ)-goniofufurone (1), 7-epi-(þ)-goniofufurone (2), (þ)-crassa-
lactones B (3a) and C (4a), and the corresponding analogues 3bed, 4bed and 5aed.
(þ)-goniofufurone (2), respectively. A number of new analogues
such as 3bed, 4bed, the corresponding 7-epimers such as epi-
3aed and epi-4aed (the structures not shown in Fig. 1) and 7-
deoxy derivatives such as 5aed have also been prepared and
their effects to the proliferation of some human malignant cell lines
have been evaluated.
2. Results and discussion
2.1. Chemistry
The conversion of (þ)-goniofufurone (1) to (þ)-crassalactone B
(3a), and 7-epi-(þ)-goniofufurone (2) to 7-epi-(þ)-crassalactone B
(epi-3a), is presented in Scheme 1. An efficient total synthesis of
starting natural products 1 and 2 was recently completed in our
laboratory starting from D-glucose [15]. This procedure enables an
efficient and fast access to 1 and 2, which fully justifies their use as
starting materials in а new synthesis of 3a and epi-3a, as well as in
the synthesis of a number of some new analogues thereof. The
benzylic hydroxyl group in 1 was selectively blocked by treatment
with tert-butyldimethylsilyl chloride whereby the key intermediate
7 was obtained in 84% yield. A minor amount of starting compound
1 (6%), along with ~10% of the corresponding bis-silyl ether (the
structure not shown) was also isolated. Subsequent treatment of
crude bis-silylated side-product with aqueous TFA afforded an
additional amount of recovered starting compound 1 so that its
total yield was increased to 14%. This further means that the desired
intermediate 7 was obtained in 95% yield calculated to reacted
starting material 1. Treatment of 7 with cinnamoyl chloride (DMAP,
MeCN) gave the corresponding cinnamic ester 8a in 90% yield.
Moreover, treatment of 8a with aqueous TFA in CH2Cl2 gave a 79%
yield of (þ)-crassalactone B (3a), along with 15% of starting com-
pound 8a. The natural product 3 was thus obtained in 93% yield
The synthesis of 5,7-di-O-cinnamoyl derivatives 6 and epi-6 is
outlined in Scheme 2. Thus, treatment of 1 with cinnamoyl chloride
in anhydrous acetonitrile, in the presence of DMAP as a proton
acceptor, gave the known [13] dicinnamoate 6 in 94% yield. Physical
constants and spectroscopic data of thus prepared sample 6
matched those previously reported [13,16]. The same synthetic
protocol was then used for the conversion of 2 in epi-6 which was
isolated after flash column chromatography in an almost quanti-
tative yield (96%). Finally, 7-deoxy derivative 9 was treated under
the similar reaction conditions, in order to prepare the 5-O-cinna-
moyl derivative 5a. The reason for the preparation of analogue 5a
arises from our recent study [20] that revealed that 7-deoxy de-
rivative 9 demonstrates potent antiproliferative activities against
calculated to reacted intermediate 8a (80% overall from 1). Our
20
optical rotation value recorded in EtOH [
a
]
þ45.7 (c 0.5)} is
D
significantly greater than the reported value for the isolated
20
(þ)-crassalactone B {lit. [2] [
optical rotation data recorded in CHCl3 {[
reasonable agreement with the value [13] reported for synthetic
a]
D þ8.0 (c 0.5, EtOH)}. However, the
20
a]
D þ35.5 (c 1.0)} was in
20
sample 3a {lit. [13] [a]
þ31.6 (c 1.0, CHCl3)}. The melting point,
D

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
239
Scheme 2. Reagents and conditions: (a) cinnamoyl chloride, DMAP, anh. MeCN, 0.5 h
at 0 ꢁC, then 30 h at rt.
[22], we first studied the influence of several solvents and found
that the best yields of the desired cinnamoates were obtained when
the reaction is performed in anhydrous ethyl acetate [16].
Thus, treatment of 7-epi-(þ)-goniofufurone 2 with cinnamic
acid or its 4-substituted derivatives, under the typical Mitsunobu
reaction conditions (DEAD, Ph3P, EtOAc), gave targets 4a (69%) 4b
(60%), 4c (66%) and 4d (66%) as the major reaction products. A high
regio- and stereoselectivity was observed whereby the cinnamic
ester group was introduced exclusively at the C-7 position, with
Walden inversion at this stereocentre. A minor amount of oxetane
10 (31%)1 was formed in all of these reactions, presumably due to a
competitive intramolecular Mitsunobu dehydration. The physical
constants and the spectral data of thus obtained sample 10 were
consistent with those previously reported by us [10]. By using a
similar methodology, (þ)-goniofufurone was converted to the
corresponding 7-epi-cinnamoates, epi-4a (59%), epi-4b (42%), epi-
4c (52%) and epi-4d (56%) as major products, accompanied with
minor amounts of oxetane epi-10 (25, 34, 32 and 35%, respectively).
The assignment of stereochemistry at the C-7 in epi-10 was
confirmed by an NOE interaction between H-3 and a proton from
the aromatic ring (AreH), indicating that H-3 and Ph group are
mutually syn oriented, on the same side of the 2,6-dioxa-bicyclo
[3.2.0]heptane ring system. The geometry of epi-10 was unambig-
uously confirmed by X-ray crystallographic analysis (for the crystal
structure of epi-10, see the Supplementary data). The distance be-
tween H-3 and AreH (2.48 Å) in the molecular structure of epi-10 is
consistent with NOE results.
Scheme 1. Reagents and conditions: (a) tBuMe2SiCl, imidazole, anh. MeCN, rt, 26 h for
1, 20 h for 2; (b) cinnamoyl chloride, DMAP, anh. CH2Cl2, 0.5 h at 0 ꢁC, then 30 h at rt;
(c) 4-substituted cinnamic acid, DCC, DMAP, anh. CH2Cl2, rt, 48 h (d) CH2Cl2/TFA/H2O
50:5:1, rt, 50 h for 8a, 22 h for epi-8a; (e) CAN, 9:1 MeCN/H2O, rt, 120 h for epi-8b, 72 h
for epi-8c, 26 h for epi-8d.
several human tumour cell lines. Accordingly compound 9 was
allowed to react with cinnamoyl chloride, under the above
described reaction conditions, to afford a 91% yield of 5-O-cinna-
moyl derivative 5a.
In another reaction we studied the esterification of 7-deoxy
derivative 9 with 4-methoxy-, 4-nitro-, or 4-fluorocinnamic acid,
under the standard Steglich conditions [17] to obtain a few novel
isosteres of 5a such as compounds 5bed (Scheme 3). Accordingly, a
treatment of 9 with 4-methoxycinnamic acid in anhydrous CH2Cl2,
in the presence of DCC and DMAP, gave the expected 5-O-(4-
methoxycinnamoyl) derivative 5b in 55% yield. Compound 9 reac-
ted with 4-nitro- or 4-fluorocinnamic acid, under the same reaction
conditions, to afford the corresponding 4-substituted cinnamoates
5c and 5d in 44 and 71% yields, respectively.
Mitsunobu-type reaction is an efficient method to introduce
ester groups with Walden inversion at the secondary carbon atom
[21e23]. We had intended to apply this reaction for the preparation
of 7-O-cinnamoyl derivatives of (þ)-goniofufurone (1) and 7-epi-
(þ)-goniofufurone (2) (Scheme 4). As it is well known that the
outcome of the Mitsunobu reaction depends on the solvent used
1
In several experiments that were performed in solvents other than ethyl acetate
(THF or CH2Cl2), the oxetanes 10 or epi-10 were isolated as major reaction products
(see the Supplementary data for details).

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
240
negative breast adenocarcinoma (MDA-MB-231), cervix carcinoma
(HeLa) and against a single normal cell line, foetal lung fibroblasts
(MRC-5). Cytotoxic activity was evaluated by using the standard
MTT assay, after exposure of cells to the tested compounds for 72 h.
The commercial antitumour agent doxorubicin (DOX) was used as a
positive control in this assay. The results are presented in Table 1.
According to the resulting IC50 values of the cytotoxic assay
(Table 1), the K562, Jurkat, Raji and HeLa cell lines are sensitive to
all of the synthesized analogues. The highest potency in the culture
of K562 cells was recorded after treatment with deoxygenated
isostere 5d (IC50 0.25 mM), which demonstrated the same potency
as the commercial drug doxorubicin in the same cell line. This
analogue was nearly 19-fold more active than the parent com-
pound 5a in the culture of same cells. The potent antiproliferative
activity in the low micromolar range was recorded after treatment
of K562 cells with analogues epi-3c (IC50 1.12
mM), 5b (IC50 1.02 mM)
and 5c (IC50 0.98 M). With the exception of natural product 4a all
m
remaining compounds show strong growth inhibition of HL-60
tumour cells. The most active compound against this cell line was
analogue epi-3a, which was 46-fold more potent than the com-
mercial antitumour agent doxorubicin. In the same time, this
molecule represents the most active compound synthesised in this
work. Additionally, a potent submicromolar antiproliferative ac-
tivities were recorded after treatment of HL-60 cells with analogues
Scheme 3. Reagents and conditions: (a) 4-methoxycinnamic acid, DCC, DMAP, anh.
CH2Cl2, rt, 22 h; (b) 4-nitrocinnamic acid, DCC, DMAP, anh. CH2Cl2, rt, 20 h; (c) 4-
fluorocinnamic acid, DCC, DMAP, anh. CH2Cl2, rt, 20 h.
2.2. Biological evaluation and SAR
epi-3b (IC50 0.11
m
M), epi-3c (IC50 0.36
mM), 5b (IC50 0.27 mM), 5c
After completion of the synthesis, all synthesized compounds
were evaluated for their in vitro cytotoxic activity against a panel of
seven human malignant cell lines, including myelogenous
(IC50 0.14 M) and epi-6 (IC50 0.15
m
mM). Comparing to DOX, these
analogues have exhibited 2.5e8-fold higher potency in the culture
of HeLa cells. Several compounds exhibited submicromolar activ-
leukaemia (K562), promyelocytic leukaemia (HL-60),
T cell
ities toward the Jurkat cells. These are epi-3d (IC50 0.69
(IC50 0.29 M), 5b (IC50 0.84 M) and epi-6 (IC50 0.31 M). However,
the potencies of these analogues are significantly lower than that
recorded for DOX in the same cell line (IC50 0.03 M). The most
active molecule in the culture of Raji cells is analogue epi-3c (IC50
1.02 M), that exhibited about 14- and 3-fold higher potency than
mM), 5a
leukaemia (Jurkat), Burkitt's lymphoma (Raji), oestrogen receptor
positive breast adenocarcinoma (MCF-7), oestrogen receptor
m
m
m
m
m
control compounds epi-3a and DOX, respectively. MCF-7 and MDA-
MB 231 cells were shown to be the least sensitive to the tested
analogues. Submicromolar activities have not been recorded in
cultures of these cells. The most potent compound against MCF-7
and MDA-MB 231 cells are epi-6 (IC50 1.64
1.32 M), respectively. The most active compounds in the culture of
HeLa cells are analogues epi-4a (IC50 0.69 M), 5b (IC50 0.94 M)
and 5c (IC50 0.87 M) that exhibited the similar potencies as the
control molecule 5a (IC50 1.25 M). Remarkably, the most of syn-
mM) and epi-3d (IC50
m
m
m
m
m
thesized styryl lactones including the natural products 3a and 4a
were completely inactive toward normal MRC-5 cells, with the
exception of dicinnamoate 6 which showed a weak cytotoxicity
(IC50 48.64
mM). In contrast, the commercial antitumour agent
doxorubicin (DOX) exhibits a potent cytotoxic activity (IC50 0.1
mM)
against this cell line. These findings suggest to a higher selectivity of
the synthetic analogues when compared to DOX, but this
assumption should be verified by additional in vitro experiments
with different normal cell lines.
In order to correlate the structures of synthesized styryl lactones
with their cytotoxic activities [16], we first considered the influence
of selected electron-donating (OMe) or electron-withdrawing (NO2
and F) groups attached in the para position of cinnamoate aromatic
ring. Non-substituted cinnamoates 3a, 4a, epi-3a, epi-4a, and 5a
have been applied as controls in this SAR analysis. As the data in
Table 1 reveal the introduction of either electron-withdrawing
groups (such as F and NO2) or an electron-donating group (such
as OMe), in the para position of the aromatic ring of 3a, failed to
improve antiproliferative activity against the majority of tumour
cell lines under evaluation. However, several interesting exceptions
deserve to be commented: Insertion of the above-mentioned sub-
stituents (OMe, NO2, and F) in the para position of the cinnamic acid
Scheme 4. Reagents and conditions: (a) cinnamic acid, DEAD, Ph3P, EtOAc, 1 h at 0 ꢁC,
then 3 h at rt; (b) 4-methoxycinnamic acid, DEAD, Ph3P, EtOAc, 1 h at 0 ꢁC, then rt, 2 h
for 4b, 24 h for epi-4b; (c) 4-nitrocinnamic acid, DEAD, Ph3P, EtOAc, 1 h at 0 ꢁC, then rt,
2 h for 4c, 4 h for epi-4c; (d) 4-fluorocinnamic acid, DEAD, Ph3P, EtOAc, 1 h at 0 ꢁC, then
rt 2 h for 4d, 4 h for epi-4d.

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
241
Table 1
In vitro cytotoxicity of (þ)-crassalactones B (3a) and C (4a), the corresponding analogues 3be3d, 4be4d, 5be5d and DOX.
Compounds
IC50
K562
(m
M)a
HL-60
2.54
50.21
48.25
11.52
Jurkat
Raji
MCF-7
0.85
11.08
>100
14.87
7.34
12.32
5.23
18.66
0.22
20.21
56.21
84.54
9.47
9.64
4.15
5.36
58.64
60.96
23.16
28.99
MDA-MB 231
HeLa
MRC-5
3a
3b
3c
3d
4a
4b
4c
4d
epi-3a
epi-3b
epi-3c
epi-3d
epi-4a
epi-4b
epi-4c
epi-4d
5a
5b
5c
5d
6
0.78
4.21
14.63
10.24
3.56
3.25
2.25
4.58
3.33
2.63
1.12
0.85
2.01
11.54
14.23
15.69
4.67
1.02
0.98
0.25
21.36
2.15
0.25
0.12
13.37
44.69
17.56
25.45
42.31
10.54
18.09
0.87
1.12
3.52
0.69
1.03
18.64
24.58
19.64
0.29
0.84
3.74
2.63
2.65
0.31
0.03
14.61
5.69
8.23
11.08
15.46
4.37
16.38
14.45
14.52
2.34
1.02
1.74
1.31
8.45
4.36
12.67
0.88
3.58
2.69
5.32
>100
5.36
25.66
87.98
>100
33.41
29.64
8.64
7.32
2.64
1.32
3.37
28.64
64.25
31.55
4.32
4.34
5.32
0.98
18.63
21.36
29.88
11.25
24.88
35.69
24.11
2.47
3.21
1.66
4.12
0.69
11.02
4.01
4.36
1.25
0.94
0.87
1.02
12.24
1.45
0.065
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
41.39
14.32
9.45
0.02
0.11
0.36
2.11
1.34
8.79
10.47
10.96
2.21
0.27
0.14
1.02
3.33
0.15
0.92
1.52
8.22
>100
6.69
11.01
10.24
2.98
>100
48.64
>100
0.10
epi-6
DOX
1.64
0.20
0.09
a
IC50 is the concentration of compound required to inhibit the cell growth by 50% compared to an untreated control. Values are means of three independent experiments.
Coefficients of variation were less than 10%.
residue in 3a resulted in an increased activity of the corresponding
isosteres (3bed) in culture of Raji cells, The same structural
modification of natural product 4a increased the potency of
resulting analogues (4bed) toward HL-60 cells. Insertion of both
electron-withdrawing groups (NO2 and F) in 4a increases the
antitumour potency in both Jurkat and MDA-MB 231 cell cultures,
whereas an introduction of the electron-donating group (OMe) in
4a gave the less active analogue (4b) against the same tumour cell
lines. The cinnamic isosteres of epi-3a (compounds epi-3b, epi-3c
and epi-3d) displayed the increased antitumour potency with
respect to that originally shown by the control molecule epi-3a in
K562, Raji and MDA-MB 231 cell cultures. Analogues with electron-
withdrawing groups (epi-4c and epi-4d) displayed the increased
antitumour potencies with respect to that originally displayed by
the control molecule epi-4a in the culture of MCF-7 tumour cells,
whereas an introduction of the electron-donating group (OMe) in
epi-4a decreases the antitumour potency against the same tumour
cell line. The best results were obtained after isosteric replacement
in the structure of lead 5a. Namely, insertion of all three sub-
stituents (OMe, NO2, and F) in the para position of the cinnamic acid
residue in 5a resulted in an increased activity of the corresponding
analogues (5bed) in cultures of three malignant cell lines (K562,
HL-60 and HeLa). In addition, the presence of electron-
withdrawing groups (NO2 and F) in the molecules of isosteres 5c
and d, increases their antiproliferative potency with respect to that
originally displayed by the control molecule 5a, whereas the
introduction of an electron-donating group (OMe) in 5a gave the
less active analogue (5b) in MCF-7 cells culture.
and epi-4b, 4c and epi-4c, 4d and epi-4d, 6 and epi-6), each of which
contains exactly the same substituents and differs only in their
absolute stereochemistry at C-7. Stereoisomers of (7R)-absolute
configuration (3aed, 4aed and 6) are considered as controls in this
part of SAR study. As the results shown in Table 1 indicate, in the
great majority of cases, the (7S)-configured isosteres (epi-3aed,
epi-4aed, and epi-6) showed a more potent cytotoxicity than the
stereoisomers of (7R)-series (3aed, 4aed and 6). These findings
strongly support the hypothesis that the (7S)-absolute stereo-
chemistry is beneficial for the potent antiproliferative activity of
styryl lactones bearing a furanofuranone scaffold.
Finally, we set a goal to examine the impact of deoxygenation at
the C-7 position on the antitumour activity of analogues. The re-
lationships between these structural changes and antiproliferative
potencies were established by comparing the IC50 values of 7-deoxy
derivatives 5aed with those recorded for the corresponding 7-
hydroxylated derivatives that are arbitrarily used as control mole-
cules (compounds 3aed or epi-3aed). As the data in Table 1 reveal
the introduction of a deoxy function at the C-7 position increases
the antiproliferative activity of the resulting analogues against
majority of tumour cell lines under evaluation. Remarkably, deox-
ygenated isostere of 3c (compound 5c) displayed the increased
antitumour potency with respect to that originally shown by the
control molecule 3c against the all tested tumour cell lines.
Furthermore, insertion of a deoxy functionality at the C-7 position
of 3b resulted in an increased activity of the corresponding isostere
5b against six of seven studied tumour cell lines. The similar trend
was observed when 3d is deoxygenated. Finally, deoxygenation of
epi-3b, epi-3c and epi-3d resulted in an increased activity of the
corresponding isosteres 5bed toward four to five malignant cell
lines.
Additional structural modifications undertaken to improve the
anti-proliferative activities were performed at the C-7 position. We
first wanted to explore the effects of absolute stereochemistry at C-
7 to antiproliferative activity of analogues. Our previous studies
[15,24] indicated that the styryl lactones having the (7S)-stereo-
chemistry represent more potent cytotoxic agents with respect to
the corresponding (7R)-epimers. To further verify this assumption,
we compared the IC50 values of nine pairs of epimers (3a and epi-
3a, 3b and epi-3b, 3c and epi-3c, 3d and epi-3d, 4a and epi-4a, 4b
All these biological results can be helpful for the further devel-
opment of new antitumour agents derived from (þ)-crassalactones
B and C and from related styryl-lactones.

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
242
3. Conclusions
(m, 1H, H-3), 5.24 (d, 1H, J6,7 ¼ 3.0 Hz, H-7), 7.30e7.43 (m, 5H, Ph).
13C NMR (CDCl3):
25.5 (SiMe2CMe3), 36.2 (C-2), 74.2 (C-5), 76.8 (C-7), 77.0 (C-3), 82.5
(C-6), 87.5 (C-4), 125.9, 128.2, 128.6, 139.4 (Ph), 175.4 (C-1). HRMS
(ESI): m/z 403.1343 (MþþK), calcd for C19H28KO5Si: 403.1338.
d
ꢀ5.4 and ꢀ5.1 (SiMe2CMe3), 18.0 (SiMe2CMe3),
In conclusion, an efficient semi-syntheses of (þ)-crassalactones
B (3a) and C (4a), as well as the corresponding 5,7-dicinamoates 6
and epi-6 have been achieved starting from (þ)-goniofufurone (1)
or from 7-epi-(þ)-goniofufurone (2), respectively. A number of
novel isosteres such as 3bed or 4bed, the corresponding 7-
epimers such as epi-3aed and epi-4aed, as well as 7-deoxy de-
rivatives such as 5aed have also been prepared starting from 1 or 2.
All the synthesized compoundsweretestedinvitrotoward a panel
of human tumour cell lines, and the preliminary structureeactivity
relationships are briefly discussed. Some of the synthesized
compounds showed potent antitumour activity, especially analogues
4.1.1.2. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-2-deoxy-7-C-
phenyl-L-glycero-D-ido-heptono-1,4-lactone (epi-7). Yield 92%. Col-
ourless powder, mp 137e138 ꢁC (Et2O/wet light petroleum),
20
[a
]
D þ113.4 (c 0.5, CHCl3); Rf ¼ 0.26 (3:2 Et2O/light petroleum). IR
(CHCl3): nmax 3439 (OH), 1785 (C]O, lactone). 1H NMR (CDCl3):
d
ꢀ0.17 and 0.05 (2ꢂ s, 3H each, SiMe2CMe3), 0.87 (s, 9H,
SiMe2CMe3), 1.27 (br. s, 1H, OH), 2.63 (br d, 1H, J2a,2b ¼ 18.8 Hz, H-
2a), 2.75 (dd, 1H, J2a,2b ¼ 18.9, J2b,3 ¼ 5.9 Hz, H-2b), 4.02 (dd, 1H,
J5,6 ¼ 3.2, J6,7 ¼ 5.1 Hz, H-6), 4.17 (d, 1H, J5,6 ¼ 2.9 Hz, H-5), 4.83 (d,
1H, J3,4 ¼ 4.6 Hz, H-4), 4.99 (d, 1H, J6,7 ¼ 5.1 Hz, H-7), 5.06 (m, 1H, H-
epi-3a (IC50 0.02 mM against HL-60) and epi-3b (IC50 0.11 mM against
HL-60) which displayed the highest activity of all compounds under
evaluation.
A brief SAR analysis suggested the structural features that may
affect the anti-proliferative activity of synthesized styryl lactones:
(A) introduction of methoxy, nitro, or fluoro isosteric functions at
the C-4 of the aromatic rings of cinnamoyl moieties in most cases
decreases the antitumour potency originally displayed by lead
molecules bearing the non-substituted phenyl groups; (B) styryl
lactones having the (7S)-stereochemistry represent more potent
cytotoxic agents than the corresponding (7R)-epimers; (C) intro-
duction of a deoxy function at the C-7 position increases the anti-
proliferative activity of the resulting analogues against majority of
tumour cell lines under evaluation.
3), 7.28e7.46 (m, 5H, Ph). 13C NMR (CDCl3):
d
ꢀ5.0 and ꢀ4.5
(SiMe2CMe3), 18.1 (SiMe2CMe3), 25.7 (SiMe2CMe3), 36.0 (C-2), 74.7
(C-7), 76.1 (C-5), 76.5 (C-3), 83.9 (C-6), 87.9 (C-4), 127.1, 128.1, 128.3,
140.8 (Ph), 175.7 (C-1). HRMS (ESI): m/z 403.1347 (MþþK), calcd for
C
19H28KO5Si: 403.1338.
4.1.2. General procedure for esterification with cinnamoyl chloride
To a cooled (0 ꢁC) and stirred solution of 1, 2, 7, epi-7 or 9
(1 equiv) in dry MeCN (0.02 M solution of 1, 2, and 9, 0.03 M for epi-
7, 0.05 M for 7) was added trans-cinnamoyl chloride (2.2e2.3 equiv
for 1, 2, 7 and epi-7, 1.3 equiv for 9) and DMAP (2.6e2.8 equiv for 1,
2, 7 and epi-7, 1.5 equiv for 9). The mixture was stirred at 0 ꢁC for
0.5 h and then at room temperature for 30 h. The mixture was
poured in water and extracted with EtOAc. The combined extracts
were washed with brine, dried and evaporated. The residue was
purified by flash column chromatography (2:1 Et2O/hexane for 6,
99:1 CH2Cl2/EtOAc for epi-6, 2:1 Et2O/light petroleum for 8a, 7:3
light petroleum/Et2O for epi-8a, 1:1 Et2O/light petroleum for 5a).
4. Experimental section
4.1. General experimental procedures
Melting points were determined on a Büchi 510 apparatus 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 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 synthe-
sized compounds were acquired on an Agilent technologies 6210
TOF LC/MS instrument (LC series 1200). Flash column chromatog-
raphy was performed using Kieselgel 60 (0.040e0.063, E. Merck).
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.
4.1.2.1. 3,6-Anhydro-7-O-(terc-butyldimethylsilyl)-5-O-cinnamoyl-2-
deoxy-7-C-phenyl-
Yield 90%. Colourless syrup, [
(CH2Cl2). IR (film): nmax 1793 (C]O, lactone), 1717 (C]O, ester),
1637 (C]C, cinnamate). 1H NMR (CDCl3):
D
-glycero-
D
-ido-heptono-1,4-lactone
(8a).
20
a]
þ9.6 (c 0.5, CHCl3); Rf ¼ 0.27
D
d
ꢀ0.37 and ꢀ0.05 (2ꢂ s,
3H each, SiMe2CMe3), 0.79 (s, 9H, SiMe2CMe3), 2.52 (br d, 1H,
J2a,2b ¼ 19.0 Hz, H-2a), 2.66 (dd, 1H, J2a,2b ¼ 19.0, J2b,3 ¼ 6.1 Hz, H-
2b), 4.29 (dd, 1H, J5,6 ¼ 2.8, J6,7 ¼ 8.8 Hz, H-6), 4.87 (d, 1H,
J6,7 ¼ 8.8 Hz, H-7), 4.94 (m, 1H, H-3), 4.99 (d, 1H, J3,4 ¼ 4.7 Hz, H-4),
5.65 (d, 1H, J5,6 ¼ 2.7 Hz, H-5), 6.59 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20),
0
0
7.28e7.68 (m, 10H, 2ꢂ Ph), 7.82 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-30). 13C
0
0
4.1.1. General procedure for the selective silylation of 1 and 2
To a solution of lactones 1 or 2 (1 equiv) in dry MeCN (0.04 M)
NMR (CDCl3):
d
ꢀ5.3 and ꢀ4.5 (SiMe2CMe3), 17.7 (SiMe2CMe3), 25.5
(SiMe2CMe3), 35.6 (C-2), 71.9 (C-7), 75.2 (C-5), 76.9 (C-3), 83.3 (C-6),
84.8 (C-4), 116.4 (C-20), 127.2, 128.16, 128.2, 128.24, 129.0, 130.8,
133.9, 141.7 (2ꢂ Ph), 146.5 (C-30), 165.5 (C-10), 175.0 (C-1). HRMS
(ESI): m/z 495.2204 (MþþH), calcd for C28H35O6Si: 495.2197.
t
was added BuMe2SiCl (4.5 equiv) and imidazole (4.5 equiv). The
mixture was stirred at room temperature until the starting mate-
rials were consumed (TLC, 26 h for 1, 20 h for 2). The mixture was
poured into 10% aq NH4Cl and extracted with EtOAc. The combined
extracts were washed with brine, dried and evaporated. The res-
idue was purified by flash column chromatography (3:2 Et2O/light
petroleum for 7, 13:7 Et2O/light petroleum for epi-7).
4.1.2.2. 3,6-Anhydro-7-O-(terc-butyldimethylsilyl)-5-O-cinnamoyl-
2-deoxy-7-C-phenyl-
Yield 97%. Colourless syrup, [
(CH2Cl2). IR (film): nmax 1793 (C]O, lactone), 1720 (C]O, ester),
1636 (C]C, cinnamate). 1H NMR (CDCl3):
L
-glycero-
D
-ido-heptono-1,4-lactone
(epi-8a).
20
a
]
þ4.8 (c 0.5, CHCl3); Rf ¼ 0.26
D
4.1.1.1. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-2-deoxy-7-C-
d
ꢀ0.10 and 0.09 (2ꢂ s, 3H
phenyl-
recovered starting compound 1). Colourless syrup, [
0.5, CHCl3); Rf ¼ 0.27 (CH2Cl2). IR (film): nmax 3426 (OH), 1789 (C]
O, lactone), 1717 (C]O, ester). 1H NMR (CDCl3):
D-glycero-
D
-ido-heptono-1,4-lactone (7). Yield 95% (based on
each, SiMe2CMe3), 0.87 (s, 9H, SiMe2CMe3), 2.76 (dd, 1H,
J2a,2b ¼ 19.0, J2b,3 ¼ 2.5 Hz, H-2a), 2.83 (dd, 1H, J2a,2b ¼ 19.0,
J2b,3 ¼ 5.5 Hz, H-2b), 4.32 (dd, 1H, J5,6 ¼ 3.8, J6,7 ¼ 6.8 Hz, H-6), 4.86
(d, 1H, J6,7 ¼ 6.6 Hz, H-7), 4.99e5.13 (m, 2H, H-4 and H-5), 5.13 (td,
20
a
]
D þ103.0 (c
d
ꢀ0.02 and 0.13
1H, J ¼ 5.3 Hz, J2a,3 ¼ 2.5 Hz, H-3), 6.51 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20),
0
0
(2ꢂ s, 3H each, SiMe2CMe3), 0.91 (s, 9H, SiMe2CMe3), 1.27 (br s, 1H,
OH), 2.67 (dd, 1H, J2a,3 ¼ 1.9, J2a,2b ¼ 18.9 Hz, H-2a), 2.75 (dd, 1H,
J2a,2b ¼ 18.7, J2b,3 ¼ 4.9 Hz, H-2b), 3.91 (t,1H, J5,6 ¼ J6,7 ¼ 2.8 Hz, H-6),
4.29 (br d, 1H, J5,6 ¼ 2.0 Hz, H-5), 4.81 (d, 1H, J3,4 ¼ 4.0 Hz, H-4), 5.07
7.20e7.64 (m, 10H, 2ꢂ Ph), 7.77 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-30). 13C
0
0
NMR (CDCl3):
d
ꢀ4.73 and ꢀ4.7 (SiMe2CMe3), 18.2 (SiMe2CMe3),
25.8 (SiMe2CMe3), 36.0 (C-2), 74.3 (C-7), 76.2 (C-5), 76.7 (C-3), 84.1

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
243
(C-6), 85.6 (C-4),116.4 (C-20),126.8,128.2,128.3,128.34,129.1,130.9,
133.9, 140.4 (2ꢂ Ph), 146.8 (C-30), 165.3 (C-10), 175.1 (C-1). HRMS
(ESI): m/z 533.1762 (MþþK), calcd for C28H34KO6Si: 533.1756.
4.1.3.1. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-5-O-(4-
methoxycinnamoyl)-2-deoxy-7-C-phenyl-
1,4-lactone (8b). Yield 91%. Colourless syrup, [
D
-glycero-
D
-ido-heptono-
þ7.0 (c 0.1,
20
a
]
D
CHCl3); Rf ¼ 0.33 (997:3 CH2Cl2/Me2CO). IR (film): nmax 1791 (C]O,
lactone), 1716 (C]O, ester), 1633 (C]C, cinnamate). 1H NMR
4.1.2.3. 3,6-Anhydro-5,7-bis-(O-cinnamoyl)-2-deoxy-7-C-phenyl-D-
(CDCl3):
d
ꢀ0.05 and ꢀ0.37 (2ꢂ s, 3H each, SiMe2CMe3), 0.79 (s, 9H,
glycero- -gluco-heptono-1,4-lactone (6). Yield 94%. Colourless
D
SiMe2CMe3). 2.49 (br d, 1H, J2a,2b ¼ 18.9 Hz, H-2a), 2.66 (dd, 1H,
J2a,2b ¼ 19.0, J2b,3 ¼ 6.1 Hz, H-2b), 3.86 (s, 3H, OMe), 4.27 (dd, 1H,
J5,6 ¼ 2.8, J6,7 ¼ 8.8 Hz, H-6), 4.87 (d, 1H, J6,7 ¼ 8.8 Hz, H-7),
4.89e5.01 (m, 2H, H-3 and H-4), 5.63 (d, 1H, J5,6 ¼ 2.7 Hz, H-5), 6.43
20
powder, mp 200 ꢁC (CH2Cl2/hexane), [
a
]
ꢀ99.6 (c 0.25, CHCl3),
D
20
lit. [13] mp 195ꢀ196 ꢁC, [
a
]
D ꢀ102.7 (c 0.24, CHCl3); Rf ¼ 0.36 (2:1
Et2O/hexane). IR (CHCl3): nmax 1791 (C]O, lactone), 1716 (C]O,
ester), 1634 (C]C, cinnamate). For 1H and 13C NMR spectral data
see the Supplementary Data. HRMS (ESI): m/z 533.1578 (MþþNa),
calcd for C31H26NaO7: 533.1571.
(d, 1H, J2 ,3 ¼ 15.9 Hz, H-20), 7.29e7.60 (m, 9H, 2ꢂ Ph), 7.78 (d, 1H,
0
0
J2 ,3 ¼ 15.9 Hz, H-30). 13C NMR (CDCl3):
d
ꢀ5.3 and ꢀ4.6
0
0
(SiMe2CMe3), 17.7 (SiMe2CMe3), 25.4 (SiMe2CMe3), 35.5 (C-2), 55.3
(OMe), 71.9 (C-7), 75.0 (C-5), 76.8 (C-3), 83.3 (C-6), 84.9 (C-4), 114.1
(C-20) 114.4, 126.6, 127.2, 128.1, 128.2, 129.9, 141.7, 161.8 (2ꢂ Ph),
146.1 (C-30), 165.8 (C-10), 175.0 (C-1). HRMS (ESI): m/z 542.2561
(MþþNH4), calcd for C29H40NO7Si: 542.2569.
4.1.2.4. 3,6-Anhydro-5,7-bis-(O-cinnamoyl)-2-deoxy-7-C-phenyl-L-
glycero- -gluco-heptono-1,4-lactone (epi-6). Yield 96%. Colourless
D
20
powder, mp 208ꢀ209 ꢁC (CH2Cl2/hexane), [
a
]
ꢀ67.6 (c 0.5,
D
CHCl3); Rf ¼ 0.35 (99:1 CH2Cl2/EtOAc). IR (CHCl3): nmax 1792 (C]O,
lactone), 1716 (C]O, ester), 1635 (C]C, cinnamate). 1H NMR
4.1.3.2. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-5-O-(4-
(CDCl3):
d
2.83 (m, 2H, 2ꢂ H-2), 4.74 (dd, 1H, J5,6 ¼ 4.0, J6,7 ¼ 8.2 Hz,
nitrocinnamoyl)-2-deoxy-7-C-phenyl-D-glycero-D-ido-heptono-1,4-
H-6), 4.94 (d,1H, J3,4 ¼ 4.4 Hz, H-4), 5.11e5.20 (m, 2H, H-3 and H-5),
6.22 (d, 1H, J6,7 ¼ 8.2 Hz, H-7), 6.52 and 6.54 (2ꢂ d, 2H,
lactone (8c). Yield 97%. Colourless powder, mp 65 ꢁC (EtOAc/light
20
petroleum), [
a
]
þ1.0 (c 0.1, CHCl3); Rf ¼ 0.35 (7:3 light petro-
D
0
0
0
J2 ,3 ¼ 16.0 Hz, H-2 ), 7.29e7.650(m,15H, 3ꢂ Ph), 7.73 and 7.79 (2ꢂ d,
leum/EtOAc). IR (CHCl3): nmax 1793 (C]O, lactone), 1724 (C]O,
1H each, J2 ,3 ¼ 16.0 Hz, 2ꢂ H-3 ). 13C NMR (CDCl3):
d
35.9 (C-2), 74.6
ester), 1641 (C]C, cinnamate), 1522 and 1346 (NO2). 1H NMR
0
0
(C-7), 75.3 (C-5), 77.6 (C-3), 81.7 (C-6), 85.2 (C-4), 116.0 and 117.6
(2ꢂ C-20), 127.3, 128.1, 128.4, 128.6, 129.0, 129.2, 129.9, 130.4, 131.0,
133.7, 134.2, 135.9 (3ꢂ Ph), 147.1 (C-30), 165.0 and 165.8 (2ꢂ C-10),
174.7 (C-1). HRMS (ESI): m/z 533.1573 (MþþNa), calcd for
(CDCl3):
d
ꢀ0.08 and ꢀ0.39 (2ꢂ s, 3H each, SiMe2CMe3), 0.78 (s, 9H,
SiMe2CMe3), 2.52 (br d, 1H, J2a,2b ¼ 19.0 Hz, H-2a), 2.69 (dd, 1H,
J2a,2b ¼ 19.2, J2b,3 ¼ 5.8 Hz, H-2b), 4.27 (dd, 1H, J5,6 ¼ 2.5,
J6,7 ¼ 8.6 Hz, H-6), 4.84 (d, 1H, J6,7 ¼ 8.7 Hz, H-7), 4.90e5.01 (m, 2H,
H-3 and H-4), 5.65 (d, 1H, J5,6 ¼ 2.6 Hz, H-5), 6.70 (d, 1H,
C
31H26NaO7: 533.1571.
J2 ,3 ¼ 16.1 Hz, H-20), 7.29e8.36 (m, 9H, 2ꢂ Ph), 7.85 (d, 1H,
0
0
J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d
ꢀ5.3 and ꢀ4.5
0
0
4.1.2.5. 3,6-Anhydro-5-O-cinnamoyl-2,7-dideoxy-7-C-phenyl-
D-
(SiMe2CMe3), 17.7 (SiMe2CMe3), 25.5 (SiMe2CMe3), 35.5 (C-2), 71.9
(C-7), 75.6 (C-5), 76.9 (C-3), 83.2 (C-6), 84.7 (C-4),121.0 (C-20),124.2,
127.2, 128.2, 128.3, 128.8, 139.9, 141.5, 148.7 (2ꢂ Ph), 143.5 (C-30),
164.6 (C-10),174.8 (C-1). HRMS (ESI): m/z 557.2314 (MþþNH4), calcd
for C28H37N2O8Si: 557.2314.
gluco-heptono-1,4-lactone (5a). Yield 91%. Colourless needles, mp
20
134 ꢁC (CH2Cl2/hexane), [
Et2O/light petroleum). IR (CHCl3): nmax 1791 (C]O, lactone), 1716
(C]O, ester), 1636 (C]C, cinnamate). 1H NMR (CDCl3):
2.67 (dd,
a
]
ꢀ19.1 (c 1.0, CHCl3); Rf ¼ 0.29 (1:1
D
d
1H, J2a,2b ¼ 19.0, J2a,3 ¼ 1.2 Hz, H-2a), 2.79 (dd, 1H, J2a,2b ¼ 18.9,
J2b,3 ¼ 5.8 Hz, H-2b), 2.97 (dd, 1H, J7a,7b ¼ 14.0, J6,7a ¼ 7.7 Hz, H-7a),
3.04 (dd, 1H, J7a,7b ¼ 14.1, J6,7b ¼ 7.2 Hz, H-7b), 4.42 (td, 1H, J6,7 ¼ 7.0,
J5,6 ¼ 3.0 Hz, H-6), 5.00 (d, 1H, J3,4 ¼ 5.8 Hz, H-4), 5.05 (td, 1H,
J3,4 ¼ 5.8, J2a,3 ¼ 1.4, J2b,3 ¼ 5.8 Hz,0H-3), 5.46 (d, 1H, J5,6 ¼ 2.9 Hz, H-
4.1.3.3. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-5-O-(4-
fluorocinnamoyl)-2-deoxy-7-C-phenyl-D-glycero-D-ido-heptono-1,4-
lactone (8d). Yield 96%. Colourless powder, mp 95 ꢁC (light petro-
20
leum), [
a]
D þ7.0 (c 0.1, CHCl3); Rf ¼ 0.29 (CH2Cl2). IR (CHCl3): nmax
0
0
5), 6.57 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-2 ), 7.15e7.66 (m, 10H, 2ꢂ Ph), 7.84
1794 (C]O, lactone), 1724 (C]O, ester), 1638 (C]C, cinnamate). 1H
NMR (CDCl3):
(d, 1H, J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d
34.6 (C-7), 35.6 (C-
0
0
d
ꢀ0.37 and ꢀ0.06 (2ꢂ s, 3H each, SiMe2CMe3), 0.79
2), 75.5 (C-5), 76.4 (C-3), 80.4 (C-6), 85.5 (C-4), 116.4 (C-20), 126.7,
128.2, 128.5, 128.8, 128.9, 130.8, 133.8, 137.0 (2ꢂ Ph), 146.7 (C-30),
165.4 (C-10), 174.9 (C-1). HRMS (ESI): m/z 387.1220 (MþþNa), calcd
for C22H20NaO5: 387.1203.
(s, 9H, SiMe2CMe3), 2.51 (br d, 1H, J2a,2b ¼ 18.9 Hz, H-2a), 2.67 (dd,
1H, J2a,2b ¼ 19.0, J2b,3 ¼ 6.1 Hz, H-2b), 4.28 (dd, 1H, J5,6 ¼ 2.8,
J6,7 ¼ 8.7 Hz, H-6), 4.86 (d, 1H, J6,7 ¼ 8.8 Hz, H-7), 4.90e5.01 (m, 2H,
H-3 and H-4), 5.65 (d, 1H, J5,6 ¼ 2.6 Hz, H-5), 6.70 (d, 1H,
J2 ,3 ¼ 16.1 Hz, H-20), 7.29e8.36 (m, 9H, 2ꢂ Ph), 7.85 (d, 1H,
0
0
J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d
ꢀ5.3 and ꢀ4.5
0
0
4.1.3. General procedure for the Steglich esterification protocol
To a solution of 7, epi-7 or 9 (1 equiv) in anhydrous CH2Cl2
(0.01 M) were successively added 4-substituted cinnamic acid
(2 equiv for 5aec, 8bed; 4 equiv for epi-8b, epi-8c and epi-8d), DCC
(2.2 equiv for 5aec, 8bed; 4.4 equiv for epi-8b, epi-8c and epi-8d)
and DMAP (4 equiv for 5aec, 8bed; 8 equiv for epi-8b, epi-8c and
epi-8d). The mixture was stirred at room temperature until the
starting materials were consumed (TLC, 22 h for 5aec; 48 h for
8bed; 72 h for epi-8b, epi-8c and epi-8d). The mixture was poured
to water (80 mL) and extracted with CH2Cl2. The combined organic
phases were washed with 10% NaCl, dried and evaporated, and the
residue was purified by flash column chromatography (CH2Cl2 for
8b and 8d, 17:3 light petroleum/EtOAc for 8c, 3:2 hexane/Et2O for
epi-8b and epi-8d, 1:1 hexane/Et2O for epi-8c, 47:3 toluene/EtOAc
for 5b, CH2Cl2 for 5c, 19:1 toluene/EtOAc for 5d) and in few cases by
additional preparative TLC (iPr2O, 3 successive developments, for
5b and 5c).
(SiMe2CMe3), 17.7 (SiMe2CMe3), 25.5 (SiMe2CMe3), 35.5 (C-2), 71.9
(C-7), 75.2 (C-5), 76.9 (C-3), 83.3 (C-6), 84.8 (C-4), 116.5 (C-20), 116.0,
116.4, 116.5, 127.2, 128.2, 128.3, 130.1, 130.2, 141.6, 162.1, 166.1 (2ꢂ
Ph), 145.2 (C-30), 165.4 (C-10), 174.9 (C-1). HRMS (ESI): m/z 530.2365
(MþþNH4), calcd for C28H37FNO6Si: 530.2369.
4.1.3.4. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-5-O-(4-
methoxycinnamoyl)-2-deoxy-7-C-phenyl-
1,4-lactone (epi-8b). Yield 87%. Colourless glass, [
L-glycero-D-ido-heptono-
20
a
]
D þ12.0 (c 0.1,
CHCl3); Rf ¼ 0.34 (1:1 Et2O/hexane). IR (CHCl3): nmax 1793 (C]O,
lactone), 1717 (C]O, ester), 1633 (C]C, cinnamate). 1H NMR
(CDCl3):
d
ꢀ0.09 and 0.10 (2ꢂ s, 3H each, SiMe2CMe3), 0.89 (s, 9H,
SiMe2CMe3), 2.72 (dd, 1H, J2a,2b ¼ 19.0, J2a,3 ¼ 2.4 Hz, H-2a), 2.83
(dd, 1H, J2a,2b ¼ 19.0, J2b,3 ¼ 5.5 Hz, H-2b), 3.87 (s, 3H, OMe), 4.32
(dd, 1H, J5,6 ¼ 4.0, J6,7 ¼ 6.4 Hz, H-6), 4.88 (d, 1H, J6,7 ¼ 6.7 Hz, H-7),
4.91e5.03 (m, 2H, H-4 and H-5), 5.13 (m, 1H, H-3), 6.39 (d, 1H,

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
244
J2 ,3 ¼ 16.0 Hz, H-20), 6.83e7.74 (m, 9H, 2ꢂ Ph), 7.74 (d, 1H,
(td, 1H, J6,7 ¼ 6.9, J5,6 ¼ 2.9 Hz, H-6), 4.99 (d, 1H, J3,4 ¼ 4.7 Hz, H-4),
5.06 (m, 1H, J3,4 ¼ 4.7, J2b,3 ¼ 5.7 Hz, H-3), 5.47 (d, 1H, J5,6 ¼ 2.8 Hz,
0
0
J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d
ꢀ4.8 and ꢀ4.7
0
0
H-5), 6.66 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20), 7.05e8.35 (m, 10H, 2ꢂ Ph
0
0
(SiMe2CMe3), 18.2 (SiMe2CMe3), 25.7 (SiMe2CMe3), 36.0 (C-2), 55.4
(OMe), 74.3 (C-7), 75.9 (C-5), 76.5 (C-3), 84.1 (C-6), 85.6 (C-4), 113.8
(C-20), 114.5, 126.6, 126.9, 128.2, 128.3, 130.0, 132.6, 140.4, 161.9 (2ꢂ
Ph), 146.4 (C-30), 165.6 (C-10), 175.1 (C-1). HRMS (ESI): m/z 542.2567
(MþþNH4), calcd for C29H40NO7Si: 542.2569.
and H-30). 13C NMR (CDCl3):
d 34.7 (C-7), 35.7 (C-2), 76.1 (C-5), 76.5
(C-3), 80.3 (C-6), 85.4 (C-4), 120.8 (C-20), 124.3, 126.9, 128.7, 128.87,
128.9, 136.9, 139.9, 148.9 (2ꢂ Ph), 143.8 (C-30), 164.6 (C-10), 174.8 (C-
1). HRMS (ESI): m/z 427.1501 (MþþNH4), calcd for C22H23N2O7:
427.1500.
4.1.3.5. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-5-O-(4-
nitrocinnamoyl)-2-deoxy-7-C-phenyl-
L
-glycero-
D
-ido-heptono-1,4-
4.1.3.9. 3,6-Anhydro-2,7-dideoxy-5-O-(4-fluorocinnamoyl)-7-C-
lactone (epi-8c). Yield 80%. Pale yellow powder, mp 148e150 ꢁC
phenyl-
needles, mp 129e131 ꢁC (CH2Cl2/hexane), [
CHCl3); Rf ¼ 0.22 (19:1 toluene/EtOAc). IR (CHCl3): nmax 1791 (C]O,
lactone), 1717 (C]O, ester). 1H NMR (CDCl3):
2.66 (br d, 1H,
D
-gluco-heptono-1,4-lactone (5d). Yield 71%. Colourless
20
20
(CH2Cl2/hexane), [
a
]
D þ2.0 (c 0.1, CHCl3); Rf ¼ 0.27 (3:2 Et2O/light
a
]
ꢀ17.4 (c 0.5,
D
petroleum). IR (CHCl3): nmax 1788 (C]O, lactone), 1721 (C]O,
ester), 1634 (C]C, cinnamate). 1H NMR (CDCl3):
d
ꢀ0.12 and 0.08
d
(2ꢂ s, 3H each, SiMe2CMe3), 0.87 (s, 9H, SiMe2CMe3), 2.73 (dd, 1H,
J2a,2b ¼ 19.0, J2a,3 ¼ 1.9 Hz, H-2a), 2.83 (dd, 1H, J2a,2b ¼ 19.0,
J2b,3 ¼ 6.1 Hz, H-2b), 4.32 (dd, 1H, J5,6 ¼ 3.7, J6,7 ¼ 6.4 Hz, H-6), 4.83
(d, 1H, J6,7 ¼ 6.4 Hz, H-7), 4.92e5.05 (m0, 2H, H-4 and H-5), 5.14 (m,
J2a,2b ¼ 18.9 Hz, H-2a), 2.75 (dd, 1H, J2a,2b ¼ 19.0, J2b,3 ¼ 5.8 Hz, H-
2b), 2.98 (d, 2H, J6,7 ¼ 6.5 Hz, H-7), 4.40 (td, 1H, J6,7 ¼ 6.8,
J5,6 ¼ 2.9 Hz, H-6), 4.98 (d, 1H, J3,4 ¼ 4.7 Hz, H-4), 5.04 (m, 1H,
J3,4 ¼ 4.7, J2b,3 ¼ 5.6 Hz, H-3), 5.44 (d, 1H, J5,6 ¼ 2.8 Hz, H-5), 6.45 (d,
1H, H-3), 6.62 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-2 ), 7.09e8.33 (m,10H, 2ꢂ Ph
1H, J2 ,3 ¼ 16.0 Hz, H-20), 6.95e7.67 (m, 9H, 2ꢂ Ph), 7.77 (d, 1H,
0
0
0
0
and H-30). 13C NMR (CDCl3):
d
ꢀ4.8 and ꢀ4.7 (SiMe2CMe3), 18.1
J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d 34.6 (C-7), 35.7 (C-2), 75.6
0
0
(SiMe2CMe3), 25.7 (SiMe2CMe3), 35.9 (C-2), 74.2 (C-7), 76.5 (C-5),
76.7 (C-3), 83.9 (C-6), 85.4 (C-4), 120.6 (C-20), 124.2, 126.7, 128.2,
128.3, 128.9, 139.8, 140.2, 148.8 (2ꢂ Ph), 143.7 (C-30), 164.4 (C-10),
175.0 (C-1). HRMS (ESI): m/z 557.2306 (MþþNH4), calcd for
(C-5), 76.4 (C-3), 80.4 (C-6), 85.5 (C-4), 116.4 (C-20), 116.0, 116.15,
116.2, 126.7, 128.6, 128.8, 130.2, 130.3, 137.0, 162.2, 165.3 (2ꢂ Ph),
145.4 (C-30), 166.2 (C-10), 174.9 (C-1). HRMS (ESI): m/z 400.1550
(MþþNH4), calcd for C22H23FNO5: 400.1555.
C
28H37N2O8Si: 557.2314.
4.1.4. General procedure for the silyl deprotection with TFA
To a stirred solution of 8a, epi-8a, 8b, 8c, or 8d (1 equiv) in 50:1
mixture of CH2Cl2/H2O (0.01e0.03 M) was added cooled (þ4 ꢁC)
TFA in portions (2 ꢂ 0.2 mL for 8a and epi-8a, 3 ꢂ 0.2 mL for 8b,
4 ꢂ 0.15 mL for 8c and 8d). The mixture was stirred at room tem-
perature until the starting materials were consumed (TLC, 50 h for
8a, 22 h for epi-8a, 48 h for 8bed). The mixture was evaporated by
co-distillation with toluene and the residue was purified by flash
column chromatography (49:1 / 24:1 CH2Cl2/Me2CO for 3a, 17:3
CH2Cl2/EtOAc for epi-3a, 24:1 CH2Cl2/Me2CO for 3b, 2:1 light pe-
troleum/EtOAc for 3c and 3d).
4.1.3.6. 3,6-Anhydro-7-O-(tert-butyldimethylsilyl)-5-O-(4-
fluorocinnamoyl)-2-deoxy-7-C-phenyl-l-glycero- -ido-heptono-1,4-
D
20
lactone (epi-8d). Yield 75%. Colourless glass, [
a
]
þ10.0 (c 0.1,
D
CHCl3); Rf ¼ 0.35 (1:1 Et2O/hexane). IR (CHCl3): nmax 1793 (C]O,
lactone), 1720 (C]O, ester), 1637 (C]C, cinnamate). 1H NMR
(CDCl3):
d
ꢀ0.10 and 0.10 (2ꢂ s, 3H each, SiMe2CMe3), 0.89 (s, 9H,
SiMe2CMe3), 2.73 (dd, 1H, J2a,2b ¼ 19.0, J2a,3 ¼ 2.4 Hz, H-2a), 2.83
(dd, 1H, J2a,2b ¼ 19.0, J2b,3 ¼ 5.5 Hz, H-2b), 4.32 (dd, 1H, J5,6 ¼ 3.5,
J6,7 ¼ 6.5 Hz, H-6), 4.86 (d, 1H, J6,7 ¼ 6.5 Hz, H-7), 4.93e5.02 (m, 2H,
H-4 and H-5), 5.13 (m, 1H, H-3), 6.44 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20),
0
0
7.13e7.68 (m, 9H, 2ꢂ Ph), 7.75 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-3 ). 13C NMR
0
0
0
(CDCl3):
d
ꢀ4.8 and ꢀ4.7 (SiMe2CMe3), 18.2 (SiMe2CMe3), 25.7
4.1.4.1. (þ)-Crassalactone B (3a). Yield 79% (93% when calculated to
(SiMe2CMe3), 36.0 (C-2), 74.2 (C-7), 76.3 (C-5), 76.5 (C-3), 84.0 (C-6),
85.5 (C-4), 116.4 (C-20), 116.0, 126.7, 128.2, 128.3, 129.0, 130.2, 130.3,
140.3, 162.2, 165.2 (2ꢂ Ph), 145.4 (C-30), 166.2 (C-10), 175.0 (C-1).
HRMS (ESI): m/z 530.2358 (MþþNH4), calcd for C28H37FNO6Si:
530.2369.
reacted intermediate 8a; 15.1% of 8a is recovered). Colourless
20
powder, mp 173ꢀ174 ꢁC (Et2O), [
a]
þ35.5 (c 1.0, CHCl3), lit. [2]
D
20
171ꢀ173 ꢁC (EtOH), [
a
]
þ8 (c 0.5, EtOH), lit. [13] 168ꢀ171 ꢁC
D
20
(EtOH), [
a
]
D þ31.6 (c 1.0, CHCl3); Rf ¼ 0.38 (97:3 CH2Cl2/Me2CO).
IR (KBr): nmax 3444 (OH), 1789 (C]O, lactone), 1717 (C]O, ester),
1636 (C]C, cinnamate). For 1H and 13C NMR spectra see the
Supplementary Data. HRMS (ESI): m/z 403.1143 (MþþNa), calcd for
4.1.3.7. 3,6-Anhydro-2,7-dideoxy-5-O-(4-methoxycinnamoyl)-7-C-
phenyl-
syrup, [
(film): nmax 1791 (C]O, lactone), 1714 (C]O, ester). 1H NMR
(CDCl3):
D
-gluco-heptono-1,4-lactone (5b). Yield 55%. Colourless
C22H20NaO6: 403.1152.
20
a
]
D ꢀ17.2 (c 0.5, CHCl3); Rf ¼ 0.23 (19:1 toluene/EtOAc). IR
4.1.4.2. 7-epi-(þ)-Crassalactone B (epi-3a). Yield 85%. Colourless
20
d
2.67 (br d, 1H, J2a,2b ¼ 19.0 Hz, H-2a), 2.78 (dd, 1H,
powder, mp 128ꢀ129 ꢁC (EtOAc/hexane), [
a]
ꢀ47.6 (c 0.5,
D
J2a,2b ¼ 18.9, J2b,3 ¼ 5.7 Hz, H-2b), 2.98 (d, 2H, J6,7 ¼ 6.8 Hz, H-7), 3.87
(s, 3H, OMe), 4.41 (td, 1H, J6,7 ¼ 6.8, J5,6 ¼ 2.9 Hz, H-6), 4.98 (d, 1H,
J3,4 ¼ 4.7 Hz, H-4), 5.04 (m, 1H, J3,4 ¼ 4.6, J2b,3 ¼ 5.6 Hz, H-3), 5.44 (d,
CHCl3); Rf ¼ 0.26 (9:1 CH2Cl2/EtOAc). IR (KBr): nmax 3424 (OH), 1791
(C]O, lactone), 1716 (C]O, ester), 1636 (C]C, cinnamate). 1H NMR
(CDCl3):
d
2.58 (br s, 1H, OH), 2.80 (m, 2H, 2ꢂ H-2), 4.39 (dd, 1H,
1H, J5,6 ¼ 2.8 Hz, H-5), 6.40 (d, 1H, J2 ,3 ¼ 15.9 Hz, H-20), 6.83e7.60
J5,6 ¼ 3.3, J6,7 ¼ 7.8 Hz, H-6), 4.94 (d, 1H, J3,4 ¼ 4.6 Hz, H-4), 5.01 (d,
0
0
(m, 9H, 2ꢂ Ph), 7.77 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
1H, J6,7 ¼ 7.8 Hz, H-7), 5.07e5.15 (m, 2H, H-3 and H-5), 6.22 (d, 1H,
0
0
0
0
d
34.7 (C-7), 35.7 (C-2), 55.4 (OMe), 75.3 (C-5), 76.4 (C-3), 80.5 (C-6),
J6,7 ¼ 8.2 Hz, H-7), 6.52 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20), 7.29e7.67 (m,
85.6 (C-4), 113.8 (C-20), 114.4, 126.7, 128.6, 128.9, 130.0, 132.5, 137.1,
161.8 (2ꢂ Ph), 146.4 (C-30), 165.7 (C-10), 174.9 (C-1). HRMS (ESI): m/z
395.1484 (MþþH), calcd for C23H23O6: 395.1489.
10H, 2ꢂ Ph), 7.79 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
0
0
d
35.8 (C-2), 72.9 (C-7), 75.1 (C-5), 77.3 (C-3), 83.8 (C-6), 85.5 (C-4),
116.1 (C-20), 126.6, 128.3, 128.7, 128.8, 129.0, 131.0, 133.7, 138.8 (2ꢂ
Ph), 147.0 (C-30), 165.0 (C-10), 174.6 (C-1). HRMS (ESI): m/z 403.1148
(MþþNa), calcd for C22H20NaO6: 403.1152.
4.1.3.8. 3,6-Anhydro-2,7-dideoxy-5-O-(4-nitrocinnamoyl)-7-C-
phenyl-
syrup, [
1790 (C]O, lactone), 1728 (C]O, ester), 1520 and 1346 (NO2). 1H
NMR (CDCl3):
2.69 (br d, 1H, J2a,2b ¼ 19.0 Hz, H-2a), 2.81 (dd, 1H,
J2a,2b ¼ 19.0, J2b,3 ¼ 6.0 Hz, H-2b), 2.98 (d, 2H, J6,7 ¼ 6.8 Hz, H-7), 4.41
D
-gluco-heptono-1,4-lactone (5c). Yield 44%. Colourless
20
a
]
D ꢀ2.6 (c 0.5, CHCl3); Rf ¼ 0.38 (CH2Cl2). IR (film): nmax
4.1.4.3. 3,6-Anhydro-5-O-(4-methoxycinnamoyl)-2-deoxy-7-C-
phenyl-
ourless powder, mp 155 ꢁC (CH2Cl2/trace Et2O), [
CHCl3); Rf ¼ 0.35 (24:1 CH2Cl2/Me2CO). IR (CHCl3): nmax 3487 (OH),
D-glycero-D-ido-heptono-1,4-lactone (3b). Yield 85%. Col-
20
d
a
]
D þ59.0 (c 0.1,

ꢀ
G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
245
1789 (C]O, lactone), 1715 (C]O, ester), 1633 (C]C, cinnamate). 1H
NMR (CDCl3):
J2,3 ¼ 3.8 Hz, 2ꢂ H-2), 3.86 (s, 3H, OMe), 4.38 (dd, 1H, J6,7 ¼ 7.7,
J5,6 ¼ 3.3 Hz, H-6), 4.93 (d, 1H, J3,4 ¼ 4.4 Hz, H-4), 4.99 (d, 1H,
J6,7 ¼ 7.7 Hz, H-7), 5.06e5.11 (m, 2H, H-3 and H-5), 6.36 (d, 1H,
d
2.59 (d, 1H, J2a,2b ¼ 18.9 Hz, H-2a), 2.73 (dd, 1H,
J2a,2b ¼ 18.9, J2b,3 ¼ 5.2 Hz, H-2b), 3.20 (d, 1H, JOH,7 ¼ 2.6 Hz), 3.87 (s,
3H, OMe), 4.25 (dd, 1H, J5,6 ¼ 2.6, J6,7 ¼ 8.6 Hz, H-6), 4.47 (dd, 1H,
J6,7 ¼ 8.7, JOH,7 ¼ 2.6 Hz, H-7), 4.95e5.04 (m, 2H, H-3 and H-4), 5.72
J2 ,3 ¼ 15.9 Hz, H-20), 6.91e7.63 (m, 9H, Ph), 7.72 (d, 1H,
0
0
J2 ,3 ¼ 16.0 Hz, H-3 ). 13C NMR (CDCl3):
d
35.9 (C-2), 55.4 (OMe), 72.9
0
0
0
(d, 1H, J5,6 ¼ 2.5 Hz, H-5), 6.41 (d, 1H, J2 ,3 ¼ 15.9 0 Hz, H-20),
(C-7), 75.0 (C-5), 77.3 (C-3), 83.9 (C-6), 85.6 (C-4), 113.5 (C-20), 114.5,
126.5, 126.6, 128.8, 130.1, 138.9, 162.0 (2ꢂ Ph), 146.6 (C-30), 165.4 (C-
10), 174.7 (C-1). HRMS (ESI): m/z 428.1704 (MþþNH4), calcd for
0
0
6.90e7.56 (m, 9H, 2ꢂ Ph), 7.79 (d, 1H, J2 ,3 ¼ 15.9 Hz, H-3 ). 13C NMR
0
0
(CDCl3):
d 35.5 (C-2), 55.4 (OMe), 70.7 (C-7), 75.2 (C-5), 77.0 (C-3),
83.1 (C-6), 85.3 (C-4), 114.4 (C-20), 113.2, 126.4, 126.8, 128.2, 128.4,
130.2, 140.4, 162.0 (2ꢂ Ph), 147.2 (C-30), 166.8 (C-10), 174.8 (C-1).
HRMS (ESI): m/z 428.1700 (MþþNH4), calcd for C23H26NO7:
428.1704.
C23H26NO7: 428.1704.
4.1.5.2. 3,6-Anhydro-5-O-(4-nitrocinnamoyl)-2-deoxy-7-C-phenyl-
L-
glycero- -ido-heptono-1,4-lactone (epi-3c). Yield 89% after crystal-
D
lization of crude reaction mixture (without column chromatog-
4.1.4.4. 3,6-Anhydro-5-O-(4-nitrocinnamoyl)-2-deoxy-7-C-phenyl-
glycero- -ido-heptono-1,4-lactone (3c). Yield 68% (74% when
calculated to reacted intermediate 8c; 7.7% of 8c is recovered).
D-
raphy). Pale yellow powder, mp 219e223 ꢁC (CH2Cl2/hexane),
D
20
[a
]
ꢀ36.0 (c 0.3, Me2CO); Rf ¼ 0.32 (19:1 CH2Cl2/Me2CO). IR
D
(KBr): nmax 3486 (OH), 1785 (C]O, lactone), 1731 and 1719 (C]O,
20
Colourless powder, mp 137e140 ꢁC (Et2O), [
a
]
þ37.0 (c 0.1,
D
ester), 1644 (C]C, cinnamate), 1522 and 1348 (NO2). 1H NMR
CHCl3); Rf ¼ 0.25 (13:7 light petroleum/EtOAc). IR (CHCl3): nmax
(acetone-d6):
d
2.70e3.00 (m, 2ꢂ H-2 overlapped with H2O from
3500 (OH), 1790 (C]O, lactone), 1723 (C]O, ester), 1642 (C]C,
acetone-d6), 4.41 (dd, 1H, J6,7 ¼ 7.1, J5,6 ¼ 3.7 Hz, H-6), 4.61 (bs, 1H,
OH), 4.94 (d, 1H, J5,6 ¼ 3.5 Hz, H-5), 4.99 (d, 1H, J6,7 ¼ 7.0 Hz, H-7),
5.08 (d, 1H, J3,4 ¼ 4.8 Hz, H-4), 5.21 (m, 1H, H-3), 6.92 (d, 1H,
cinnamate), 1520 and 1346 (NO2). 1H NMR (CDCl3):
d 2.59 (d, 1H,
J2a,2b ¼ 18.8 Hz, H-2a), 2.73 (m, 2H, J2a,2b ¼ 19.0, J2b,3 ¼ 2.9 Hz, H-2b
and OH), 4.28 (dd, 1H, J5,6 ¼ 2.8, J6,7 ¼ 8.5 Hz, H-6), 4.73 (d, 1H,
J6,7 ¼ 8.5 Hz, H-7), 5.03 (m, 2H, H-3 and H-4), 5.76 (d, 1H,
0
0
0
0
0
0
J2 ,3 ¼ 16.1 Hz, H-2 ), 7.17e8.36 (m,10H, J2 ,3 ¼ 16.1 Hz, Ph and H-3 ).
13C NMR (acetone-d6):
d 36.1 (C-2), 73.3 (C-7), 77.1 (C-5), 78.3 (C-3),
J5,6 ¼ 2.7 Hz, H-5), 6.67 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20), 7.29e8.33 (m,
0
0
84.6 (C-6), 86.4 (C-4), 122.3 (C-20), 124.8, 127.7, 128.7, 129.0, 130.3,
141.4, 142.0, 149.6 (2ꢂ Ph), 144.0 (C-30), 165.4 (C-10), 175.7 (C-1).
HRMS (ESI): m/z 443.1452 (MþþNH4), calcd for C22H23N2O8:
443.1449.
9H, 2ꢂ Ph), 7.85 (d,1H, J2 ,3 ¼ 16.0 Hz, H-3 ). 13C NMR (CDCl3):
d
35.5
0
0
0
(C-2), 71.0 (C-7), 75.8 (C-5), 77.2 (C-3), 82.8 (C-6), 85.1 (C-4), 120.6
(C-20), 124.2, 126.7, 128.5, 128.6, 128.0, 139.8, 140.4, 148.8 (2ꢂ Ph),
144.2 (C-30), 165.3 (C-10), 174.63 (C-1). HRMS (ESI): m/z 443.1448
(MþþNH4), calcd for C28H23N2O8: 443.1449.
4.1.5.3. 3,6-Anhydro-5-O-(4-fluorocinnamoyl)-2-deoxy-7-C-phenyl-
4.1.4.5. 3,6-Anhydro-5-O-(4-fluorocinnamoyl)-2-deoxy-7-C-phenyl-
L
-glycero-
D
-ido-heptono-1,4-lactone (epi-3d). Yield 62%. Colourless
20
D-glycero-D-ido-heptono-1,4-lactone (3d). Yield 90% (98% when
needles, mp 160e163 ꢁC (CH2Cl2/hexane), [
a]
D þ4.0 (c 0.1, CHCl3);
calculated to reacted 8d; 5% of starting 8d is recovered). Colourless
Rf ¼ 0.29 (24:1 CH2Cl2/Me2CO. IR (KBr): nmax 3497e3407 (OH), 1791
20
powder, mp 176 ꢁC (CH2Cl2/hexane), [
a
]
þ47.0 (c 0.1, CHCl3);
(C]O, lactone), 1720 (C]O, ester), 1638 (C]C, cinnamate). 1H NMR
D
Rf ¼ 0.30 (13:7 light petroleum/EtOAc). IR (CHCl3): nmax 3470 (OH),
(CDCl3):
d
2.36 (bs, 1H, OH), 2.81 (d, 2H, J2,3 ¼ 3.7 Hz, 2ꢂ H-2), 4.37
1789 (C]O, lactone), 1720 (C]O, ester), 1637 (C]C, cinnamate). 1H
(dd, 1H, J6,7 ¼ 7.7, J5,6 ¼ 3.3 Hz, H-6), 4.93 (d, 1H, J3,4 ¼ 5.2 Hz, H-4),
4.99 (d, 1H, J6,7 ¼ 7.7 Hz, H-7), 5.07e5.16 (m, 2H, H-3 and H-5), 6.42
NMR (CDCl3):
d
2.57 (br d, 1H, J2a,2b ¼ 19.0 Hz, H-2a), 2.72 (dd, 1H,
(d, 1H, J2 ,3 ¼ 15.9 Hz, H-20), 7.02e7.67 (m, 9H, Ph), 7.74 (d, 1H,
0
0
J2a,2b ¼ 19.0, J2b,3 ¼ 5.0 Hz, H-2b), 3.01 (br s, 1H, OH), 4.26 (dd, 1H,
J5,6 ¼ 2.2, J6,7 ¼ 8.6 Hz, H-6), 4.69 (d, 1H, J6,7 ¼ 8.6 Hz, H-7),
4.97e5.07 (m, 2H, H-3 and H-4), 5.73 (d, 1H, J5,6 ¼ 2.6 Hz, H-5), 6.47
J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d
35.9 (C-2), 72.9 (C-7), 75.2
0
0
(C-5), 77.4 (C-3), 83.9 (C-6), 85.6 (C-4), 116.0 (C-20), 116.2, 116.5,
126.6, 128.8, 130.1, 130.3, 130.4, 138.8, 162.4, 165.0 (2ꢂ Ph), 145.7 (C-
30), 166.4 (C-10), 174.6 (C-1). HRMS (ESI): m/z 416.1501 (MþþNH4),
calcd for C22H23FNO6: 416.1504.
(d, 1H, J2 ,3 ¼ 16.0 Hz, H-20), 7.07e7.65 (m, 9H, Ph), 7.80 (d, 1H,
0
0
J2 ,3 ¼ 16.0 Hz, H-30). 13C NMR (CDCl3):
d
35.5 (C-2), 70.8 (C-7), 75.4
0
0
(C-5), 77.0 (C-3), 82.9 (C-6), 85.2 (C-4), 115.8 (C-20), 116.0, 116.4,
126.8, 128.3, 128.5, 130.0, 130.05, 130.3, 130.4, 140.4, 162.2, 166.3
(2ꢂ Ph), 146.1 (C-30), 165.4 (C-10), 174.9 (C-1). HRMS (ESI): m/z
416.1501 (MþþNH4), calcd for C22H23FNO6: 416.1504.
4.1.6. General procedure for the Mitsunobu reaction
To a stirred and cooled (0 ꢁC) solution of 1 or 2 (1 equiv) in
anhydrous EtOAc (0.02e0.04 M) were successively added cinnamic
acid or 4-substituted cinnamic acid (2e3 equiv), Ph3P (2e3 equiv)
and 40% solution of DEAD in toluene (2e3 equiv) and the mixture
was stirred at 0 ꢁC for 1 h and then at room temperature until the
starting materials were consumed (TLC, 2 h for 4b, 4c and 4d, 3 h for
4a and epi-4a, 4 h for epi-4c, and epi-4d, 24 h for epi-4b). The
mixture was evaporated and the residue was purified by flash
column chromatography (7:3 Et2O/light petroleum for 4a and epi-
4a, 4:1 Et2O/light petroleum for 4b, 9:1 / 3:1 Et2O/light petroleum
for 4c 4:1 / 3:1 Et2O/light petroleum for 4d, 1:1 light petroleum/
EtOAc, 19:1 Et2O/light petroleum for epi-4b, 9:1 / 19:1 Et2O/light
petroleum for epi-4c, 9:1 Et2O/light petroleum for epi-4d).
4.1.5. General procedure for the silyl deprotection with CAN
To a stirred solution of epi-8b, epi-8c, or epi-8d (1 equiv) in 9:1
mixture of MeCN/H2O (0.005 M) was added CAN (1.2 equiv). The
mixture was stirred at room temperature until the starting mate-
rials were consumed (TLC, 120 h for epi-8b, 72 h for epi-8c, 26 h for
epi-8d). The mixture was partitioned between CHCl3 and 10%
NaHCO3, the organic layer was separated, dried and evaporated.
The residue was purified by flash column chromatography (19:1
CH2Cl2/Me2CO for epi-3b, 47:3 CH2Cl2/Me2CO for epi-3d), or in the
case of epi-3c by direct crystallization of crude reaction mixture
from CH2Cl2/hexane.
4.1.5.1. 3,6-Anhydro-5-O-(4-methoxycinnamoyl)-2-deoxy-7-C-
phenyl-
L
-glycero-
D
-ido-heptono-1,4-lactone (epi-3b). Yield 73%.
4.1.6.1. (þ)-Crassalactone C (4a) and 3,6:5,7-dianhydro-2-deoxy-7-
20
Colourless plates, mp 147e149 ꢁC (CH2Cl2/hexane), [
a
]
D ꢀ76.7 (c
C-phenyl-D-glycero-D-ido-heptono-1,4-lactone (10). Yields: 69% of
0.3, CHCl3); Rf ¼ 0.39 (19:1 CH2Cl2/Me2CO). IR (KBr): nmax 3498 and
3406 (OH), 1790 (C]O, lactone), 1716 (C]O, ester), 1631 (C]C,
4a, 31% of 10. Physical constants and spectroscopic data of thus
prepared samples 4a and 10 matched those previously reported by
us [10].
cinnamate). 1H NMR (CDCl3):
d 2.46 (bs, 1H, OH), 2.79 (d, 2H,

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
246
4.1.6.2. 3,6-Anhydro-7-O-(4-methoxycinnamoyl)-2-deoxy-7-C-
phenyl- -glycero- -ido-heptono-1,4-lactone (4b). Yield 60% (31% of
J5,6 ¼ J6,7 ¼ 4.4 Hz, H-6), 5.56 (d, 1H, J5,6 ¼ 3.9 Hz, H-5), 5.88 (d, 1H,
J6,7 ¼ 4.8 Hz, H-7), 7.24e7.46 (m, 5H, Ph). NOE contact: H-3 and Ph.
13C NMR (CDCl3): 36.3 (C-2), 80.4 (C-3), 81.0 (C-6), 84.8 (C-4), 85.3
(C-5), 86.1 (C-7), 124.4, 127.7, 128.4, 136.7 (Ph), 174.2 (C-1). HRMS
(ESI): m/z 233.0810 (MþþH), calcd for C13H13O4: 233.0808.
D
D
oxetane 10 was also isolated). Compound 4b: Colourless powder,
20
mp 89ꢀ90 ꢁC (Et2O), [
a]
D þ143.0 (c 0.1, CHCl3); Rf ¼ 0.37 (Et2O). IR
(CHCl3): nmax 3445 (OH), 1789 (C]O, lactone), 1712 (C]O, ester),
1633 (C]C, cinnamate). 1H NMR (CDCl3):
d 2.54 (d, 1H,
J2a,2b ¼ 18.8 Hz, H-2a), 2.70 (dd, 1H, J2a,2b ¼ 18.8, J2b,3 ¼ 4.9 Hz, H-
2b), 3.84 (s, 3H, Me), 4.25 (dd, 1H, J5,6 ¼ 2.1, J6,7 ¼ 9.2 Hz, H-6), 4.26
(br s, 1H, OH), 4.43 (br d, 1H, J5,6 ¼ 1.8 Hz, H-5), 4.92e5.10 (m, 2H, H-
4.1.6.6. 3,6-Anhydro-7-O-(4-methoxycinnamoyl)-2-deoxy-7-C-
phenyl-
L-glycero-D-ido-heptono-1,4-lactone (epi-4b). Yield 42% (34%
of oxetane epi-10 was also isolated). Compound epi-4b: Colourless
20
0
0
3 and H-4), 6.00 (d,1H, J6,7 ¼ 9.2 Hz, H-7), 6.32 (d,1H, J2 ,3 ¼ 15.9 Hz,
powder, mp 93e95 ꢁC (Et2O/hexane), [
Rf ¼ 0.32 (Et2O). IR (CHCl3): nmax 3454 (OH), 1789 (C]O, lactone),
1714 (C]O, ester), 1633 (C]C, cinnamate). 1H NMR (CDCl3):
2.74
a]
ꢀ36.0 (c 0.1, CHCl3);
D
H-20), 7.85e8.39 (m, 9H, 2ꢂ Ph), 7.71 (d, 1H, J2 ,3 ¼ 15.9 Hz, H-30).
0
0
13C NMR (CDCl3):
d
35.8 (C-2), 55.4 (Me), 72.6 (C-5), 73.1 (C-7), 77.0
d
(C-3), 82.5 (C-6), 87.0 (C-4), 113.9 (C-20), 114.4, 126.4, 127.7, 128.6,
128.8, 130.1, 136.9, 161.9 (2ꢂ Ph), 146.9 (C-30), 167.8 (C-10), 175.4 (C-
1). HRMS (ESI): m/z 411.1435 (MþþH), calcd for C23H23O7: 411.1438.
(d, 2H, J2,3 ¼ 3.5 Hz, 2ꢂ H-2), 3.25 (br d, 1H, J5,OH ¼ 5.9 Hz, OH), 3.82
(s, 3H, OMe), 4.02 (br s,1H, H-5), 4.41 (dd,1H, J5,6 ¼ 2.9, J6,7 ¼ 8.8 Hz,
H-6), 4.85 (d, 1H, J3,4 ¼ 3.3 Hz, H-4), 5.09 (m, 1H, H-3), 6.19 (d, 1H,
J6,7 ¼ 8.8 Hz, H-7), 6.36 (d, 1H, J2 ,3 ¼ 15.9 Hz, H-20), 6.84e7.54 (m,
0
0
9H, 2ꢂ Ph), 7.64 (d,1H, J2 ,3 ¼ 16.0 Hz, H-3 ). 13C NMR (CDCl3):
d 36.0
0
0
0
4.1.6.3. 3,6-Anhydro-7-O-(4-nitrocinnamoyl)-2-deoxy-7-C-phenyl-
D
-
glycero-
D
-ido-heptono-1,4-lactone (4c). Yield 66% (31% of oxetane
(C-2), 55.3 (OMe), 73.9 (C-5), 74.6 (C-7), 77.3 (C-3), 83.0 (C-6), 87.6
(C-4), 115.1 (C-20), 114.3, 126.9, 127.5, 128.7, 128.8, 129.9, 136.8, 161.5
(2ꢂ Ph), 145.4 (C-30), 166.6 (C-10), 175.63 (C-1). HRMS (ESI): m/z
449.0996 (MþþK), calcd for C23H22KO7: 449.0997.
10 was also isolated). Compound 4c: Pale yellow powder, mp 195 ꢁC
20
(EtOAc/hexane), [
a]
þ133.0 (c 0.1, CHCl3); Rf ¼ 0.40 (3:2 EtOAc/
D
cyclohexane). IR (CHCl3): nmax 3479 (OH), 1786 (C]O, lactone), 1716
(C]O, ester), 1639 (C]C, cinnamate), 1520 and 1346 (NO2). 1H
NMR (CDCl3):
d
2.55 (d, 1H, J2a,2b ¼ 18.8 Hz, H-2a), 2.70 (dd, 1H,
4.1.6.7. 3,6-Anhydro-7-O-(4-nitrocinnamoyl)-2-deoxy-7-C-phenyl-
glycero- -ido-heptono-1,4-lactone (epi-4c). Yield 52% (32% of oxe-
tane epi-10 was also isolated). Compound epi-4c: Colourless pow-
L-
J2a,2b ¼ 19.0, J2b,3 ¼ 5.1 Hz, H-2b), 3.90 (d, 1H, J5,OH ¼ 3.6 Hz, OH),
4.29 (dd, 1H, J5,6 ¼ 2.3, J6,7 ¼ 9.2 Hz, H-6), 4.46 (dd, 1H, J5,6 ¼ 2.6,
J5,OH ¼ 2.9 Hz, H-5), 4.93e5.06 (m, 2H, H-3 and H-4), 6.03 (d, 1H,
D
20
der, mp 103e105 ꢁC (Et2O/hexane), [
a
]
ꢀ10.0 (c 0.1, CHCl3);
D
J6,7 ¼ 9.2 Hz, H-7), 6.59 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20), 7.31e8.39 (m,
Rf ¼ 0.32 (Et2O). IR (CHCl3): nmax 3441 (OH), 1787 (C]O, lactone),
0
0
9H, 2ꢂ Ph), 7.76 (d, 1H, J2 3 ¼ 16.0 Hz, H-3 ). 13C NMR (CDCl3):
d
35.8
1716 (C]O, ester), 1640 (C]C, cinnamate), 1521 and 1345 (NO2). 1H
0
0
0
(C-2), 73.2 (C-5), 73.3 (C-7), 77.1 (C-3), 82.2 (C-6), 87.0 (C-4), 121.0
(C-20), 124.2, 127.7, 128.7, 128.9, 129.1, 136.4, 139.7, 148.9 (2ꢂ Ph),
143.9 (C-30), 166.2 (C-10), 175.3 (C-1). HRMS (ESI): m/z 443.1448
(MþþNH4), calcd for C28H23N2O8: 443.1449.
NMR (CDCl3):
d
2.74 (d, 2H, J2,3 ¼ 3.2 Hz, 2ꢂ H-2), 3.98 (br s, 2H, H-5
and OH), 4.42 (dd, 1H, J5,6 ¼ 2.9, J6,7 ¼ 9.0 Hz, H-6), 4.84 (d, 1H,
J3,4 ¼ 4.2 Hz, H-4), 5.08 (m, 1H, H-3), 6.21 (d, 1H, J6,7 ¼ 9.0 Hz, H-7),
6.60 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20), 7.27e8.26 (m, 10H, 2ꢂ Ph and H-
0
0
30). 13C NMR (CDCl3):
d 36.0 (C-2), 73.7 (C-5), 75.4 (C-7), 77.4 (C-3),
4.1.6.4. 3,6-Anhydro-7-O-(4-fluorocinnamoyl)-2-deoxy-7-C-phenyl-
82.9 (C-6), 87.5 (C-4), 122.1 (C-20), 124.1, 127.6, 127.7, 128.7, 129.0,
136.3, 140.3, 148.5 (2ꢂ Ph), 142.5 (C-30), 165.2 (C-10), 175.6 (C-1).
HRMS (ESI): m/z 464.0739 (MþþK), calcd for C22H19KNO8:
464.0742.
D
-glycero-D-ido-heptono-1,4-lactone (4d). Yield 66% (31% of oxetane
10 was also isolated). Compound 4d: Colourless powder, mp 155 ꢁC
(EtOAc/hexane), [
20
a
]
þ129.0 (c 0.1, CHCl3); Rf ¼ 0.45 (3:2 EtOAc/
D
cyclohexane). IR (KBr): nmax 3451 (OH), 1775 (C]O, lactone), 1688
(C]O, ester), 1634 (C]C, cinnamate). 1H NMR (CDCl3):
d
2.54 (d,
4.1.6.8. 3,6-Anhydro-7-O-(4-fluorocinnamoyl)-2-deoxy-7-C-phenyl-
1H, J2a,2b ¼ 18.8 Hz, H-2a), 2.68 (dd, 1H, J2a,2b ¼ 19.0, J2b,3 ¼ 4.9 Hz,
H-2b), 4.17 (d, 1H, J5,OH ¼ 3.3 Hz, OH), 4.27 (dd, 1H, J5,6 ¼ 2.2,
J6,7 ¼ 9.2 Hz, H-6), 4.44 (br s,1H, H-5), 4.92e5.04 (m, 2H, H-3 and H-
L-glycero-D-ido-heptono-1,4-lactone (epi-4d). Yield 50% (56% based
on reacted 1; 35% of oxetane epi-10 was also isolated, along with
11% of recovered 1). Compound epi-4d: Colourless powder, mp
20
4), 6.01 (d, 1H, J6,7 ¼ 9.2 Hz, H-7), 6.38 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-20),
85e87 ꢁC (Et2O/hexane), [
a]
D ꢀ7.0 (c 0.1, CHCl3); Rf ¼ 0.37 (Et2O).
0
0
6.97e7.59 (m, 9H, 2ꢂ Ph), 7.71 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-3 ). 13C NMR
IR (CHCl3): nmax 3456 (OH), 1787 (C]O, lactone), 1710 (C]O, ester),
0
0
0
(CDCl3):
d
35.8 (C-2), 72.8 (C-5), 73.1 (C-7), 77.1 (C-3), 82.4 (C-6),
1636 (C]C, cinnamate). 1H NMR (CDCl3):
d
2.68 (br. s, 1H, OH), 2.73
87.0 (C-4), 116.4 (C-20), 116.0, 116.4, 127.7, 128.6, 128.8, 128.9, 130.2,
130.3, 136.7, 162.1, 166.2 (2ꢂ Ph), 145.8 (C-30), 167.2 (C-10), 175.4 (C-
1). HRMS (ESI): m/z 416.1504 (MþþNH4), calcd for C22H23FNO6:
416.1504.
(d, 2H, J2,3 ¼ 3.2 Hz, 2ꢂ H-2), 4.03 (br. s, 1H, H-5), 4.45 (dd, 1H,
J5,6 ¼ 2.9, J6,7 ¼ 8.8 Hz, H-6), 4.83 (d, 1H, J3,4 ¼ 4.3 Hz, H-4), 5.13 (m,
0
0
1H, H-3), 6.18 (d, 1H, J6,7 ¼ 8.8 Hz, H-7), 6.43 (d, 1H, J2 ,3 ¼ 16.0 Hz,
H-20), 6.96e7.59 (m, 9H, 2ꢂ Ph), 7.66 (d, 1H, J2 ,3 ¼ 16.0 Hz, H-30).
0
0
13C NMR (CDCl3):
d 36.0 (C-2), 74.0 (C-5), 74.8 (C-7), 77.4 (C-3), 83.0
4.1.6.5. 7-epi-crassalactone C (epi-4a) and 3,6:5,7-dianhydro-2-
(C-6), 87.4 (C-4), 117.5 (C-20), 115.9, 116.2, 127.6, 128.8, 129.0, 130.0,
130.1, 130.4, 130.5, 136.6, 162.0 (2ꢂ Ph), 144.3 (C-30), 166.9 (C-10),
175.4 (C-1). HRMS (ESI): m/z 437.0792 (MþþK), calcd for
deoxy-7-C-phenyl-
L
-glycero-
D
-ido-heptono-1,4-lactone
(epi-10).
Compound epi-4a: Yield 59%. Colourless powder, mp 120 ꢁC (phase
20
change at 75 ꢁC, CH2Cl2/hexane), [
a
]
ꢀ29.0 (c 0.5, EtOH), for
C22H19FKO6: 437.0797.
D
(þ)-enantiomer: lit. [14] mp 134e137 ꢁC (from EtOAc/hexane),
20
[
a
]
D þ16.4 (c 0.5, EtOH), Rf ¼ 0.18 (9:1 CH2Cl2/EtOAc). IR (CHCl3):
4.2. Cell lines
nmax 3458 (OH), 1786 (C]O, lactone), 1709 (C]O, ester), 1636 (C]
C, cinnamate). For 1H and 13C NMR spectral data see the
Supplementary Data. HRMS (ESI): m/z 403.1140 (MþþNa), calcd for
Human chronic myelogenous leukaemia (K562), human pro-
myelocytic leukaemia (HL-60), human T cell leukaemia (Jurkat) and
Burkitt's lymphoma (Raji) were grown in RPMI 1640, while ERþ
breast adenocarcinoma (MCF-7), ERꢀ breast adenocarcinoma
(MDA-MB-231), cervix carcinoma (HeLa) malignant cells and
normal foetal lung fibroblasts (MRC-5) were grown in DMEM me-
dium. Both media were supplemented with 10% of foetal calf serum
(FTS, NIVNS) and antibiotics (100 IU/mL of penicillin and 100 mg/
C
22H20NaO6: 403.1152.
Compound epi-10: Yield 25%. Colourless needles, mp 118ꢀ119 ꢁC
20
(CH2Cl2/hexane), [
a
]
D þ122.6 (c 0.5, CHCl3); Rf ¼ 0.27 (CH2Cl2). IR
(CHCl3): nmax 1789 (C]O), 1604 (Ph). 1H NMR (CDCl3): 2.55 (br d,
1H, J2a, 2b ¼ 18.2 Hz, H-2a), 2.64 (dd, 1H, J2a, 2b ¼ 18.3, J2b,3 ¼ 4.0 Hz,
H-2b), 4.67 (m, 1H, H-3), 5.00 (d, 1H, J3,4 ¼ 3.8 Hz, H-4), 5.31 (t, 1H,

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G. Benedekovic et al. / European Journal of Medicinal Chemistry 87 (2014) 237e247
247
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Acknowledgement
lactones: (þ)-crassalactones
B
and C, (þ)-howiionol A, (þ)-tricinnamate,
Financial support from the Ministry of Education, Science and
Technological Development of the Republic of Serbia (Grant No
172006) is gratefully acknowledged.
(þ)-goniofufurone and (þ)-dicinnamoyl goniofufurone, Tetrahedron Asym-
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