Synthesis and antiproliferative activity of two diastereomeric lignan amides serving as dimeric caffeic acid-l-DOPA hybrids

Article abstract of DOI:10.1016/j.bioorg.2016.04.003

Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-l-DOPA hybrids were synthesized. The synthesis involved the FeCl₃-mediated phenol oxidative coupling of methyl caffeate to afford trans-diester 1a as a mixture of en

Full text of DOI:10.1016/j.bioorg.2016.04.003

Bioorganic Chemistry 66 (2016) 132–144
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and antiproliferative activity of two diastereomeric lignan
amides serving as dimeric caffeic acid-L-DOPA hybrids
a
George E. Magoulas a, , Andreas Rigopoulos , Zoi Piperigkou b,c, Chrysostomi Gialeli b,d
,
⇑
Nikos K. Karamanos b,c, , Panteleimon G. Takis e, Anastassios N. Troganis e,
⇑
Athanassios Chrissanthopoulos f,1, George Maroulis f, Dionissios Papaioannou a
a Laboratory of Synthetic Organic Chemistry, Department of Chemistry, University of Patras, 26504 Patras, Greece
b Biochemistry, Biochemical Analysis & Matrix Pathobiology Research Group, Laboratory of Biochemistry, Department of Chemistry, University of Patras, Patras, Greece
c Foundation for Research and Technology-Hellas (FORTH)/Institute of Chemical Engineering Sciences (ICE-HT), Patras, Greece
d Lund University, Department of Laboratory Medicine, Malmö University Hospital, Malmö, Sweden
e Department of Biological Applications and Technology, University of Ioannina, Ioannina 45110, Greece
f Laboratory of Theoretical Chemistry, Department of Chemistry, University of Patras, 26504 Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Two new diastereomeric lignan amides (4 and 5) serving as dimeric caffeic acid-L-DOPA hybrids were
synthesized. The synthesis involved the FeCl3-mediated phenol oxidative coupling of methyl caffeate
to afford trans-diester 1a as a mixture of enantiomers, protection of the catechol units, regioselective
saponification, coupling with a suitably protected L-DOPA derivative, separation of the two diastereomers
Received 24 February 2016
Accepted 23 April 2016
Available online 23 April 2016
thus obtained by flash column chromatography and finally global chemoselective deprotection of the cat-
echol units. The effect of hybrids 4 and 5 and related compounds on the proliferation of two breast cancer
cell lines with different metastatic potential and estrogen receptor status (MDA-MB-231 and MCF-7) and
of one epithelial lung cancer cell line, namely A-549, was evaluated for concentrations ranging from 1 to
Keywords:
Lignans
Amides
L-DOPA
256
tent antiproliferative activities (IC50 64–70
but they were more selective and much more potent (IC50 4–16
treatment. The highest activity for both hybrids and both breast cancer lines was observed after 72 h
l
M and periods of treatment of 24, 48 and 72 h. Both hybrids showed interesting and almost equipo-
M) for the MDA-MB-231 cell line after 24–48 h of treatment,
M) for the MCF-7 cells after 48 h of
Phenol oxidative coupling
Antiproliferative activity
MDA-MB-231
MCF-7
A-549
l
l
of treatment (IC50 1–2
l
M), probably as the result of slow hydrolysis of their methyl ester functions.
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
biological activity. Lignans comprise a large family of optically
active compounds isolated from plant extracts sharing a common
feature, that is, two phenylpropanoid units coupled through the
central carbons of their propane side chains. In plants, the biosyn-
Nature has been always considered as a huge ‘‘laboratory”
providing constantly innumerous compounds with significant
thesis of lignans is probably realized through
a peroxidase
enzyme-catalyzed free radical dimerization of HCA derivatives
[1] or of their reduced analogs. This procedure, also known as
phenol oxidative coupling (POC), generates a large library of com-
pounds with structural diversity and hence an array of different
biological activities [2], with the anticancer activity being probably
the most important one. Indeed, polyphenolic compounds gener-
ated in nature exhibit a wide range of biological activities including
anti-inflammatory, antioxidant, antiapoptotic and cytotoxic
actions [3]. The naturally occurring lignan podophyllotoxin is the
starting material for the synthesis of etoposide which is used
against small cell lung cancer, lymphomas, leukemia or brain
tumor [4].
Abbreviations: Boc, tert- butoxycarbonyl; CAPE, caffeic acid phenethyl ester; L-
DOPA, (S)-2-amino-3-(3,4-dihydroxyphenyl)propanoic acid; ER, estrogen receptor;
FCC, flash column chromatography; FBS, fetal bovine serum; HCA, hydroxycinnamic
acid; PBS, phosphate-buffered saline; POC, phenol oxidative coupling; PyBrOP, br
omotripyrrolidinophosphonium hexafluorophosphate; TFA, trifluoroacetic acid.
⇑
Corresponding authors at: Institute of Biology, Medicinal Chemistry and
Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou
Avenue, 11635 Athens, Greece (G.E. Magoulas) and Laboratory of Biochemistry,
Department of Chemistry, University of Patras, 26504 Patras, Greece (N.K.
Karamanos).
E-mail addresses: magoulas@upatras.gr (G.E. Magoulas), n.k.karamanos@
upatras.gr (N.K. Karamanos).
1
Permanent address: Department of Chemistry, National and Kapodistrian
University of Athens, Athens 15771, Greece.
http://dx.doi.org/10.1016/j.bioorg.2016.04.003
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
133
Caffeic acid is one of the most prominent members of the HCA
family. It is considered one of the constituents responsible for the
antiproliferative effects of honeybee propolis [5]. In addition, many
studies have further established the wide range of its potential
antitumor activities. Its phenethyl ester (CAPE) can arrest the
growth of human leukemia HL-60 cells [6], it can act as a potential
antimetastatic agent [7] or it can play an important role for the
treatment and/or prevention of estrogen-related diseases, such as
breast or uterine cancer [8,9]. On the other hand, 1-aryl-1,
2-dihydronaphthalene 1a (Fig. 1), which can be derived from the
ferrous chloride-mediated oxidative dimerization of methyl caf-
feate, has shown a very interesting biological profile as a topoiso-
merase inhibitor [10] and was also found to be remarkably more
active than caffeic acid against breast cancer cells [11].
0 °C, followed by FCC purification, compound 1a was isolated as
the main product in 38% yield (75% based on recovered starting
material 10), in the form of a mixture of enantiomers (only one
enantiomer is drawn in the scheme).
The relative stereochemistry (trans) of the stereogenic centers
C1 and C2 in dimer 1a was established by measuring the coupling
constant (J = 2.8 Hz) of protons H1 and H2 in the 1H NMR spectrum
of the synthesized dimer 1a. This coupling constant is consistent
with J values reported for other trans-aryldihydronaphthalene
lignans [16,17]. Interestingly, quantum mechanical calculations
(see Supplementary material) of the structures of the two possible
diastereomers 1a and 1b (again only one enantiomer is drawn in
the scheme) anticipated as POC products from this experiment,
revealed that the dihedral angles H1-C1-C2-H2 (H31-C7-C8-H32
in Fig. 2) are ꢁ81.3 and 56.3°, respectively. The corresponding cal-
culated J values for the two diastereomers 1a (trans) and 1b (cis)
are 1.43 and 5.57 Hz, thus verifying the correctness of the proposed
structure 1a for the main product of POC of methyl caffeate. The
use of quantum chemical methods in this work aims at the calcu-
lation of stable molecular structures. The use of rigorous quantum
mechanical methods in recent years has added new impetus to
extended scope disciplines as organic synthesis [18] and medicinal
chemistry [19]. Highly performant quantum chemical methods can
provide rational interpretations to chemical phenomena. What is
more, the determination of accurate molecular descriptors via
the calculation of molecular properties enables the development
of realistic Quantitative Structure-Activity (QSAR) and Structure-
Property (QSPR) Relationships [20]. The choice of level of theory
usually employed in large molecular architectures depends on
their size. As the size of the molecules increases, the computational
cost of very accurate, time-consuming methods and large, flexible
basis sets becomes prohibitive [21–24].
Furthermore, N-trans-caffeoyl-L-DOPA (2), also known as
Clovamide, belongs to the class of N-phenylpropenoyl amino acids
and it was identified in the antioxidant polyphenolic fraction of
Theobroma cacao L. [12]. Clovamide or its methyl ester 3 have
shown a significant binding affinity for the p56lck SH2 domain,
highlighting that way interesting properties toward the treatment
of a wide range of diseases such as cancer, osteoporosis or chronic
inflammatory disease [13].
Over the last years, the synthesis of new compounds called
hybrids has gained ground among researchers. Hybrid molecules
can be derived from the covalent connection (through cleavable
or non-cleavable bonds) of two or more different chemical entities
with the intention to enhance the biological activity that each one
of these pharmacophores exhibits [14].
Prompted by the afore mentioned significant biological profiles
of both caffeic acid dimer 1a and hybrid compound Clovamide (2),
we thought of interest to synthesize the diastereomeric lignan
amides 4 and 5 (Fig. 1), which could serve as dimeric caffeic
acid-
L
-DOPA hybrids, and to further explore their antiproliferative
Furthermore, treatment of 1a with benzyl bromide in refluxing
acetone in the presence of K2CO3 led to the fully protected dimer
11 in 74% yield. Diester 11 was then subjected to regioselective
saponification with 1.0 equivalent LiOH in THF/MeOH/H2O to give
mono-acid ( )ꢁ12 in 55% yield.
activity initially on two breast cancer cell lines, namely MDA-MB-
231 and MCF-7 with different metastatic potential and estrogen
receptor (ER) status, and one epithelial lung cancer cell line,
namely A-549.
Having both key-intermediates 9 and 12 at hand, the assembly
of the projected skeleton was readily realized using PyBrOP as cou-
pling agent in the presence of Et3N. That way, the fully protected
lignan amides 13 and 14 were obtained in 63% yield as a mixture
of diastereomers. Complete separation and isolation of amides 13
and 14 in almost equimolar amounts was accomplished through
FCC (Scheme 3). The final step of the synthesis involved the global
deprotection of all catechol units. Due to the presence of the dou-
ble bond in the dihydronaphthalene moiety, the obvious use of H2
in the presence of Pd/C was not examined as the method of choice
for the chemoselective removal of the benzyl groups. Interestingly,
Lamidey et al. [25] had described a very efficient silane-promoted
Pd-mediated hydrogenolysis en route the synthesis of (-)-(2R,3R)-
chicoric acid. By applying this particular methodology to each of
the diastereomeric amides 13 and 14, the projected compounds 4
and 5 were obtained in 68% and 72% yield, respectively, following
FCC purification.
2. Results and discussion
2.1. Chemistry
The synthesis of the projected lignan amides 4 and 5 was
accomplished using a convergent methodology. The two requested,
suitably protected, building blocks 9 and 12 for the assembly of the
skeleton of these amides (Scheme 3) were synthesized from
L-DOPA (Scheme 1) and caffeic acid (Scheme 2), respectively. Com-
pound 9 was obtained after TFA deprotection of fully protected
compound 8 which was synthesized according to a published pro-
cedure [15] (see Supplementary material). Thus, commercially
available L-DOPA was converted to its methyl ester 6 in 95% yield,
after treatment with SOCl2 in MeOH at ambient temperature. The
latter was protected at its amino function with the Boc group,
using di-tert-butyl dicarbonate in THF in the presence of a satu-
rated aqueous solution of NaHCO3 as base. That way, compound
7 was obtained in 85% yield. The next step involved the protection
of the catechol unit, which was realized upon treatment of com-
pound 7 with benzyl bromide in refluxing acetone in the presence
of K2CO3. Following routine purification by FCC, the fully protected
compound 8 was obtained in 92% yield. Finally, TFA-mediated
N-deprotection provided the trifluoroacetate salt 9 in 93% yield.
On the other hand, compound 1a was readily synthesized
according to a published procedure [16]. Thus, methyl caffeate
10 [17] was subjected to a POC reaction with an aqueous solution
of FeCl3ꢀ6H2O in acetone (Scheme 2). After overnight stirring at
The structure of compounds 4 and 5 was established by
analytical and spectroscopic methods and in particular NMR.
Table S5 (in supplementary material) contains the complete proton
and carbon assignment of compounds 4 and 5. In particular, the
absolute stereochemistry (configuration) of the two stereogenic
centers at C1 and C2 (the third stereogenic center at C11 is of the
S configuration due to the L-DOPA used as starting material) of
the diastereomers 4 and 5 was determined on the basis of NMR
studies as well as quantum mechanical calculations of the struc-
tures of the two configurational arrangements, that is 1S,2R,11S
(diastereomer 4) and 1R,2S,11S (diastereomer 5). A noticeable
difference, which seems to be of particular diagnostic interest, is

134
G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
Fig. 1. Structures of compounds related to the present work.
Scheme 1. Synthetic route for the preparation of suitably protected
L-DOPA key-intermediate 9. Reagents and conditions: (i) SOCl2, MeOH, 16 h, RT, 95%; (ii) Boc2O, sat. aq.
NaHCO3, THF, 1 h, RT, 85%; (iii) PhCH2Br, K2CO3, Acetone, 5 h, reflux, 92%; and (iv) 50% TFA/CH2Cl2, 1 h, 93%.
in the chemical shifts and coupling patterns of protons H11 and
H12a and H12b in the -DOPA moiety of the two diastereomers.
mechanical calculation of the two configurational arrangements,
L
that is 1S, 2R, 11S (diastereomer 4) and 1R, 2S, 11S (diastereomer
5), showed (Fig. 3 and supplementary material) that restricted
rotation is indeed imposed in the diastereomer 5 and thus the less
polar diastereomer should be correlated to structure 4 and accord-
ingly, the more polar diastereomer should be correlated to
structure 5.
Thus, in the 1H NMR spectrum of the less polar diastereomer
(Rf = 0.22, see experimental section), these particular protons res-
onate at d 4.499 and 2.815 ppm and appear as triplet (J = 6.4 Hz)
and doublet (J = 6.4 Hz), respectively. On the other hand, in the
1H NMR spectrum of the more polar diastereomer (Rf = 0.13, see
experimental section), the H11 proton resonate at d 4.476 as dd
(J = 5.6 and 7.6 Hz) and the H12a and H12b protons resonate at
2.871 ppm as dd (J = 5.6 and 14 Hz) and at 2.746 also as dd
(J = 7.6 and 14 Hz).
2.2. Biology
In order to determine possible antiproliferative effects of the
diastereomeric lignan amides 4 and 5 against cancer cells, the cell
proliferation/viability rates in three cancer biological models,
including two breast and one lung cancer cell lines, was evaluated.
This is taken to mean that in the more polar diastereomer
restricted rotation is imposed on the
L-DOPA aliphatic chain, thus
making the H12a and H12b protons diasterotopic. Quantum

G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
135
Scheme 2. Synthetic route for the preparation of mono-acid ( )-12. Reagents and conditions: (i) H2SO4, MeOH, 1 h, reflux, 88%; (ii) FeCl3ꢀ6H2O, Acetone, H2O, overnight, 0 °C,
38% (75% based on recovered 10); (iii) PhCH2Br, K2CO3, Acetone, 16 h, reflux, 74%; and (iv) LiOH, THF/MeOH/H2O (3:1:1), 16 h, RT, 55%.
Fig. 2. Minimum energy structures of diastereomers 1a (trans) and 1b (cis) as obtained through quantum mechanical calculations.
We included in these studies the dimeric methyl caffeate 1a, the
methyl ester 3 of the amide hybrid caffeic acid- -DOPA (Clo-
vamide), and the methyl esters 6 and 10 of -DOPA and caffeic acid,
respectively, in order to draw meaningful conclusions on the effect
of dimerization of caffeic acid on the potential antiproliferative
activity of these compounds. The effects on cell proliferation were
determined upon treatment of cancer cells with these compounds
increasingly regarded as oncoprotective [26], whereas compounds
3 and 6 exhibit anticancer activity. Indeed, it was generally
observed that after 24 h of treatment with compounds 3 and 6,
cancer cells exhibited a dose-dependent response; this effect was
more profound in higher concentrations. Furthermore, compounds
1a and 10 induced a dose-dependent effect in the three cancer cell
lines, after a 24 h treatment period with elevated concentrations of
L
L
in increasing concentrations, ranging between 1 and 256
l
M, and
these compounds (1–256
more aggressive breast cancer cell line MDA-MB-231 with an IC50
value at low concentrations (16 M). In addition, as for the two
dimeric caffeic acid- -DOPA hybrids, 4 and 5, the effect on MDA-
MB-231 breast cancer cell line was more intense than in MCF-7
low metastatic breast cancer cells.
In particular, the involvement of all tested compounds in the
mediation of the proliferation capacity of A-549 lung cancer cells
was first assessed. This assessment revealed that the compounds
exhibited antiproliferative activity even at low concentrations
(Fig. 4). More specifically, it was observed that compounds 3, 5
and 6 exhibited dose-dependent effects on cell proliferation rates
lM). This effect was more intense in the
for time periods of 24, 48 and 72 h. In the present study, 0.1%
DMSO in H2O for dissolution and dilution of the sample to be
tested was used. This concentration of DMSO was utilized since fol-
lowing pilot experiments no cytotoxic effects were found. The
breast cancer cell lines used were MDA-MB-231 (highly metastatic
breast cancer cell line) and MCF-7 (low metastatic potential breast
cancer cell line), whereas that for lung cancer was A-549 epithelial
lung cancer cell line.
l
L
The initial biological evaluation generally revealed that the
majority of the compounds tested in the three cancer cell lines
exhibited statistically significant antiproliferative effects in
a
dose- and in some cases time-dependent manner at most of the
doses applied. It has been reported that the role of caffeic acid is
with IC50 values of 130
sented antiproliferative action even at the lowest concentration
lM (Table 1). However, the hybrid 5 pre-

136
G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
Scheme 3. Synthesis of lignan amides 4 and 5. Reagents and conditions: (i) Et3N, PyBrOP, CHCl3, 40 min, RT, 63%; and (ii) Pd(OAc)2, Et3N, Et3SiH, CH2Cl2, overnight, RT, 68%
(for 4), 72% (for 5).
Fig. 3. Minimum energy structures of diastereomers 4 and 5 as obtained through quantum mechanical calculations.
tested (1
l
M), as it reduced significantly cell viability (40%). On the
plays a significant role in activity, with 4 being much less active
than 5.
other hand, hybrid 4 did not cause significant reduction of cell pro-
liferation of lung cancer cells. Moreover, compounds 1a and 10
exhibited stronger effects in a dose-dependent manner (IC50 32
As far as MDA-MB-231 breast cancer cells are concerned
(Fig. 5), the obtained data revealed that, following 24 h incubation
period, the hybrids 4 and 5 as well as compound 6 exhibited a
dose-dependent effect in breast cancer cell proliferation, with
IC50 values at higher concentrations. Although compound 3 exhib-
ited a slight antiproliferative effect, it reduced significantly cell
proliferation rates of the highly metastatic breast cancer cells
(60%), as compared to untreated cells, at the highest concentration
and 40 lM, respectively).
Thus, with the exception of 4, there is a strong anticancer action
of these compounds on A-549 lung cancer cells, after 24 h of treat-
ment, a fact that can be correlated with the antiproliferative poten-
tial on our cancer cellular system, verifying the already known
literature results mentioned above. Interestingly, dimerization of
CA leads to a slightly better antiproliferative effect, whereas conju-
gation of
DOPA but it lowers ca. 3 times the activity of CA. In addition,
although conjugation of dimeric CA (1a) to -DOPA, creating hybrid
tested (256
more intense effects among all the derivatives tested, as their
IC50 values are 16 M (Table 1).
Interestingly, dimerization of CA does not change the antiprolif-
erative effect of the monomer (CA), whereas conjugation of -DOPA
to CA lowers ca. 2 times the activity of -DOPA and 16 times the
lM). As for compounds 1a and 10, they pinpointed
L-DOPA to CA does not offer any particular advantage to L-
l
L
5, leads to a less active compound compared to 1a itself. It appears
that the relative stereochemistry of diastereomeric hybrids 4 and 5
L
L

G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
137
Fig. 4. Dose-dependent responses of A-549 lung cancer cells to the tested compounds. The concentration range was from 1 to 256
l
M and the incubation time 24 h. The
results are expressed as mean SD from experiments in triplicate. Symbols mark the statistically significant levels as follows: (⁄), (⁄⁄) and (⁄⁄⁄) indicate p < 0.05, p < 0.01 and
p < 0.005, respectively, as compared to control cells.
activity of CA. In addition, conjugation of dimeric CA (1a) to L-
Table 1
DOPA, creating hybrid 4 or 5, leads to a 4 times less active com-
pound compared to 1a itself, it appears that in this cancer line
the relative stereochemistry of diastereomeric hybrids 4 and 5
does not play any role in activity.
Treatment of MCF-7 breast cancer cells with these compounds
for 24 h induced only slight effects in cell proliferation rates
(Fig. 6). More specifically, in the presence of the hybrid 5 and also
of compounds 3 and 6 a reduction in cell proliferation up to 40%, as
compared to control cells, was observed. Moreover, cell prolifera-
tion reduced up to 50%, as compared to untreated cells, in the pres-
ence of both 5 and 1a only near the highest concentration tested
IC50 values of three cancer cell lines upon treatment with the tested compounds for
24 h.
Compound
Cancer Cell Lines
A-549
MDA-MB-231
MCF-7
IC50 Values (lM)
1a
3
6
10
4
5
32
130
130
40
>256
130
16
256
130
16
64
64
128
>256
>256
60
>256
256
(256
lM). Only in the case of 10 there is a strong antiproliferative
effect in the proliferation capacity of MCF-7 breast cancer cells.

138
G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
Fig. 5. Dose-dependent responses of MDA-MB-231 breast cancer cells to the tested compounds. The concentration range was from 1 to 256 lM and the incubation time 24 h.
The results are expressed as mean SD from experiments in triplicate. Symbols mark the statistically significant levels as follows: (⁄⁄) and (⁄⁄⁄) indicate p < 0.01 and p < 0.005,
respectively, as compared to control cells.
Interestingly, dimerization of CA almost halves the antiprolifer-
ative effect of the monomer (CA), whereas conjugation of -DOPA to
CA does not seem to offer any particular advantage on the activity of
-DOPA. In addition, conjugation of dimeric CA (1a) to -DOPA, cre-
ating hybrid 4 or 5, almost halves the activity of 1a itself. Also, in
this cancer line the relative stereochemistry of diastereomeric
hybrids 4 and 5 does not seem to play any noticeable role in activity.
Taking into consideration these data, it is plausible to suggest
that the anticancer effect of the derivatives tested for a short-
term treatment is related with the metastatic potential of breast
cancer cells and that there is a potential for more intense effects
after long-term treatment with these compounds.
[27–29]. This discrimination may be correlated with the differenti-
ated metastatic potential of these two breast cancer cell lines.
L
While ER
has been thoroughly studied, the role of its isoform ERb is less elu-
cidated. In addition, it is documented that both ER and ERb pre-
a contribution in breast cancer growth and development
L
L
a
sent antagonistic actions, regulating the cell behavior in breast
cancer initiation and differentiation [30,31].
Taking into account the fact that the methyl esters are slowly
catabolized in the body, the antiproliferative effect of the two
new hybrids 4 and 5 after a long-term treatment (48, 72 h) was
evaluated. We focused on the breast cancer cell lines, in order to
highlight the possible anticancer effect in the breast cancer cellular
model. We expected that the IC50 of the hybrids will be lower than
that observed following a 24 h treatment. Indeed, it was found that
after 48 h treatment with hybrids 4 and 5, the IC50 values (Table 2)
were significantly lower than those of 24 h of treatment. More
It is well established that MDA-MB-231 and MCF-7 breast can-
cer cells are characterized by different ER status. More specifically,
MCF-7 breast cancer cells are indicated as ER
a positive, whereas
MDA-MB-231 cells are positive for ERb and negative for ER
a

G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
139
Fig. 6. Dose-dependent responses of MCF-7 breast cancer cells to the tested compounds. The concentration range was from 1 to 256
lM and the incubation time 24 h. The
results are expressed as mean SD from experiments in triplicate. Symbols mark the statistically significant levels as follows: (⁄), (⁄⁄) and (⁄⁄⁄) indicate p < 0.05, p < 0.01 and
p < 0.005, respectively, as compared to control cells.
Table 2
specifically, as far as MDA-MB-231 breast cancer cells are
IC50 values of breast cancer cell lines (MDA-MB-231 and MCF-7) upon treatment with
the tested compound for 48 and 72 h.
concerned, it was observed that 48 h of treatment with both 4
and 5 resulted in a dose-dependent decrease in proliferation rates
with increased concentration (Fig. 7).
This effect becomes more profound when highly metastatic
breast cancer cells are treated with the tested materials for 72 h
Compound
Cancer Cell Lines
MDA-MB-231
MCF-7
48 h
48 h
72 h
lM)
72 h
(Fig. 8). In this case, a 1
lated. This is significantly lower compared to that following a 48 h
treatment (IC50 value was 70 M for both tested hybrids). As for
MCF-7 breast cancer cell line, our data revealed that the IC50 values
after 48 h treatment were approximately 16 M and 4 M regard-
lM IC50 value for both 4 and 5 was calcu-
IC50 Values (
4
5
70
70
1
1
16
4
2
1
l
l
l
ing 4 and 5, respectively (Fig. 7). On the other hand, when these
cells were long-term-treated with the tested materials (72 h) the
relative stereochemistry of the two hybrids does not play any sig-
nificant role in the highly metastatic cancer lines after long-term
treatment whereas for the MCF-7 cancer lines hybrid 4 seems to
IC50 values become significantly lower (2
lM and 1 lM regarding
4 and 5, respectively) (Fig. 6). It is therefore apparent that the

140
G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
Fig. 7. Dose-dependent responses of MDA-MB-231 and MCF-7 breast cancer cells to the tested diastereomeric lignan amides 4 and 5. The concentration range was from 1 to
256
M and the incubation time 48 h. The results are expressed as mean SD from experiments in triplicate. Symbols mark the statistically significant levels as follows: (⁄⁄
indicate p < 0.01, as compared to control cells.
l
)
Fig. 8. Dose-dependent responses of MDA-MB-231 and MCF-7 breast cancer cells to the tested diastereomeric lignan amides 4 and 5. The concentration range was from 1 to
256
M and the incubation time 72 h. The results are expressed as mean SD from experiments in triplicate. Symbols mark the statistically significant levels as follows: (⁄⁄
indicate p < 0.01, as compared to control cells.
l
)

G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
141
be 4 and 2 times more active than hybrid 5 after 48 and 72 h treat-
ment, respectively.
unless otherwise stated, on a Perkin-Elmer 16PC FT-IR spectropho-
tometer. Routine 1H NMR spectra were recorded at 400.13 MHz
and 13C NMR spectra at 100.62 MHz on a Bruker DPX spectrometer.
Tetramethylsilane (TMS) was used as internal standard. Chemical
shifts are reported in d units, parts per million (ppm) downfield
from TMS. 2D homonuclear and heteronuclear NMR experiments
were performed on a Bruker AV-500, equipped with a cryo-
probe. Electro-spray ionization (ESI) mass spectra were recorded
on a Micromass-Platform LC spectrometer using MeOH as solvent.
Elemental analyses were determined on a Carlo Erba EA 1108
CHNS elemental analyzer in the Instrumental Analysis Centre of
the University of Patras and the results are within 0.4% of the cal-
culated values. Optical rotations were determined with a half-
automatic Schmidt + Haensch Polatronic D MHZ-8 at the sodium
D line (589 nm) using a 500 mm path-length cell and are reported
as follows: ½aꢂ2D5, concentration (g/100 mL) and solvent. FCC was
performed on Merck silica gel 60 (230–400 mesh) and TLC on 60
Merck 60 F254 films (0.2 mm) precoated on aluminum foil. Spots
were visualized with UV light at 254 nm and the ninhydrin or
the charring agent. All solvents were dried and/or purified accord-
ing to standard procedures prior to use. Anhydrous Na2SO4 was
used for drying organic solvents and subsequently solvents were
routinely removed at ca. 40 °C under reduced pressure (water aspi-
rator). All reagents employed in the present work were purchased
from commercial suppliers and used without further purification.
Reactions were run in flame-dried glassware under an atmosphere
of argon with the exception of those involving aqueous solutions.
Clovamide methyl ester (3) [13] was synthesized according to pub-
lished procedure.
3. Conclusions
Two diastereomeric amide-type hybrids 4 and 5 of POC-derived
dimeric CA with L-DOPA were efficiently synthesized using a con-
vergent methodology. The two key-intermediates required for
the assembly of the skeleton of the hybrids, namely compounds
9 and 12 were obtained in ca. 30% and 69% overall yield from the
commercially available L-DOPA and caffeic acid, respectively. Cou-
pling of these intermediates, afforded two diastereomeric com-
pounds (13 and 14) which could be easily separated by FCC. Each
one of these fully protected compounds was subjected to a
palladium-mediated chemoselective deprotection of the catechol
functionalities providing an almost equimolar mixture of hybrids
4 and 5, in 44% yield for the two steps.
The antiproliferation effect of the hybrids 4 and 5, the dimeric
methyl caffeate 1a, the methyl ester 3 of the CA-L-DOPA (Clo-
vamide), and the methyl esters 6 and 10 of -DOPA and CA, respec-
L
tively, on the epithelial lung cancer cell line A-549, the highly
metastatic breast cancer cell line MDA-MB-231 and the low meta-
static potential breast cancer cell line MCF-7 was evaluated for con-
centrations ranging from 1 to 256 lM and for periods of treatment
of 24, 48 and 72 h. The preliminary biological evaluation of the
compounds at 24 h revealed that hybrids 4 and 5 were particularly
active and equipotent on the MDA-MB-231 cell line and of very low
activity on the MCF-7 cell line, whereas on the A-549 cell line
hybrid 5 was substantially more active than hybrid 4. Interestingly,
most active compounds in on all three cell lines examined were the
methyl esters of monomeric (10) and the dimeric (1a) CA, thus
4.1.1. Dimethyl 1-(3,4-dihydroxyphenyl)-6,7-dihydroxy-1,2-
dihydronaphthalene-2,3-dicarboxylate [( )-1a]
showing that their conjugation to L-DOPA does not offers any
advantage on their antiproliferative potential. Also, dimerization
of CA seems not to lead to a particularly more active compound.
In particular, as concerns the breast cancer cells, the antiprolif-
erative effects of these hybrids were much stronger in MCF-7
breast cancer cells after 48 and 72 h treatment, but in both breast
cancer cell lines, there was a dose-dependent effect, as it was con-
ducted by the rudimentary biological evaluation. It has been
reported that CA derivatives, such as CAPE, have estrogenic effects
and it seems to have selective binding affinity to ERb rather than
To a solution of ester 10 (3.88 g, 20 mmol) in acetone (120 mL),
a solution of FeCl3ꢀH2O (6.05 g, 22.4 mmol) in H2O (5.2 mL) was
added at 5 °C over 1.5 h and the mixture was kept at 0 °C over-
night. Then, the reaction mixture was evaporated to a minimum
volume and extracted thrice with Hex/EtOAc (1:1). The combined
organic layers were washed twice with water, dried over Na2SO4
and evaporated to dryness. The residue was subjected to FCC using
CHCl3/MeOH (95:5) as eluent to collect unreacted caffeic acid
(1.54 g) and then CHCl3/MeOH (9:1) to obtain pure ( )-1a.
Yield: 1.47 g (38%); white solid, mp: 238–240 °C, lit [16]. mp:
239–242 °C; Rf (CHCl3/MeOH 9:1): 0.26; IR (KBr, cmꢁ1): 3384,
1716, 1686, 1610, 1458, 1266; MS (ESI, 30 eV): m/z 795.19 [2 M
+Na], 425.30 [M+K], 409.37 [M+Na]; 1H NMR (d6-DMSO): d 7.56
(s, 1H), 6.88 (s, 1H), 6.56 (d, J = 8.0 Hz, 1H), 6.48 (s, 1H), 6.30 (d,
J = 2.0 Hz, 1H), 6.25 (dd, J = 2.0 and 8.0 Hz, 1H), 4.27 (d, J = 2.8 Hz,
1H), 3.72 (d, J = 2.8 Hz, 1H), 3.65 (s, 3H), 3.53 (s, 3H); 13C NMR
(d6-DMSO): d 172.8, 167.0, 148.4, 145.3, 144.8, 144.4, 138.5,
134.6, 129.5, 123.0, 120.9, 118.4, 116.9, 116.6, 115.9, 115.0, 52.6,
52.1, 47.5, 45.0.
ERa [9]. Indeed, we observed that our compounds exhibited higher
IC50 values for the MDA-MB-231 than the MCF-7 breast cancer
cells, but only for the 24 h treatment, whereas for longer periods
of treatment, that is 48 h, both hybrids showed much higher activ-
ity for the MCF-7 cell line with hybrid 4 being 4 times less active
than hybrid 5. A powerful antiproliferative effect is however
observed after 72 h of treatment for both hybrids and both cell
lines with hybrid 4 being more selective for MDA-MB-231. The
higher antiproliferative activity of hybrids 4 and 5 after longer
periods of treatment is probably due to the slow hydrolysis of their
methyl ester moieties, indicating that these compounds act as pro-
drugs. The observed differentiated effects of these compounds
among the two breast cancer cell lines suggest that there is a cor-
relation with the different ER status among them. This generates
4.1.2. Dimethyl 6,7-bis(benzyloxy)-1-(3,4-bis(benzyloxy)phenyl)-1,2-
dihydronaphthalene-2,3-dicarboxylate [( )-11]
To a suspension of ( )-1 (0.96 g, 2.48 mmol) and K2CO3 (2.73 g,
19.86 mmol) in acetone (10 mL), benzyl bromide (2.36 mL,
19.86 mmol) was added. The reaction mixture was refluxed over-
night and then filtrated under vacuo. The filtrate was evaporated;
the residue was taken up in CH2Cl2, washed twice with water,
dried over dried over Na2SO4 and evaporated to dryness. The oily
residue was subjected to FCC using Hex/EtOAc (9:1) to elute excess
benzyl bromide and then Hex/EtOAc (7:3) to obtain compound ( )-
11.
the suggestion that the present dimeric CA-L-DOPA hybrids serve
as selective estrogen receptor modulators, which identifies them
as effective tools for breast cancer focused treatment approaches.
4. Material and methods
4.1. Chemistry
Melting points were determined with a Buchi SMP-20 appara-
tus and are uncorrected. IR spectra were recorded as KBr pellets,
Yield: 1.37 g (74%); light yellow oil; Rf (Hex/EtOAc7:3): 0.25; IR
(KBr, cmꢁ1): 3030, 2950, 1734, 1634, 1508, 1238, 1012, 738, 696;

142
G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
MS (ESI, 30 eV): m/z 785.23 [M+K], 769.30 [M+Na], 747.19 [M+H];
1H NMR (CDCl3): d 7.53 (s, 1H), 7.50–7.31 (m, 20H), 6.92 (s, 1H),
6.79 (d, J = 8.0 Hz, 1H), 6.78 (s, 1H), 6.54 (d, J = 8.0 Hz, 1H), 6.52
(s, 1H), 5.20 (s, 2H), 5.13 (s, 2H), 5.06 (s, 2H), 5.02 (s, 2H), 4.55
(d, J = 2.8 Hz, 1H), 3.95 (d, J = 2.8 Hz, 1H), 3.76 (s, 3H), 3.62 (s,
3H); 13C NMR (CDCl3): d 172.8, 166.9, 150.9, 148.5, 148.0, 147.8,
137.5, 137.4, 137.2, 137.1, 136.6, 135.6, 130.9, 128.6 (two C),
128.5 (four C), 128.4 (two C), 127.9 (two C), 127.7 (two C), 127.4
(two C), 127.3 (two C), 127.2 (four C), 124.6, 122.6, 120.5,115.6,
115.0, 114.9, 114.7, 71.5, 71.3, 71.1, 70.9, 52.4, 51.9, 47.2, 45.5.
4.1.4.2. Methyl (1R,2S)-6,7-bis(benzyloxy)-1-(3,4-bis(benzyloxy)phe-
nyl)-2-(((S)-3-(3,4-bis(benzyloxy)phenyl)-1-methoxy-1-oxopropan-
2-yl)carbamoyl)-1,2-dihydronaphthalene-3-carboxylate (14). Yield:
25
0.27 g (30%); colorless oil; ½aꢂD = ꢁ142 (c 0.66, CHCl3); Rf (Hex/
EtOAc 6:4): 0.15; IR (KBr, cmꢁ1): 3029, 2930, 1735, 1671, 1517,
1248, 1136, 1022, 741, 696; MS (ESI, 30 eV): m/z 1144.23 [M+K],
1128.31 [M+Na], 1106.35 [M+H]; 1H NMR (CDCl3): d 7.53 (s, 1H),
7.48–7.29 (m, 30H), 6.88 (d, J = 8.0 Hz, 1H), 6.87 (s, 1H), 6.78–
6.72 (m, 3H), 6.68 (d, J = 8.0 Hz, 1H), 6.59 (unresolved dd, 1H),
6.51 (unresolved dd, 1H), 6.48 (unresolved d, 1H), 5.18–4.96 (m,
12H), 4.72–4.66 (m, 1H), 4.56 (unresolved d, 1H), 3.75 (unresolved
d, 1H), 3.70 (s, 3H), 3.51 (s, 3H), 3.00–2.96 (m, 2H); 13C NMR
(CDCl3): d 171.4, 170.9, 167.5, 151.5, 148.9, 148.3, 148.0, 147.6,
147.5, 138.7, 137.3, 137.2, 137.1, 137.0 (two C), 136.5, 136.3,
132.1, 129.2, 128.5–127.1 (14 signals, thirty C), 123.6, 122.2,
121.5, 120.3, 115.9, 115.6, 115.0, 114.6, 114.5 (two C), 71.4, 71.3,
71.2, 71.1, 70.9, 70.6, 53.2, 52.0, 51.9, 48.3, 44.8, 37.2.
4.1.3. 6,7-bis(benzyloxy)-1-(3,4-bis(benzyloxy)phenyl)-3-
(methoxycarbonyl)-1,2-dihydronaphthalene-2-carboxylic acid [( )-
12]
To an ice-cold solution of ( )-11 (1.3 g, 1.74 mmol) in THF/
MeOH (3:1, 14 mL), a solution of LiOH (0.042 g, 1.74 mmol) in
H2O (3.5 mL) was added and the reaction mixture was stirred at
ambient temperature overnight. Then, it was acidified with 0.5 N
HCl and extracted twice with EtOAc. The combined organic layers
were washed twice with water, dried over Na2SO4 and evaporated
to dryness. The residue was subjected to FCC using CHCl3/MeOH
(95:5) as eluent to afford compound ( )-12. Unreacted starting
material can be recovered from FCC and subjected once again to
saponification.
Yield: 0.70 g (55%); colorless oil; Rf (CHCl3/MeOH 95:5): 0.20; IR
(KBr, cmꢁ1): 3300–2900, 1704, 1602, 1508, 1240, 1134, 1014, 737,
696; MS (ESI, 30 eV): m/z 771.25 [M+K], 755.19 [M+Na]; 1H NMR
(CDCl3): d 7.54 (s, 1H), 7.49–7.29 (m, 20H), 6.92 (s, 1H), 6.77 (d,
J = 8.4 Hz, 1H), 6.71 (s, 1H), 6.49 (d, J = 8.4 Hz, 1H), 6.47 (s, 1H),
5.18 (s, 2H), 5.12 (s, 2H), 5.01 (s, 2H), 5.00 (s, 2H), 4.89 (d,
J = 2.8 Hz, 1H), 3.92 (d, J = 2.8 Hz, 1H), 3.76 (s, 3H); 13C NMR
(CDCl3): d 177.0, 167.6, 151.2, 148.5, 148.1, 147.8, 138.1, 137.4,
137.1, 137.0, 136.5, 135.6, 130.8, 128.6 (two C), 128.5 (four C),
128.4 (two C), 128.0 (two C), 127.8 (two C), 127.5 (two C), 127.3
(two C), 127.2 (four C), 124.2, 121.2, 120.3, 115.7, 115.0, 114.8,
114.6, 71.6, 71.2, 71.1, 70.9, 52.2, 47.1, 44.9.
4.1.5. Global deprotection – Synthesis of hybrids 4 and 5
To a solution of Pd(OAc)2 (0.081 g, 0.36 mmol) in CH2Cl2
(2.5 mL) Et3N (50 lL, 0.36 mmol) was added and the mixture was
stirred for 10 min. Then, a solution of 13 or 14 (0.17 g, 0.15 mmol)
in CH2Cl2 (2.5 mL) was added dropwise over 30 min. The resulting
mixture was stirred for 10 min followed by the dropwise addition
of Et3SiH (0.58 mL, 3.6 mmol) over 1 h. Finally, the reaction was
stirred at ambient temperature overnight. Addition of MeOH fol-
lowed by filtration and evaporation of the filtrate gave an oily resi-
due which was diluted EtOAc, washed twice with an aqueous
solution 5% citric acid and once with water, dried over Na2SO4
and evaporated to dryness. Final products were obtained after
FCC purification using CHCl3/MeOH (8:2) as eluent.
4.1.5.1. Methyl (1S,2R)-1-(3,4-dihydroxyphenyl)-2-(((S)-3-(3,4-dihy-
droxyphenyl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-6,7-dihy-
droxy-1,2-dihydronaphthalene-3-carboxylate (4). Yield: 0.057 g
25
(68%); yellowish solid, mp: 234–237 °C (decomp.); ½aꢂD = +197 (c
0.6, MeOH); Rf (CHCl3/MeOH 8:2): 0.22; IR (KBr, cmꢁ1): 3380,
2924, 1726, 1692, 1642, 1606, 1518, 1440, 1336, 1262; MS (ESI,
30 eV): m/z 604.23 [M+K], 588.23 [M+Na], 566.38 [M+H]; 1H
NMR (d4-MeOH): d 7.61 (s, 1H), 6.86 (s, 1H), 6.61 (d, J = 2.0 Hz,
1H), 6.59 (d, J = 2.0 Hz, 1H), 6.52 (s, 1H), 6.47 (d, J = 8.0 Hz, 1H),
6.40 (d, J = 8.0 Hz, 1H), 6.38 (dd, J = 2.0 and 8.0 Hz, 1H), 6.15 (dd,
J = 2.0 and 8.0 Hz, 1H), 4.50 (t, J = 6.4 Hz, 1H), 4.27 (d, J = 2.8 Hz,
1H), 3.71 (d, J = 2.8 Hz, 1H), 3.67 (s, 3H), 3.65 (s, 3H), 2.81 (d,
J = 6.4 Hz, 2H); 13C NMR (d4-MeOH): d 172.8, 171.8, 167.8, 148.1,
144.8, 144.6, 144.1, 143.9, 143.5, 139.7, 135.2, 130.6, 127.4,
123.2, 120.8, 120.3, 118.6, 116.1, 116.0, 115.7, 115.0, 114.8,
114.4, 53.7, 51.3, 51.1, 49.0, 45.7, 36.2.
4.1.4. Synthesis of lignan amides 13 and 14
To an ice-cold solution of amine 9 (0.54 g, 1.05 mmol), acid ( )-
12 (0.6 g, 0.81 mmol) and Et3N (0.45 mL, 3.24 mmol) in CHCl3
(1.2 mL), PyBrOP (0.48 g, 1.05 mmol) was added. The mixture
was stirred at 0 °C for 10 min and then at ambient temperature
for additional 30 min. Then, the reaction mixture was diluted with
CHCl3, washed twice with water, dried over Na2SO4 and evapo-
rated to dryness to give a mixture of diastereomers which were
separated by routine FCC using Hex/EtOAc (6:4) as eluent.
4.1.4.1. Methyl (1S,2R)-6,7-bis(benzyloxy)-1-(3,4-bis(benzyloxy)phe-
nyl)-2-(((S)-3-(3,4-bis(benzyloxy)phenyl)-1-methoxy-1-oxopropan-
2-yl)carbamoyl)-1,2-dihydronaphthalene-3-carboxylate (13). Yield:
4.1.5.2. Methyl (1R,2S)-1-(3,4-dihydroxyphenyl)-2-(((S)-3-(3,4-dihy-
droxyphenyl)-1-methoxy-1-oxopropan-2-yl)carbamoyl)-6,7-dihy-
droxy-1,2-dihydronaphthalene-3-carboxylate (5). Yield: 0.061 g
25
0.30 g (33%); White foam; ½aꢂD = +132 (c 1.0, CHCl3); Rf (Hex/EtOAc
6:4): 0.26; IR (KBr, cmꢁ1): 3033, 2924, 1740, 1686, 1508, 1240,
1136, 1018, 736, 696; MS (ESI, 30 eV): m/z 1144.39 [M+K],
1128.39 [M+Na], 1106.03 [M+H]; 1H NMR (CDCl3): d 7.47–7.27
(m, 32H), 6.85 (s, 1H), 6.80 (d, J = 7.2 Hz, 1H), 6.77 (d, J = 5.6 Hz,
1H), 6.74 (d, J = 8.0 Hz), 6.64 (d, J = 1.6 Hz, 1H), 6.48 (s, 1H), 6.47
(d, J = 7.2 Hz, 1H), 6.31 (dd, J = 1.6 and 8.0 Hz, 1H), 5.13–4.99 (m,
12H), 4.75 (m, 1H), 4.61 (unresolved d, 1H), 3.74 (unresolved d,
1H), 3.67 (s, 3H), 3.66 (s, 3H), 2.97 (dd, J = 5.2 and 14.0 Hz, 1H),
2.86 (dd, J = 6.8 and 14.0 Hz, 1H); 13C NMR (CDCl3): d 171.8,
170.8, 167.7, 151.6, 148.9, 148.3, 147.8, 147.6, 147.5, 138.6,
137.3, 137.2, 137.1, 137.0, 136.9, 136.5, 136.2, 132.4, 129.0,
128.4–127.1 (13 signals, thirty C), 123.6, 122.0, 121.3, 120.3,
115.7, 115.6, 114.9, 114.7, 114.6, 114.5, 71.4, 71.2, 71.1, 71.0,
70.9, 70.6, 53.0, 52.1, 52.0, 48.1, 44.5, 37.0.
25
(72%); yellowish solid, mp: 204–207 °C (decomp.); ½aꢂD = ꢁ170 (c
0.6, MeOH); Rf (CHCl3/MeOH 8:2): 0.13; IR (KBr, cmꢁ1): 3382,
2927, 1734, 1690, 1642, 1518, 1444, 1337, 1262; MS (ESI, 30 eV):
m/z 604.26 [M+K], 588.27 [M+Na], 566.43 [M+H]; 1H NMR (d4-
MeOH): d 7.59 (s, 1H), 6.79 (s, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.61
(d, J = 8.0 Hz, 1H), 6.51 (d, J = 2.0 Hz, 1H), 6.44 (s, 1H), 6.40–6.38
(m, 2H), 6.36 (dd, J = 2.0 and 8.0 Hz, 1H), 4.48 (dd, J = 5.6 and
7.6 Hz, 1H), 4.06 (d, J = 2.8 Hz, 1H), 3.69 (s, 3H), 3.67 (d,
J = 2.8 Hz, 1H), 3.56 (s, 3H), 2.87 (dd, J = 5.6 and 14 Hz, 1H), 2.75
(dd, J = 7.6 and 14 Hz, 1H); 13C NMR (d4-MeOH): d 173.0, 171.8,
167.6, 147.9, 145.0, 144.6, 144.1, 143.9, 143.6, 139.7, 135.2,
130.4, 127.6, 123.2, 121.0, 120.4, 118.7, 116.0, 115.9, 115.7,
115.1, 114.8, 114.4, 53.5, 51.2, 51.1, 49.3, 46.2, 36.3.

G.E. Magoulas et al. / Bioorganic Chemistry 66 (2016) 132–144
143
4.2. Biology
method for quadrature detection, while COSY 2D spectra were
acquired using QF method. The 2D measurements were recorded
using 512 increments of 2 K complex data points and 16 scans
per increment for 2D 1H ROESY, 8 scans for 2D 1H TOCSY and 4 scans
for 2D COSY experiments, respectively. The mixing time for ROESY
spectra was 200 ms, and that for TOCSY was 100 ms. 1H–13C HSQC
and HMBC 2D spectra were acquired using echo-antiecho and QF
method for detection, respectively. Hetero-nuclear experiments
were recorded using 1 K increments of 2 K complex data points
and 4 scans per increment for 1H–13C HSQC and 32 scans for
1H–13C HMBC experiments, respectively. Data were processed
using the TOPSPIN standard software. The t1 dimension for both
homonuclear and heteronuclear experiments was zero-filled to
1 K real data points, and 60° phase-shifted square sine bell window
functions were applied in both dimensions, except for COSY and
HMBC experiments where 0° phase-shifted sine bell window func-
tions were applied in both dimensions.
4.2.1. Cell cultures
MCF-7 and MDA-MB-231 breast cancer cell lines were obtained
from the American Type Culture Collection (ATCC). A-549 human
lung carcinoma cell line was obtained from Cell Lines Service
(CLS, Germany). Breast cancer cells were cultured in complete
medium Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS). Lung cancer cells were
cultured as monolayers in DMEM Ham’s F12 supplemented with
5% (v/v) FBS. Cell culture mediums were supplemented with
1.0 mM sodium pyruvate, 2 mM
antimicrobial agents (100 IU/mL penicillin, 100
mycin, 10 g/mL gentamycin sulfate and 2.5 g/mL amphotericin
L
-glutamine and a cocktail of
lg/mL strepto-
l
l
B). Cells were routinely grown at 37 °C in a humidified atmosphere
of 5% (v/v) CO2 and 95% air. Culture medium was changed every
48–72 h and the cultures were not left to become confluent. Cells
were harvested by trypsinization with 0.05% (w/v) trypsin in PBS
containing 0.02% (w/v) Na2EDTA.
Acknowledgements
4.2.2. Proliferation assay
We thank the Research Committee of the University of Patras
for funding the present work. We also thank Dr. M. Fousteris and
Mrs Dimanthi Pliatsika, Department of Pharmacy, University of
Patras for their help with the optical rotation measurements. We
are grateful to Dr C. Tsiafoulis (NMR Center, University of Ioannina)
for his help with NMR spectra collection using the Bruker AV-500
spectrometer.
MDA-MB-231 and MCF-7 breast cancer cells and A-549 lung
cancer cells were seeded in the presence of serum into 96-well
plates at a density of 5000, 7500 and 4500 cells/well, respectively,
and then the agents were added according to the experimental
plan, in elevated concentrations (1, 2, 4, 8, 16, 32, 64, 128,
256 lM) in serum-free culture medium. After 24 h, 48 h or 72 h
of incubation, Premix WST-1 (water-soluble tetrazolium salt) Cell
Proliferation Assay System (Takara Bio Inc., Japan) was added at
a ratio 1:10. The assay is based on the reduction of WST-1 by viable
cells, producing a soluble formazan salt absorbing at 450 nm
(reference wavelength at 650 nm).
Appendix A. Supplementary material
Experimental procedures for the synthesis of compound 9 and
full characterization of all intermediates. Quantum chemical calcu-
lations for compounds 1a, 1b, and diastereomeric lignan amides 4
and 5. Selected 1H and 13C NMR spectra of key intermediates and
final products.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bioorg.2016.04.
003. These data include MOL files and InChiKeys of the most
important compounds described in this article.
4.2.3. Statistical analysis
Reported values are expressed as mean standard deviation
(SD) of experiments in triplicate. Statistically significant differ-
ences were evaluated using the analysis of variance (ANOVA) test
and were considered statistically significant at the level of at least
p 6 0.05.
4.3. Theoretical calculations
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C, N and O centers [33]. The exponent of the d-GTF is
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