Dihydrobenzofuran Neolignanamides: Laccase-Mediated Biomimetic Synthesis and Antiproliferative Activity

Article abstract of DOI:10.1021/acs.jnatprod.6b00577

The biomimetic synthesis of a small library of dihydrobenzofuran neolignanamides (the natural trans-grossamide (4) and the related compounds 21-28) has been carried out through an eco-friendly oxidative coupling reaction mediated by Trametes versicolor laccase. These products, after complete spectroscopic characterization, were evaluated for their antiproliferative activity against Caco-2 (colon carcinoma), MCF-7 (mammary adenocarcinoma), and PC-3 (prostate cancer) human cells, using an MTT bioassay. The racemic neolignamides (±)-21 and (±)-27, in being the most lipophilic in the series, were potently active, with GI₅₀ values comparable to or even lower than that of the positive control 5-FU. The racemates were resolved through chiral HPLC, and the pure enantiomers were subjected to ECD measurements to establish their absolute configurations at C-2 and C-3. All enantiomers showed potent antiproliferative activity, with, in particular, a GI₅₀ value of 1.1 μM obtained for (2R,3R)-21. The effect of (±)-21 on the Caco-2 cell cycle was evaluated by flow cytometry, and it was demonstrated that (±)-21 exerts its antiproliferative activity by inducing cell cycle arrest and apoptosis.

Full text of DOI:10.1021/acs.jnatprod.6b00577

Article
pubs.acs.org/jnp
Dihydrobenzofuran Neolignanamides: Laccase-Mediated Biomimetic
Synthesis and Antiproliferative Activity
†
†
,†
‡
‡
̀
Nunzio Cardullo, Luana Pulvirenti, Carmela Spatafora,* Nicolo Musso, Vincenza Barresi,
‡
†
Daniele Filippo Condorelli, and Corrado Tringali
†
‡
Dipartimento di Scienze Chimiche and Dipartimento di Scienze Biomediche e Biotecnologiche, Sezione di Biochimica Medica,
Universita degli Studi di Catania, Viale A. Doria 6, I-95125 Catania, Italy
̀
*
S Supporting Information
ABSTRACT: The biomimetic synthesis of a small library of dihydrobenzofuran neolignanamides (the natural trans-grossamide
4) and the related compounds 21−28) has been carried out through an eco-friendly oxidative coupling reaction mediated by
(
Trametes versicolor laccase. These products, after complete spectroscopic characterization, were evaluated for their
antiproliferative activity against Caco-2 (colon carcinoma), MCF-7 (mammary adenocarcinoma), and PC-3 (prostate cancer)
human cells, using an MTT bioassay. The racemic neolignamides (±)-21 and (±)-27, in being the most lipophilic in the series,
were potently active, with GI50 values comparable to or even lower than that of the positive control 5-FU. The racemates were
resolved through chiral HPLC, and the pure enantiomers were subjected to ECD measurements to establish their absolute
configurations at C-2 and C-3. All enantiomers showed potent antiproliferative activity, with, in particular, a GI value of 1.1 μM
50
obtained for (2R,3R)-21. The effect of (±)-21 on the Caco-2 cell cycle was evaluated by flow cytometry, and it was demonstrated
that (±)-21 exerts its antiproliferative activity by inducing cell cycle arrest and apoptosis.
4
5
ne of the reasons for the renewed interest in small
molecules of natural origin is their structural diversity,
which, as the result of millions of years of evolution, affords
selected molecules with biological properties that are advanta-
geous for the organism producing them. A clear example of this
is the family of lignans, as well as related compounds
their biological activities, including antioxidant, antibacterial,
6
7
4,8,9
O
anti-inflammatory, cardiovascular, and cytotoxic effects.
Frequently cited examples are the cytotoxic and antiangio-
genic 3′,4-di-O-methylcedrusin (1), the active principle of
Dragon’s blood (Croton spp.), and licarin A (2), found in the
10
11
1
2
wood of Licaria aritu and reported as having antioxidant,
1
3
12,13
14
(
neolignans, oxyneolignans, and mixed lignans), distributed
antiviral, cytotoxic,
and neuroprotective activities.
widely within the plant kingdom, which have an extraordinary
variety of structures and biological properties. These com-
pounds share a common origin from the shikimate pathway,
but different biosynthetic mechanisms, normally through
oxidative coupling of two phenylpropanoid (C C ) units, lead
These biological reports make dihydrobenzofuran neolignans
attractive targets for chemical synthesis or modification. In
particular, biomimetic oxidative coupling reactions may afford
“unnatural” products, maintaining a basic “natural” skeleton.
Due to the lack of stereocontrol in both metal- and enzyme-
mediated radical coupling, racemic mixtures are frequently
obtained. Nevertheless, several interesting products have been
obtained in such a manner, and in some cases a bioactive
racemate has been resolved to obtain the more active
enantiomer. A representative example is the synthetic neolignan
6
3
to remarkably different carbon skeletons. According to the
15
1
IUPAC recommendations, the term “lignan” refers to a dimer
generated by a β−β′ (8−8′) oxidative coupling of two
propylbenzene residues, whereas the term “neolignan” should
be used for a compound formed by other than 8−8′ coupling.
One of the most well-documented bioactive lignans is
podophyllotoxin, from which the optimization has afforded
anticancer drugs such as etoposide, teniposide, and etopo-
3
, obtained by oxidative dimerization of caffeic acid methyl
ester (here only the 2R,3R-enantiomer is reported), and (±)-3
2
,3
phos. Among the neolignans, those with a dihydrobenzofuran
core are worthy of particular attention for the wide range of
Received: June 23, 2016
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00577
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
1
showed antiangiogenic and nanomolar antiproliferative
(dimethylamino)]phosphonium hexafluorophosphate (BOP)
as a carboxyl activating agent. According to Scheme 1, nine
amides (12−20) were synthesized, and, among these, N-trans-
feruloyl tyramine (12), N-trans-caffeoyl tyramine (15), and N-
trans-p-coumaroyl tyramine (18) are bioactive naturally
occurring products. The other hydroxycinnamoyl amides have
not been reported previously. These amides were used as
substrates for a biomimetic dimerization reaction. To explore
the catalytic activity of commercially available oxidases, amides
8
potency toward HL 60 human leukemia cells. When the
enantiomers isolated were evaluated for growth inhibition of
HL 60 cells, (2R,3R)-3 showed higher inhibitory activity than
2S,3S)-3. Dihydrobenzofurans bearing amide functions (neo-
lignanamides) are infrequently isolated from natural sources
and include trans-grossamide (4), isolated from Hyoscyamus
niger and other plants, which has shown anti-inflammatory
and mild cytotoxic activity, and the antifungal and
(
16
1
7
18
19
1
2−20 were employed in a preliminary screening on an
adrenergic receptor antagonist hordatine A (5), isolated
initially from barley seedlings (Hordeum vulgare).
analytical scale with three basidiomycete laccases, namely, those
from Agaricus bisporus (AbL), Pleurotus ostreatus (PoL), and
Trametes versicolor (TvL), and with horseradish peroxidase
Given their relative rarity in Nature and the limited number
of biological studies concerning these compounds, neo-
lignanamides are worthy of deeper investigation. In addition,
it is known that amides generally show a higher metabolic
stability than their isosteric esters, and this may be of crucial
importance if the compounds under study can be degraded by
intracellular esterases before reaching their biological target.
Hence, as a continuation of studies on biomimetic synthesis of
(
HRP). Two sets of experiments were carried out in a biphasic
system using acetate buffer (where the enzyme was solubilized),
and ethyl acetate (EtOAc) or dichloromethane (CH Cl ). A
2
2
further set of reactions was carried out in a monophasic system
consisting of 1% dimethyl sulfoxide (DMSO) in acetate buffer.
The small-scale reactions were carried out at the optimal
28
20−24
25
enzyme activity, namely, at 25 °C and pH 4.5. The reactions
were monitored by HPLC-UV on a C18 reversed-phase silica
gel column at regular time intervals in order to find the best
reaction conditions. For example, the HPLC-UV profiles for
the reactions of 13 in the presence of the four enzymes,
employing three different cosolvents, are reported in Figure
lignans and neolignans with antitumor,
antiangiogenic properties,
antioxidant, and
2
6,27
we have now synthesized a small
library of neolignanamides through an eco-friendly, enzyme-
mediated oxidative coupling reaction. As detailed below, a
preliminary screening with different oxidases indicated Trametes
versicolor laccase as the most convenient enzyme for these
syntheses. The products were purified, characterized, and
evaluated for their antiproliferative activity against Caco-2
1
a−d. No products were obtained by AbL-mediated reaction
even after 24 h, either for amide 13 (Figure 1a) or for the other
substrates evaluated. The HRP-mediated reaction (Figure 1b)
gave a low conversion of 13 into a less polar product when the
reaction was carried out with EtOAc, and even less promising
results were observed in CH Cl and DMSO. Poor results were
(
human colon cancer cells), MCF-7 (breast adenocarcinoma
cells), and PC-3 (human prostate cancer cells). The results and
some structure−activity relationship considerations are re-
ported herein.
2
2
obtained using HRP also with the other substrates. The PoL-
mediated reaction of 13 (Figure 1c) showed the formation of a
main product, less polar than the substrate, with the conversion
only partial with EtOAc and poor with the other cosolvents
used. Analogous results were obtained with other amides. The
best results were obtained with a TvL-mediated reaction
(
Figure 1d), giving almost complete conversion of 13 into a
major product within 4 h, when EtOAc was used as cosolvent.
Also in this case, the reactions were inferior with CH Cl and
2
2
DMSO. Analogously, the other feruloyl amides 12 and 14, as
well as compounds 18−20 (p-coumaroyl amides), gave a major
product in the EtOAc biphasic system, whereas the caffeoyl
amides 15−17 afforded a main product in the CH Cl biphasic
2
2
system.
On the basis of this preliminary screening, Trametes versicolor
laccase was employed for preparative reactions on amides 12−
20. These afforded the natural trans-grossamide (4) and the
previously unreported dimeric neolignanamides 21−28
(
Scheme 1).
As detailed below, the analysis of spectroscopic data for these
compounds clearly indicated the formation of dihydrobenzo-
furan dimers. The mechanism of formation of synthetic
dihydrobenzofuran neolignans in both metal- and enzyme-
RESULTS AND DISCUSSION
■
A small library of new bioinspired neolignanamides was
prepared by suitable monomeric amides by an amidation
reaction of natural phenolic acids, namely, p-coumaric acid (6),
caffeic acid (7), and ferulic acid (8). We employed three amines
characterized by different steric hindrance and lipophilicity
parameters, namely, 2-(4-hydroxyphenyl)ethylamine (9, a
natural product also known as tyramine), 4-methylbenzylamine
10), and 3,4,5-trimethoxybenzylamine (11). Reactions were
carried out under basic conditions (triethylamine, TEA) and in
the presence of (1H-benzotriazol-1-yloxy)[tris-
29,30
mediated reactions has been previously investigated
and is
based on 8−5′ radical coupling, according to Scheme 2. A
reactive quinone-methide intermediate undergoes intramolec-
ular cyclization, giving rise to the dihydrobenzofuran core. It is
worth highlighting that this dimerization, even in enzyme-
mediated reactions, occurs with regio- and diastereoselectivity,
29
(
but not enantioselectivity, and consequently affords trans-
substituted racemic mixtures. In Schemes 1 and 2 only one of
the two enantiomers is reported. The observed lack of
B
DOI: 10.1021/acs.jnatprod.6b00577
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Journal of Natural Products
Article
a
Scheme 1. Synthetic Procedure for Neolignanamides (± )-4 and (± )-21−(± )-28
a
Conditions: (a) DMF, TEA, 0 °C, 15 min, BOP solution (CH Cl ) 0 °C, 30 min, rt, 24 h; (b) TvL solution (acetate buffer, 0.1 M, pH = 4.7),
2
2
EtOAc, 4 h, for 4, 21, 22; (c) TvL solution (acetate buffer, 0.1 M, pH = 4.7), CH Cl , 2 h, for 23−25; (d) TvL solution (acetate buffer, 0.1 M, pH =
2
2
4
.7), EtOAc−DMSO, 24 h, for 26−28. Only the 2R,3R enantiomers are reported.
Figure 1. HPLC-UV chromatograms of 13 in the presence of (a) Agarigus bisporus laccase at 24 h; (b) horseradish peroxidase at 12 h; (c) Pleurotus
ostreatus laccase at 24 h; (d) Trametes versicolor laccase at 4 h.
enantioselectivity in enzyme-mediated oxidative coupling
reactions is not surprising when considering the known
biosynthetic mechanism involving a “dirigent protein” able to
control the stereochemistry of the reaction, and, in the absence
by flash chromatography in 16% yield (see Experimental
Section for details), was subjected to spectroscopic analysis, and
1
13
the ESIMS and H and C NMR data were in agreement with
32
those previously reported and allowed the identification of
this product as (±)-4.
15
of this protein, racemic mixtures are obtained. The observed
diastereoselectivity in favor of the trans-substituted diaster-
eomers is reasonably due to the lower steric hindrance in the
transition state (TS) leading to the trans product, as supported
The feruloyl amide 13, treated with TvL, gave one main
product. The molecular formula, C H N O , was determined
3
6
36
2
6
−
by elemental analysis and ESIMS, affording a [M − H] peak at
m/z 591.3. This indicated the formation of a dimeric product
and was consistent with the expected structure (±)-21. The H
and C NMR spectra were similar to those of trans-grossamide,
23,31
by previous literature data for similar coupling mechanisms.
1
trans-Grossamide (4), bearing two tyramine pendant groups,
was obtained by treating the monomeric feruloyl amide 12 with
TvL in ethyl acetate−acetate buffer. The main product, isolated
13
1
13
(±)-4. However, (±)-21 was subjected to extensive H and C
C
DOI: 10.1021/acs.jnatprod.6b00577
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Journal of Natural Products
Article
Scheme 2. Mechanism of Formation of (± )-4 and (± )-21−(± )-28
NMR analysis, including two-dimensional methods (COSY,
HSQC, and HMBC), in order to establish unambiguously its
structure and assign all the H and C NMR signals, as
reported in Tables 1 and 2. Thus, a dihydrobenzofuran core
was suggested by two mutually coupled proton signals
resonating at 6.07 (H-2) and 4.34 ppm (H-3) and by the
corresponding carbon signals at 88.0 (C-2) and 57.0 ppm (C-
17, gave a main product (24% yield), showing an ESIMS peak
−
at m/z 715.2 ([M − H] ) and with the molecular formula,
1
13
C H N O , consistent with the expected structure (±)-25. In
38
40
2
12
1
13
all these cases, the analyses of both H and C NMR spectra
were aided by two-dimensional NMR methods, thus confirming
the structures of (±)-23, (±)-24, and (±)-25 and allowing the
unambiguous assignment of all NMR resonances (Tables 1 and
2).
3), and this was corroborated by the key HMBC correlations of
H-3, H-2′, and H-6′ with C-2; of H-2 and H-4 with C-3; and of
H-3 and H-6 with C-7a (Figure 2). Unambiguous assignments
of the resonances for the two 4-methylbenzyl pendant groups
were obtained through the following HMBC correlations: from
H-2, H-3, H-9 (NH), and H-10 to C-8; from H-2″/H-6″ to C-
The syntheses of the three p-coumaroyl neolignanamides,
(±)-26−(±)-28 (with respective yields of 16%, 29%, and 34%),
were carried out through TvL-mediated dimerization of amides
20−22 in the biphasic solvent system EtOAc−1% DMSO−
acetate buffer. Also these products were subjected to
spectroscopic characterization, in which elemental analysis
and ESIMS gave the expected molecular formulas and [M −
1
3
8
0; from H-10 and H-3″/H-5″ to C-1″; from H-2″/H-6″, H-
″/H-5″, and OCH -4″ to C-4″ (C-8 pendant); from H-7′, H-
3
−
′, H-10′ (NH), and H-11′ to C-9′; from H-2‴/H-6‴ to C-11′;
H] peaks, respectively, at m/z 563.3 (26), 531.3 (27), and
from H-11′ and H-3‴/H-5‴ to C-1‴; from H-2‴/H-6‴, H-3‴/
683.4 (28). The dimeric structures were confirmed by analysis
1
13
H-5‴, and OCH -4‴ to C-4‴ (C-9′ pendant). The trans
of H and C NMR spectra aided by two-dimensional
experiments, thus allowing the assignments of all NMR peaks
(Tables 1 and 2).
3
relative configuration of the C-2 and C-3 substituents was
established on the basis of the J value of 8.3 Hz for the H-2/H-
3
3
3
8
7
coupling. The expected E configuration of the C-7′ and C-
′ double bond was confirmed by the J value of 15.5 Hz for H-
′/H-8′. Thus, structure (±)-21 was established for this
The above-mentioned racemic neolignanamides (±)-4 and
(±)-21−(±)-28 were evaluated for their antiproliferative
activity against three human cancer cell lines, namely, Caco-2
(human colon carcinoma), MCF-7 (human mammary
adenocarcinoma), and PC-3 (human prostate cancer) cells,
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
dimeric neolignanamide.
The feruloyl amide 14, treated as above, afforded one main
product (17% yield), obtained after purification. The molecular
formula was found to be C H N O , based on elemental
34
mide (MTT), according to the Mosmann procedure. In
Table 3, only results for compounds showing at least a GI50
value lower than 10 μM are included. The anticancer drug 5-
fluorouracil (5-FU) was used as positive control.
4
0
44
2
12
−
analysis and the ESIMS spectrum ([M − H] peak at m/z
43.3), indicating the formation of a dimer. The structure of
7
the expected neolignanamide (±)-22, with 3,4,5-trimethox-
ybenzyl pendant substituents, was established on the basis of
Notwithstanding the close structural analogy of the
compounds under study, their GI50 values showed a significant
variation and suggested some structure−biological activity
relationship information. Racemic neolignanamides bearing
tyramine (4, 23, and 26) or 3,4,5-trimethoxybenzyl pendants
(22, 25, and 28) were inactive or showed only moderate
growth inhibition. In particular, the natural trans-grossamide
[(±)-4] was inactive toward Caco-2 cells (GI > 100 μM) and
1
13
the analysis of the H and C NMR spectra as supported by
two-dimensional methods, which allowed also the complete
1
13
assignments of H and C NMR signals (Tables 1 and 2). The
trans substitution at C-2, C-3 and the E configuration at C-7′,
C-8′ were established as above, through coupling constant
measurements.
A similar procedure was applied for the syntheses of the
50
caffeoyl neolignanamides, employing TvL in a CH Cl −acetate
only moderately active against MCF-7 and PC-3 cells, with GI50
values of 24.0 and 21.0 μM, respectively. A similar profile was
observed for (±)-23, which was inactive toward Caco-2 cells
and weakly active toward both PC-3 (GI50 = 66.0 μM) and
MCF-7 cells (GI50 = 39.7 μM). In turn, (±)-26 was evaluated
as moderately active, with GI50 values of 28.9 (Caco-2), 33.2
(MCF-7), and 26.0 μM (PC-3). A mild or weak activity was
observed for the racemate 22 toward MCF-7 (GI50 = 23.0),
2
2
buffer biphasic solvent system. The amide 15 gave the dimer
(
±)-23 (15% yield) with a molecular formula of C H N O ,
34 32 2 8
−
based on both elemental analysis and the ESIMS ([M − H] at
m/z 595.1). Under the same conditions, 16 afforded in 16%
yield the dimeric neolignanamide (±)-24, showing in its ESIMS
the expected peak at m/z 563.2 ([M − H] ), indicating a
molecular formula of C H N O . The third caffeoyl amide,
−
3
4
32
2
6
D
DOI: 10.1021/acs.jnatprod.6b00577
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1
Table 1. H NMR Data of Compounds (± )-21−(± )-28 (500 MHz)
a
a
b
a
a
a
c
c
(
±)-21
(±)-22
(±)-23
(±)-24
(±)-25
(±)-26
(±)-27
(±)-28
δH, mult. (J
position
δH, mult. (J Hz) δH, mult. (J Hz) δH, mult. (J Hz) δH, mult. (J Hz) δH, mult. (J Hz) δH, mult. (J Hz) δH, mult. (J Hz)
Hz)
2
3
6.07, d (8.3)
4.34, d (8.3)
6.10, d (8.3)
4.35, d (8.3)
5.00, d (6.9)
4.40, d (6.9)
5.18, d (6.5)
4.78, d (6.5)
5.34, d (5.5)
4.87, d (5.5)
6.04, d (8.0)
4.14, d (8.0)
6.69, d (8.0)
4.77, d (8.0)
6.75, d (8.3)
4.81, d
d
(
8.3)
e
f
4
6
7.16, s
7.11, s
7.13, s
7.06, s
6.73, s
6.95, s
6.84, bs
7.86, bs
7.86, s
g
f
h
i
6.83, s
6.97, s
6.97, s
7.46, dd (2.0,
7.56, d (8.0)
7.56, d (8.5)
8
.5)
7
1
6.81, d (8.5)
3.72, m
7.01, d (8.0)
4.83, d (6.0)
7.04, d (8.5)
4.98, m
0
4.46, t (5.0)
4.59, dd (7.0,
3.20, m
3.33, m
2.53, m
4.37, dd (6.0,
15.0)
4.37, dd (5.7,
15.0)
1
5.0)
4
.31, dd (6.0,
5.0)
4.21, dd (6.0,
15.0)
4.22, dd (5.7,
15.0)
3.44, m
4.81, m
1
l
1
1
2.91−2.73, m
2
.43, m
f
h
h
i
i
2′
3′
4′
5′
7.02, d (2.0)
7.04, d (2.0)
6.84, d (8.0)
7.16, d (2.0)
6.97,d (2.0)
7.14, d (2.0)
6.95, d (8.5)
7.21, d (8.5)
6.86, d (8.5)
OH 8.48, bs
6.86, d (8.5)
7.53, d (8.5)
7.22, d (8.5)
7.56, d (8.5)
7.22, d (8.5)
OH 7.69, bs
6.84, d (8.0)
7.10, dd (2.0,
6.83, d (8.0)
7.22, d (8.5)
7.53, d (8.5)
7.22, d (8.5)
7.56, d (8.5)
8.16, d
8
.4)
6
7
8
1
′
7.88, dd (2.0,
6.89, dd (2.0,
8.0)
6.97, d (8.4)
7.43, d (15.7)
6.44, d (15.7)
3.46, t (7.4)
6.76, dd (2.0,
8.0)
7.09, dd (2.0,
8.5)
7.21,d (8.5)
8.0)
′
7.49, d (15.5)
6.57, d (15.5)
4.47, d (5.5)
7.47, d (16.0)
6.38, d (16.0)
4.46, d (4.5)
7.48, d (15.5)
6.66, d (15.5)
4.45, d (5.5)
7.47, d (16.0)
6.60, d (16.0)
4.43, d (5.5)
7.45, d (16.0)
6.48, d (16.0)
3.56, t (6.5)
8.13, d (15.5)
6.92, d (15.5)
4.87, dd
(
15.5)
6.86, d
15.5)
′
(
1′
4.90, t (6.0)
(
6.5,13.5)
l
1
2
3
4
2
3
4
2′
2.75, t (7.4)
7.05, d (8.4)
2.91−2.73, m
m
n
″/6″
″/5″
″
‴/6‴
‴/5‴
‴
7.22, d (8.0)
7.13, d (8.0)
6.67, s
6.70, s
7.04, d (8.0)
6.87, d (8.0)
6.46, s
6.66, s
7.10, d (8.5)
6.83, d (8.5)
OH 8.79, bs
7.10, d (8.5)
6.78, d (8.5)
OH 8.15 bs
7.44, d (7.5)
7.17, d (7.5)
6.90, s
e
6.72, d (8.4)
g
m
n
7.24, d (8.0)
7.15, d (8.0)
6.84, d (8.5)
7.22, d (8.0)
7.12, d (8.0)
7.47, d (7.5)
7.20, d (7.5)
6.94, s
6.66, d (8.5)
Me-4″
Me-4‴
2.30, s
2.28, s
3.90, s
3.81, s
2.62, s
2.61, s
2.29, s
2.26, s
OMe-7
OMe-3′
OMe-3″/
3.90, s
3.82, s
3.83, s
3.72, s
3.76, s
5″
OMe-4″
3.72, s
3.76, s
3.67, s
3.79, s
3.93, s
3.80, s
OMe-3‴/
5‴
OMe-4‴
NH-9
3.68, s
3.69, s
3.97, s
7.95, bt (5.0)
7.53, bt (5.5)
8.07, t (6.0)
7.88, t (5.5)
7.89, t (5.7)
7.59, t (5.5)
9.72, t (6.0)
9.11, t (6.5)
NH-10′
8.05, bt (4.5)
7.49, t (6.5)
8.97, t (6.0)
a
Recorded in acetone-d . Recorded in methanol-d . Recorded in pyridine-d . d−nSignals with the same superscript are partially overlapped.
b
c
6
4
5
PC-3 cells (GI50 = 38.0 μM), and Caco-2 cells (GI50 = 59.1
μM), while compound (±)-25 was inactive toward all three cell
lines, and (±)-28 displayed weak activity, with GI50 values of
It is worthy of note that the minimal structural diversification of
synthetic (±)-21 and (±)-27 with respect to the natural trans-
grossamide, (±)-4, caused a significant enhancement of
antiproliferative activity. In cell bioassays, a number of different
factors may affect the results, among others cell membrane
permeability, diffusion, and metabolic stability; the easily
oxidizable system of caffeoyl derivatives, for instance, may
affect their metabolic stability. Lipophilicity is usually a critical
parameter, in particular with regard to cell uptake. Thus, to
highlight a possible connection between lipophilicity and
antiproliferative activity, an HPLC method was used that
correlates the capacity factor (K) of a given compound with its
42.3 (Caco-2), 44.6 (MCF-7), and 52.0 μM (PC-3). A more
effective growth inhibition was observed generally for the three
neolignanamides bearing 4-methylbenzyl pendant groups: the
caffeoyl dimer (±)-24, although being scarcely active toward
MCF-7 cells (GI50 = 42.0 μM), was more potent toward PC-3
(
GI = 12.0 μM) and Caco-2 (GI = 16.4 μM) cells; the other
50 50
dimers were potently active, with GI50 values comparable or
even lower than that of 5-FU (Table 3). Thus, the feruloyl
dimer (±)-21 gave GI50 values of 2.8 (Caco-2), 2.7 (MCF-7),
and 4.8 μM (PC-3), and the p-coumaroyl dimer (±)-27 gave
GI values of 3.5 (Caco-2), 6.9 (MCF-7), and 3.7 μM (PC-3).
35−37
lipophilicity (namely, with its log P).
This method is
particularly useful when the direct measurement of log P may
50
E
DOI: 10.1021/acs.jnatprod.6b00577
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Journal of Natural Products
Article
Table 2. 13C NMR Data of Compounds (± )-21−(± )-28 (125 MHz)
a
a
b
a
a
a
c
c
(
±)-21
(±)-22
(±)-23
(±)-24
(±)-25
(±)-26
(±)-27
(±)-28
position
δC, type
δC, type
δC, type
δC, type
δC, type
δC, type
δC, type
δC, type
2
3
3
4
5
6
7
7
8
1
1
1
2
3
4
5
6
7
8
9
1
1
1
2
3
4
1
2
3
4
88.0, CH
57.0, CH
128.4, C
111.9, CH
129.0, C
116.3, CH
144.5, C
149.5, C
169.5, C
42.7, CH2
88.0, CH
56.8, CH
128.7, C
77.9, CH
79.6, CH
123.1, C
76.9, CH
78.7, CH
127.7, C
119.9, CH
130.1, C
115.9, CH
146.1, C
146.8, C
166.7, C
42.7, CH2
75.1, CH
77.4, CH
127.7, C
119.1, CH
129.3, C
114.4, CH
144.8, C
145.3, C
166.3, C
42.6, CH2
87.2, CH
56.3, CH
127.7, C
88.9,CH
58.0, CH
128.4, C
88.4, CH
57.5, CH
127.9, C
a
a
112.8, CH
129.0, C
118.5, CH
130.6, C
125.1, CH
127.6, C
124.6, CH
130.0, C
123.7, CH
128.7, C
115.3, CH
144.5, C
115.6, CH
145.0, C
127.5, CH
109.4, CH
160.6, C
129.9, CH
110.5, CH
161.7, C
129.9, CH
110.1, CH
161.3, C
149.4, C
145.2, C
169.4, C
168.8, C
169.2, C
170.9, C
170.5, C
0
42.98, CH2
42.3, CH2
35.5, CH2
141.2, C
40.4, CH2
33.5, CH2
131.2, C
43.8, CH2
43.8, CH2
1
′
′
′
′
′
′
′
′
131.6, C
131.4, C
144.5, C
143.4, C
131.5, C
128.6, C
109.5, CH
147.4, C
109.6, CH
147.5, C
116.5, CH
146.6, C
116.2, CH
144.4, C
115.8, CH
143.2, C
115.2, CH
127.2, CH
157.4, C
116.6, CH
128.3, CH
159.4, C
116.2, CH
128.7, CH
159.1, C
146.6, C
146.7, C
147.3, C
144.4, C
143.2, C
114.8, CH
118.7, CH
139.4, CH
119.3, CH
165.1, C
114.8, CH
118.9, CH
139.6, CH
119.1, CH
164.9, C
117.3, CH
120.6, CH
141.2, CH
118.4, CH
169.9, C
115.2, CH
121.2, CH
139.8, CH
122.1, CH
165.9, C
117.1, CH
121.4, CH
138.8, CH
120.3, CH
164.9, C
127.2, CH
115.2, CH
139.9, CH
118.0, CH
166.4, C
128.3, CH
116.6, CH
140.2, CH
120.3, CH
166.5, C
128.7, CH
116.2, CH
139.9, CH
119.7, CH
165.9, C
′
1′
2′
″
″/6″
″/5″
″
42.3, CH2
42.93, CH2
42.6, CH2
35.8, CH2
131.0, C
43.1, CH2
42.9, CH2
41.0, CH2
34.5, CH2
129.58, C
129.54,CH
115.8, CH
155.8, C
43.6, CH2
43.8, CH2
136.6, C
135.26, C
104.8, CH
153.23, C
137.01, C
135.05, C
105.0, CH
153.33, C
137.26, C
136.3, C
134.9, C
104.5, CH
153.2, C
137.1, C
134.9, C
105.0, CH
153.3, C
137.2, C
136.09,C
134.7, C
105.1, CH
153.7, C
137.5, C
134.7, C
105.4, CH
153.7, C
137.7, C
127.4, CH
128.8, CH
136.15, C
136.3, C
130.7, CH
116.3, CH
156.9, C
127.8, CH
129.45, CH
136.9, C
128.1, CH
129.6, CH
137.4, C
‴
131.3, C
136.7, C
129.9, C
136.8, C
‴/6‴
‴/5‴
‴
127.3, CH
128.7, CH
136.06, C
19.9, CH3
19.9, CH3
55.3, CH3
55.2, CH3
130.7, CH
116.3, CH
156.8, C
128.3, CH
129.53,CH
137.4, C
129.4, CH
115.0, CH
155.6, C
128.2, CH
129.7, CH
137.4, C
Me-4″
Me-4‴
OMe-7
20.82, CH3
20.83, CH3
21.06, CH3
21.05, CH3
55.34, CH3
55.34, CH3
55.34, CH3
59.51, CH3
55.34, CH3
59.40, CH3
OMe-3′
OMe-3″/5″
OMe-4″
OMe-3‴/5‴
OMe-4‴
55.27, CH3
59.3, CH3
55.30, CH3
59.4, CH3
55.7, CH3
60.25, CH3
55.7, CH3
60.28, CH3
a
b
c
Recorded in acetone-d . Recorded in methanol-d . Recorded in pyridine-d .
6
4
5
Table 3. Antiproliferative Activity of (± )-4 and (± )-21−
a
(
± )-28
b
GI50 (μM) ± SD
c
d
e
compound
Caco-2
MCF-7
PC-3
(
(
±)-21
±)-27
-FU
2.8 ± 0.3
3.5 ± 0.4
4.2 ± 0.5
2.7 ± 0.1
6.9 ± 0.7
3.6 ± 0.3
4.8 ± 0.6
3.7 ± 0.3
6.3 ± 1.1
5
a
Compounds 4, 22−26, and 28 were inactive against the tested cancer
b
cell lines or showed GI50 > 10 μM. GI calculated after 72 h of
50
Figure 2. Key HMBC correlations (from H to C) of (±)-21.
continuous exposure relative to untreated controls; values are the
c
mean (±SD) of four experiments. Caco-2: human colon carcinoma
d
e
cells. MCF-7: human mammary carcinoma cells. PC-3: human
prostate cancer cells.
2
4
be difficult for some or all compounds under study. The
HPLC profiles of neolignanamides (±)-4 and (±)-21−(±)-28
and that of ascorbic acid (AA), used as a highly polar reference
standard, are reported in the Supporting Information. On the
basis of this experimental work, the K values for the above-cited
compounds were determined and are reported in Table 4. For
the sake of completeness, calculated log P values (obtained with
ACD/Labs log P program version 11) are added in this table.
As it is evident from these data, the neolignanamides (±)-21,
(±)-24, and (±)-27, with the highest K values, also showed the
F
DOI: 10.1021/acs.jnatprod.6b00577
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Journal of Natural Products
Article
Table 4. Calculated log P and Experimental K Values of
(± )-4 and (± )-21−(± )-28
a
b
compound
log P
K
(
(
(
(
(
(
(
(
(
±)-4
2.25 ± 0.58
4.21 ± 0.63
0.50 ± 0.71
1.20 ± 0.58
3.16 ± 0.63
−0.55 ± 0.71
3.15 ± 0.49
5.11 ± 0.55
1.40 ± 0.64
2.32
8.45
6.32
0.56
7.89
5.69
5.20
8.36
6.35
±)-21
±)-22
±)-23
±)-24
±)-25
±)-26
±)-27
±)-28
a
Calculated log P values were obtained with ACD/Labs log P program
b
version 11. Capacity factors were calculated by the expression K = (t
R
−
t )/t .
0 0
highest calculated log P values and were thus the most
lipophilic in this series, presumably due to the lipophilic
character of the 4-methylbenzyl pendant substituents. On
comparing these data with those in Table 3, it is evident that
the most potent compound, (±)-21 (feruloyl dimer), is the
most lipophilic, followed by the highly active compound
(
±)-27 (p-coumaroyl dimer) showing a very similar K value,
with the third compound in order of lipophilicity, (±)-24
caffeoyl dimer), also being third in order of cell growth
(
inhibition. These data strongly suggest a possible relationship
between lipophilic character (which is in turn correlated with
cell membrane permeability) and antiproliferative activity.
However, further structural details may play a role in
interactions with the biological targets. Hence, it was decided
to evaluate the possible role of the configuration at the
stereogenic centers C-2 and C-3, for the most promising
antiproliferative compounds (±)-21 and (±)-27. To achieve
this aim, these racemic mixtures were subjected to chiral
resolution to isolate the pure enantiomers. The chiral resolution
of both (±)-21 and (±)-27 was carried out by HPLC on a Lux
Cellulose-2 chiral column eluting with n-hexane−EtOH, 30:70
and 35:65, respectively (see Experimental Section). Under
these conditions, the enantiomer 21a eluted with a shorter
retention time (11.59 min) than enantiomer 21b (13.63 min).
A similar profile was obtained for the enantiomers 27a (8.09
min) and 27b (11.42 min). To establish the absolute
configuration at C-2/C-3 of the pure enantiomers, electronic
circular dichroism spectroscopy (ECD) and optical rotation
polarimetric measurements were employed, since comparison
of ECD spectra and [α] values with those of closely related
compounds of known absolute configuration is one of the most
Figure 3. ECD spectra (CH OH) of (a) 21a and 21b; (b) 27a and
3
27b.
configuration was assigned to both 21a and 27a, with the 2R,3R
configuration assigned to both 21b and 27b. The pure
enantiomers (2S,3S)-21, (2R,3R)-21, (2S,3S)-27, and
(2R,3R)-27 were subjected to the antiproliferative activity
testing toward Caco-2, MCF-7, and PC-3 cancer cells; 5-FU
was used as positive reference also in this experiment. The
results are summarized in Table 5 as GI50 values.
Table 5. Antiproliferative Activity of (2S,3S)-21, (2R,3R)-21,
(2S,3S)-27, and (2R,3R)-27
a
GI50 (μM) ± SD
b
c
d
compound
Caco-2
MCF-7
PC-3
(2S,3S)-21
(2R,3R)-21
(2S,3S)-27
(2R,3R)-27
5-FU
1.4 ± 0.2
1.1 ± 0.1
3.3 ± 0.3
2.1 ± 0.2
3.3 ± 0.5
2.6 ± 0.1
4.4 ± 0.2
5.3 ± 0.1
1.7 ± 0.1
3.8 ± 0.4
1.6 ± 0.2
2.8 ± 0.3
2.5 ± 0.1
2.4 ± 0.1
4.1 ± 0.9
a
GI50 calculated after 72 h of continuous exposure relative to untreated
38
b
useful methods for this type of determination. In particular,
this approach has been applied previously to dimeric
controls. Values are the mean (±SD) of three experiments. Caco-2:
human colon carcinoma. MCF-7: human mammary carcinoma. PC-
3: human prostate cancer.
c
d
8
,19,39
dihydrobenzofuran neolignans.
The ECD spectrum of
enantiomer 21a (reported in Figure 3a with a blue line) showed
a positive Cotton effect in the range 250−325 nm, in
The interpretation of the data shown in Table 5 is not
straightforward: both p-coumaroyl enantiomers resulted in
comparable or higher activity than the racemic mixture, and
(2R,3R)-27 was the most potent enantiomer toward all three
cell lines, with GI50 values of 2.1 (Caco-2 cells), 1.7 (MCF-7
cells), and 2.4 μM (PC-3 cells). The least potent enantiomer,
(2S,3S)-27, was active in a micromolar range, with GI50 values
of 3.3, 5.3, and 2.5 μM (respectively Caco-2, MCF-7, and PC-3
cells). The feruloyl enantiomer (2R,3R)-21 was the most
potent against Caco-2 cells, with a GI50 value of 1.1 μM.
However, the (2S,3S)-21 enantiomer was the most potent
2
0
agreement with the experimental [α]
value of +117;
D
conversely, the ECD spectrum of 21b (reported in Figure 3a
with a red line) gave a negative Cotton effect, in agreement
with the [α]20 value of −115. Analogously, 27a showed a
D
20
positive Cotton effect (Figure 3b, blue line) and [α] value
D
(
+108), and 27b showed a negative Cotton effect (Figure 3b,
20
red line) and [α] value (−105).
On the basis of a comparison with chiro-optical data values of
closely related compounds reported in the literature, such as
D
40
8
41
19
1
, 3, (−)-ephedradine A, and hordatine A (5), the 2S,3S
G
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Figure 4. Cell cycle distribution of Caco-2 cells treated with compound (±)-21 (10 μM, 24−36 h) and with 5-FU (100 μM, 24−36 h) and analyzed
by flow cytometry and propidium iodide staining. Data are shown as means of three independent experiments.
Figure 5. (a) Typical images of Caco-2 cells analyzed with a cytofluorimeter (Amnis FlowsSigh). Each cell (event) is visible in a bright field and
stained by annexin-V positive, propidium iodide positive, and double positive cells. (b) Flow cytometric dot plot of specific cell populations in Caco-
2
cells: live (double annexin/PI negative), early apoptosis (annexin positive), late apoptosis (double annexin/PI positive), and necrosis (annexin
negative and PI positive).
against MCF-7 and PC-3 cells, showing GI50 values of 2.6
compound (±)-21 at 10 μM and 5-FU at 100 μM for 24−36 h,
and cell cycle changes were analyzed in comparison to vehicle-
treated cells (Figure 4). Treatment with (±)-21 revealed the
presence of cells in the sub-G1 phase (29.8%) as well as a
reduction of cells in both the G0/G1 (33.6% vs 51.1%) and G2
phase (19.6% vs 28.0%). In cultures treated with 5-FU, an
increase in the number of cells arrested in S phase (39.8% vs
18.0%) was observed. In addition, with respect to vehicle-
treated cells, a sub-G1 phase appeared (10.4%), although of
lesser intensity than that observed in cells treated with
compound (±)-21. As is well known, 5-FU is an antimetabolite
that acts by the irreversible inhibition of the enzyme
(
(
MCF-7 cells) and 1.6 μM (PC-3 cells). Both (2S,3S)-21 and
2R,3R)-21 were more potent than the racemate against Caco-
2
and PC-3 cells. These data indicate that a specific
configuration at C-2 and C-3 is not a structural determinant
for the antiproliferative activity of these dihydrobenzofuran
dimers and suggest that multiple targets (with different
interactions for the 2R,3R or 2S,3S enantiomer) may be
involved in their mechanism of action.
In addition, the effect of (±)-21 on the Caco-2 cell cycle was
evaluated by flow cytometry and propidium iodide (PI)
staining. Thus, Caco-2 cells were treated respectively with
H
DOI: 10.1021/acs.jnatprod.6b00577
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Journal of Natural Products
Article
optimized for a long-range 13C− H coupling constant of 8.0 Hz. All
NMR experiments, including 2D spectra, i.e., g-COSY, g-HSQCAD,
and g-HMBCAD, were performed using software supplied by the
manufacturer and acquired at constant temperature (300 K).
Chloroform-d , methanol-d , acetone-d , and pyridine-d were used
1
thymidylate synthetase, leading to a deficit of deoxythymidine
monophosphate (dTMP) and nonfunctional DNA synthesis.
42
Therefore, 5-FU is primarily an S phase specific drug that acts
on actively proliferating cells. Cytofluorimetric data have shown
that 24 h treatment with 5-FU at 100 μM produces S-phase
arrest. Conversely, the inhibition of Caco-2 proliferation by
compound (±)-21 at 10 μM could be associated with an
increased DNA fragmentation, as shown by the sub-G1 peak
area. It is known that DNA fragmentation is an important
feature in cells undergoing apoptosis. To the best of our
knowledge this is the first study to investigate the effect of
neolignanamides on cell cycle progression using colorectal
cancer cells.
1
4
6
5
as solvents. Mass spectra were acquired with an Agilent 6410 Triple
Quadrupole mass spectrometer (1200 Series; Milan, Italy) equipped
with a multimodal ionization source operating in MMI-ESI, in positive
or negative mode. Samples infused were eluted on a cartridge (Zorbax
Eclipse XDB-C18; 4.6 × 30 mm, 3.5 μm; Agilent, Milan, Italy), with
MeOH−H
parameters were used for sample ionization: gas temperature 300
C; vaporizer temperature 250 °C; gas flow 10 L/min; nebulizer 60
O−HCOOH (98:2:0.1) as solvent. The following
2
°
psi; fragmentator 135 V; capillary voltage 3500 V; charging 2000 V.
Other mass spectra were acquired with a Thermo Scientific LCQ-
DECA ion trap mass spectrometer (Waltham, MA, USA) equipped
with an ESI ion source operating in the negative ion mode. Samples
were directly infused, and electrospray mass spectra were acquired
from m/z 150 to 2000 using the following electrospray ion source
parameters: capillary temperature 220 °C; capillary voltage −18 V;
spray voltage 3.5 kV; gas flow rate 30 au. Elemental analyses were
performed on a PerkinElmer 240B microanalyzer (Milan, Italy). High-
performance liquid chromatography (HPLC) was carried out using an
Agilent Series (Milan, Italy) G1354A pump and an Agilent UV
G1315D as diode array detector. An Agilent Series 1100 G1313A
autosampler was used for sample injection; an analytical reversed-
phase column (Luna C , 5 μm; 4.6 × 250 mm; Phenomenex, Castel
Maggiore, BO, Italy) was employed to monitor the course of the
enzymatic reactions and to determine the capacity factor value (K) of
dihydrobenzofuran neolignanamides. Chiral resolution was carried out
with HPLC on a Lux Cellulose-2 column (250 × 4.6 mm, 5 μm;
Phenomenex), and the details are reported below. Preparative liquid
chromatography was performed on LiChroprep Si 60 (0.025−0.040
mm or 0.063−0.200 mm; Merck Millipore, Milan, Italy) using
different solvent systems, as reported for each compound. TLC was
carried out using precoated silica gel F254 plates (Merck Millipore);
visualization of reaction components was achieved under UV light at
wavelengths of 254 and 366 nm or by staining with a solution of
cerium sulfate and phosphomolybdic acid followed by heating.
All chemicals were of reagent grade and were used without further
purification. The enzymes used, namely, Trametes versicolor laccase
TvL, 10.0 U/mg), Pleurotus ostreatus laccase (PoL, 11.8 U/mg),
Agaricus bisporus laccase (AbL, 6.8 U/mg), and horseradish peroxidase
HRP, Type I) were purchased from Sigma-Aldrich (Milan, Italy).
General Procedure for the Synthesis of Hydroxycinnamoyl
Amides 12−20. Coumaric, caffeic, or ferulic acid (1 equiv) was
dissolved in dry dimethylformamide (DMF) (1.0 mmol in 10.0 mL)
and triethylamine (1 equiv). The solution was cooled in an ice bath for
10 min, and the suitable amine (1.3 equiv) was added; a solution of
BOP in CH Cl (1 equiv; 1 mmol in 15 mL) was added dropwise, and
the mixture was stirred at 0 °C for 30 min and at room temperature for
0 h. The reaction mixture was evaporated in vacuo to remove
CH Cl , the residue was partitioned between EtOAc (50 mL) and 1 N
In order to confirm if the inhibition of proliferation was due
to apoptosis, cells treated with (±)-21 (10 μM, for 24 h) were
evaluated by flow cytometry using Alexa Fluor 488 annexin V
and PI (Figure 5, Table 6). The results obtained demonstrated
a
Table 6. (± )-21 Induces Apoptotic Death in Caco-2 Cells
cell distribution, %
Caco-2 cells
live
CTRL
(±)-21
5-FU
95.1
3.9
33.0
58.5
5.2
36.8
56.2
5.3
annexin+
annexin+/PI+
PI+
18
1.8
1.1
0.3
0.3
a
Determined by Alexa Fluor 488; annexin V/propidium iodide
annexin/PI) staining after treatment with (±)-21 (10 μM) and 5-
(
FU (100 μM) for 24 h. The analysis was performed on 10 000 events
for each condition and expressed in percentage of total number of
events.
a decreased cell viability accompanied by an increase in cells
undergoing early apoptosis when compared to vehicle-treated
cells. The percentage of annexin-positive cells increased from
3
.9% to 58.5% in (±)-21-treated cells. 5-FU-treated cells were
used as a positive control for apoptosis induction, resulting in
strong reduction of cell viability and apoptosis induction
(
(
56.2% vs 3.9%). Under both conditions, and in vehicle-treated
(
cells, the same low percentage of late apoptosis cells (double
annexin/PI positive cells) was revealed [(±)-21, 5.2% vs 2.9%;
5-FU, 5.3% vs 2.9%]. In contrast, necrotic cells were not
detected (PI staining alone). These data showed that (±)-21
exerts an antiproliferative effect by inducing cell cycle arrest and
apoptosis.
In conclusion, the results of the present study have shown
that bioinspired neolignanamides are worthy of further
investigation to develop new potential anticancer agents,
although further work will be necessary to fully delineate the
underlying antitumor mechanisms.
2
2
2
2
2
HCl solution (2 × 25 mL), and then the organic layer was partitioned
with saturated NaHCO solution (2 × 30 mL). The organic phase was
washed with water, dried over anhydrous Na SO , filtered, and
3
2
4
evaporated to dryness. The residue was purified by flash chromatog-
raphy on silica gel and, occasionally, by further recrystallization.
N-trans-Feruloyltyramine (12). Ferulic acid (8, 201.3 mg, 1.04
mmol) was stirred with TEA (0.14 mL, 1.04 mmol) and DMF (10
mL) at 0 °C (in an ice bath) for 10 min. An excess of tyramine (9,
186.7 mg, 1.36 mmol) was added, followed by a solution of BOP in
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Rudolph Research Analytical polarimeter (Autopol
I, Linate, MI, Italy) at 20 °C and 589 nm. UV spectra were recorded
on a PerkinElmer Lambda 25 spectrometer (Milan, Italy). ECD
spectra were run on a JASCO J-810 spectropolarimeter (Cremella, LC,
Italy). NMR spectra were run on a Varian Unity Inova spectrometer
CH Cl (459.9 mg, 1.04 mmol in 15.7 mL) gradually poured in the
2 2
reaction flask. The mixture was stirred at 0 °C for 30 min and at rt
overnight. After workup, the residue was purified by flash
chromatography on silica gel eluting with petroleum ether−EtOAc
(55:45 → 30:70), affording the amide 12 (237.6 mg, 73%): white,
amorphous powder; R (TLC) 0.35 (CH Cl −MeOH, 97:3). NMR
1
13
(
Italy, Milan) operating at 499.86 ( H) and 125.70 MHz ( C) and
equipped with a gradient-enhanced, reverse-detection probe. Chemical
shifts (δ) are indirectly referred to tetramethylsilane using residual
solvent signals. The 2D g-HSQCAD experiments were performed with
matched adiabatic sweeps for coherence transfer, corresponding to a
central 13C− H J value of 146 Hz. G-HMBCAD experiments were
f
2
2
and MS spectroscopic data were in agreement with those reported in
1
43
the literature.
I
DOI: 10.1021/acs.jnatprod.6b00577
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
E)-N-(4-Methylbenzyl)-3-(4-hydroxy-3-methoxyphenyl)-
the residue was partitioned using EtOAc (100 mL) as reported in the
general procedure. The crude mixture was purified by a flash
chromatography column on silica gel eluting with petroleum ether−
EtOAc (60:40 → 30:70), and the amide 16 (364.7 mg, 65%) was
acrylamide (13). Ferulic acid (8, 230.2 mg, 1.18 mmol) was
solubilized in dry DMF (12 mL) and kept under stirring with TEA
0.17 mL, 1.18 mmol) in an ice bath for 10 min. An excess of 4-
(
methylbenzylamine (10, 0.20 mL, 1.59 mmol) was added, followed by
a gradual addition of BOP (553.5 mg, 1.25 mmol, in 20 mL of
CH Cl ). The mixture was stirred at 0 °C for 30 min and at rt
recovered as a yellow powder: R (TLC) 0.50 (petroleum ether−
f
1
EtOAc, 30:70); H NMR (methanol-d , 500 MHz) δ 7.43 (1H, d, J =
4
2
2
15.5 Hz, H-7), 7.19 (2H, d, J = 8.0 Hz, H-3′ and H-5′), 7.14 (2H, d, J
= 8.0 Hz, H-2′ and H-6′), 7.01 (1H, d, J = 2.0 Hz, H-2), 6.91 (1H, dd,
J = 2.0, 9.0 Hz, H-6), 6.76 (1H, d, J = 9.0 Hz, H-5), 6.40 (1H, d, J =
overnight. The CH Cl was removed under vacuum, and the residue
2
2
was diluted with 50 mL of EtOAc and partitioned as reported in the
general procedure. The residue was purified by column chromatog-
raphy on silica gel eluting with CH Cl −MeOH (100:0 → 95:5) to
1
3
15.5 Hz, H-8), 4.42 (2H, s, H-7′), 2.31 (3H, s, CH ); C NMR
3
2
2
(methanol-d , 125 MHz) δ 167.7 (C, C-9), 147.4 (C, C-4), 145.3 (C,
4
give 13 (287.2 mg, 82%) as a yellow oil: R (TLC) 0.37 (CH Cl −
C-3), 141.0 (CH, C-7), 136.5 (C, C-4′), 135.5 (C-1′), 128.7 (CH, C-
f
2
2
1
MeOH, 98:2); H NMR (methanol-d , 500 MHz) δ 7.49 (1H, d, J =
3′ and C-5′), 127.2 (C, C-1 and CH, C-2′ and C-6′), 126.9 (CH, C-6),
4
1
6.0 Hz, H-7), 7.18 (2H, d, J = 8.5 Hz, H-3′ and H-5′), 7.11* (2H, d, J
8.5 Hz, H-2′ and H-6′), 7.10* (1H, d, J = 2.0 Hz, H-2), 7.02 (1H,
dd, J = 2.0, 8.5 Hz, H-6), 6.80 (1H, d, J = 8.5 Hz, H-5), 6.48 (1H, d, J
16.0 Hz, H-8), 4.42 (2H, s, H-7′), 3.82 (3H, s, OCH ), 2.26 (3H, s,
120.7 (CH, C-8), 115.0 (CH, C-5), 113.7 (CH, C-2), 42.6 (CH , C-
2
−
=
7′), 19.7 (CH , CH -4′); ESIMS m/z 282.1 [M − H] (calcd for
3
3
C H NO , 282.11); anal. C 72.05, H 6.07, N 4.91%, calcd for
17
16
3
=
C H NO , C 72.07, H 6.05, N 4.94%.
3
17 17 3
CH ), the signals with identical superscript (*) were partially
overlapped; C NMR (methanol-d , 125 MHz) δ 168.9 (C, C-9),
(E)-N-(3,4,5-Trimethoxybenzyl)-3-(3,4dihydroxyphenyl)-
acrylamide (17). Caffeic acid (7, 302.4 mg, 1.68 mmol) and TEA
(0.24 mL, 1.69 mmol) were stirred in dry DMF (15.4 mL) at 0 °C for
10 min. Then, 3,4,5-trimethoxybenzylamine (11, 0.35 mL, 2.21 mmol)
and gradually the BOP solution (752.4 mg; 1.68 mmol; in 30.5 mL of
CH Cl ) were poured in the mixture. The reaction was stirred at 0 °C
3
13
4
1
1
1
1
49.8 (C, C-4), 149.2 (C, C-3), 142.3 (CH, C-7), 137.9 (C, C-4′),
36.8 (C-1′), 130.1 (CH, C-3′ and C-5′), 128.6 (CH, C-2′ and C-6′),
28.2 (C. C-1), 123.2 (CH, C-6), 118.7 (CH, C-8), 116.5 (CH, C-5),
11.6 (CH, C-2), 56.3 (CH , OCH -3), 44.0 (CH , C-7′), 21.1 (CH ,
3
3
2
3
2
2
−
CH -4′); ESIMS m/z 296.2 [M − H] (calcd for C H NO ,
for 30 min and at rt for 24 h. The CH Cl was evaporated, and the
3
18 18
3
2 2
2
96.13); anal. C 72.68, H 6.45, N 4.70%, calcd for C H NO , C
residue was diluted with 80 mL of EtOAc and partitioned as reported
in the general procedure. Flash column chromatography on silica gel
18
19
3
7
2.71, H 6.44, N 4.71%.
(
E)-N-(3,4,5-Trimethoxybenzyl)-3-(4-hydroxy-3-methoxyphenyl)-
eluted with CHCl −MeOH (99:1 → 90:10) gave amide 17 (385.9 mg,
3
acrylamide (14). Ferulic acid (8, 224.6 mg, 1.16 mmol) was
solubilized in dry DMF (10.4 mL) and TEA (0.16 mL, 1.16 mmol),
and the mixture was stirred in an ice bath for 10 min. An excess of
64%) as a yellow, amorphous powder: R (TLC) 0.22 (CHCl −
f
3
1
MeOH, 93:7); H NMR (methanol-d , 500 MHz) δ 7.44 (1H, d, J =
4
15.0 Hz, H-7), 7.01 (1H, d, J = 2.0 Hz, H-2), 6.91 (1H, dd, J = 2.0, 8.5
Hz, H-6), 6.76 (1H, d, J = 8.5 Hz, H-5), 6.42 (1H, d, J = 15.0 Hz, H-
8), 6.64 (2H, s, H-2′ and H-6′), 4.42 (2H, s, H-7′), 3.82 (6H, s,
3
,4,5-trimethoxybenzylamine (11, 0.18 mL, 1.30 mmol) and BOP
solution (513.2 mg, 1.16 mmol, in 15.8 mL of CH Cl ) were added,
and the mixture was stirred at 0 °C for 30 min and at rt for 20 h. The
CH Cl was removed under vacuum, and the residue was worked up as
reported in the general procedure. The crude extract was purified by
flash column chromatography on silica gel using CH Cl −MeOH as
eluent (99:1 → 90:10), and the product 14 was recovered (277.4 mg,
4%) as a yellow oil: R (TLC) 0.44 (petroleum ether−EtOAc, 80:20);
H NMR (chloroform-d , 500 MHz) δ 7.69 (1H, d, J = 16.0 Hz, H-7),
.08 (1H, dd, J = 1.5, 8.5 Hz, H-6), 7.01 (1H, d, J = 1.5 Hz, H-2), 6.91
2
2
1
3
OCH -3′ and OCH -5′), 3.74 (3H, s, OCH -4′); C NMR
3
3
3
2
2
(methanol-d , 125 MHz) δ 169.2 (C, C-9), 154.6 (C, C-3′ and C-
4
5′), 148.9 (C, C-4), 146.7 (C, C-3), 142.7 (CH, C-7), 138.2 (C, C-4′),
136.2 (C-1′), 128.2 (C, C-1), 122.3 (CH, C-6), 118.2 (CH, C-8),
116.5 (CH, C-5), 115.1 (CH, C-2), 106.0 (CH, C-2′ and C-6′), 61.1
(CH , OCH -4′), 56.6 (CH , OCH -3′ and OCH -5′), 44.5 (CH , C-
2
2
6
f
3
3
3
3
3
2
1
−
1
7′); ESIMS m/z 358.1 [M − H] (calcd for C H NO , 358.13);
19 20
6
7
anal. C 63.56, H 5.88, N 3.92%, calcd for C H NO , C 63.50, H 5.89,
19 21 6
(
1
1H, d, J = 8.5 Hz, H-5), 6.56 (2H, s, H-2′ and H-6′), 6.28 (1H, d, J =
N 3.90%.
6.0 Hz, H-8), 5.84 (1H, bt, J = 6.5 Hz, NH), 4.51 (2H, d, J = 6.5 Hz,
N-trans-p-Coumaroyltyramine (18). Coumaric acid (6, 478.5 mg,
2.91 mmol) was solubilized in dry DMF (25.0 mL), and the solution
was stirred with TEA (0.40 mL, 2.90 mmol) at 0 °C for 10 min.
Tyramine (9, 558.3 mg, 3.78 mmol) was added to the mixture, and
subsequently a solution of BOP (1286.2 mg, 2.91 mmol, in 50 mL of
CH Cl ) was added dropwise. The mixture was stirred at 0 °C for 30
H-7′), 3.92 (6H, s, OCH -3′ and OCH -5′), 3.86 (3H, s, OCH -3),
3
3
3
13
3
.84 (3H, s, OCH -4′); C NMR (chloroform-d , 125 MHz) δ 165.9
3
1
(
1
(
C, C-9), 153.3 (C, C-3′ and C-5), 147.5 (C, C-4), 146.7 (C, C-3),
41.5 (C, C-4′ and CH, C-7), 134.0 (C, C-1′), 127.3 (C. C-1), 122.2
CH, C-6), 117.9 (CH, C-8), 114.7 (CH, C-5), 109.6 (CH, C-2),
2
2
1
3
05.1 (CH, C-2′ and C-6′), 60.9 (CH , OCH -4′), 56.2 (CH , OCH -
min and at rt overnight. The CH Cl was removed by evaporation, and
3
3
3
3
2 2
′ and OCH -5′), 55.9 (CH , OCH -3), 44.2 (CH , C-7′); ESIMS m/
the residue diluted with EtOAc (140 mL) was extracted as described
above. The crude mixture was purified by flash chromatography on
3
3
3
2
−
z 372.1 [M − H] (calcd for C H NO , 372.14); anal. C 64.31, H
20
22
6
6
.18, N 3.77%, calcd for C H NO , C 64.33, H 6.21, N 3.75%.
silica gel eluting with CHCl −MeOH (98:2 → 80:20), and the amide
20
23
6
3
N-trans-Caffeoyltyramine (15). Caffeic acid (7, 310.2 mg, 1.72
18 (616.8 mg, 75%) was recovered as a white, amorphous powder: R
f
mmol) was stirred with TEA (0.24 mL, 1.73 mmol) in dry DMF (17.5
mL) in an ice bath for 10 min. Then, tyramine (9, 306.3 mg, 2.23
mmol) and gradually the BOP solution (760.2 mg, 1.72 mmol in 25.4
mL of CH Cl ) were added. The mixture was stirred at 0 °C for 30
min and then at rt overnight. The reaction was quenched by partition
as described above. The dried organic phase was evaporated in vacuo
(TLC) 0.36 (CHCl −MeOH, 92:8); NMR and MS data were in
3
4
3
agreement with values reported in the literature.
(E)-N-(4-Methylbenzyl)-3-(4-hydroxyphenyl)acrylamide (19).
Coumaric acid (6, 225.1 mg, 1.37 mmol) was stirred with TEA
(0.19 mL, 1.37 mmol) in dry DMF (14.2 mL) at 0 °C for 10 min.
Then, an excess of 4-methylbenzylamine (10, 0.24 mL, 1.78 mmol)
was added, and a solution of BOP in CH Cl (685.1 mg, 1.54 mmol,
2
2
and chromatographed on silica gel eluting with CHCl −MeOH (98:2
3
2
2
→
85:15); the amide 15 was obtained after crystallization in CH Cl −
23.2 mL) was added dropwise. The mixture was stirred at 0 °C for 30
min and at rt overnight. The reaction mixture was evaporated under
vacuum to remove CH Cl , and the residue was partitioned as
2
2
MeOH (396.1 mg, yield: 77%): yellow, amorphous powder; R (TLC)
f
0
.37 (CHCl −MeOH, 92:8). The acquired MS and NMR data are in
3
2
2
44
agreement with those previously reported.
reported in the general procedure. Purification by silica gel column
(
E)-N-(4-Methylbenzyl)-3-(3,4-dihydroxyphenyl)acrylamide (16).
Caffeic acid (7, 358.7 mg, 1.98 mmol) was dissolved in dry DMF
20.0 mL), and the solution was stirred with TEA (0.28 mL, 1.99
chromatography with CHCl −MeOH (100:0 → 95:5) gave the pure
3
amide 19 (322.4 mg, 88%) as a white, amorphous powder: R (TLC)
f
1
(
0.44 (CH Cl −MeOH, 93:7); H NMR (methanol-d , 500 MHz) δ
2
2
4
mmol) at 0 °C. After 10 min, amine 10 (0.25 mL, 1.98 mmol) and
BOP solution (876.9 mg, 1.98 mmol, in 30.2 mL of CH Cl ) were
added dropwise to the mixture. The reaction was stirred at 0 °C for 30
min and at rt overnight. The CH Cl was removed by evaporation, and
7.49 (1H, d, J = 15.7 Hz, H-7), 7.41 (2H, d, J = 8.5 Hz, H-2 and H-6),
7.20 (2H, d, J = 8.0 Hz, H-3′ and H-5′), 7.14 (2H, d, J = 8.0 Hz, H-2′
and H-6′), 6.79 (2H, d, J = 8.5 Hz, H-3 and H-5), 6.45 (1H, d, J = 15.7
2
2
1
3
Hz, H-8), 4.43 (2H, s, H-7′), 2.31 (3H, s, CH -4′); C NMR
2
2
3
J
DOI: 10.1021/acs.jnatprod.6b00577
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
(
1
1
(
methanol-d , 125 MHz) δ 169.1 (C, C-9), 160.5 (C, C-4), 142.1
organic phase was finally washed with water, dried, and evaporated in
vacuo. Flash chromatography with silica gel, eluted with petroleum
ether−EtOAc (65:35 → 25:75%), gave the racemate neolignanamide
4
CH, C-7), 138.0 (C, C-4′), 136.9 (C-1′), 130.6 (CH, C-2 and C-6),
30.1 (CH, C-3′ and C-5′), 128.6 (C-2′ and C-6′), 127.7 (C, C-1),
18.3 (CH, C-8), 116.7 (CH, C-3 and C-5), 44.1 (CH , C-7′), 21.1
(±)-21 (19.3 mg, 16%) as a white, amorphous powder: R (TLC) 0.46
2
f
−
CH , CH -4′); ESIMS m/z 266.1 [M − H] (calcd for C H NO ,
(CH Cl −MeOH, 93:7); UV (MeOH) λmax (log ε) 290 (4.49), 304
2 2
1 13
3
3
17 16
2
2
66.12); anal. C 76.35, H 6.43, N 5.26%, calcd for C H NO , C
(4.51), 321 (4.56) nm; H and C NMR data, see Tables 1 and 2;
1
7
17
2
−
7
6.38, H 6.41, N 5.24%.
E)-N-(3,4,5-Trimethoxybenzyl)-3-(4-hydroxyphenyl)acrylamide
20). Coumaric acid (6, 449.0 mg, 2.73 mmol) was dissolved in dry
ESIMS m/z 591.3 [M − H] (calcd for C36
H
35
N
2
O
6
, 591.25); anal. C
H N O , C 72.95, H 6.12, N
36 2 6
(
72.93, H 6.13, N 4.75%, calcd for C36
4.73%.
(
DMF (28 mL) and TEA (0.38 mL, 2.73 mmol). The mixture was
stirred in an ice bath for 10 min, and subsequently, the amine 11 (0.60
mL, 3.55 mmol) and gradually the BOP solution (1420.6 mg; 3.21
mmol in 42.4 mL of CH Cl ) were added. The mixture was stirred at 0
2-(4-Hydroxy-3-methoxyphenyl)-7-methoxy-5-[(E)-3-oxo-3-
(3,4,5trimethoxybenzyl)aminoprop-1-en-1-yl]-N-(3,4,5-trimethoxy-
benzyl)-2,3-dihydrobenzofuran-3-carboxamide [(±)-22]. Com-
pound 14 (74.6 mg, 0.20 mmol) was dissolved in EtOAc (37.3
mL), and a solution containing TvL (85.0 mg in 42.5 mL of acetate
buffer 0.1 M, pH = 4.5) was added. The resulting biphasic system was
stirred at rt for 4 h. The crude mixture was quenched by phase
separation, and the aqueous layer was partitioned with EtOAc (2 × 15
mL). The organic residue was purified by a flash chromatography
2
2
°C for 30 min and then at rt overnight. The mixture was concentrated
by evaporation and partitioned as described in the general procedure.
The flash chromatography column with silica gel, eluted with
petroleum ether−EtOAc (85:15 → 20:80), afforded the amide 20,
after crystallization in CH Cl −MeOH (615 mg, 67%), as a white,
2
2
1
column on silica gel eluting with CH Cl −MeOH (99:1 → 90:10),
amorphous powder: R (TLC) 0.41 (CH Cl −MeOH, 98:2);
NMR (methanol-d , 500 MHz) δ 7.55 (1H, d, J = 15.5 Hz, H-7), 7.41
H
2
2
f
2
2
affording the racemate (±)-22 (12.5 mg, 17%) as a yellow oil: R
f
4
1
13
(
TLC) 0.16 (CH Cl −MeOH, 95:5); H and C NMR data, see
(
5
(
2H, d, J = 8.5 Hz, H-2 and H-6), 6.79 (2H, d, J = 8.5 Hz, H-3 and H-
), 6.64 (2H, s, H-2′ and H-6′), 6.46 (1H, d, J = 15.7 Hz, H-8), 4.42
2H, s, H-7′), 3.82 (6H, s, OCH -3′ and OCH -5′), 3.74 (3H, s,
2
2
−
Tables 1 and 2; ESIMS m/z 743.3 [M − H] (calcd for C H N O ,
43.28); anal. C 64.48, H 5.97, N 3.73%, calcd for C H N O , C
40 43
2
12
7
40 44 2 12
3
3
13
64.51, H 5.95, N 3.76%.
OCH -4′); C NMR (methanol-d , 125 MHz) δ 169.1 (C, C-9),
1
C-4′), 136.2 (C-1′), 130.6 (CH, C-2 and C-6), 127.7 (C, C-1), 118.2
(
(
7
3
4
2
-(3,4-Dihydroxyphenyl)-7-hydroxy-N-(4-hydroxyphenethyl)-5-
(E)-3-(4-hydroxyphenethyl)amino-3-oxoprop-1-en-1-yl]-2,3-
dihydrobenzofuran-3-carboxamide [(±)-23]. Caffeoyl amide 15
118.2 mg, 0.40 mmol) was dissolved in CH Cl (59.2 mL), and
60.6 (C, C-4), 154.6 (C, C-3′ and C-5′), 142.3 (CH, C-7), 138.3 (C,
[
CH, C-8), 116.7 (CH, C-3 and C-5), 106.0 (C-2′ and C-6′), 61.1
(
2
2
CH , OCH -4′), 56.6 (CH , OCH -3′ and OCH -5′), 44.5 (CH , C-
3
3
3
3
3
2
the mixture was stirred with a solution of TvL (108.6 mg in 54.3 mL of
.1 M acetate buffer, pH = 4.5) at rt for 2 h. The reaction was
−
′); ESIMS m/z 342.2 [M − H] (calcd for C H NO , 342.13);
1
9
20
5
0
anal. C 66.42, H 6.18, N 4.05%, calcd for C H NO , C 66.46, H 6.16,
19
21
5
quenched by phase separation, and the aqueous phase was partitioned
N 4.08%.
with CH Cl (2 × 25 mL).The combined organic phases were washed
2
2
Biomimetic Synthesis of Dihydrobenzofuran Neolignana-
with water, dried over anhydrous Na SO , filtered, and taken to
2
4
mides (± )-4 and (± )-21−(± )-28. Preliminary Screening. Aliquots
dryness. The silica gel column chromatography eluted with CHCl −
3
(
2.0 mg) of the amides 12−20 were dissolved in three different
MeOH (97:3 → 85:15) afforded the racemate (±)-23 (18.2 mg, 15%)
as a yellow oil: R (TLC) 0.41 (CHCl −MeOH, 88:12); UV (MeOH)
solvents (1 mL), namely, EtOAc, CH Cl , and 2% DMSO in acetate
2
2
f
3
buffer (0.1 M pH = 4.5). The three different solutions of each
compound were treated with the following enzymes: TvL, PoL, AbL,
and HRP (2 mg), previously dissolved in acetate buffer (1.0 mL, 0.1M,
pH = 4.5). The reactions were stirred at rt in vials without caps except
for HRP-mediated reactions; in the latter, a 30% (v/v) H O solution
1
13
λ
(log ε) 285 (4.05), 320 (3.93) nm; H and C NMR data, see
Tables 1 and 2; ESIMS m/z 595.1 [M − H] (calcd for C H N O ,
95.21); anal. C 68.41, H 5.43, N 4.69%, calcd for C H N O , C
max
−
3
4
31
2
8
5
34 32 2 8
6
8.45, H 5.41, N 4.70%.
-(3,4-Dihydroxyphenyl)-7-hydroxy-N-(4-methylbenzyl)-5-[(E)-3-
2
2
2
(
1.0 μL) was added to each mixture, and the reactions were carried out
(
4-methylbenzyl)amino-3-oxoprop-1-en-1-yl]-2,3-dihydro-
in capped vials. For each experiment, a blank was carried out in the
same conditions, without enzyme. The reactions were monitored at
regular time intervals by HPLC with a reversed-phase column (RP-18)
benzofuran-3-carboxamide [(±)-24]. The amide 16 (131.2 mg, 0.46
mmol) was dissolved in CH Cl (65.6 mL), and a TvL solution in
2
2
acetate buffer (128.4 mg of 64.8 mL) was added. The reaction was
stirred at rt for 2 h. After the two phases were separated, the aqueous
phase was partitioned with CH Cl (2 × 25 mL), and finally the
+
+
with the following gradient of CH CN/H (99:1 v/v; B) in H O/H
3
2
(
99:1 v/v; A) at 1 mL/min: t0 min B = 30%, t30 min B = 80%. A diode
2
2
array detector was set at 254, 280, 305, and 325 nm.
combined organic phases were washed with water, dried, and
evaporated to dryness. Flash chromatography on silica gel, eluted
with CH Cl −MeOH (98:2 → 95:5), afforded the racemic dimer
Trametes versicolor laccase gave the best results in terms of both
substrate conversion and formation of a main product, and it was
employed to obtain the dimers on a preparative scale.
2
2
(
±)-24 (21.2 mg, 16%) as a yellow oil: R (TLC) 0.56 (CH Cl −
f
2
2
(
±)-trans-Grossamide (4). Feruloyl amide 12 (120.3 mg, 0.38
MeOH, 92:8); UV (MeOH) λ (log ε) 240 (4.60), 282 (4.66), 318
max
1
13
mmol) was dissolved in EtOAc (60.0 mL), and the organic solution
was stirred with acetate buffer solution containing the enzyme TvL
(4.36) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/z
−
563.2 [M − H] (calcd for C H N O , 563.22); anal. C 72.29, H
5.68, N 4.95%, calcd for C H N O , C 72.32, H 5.71, N 4.96%.
34
31
2
6
(
115.8 mg dissolved in 58.0 mL). The reaction was carried out at rt for
34 32 2 6
4
h. The reaction was quenched by phase separation, and the aqueous
2-(3,4-Dihydroxyphenyl)-7-hydroxy-5-[(E)-3-oxo-3-(3,4,5-
trimethoxybenzyl)aminoprop-1-en-1-yl]-N-(3,4,5-trimethoxy-
benzyl)-2,3-dihydrobenzofuran-3-carboxamide [(±)-25]. The amide
phase was partitioned with EtOAc (2 × 25 mL). The combined
organic phases were washed with water, dried over anhydrous Na SO ,
2
4
and filtered, and then the solvent was evaporated. Purification by flash
17 (105.8 mg, 0.29 mmol) was solubilized in CH Cl (53.0 mL). The
2 2
column chromatography on silica gel with CHCl −MeOH (98:2 →
mixture was stirred with a TvL solution previously prepared (104.2
mg, in 52.2 mL of acetate buffer, pH = 4.5) at rt for 2 h. The reaction
was quenched by separation of the two phases, with the aqueous phase
partitioned with EtOAc (2 × 25 mL), and the total organic phase was
3
9
0:10) afforded the dihydrobenzofuran (±)-4 as a racemic mixture of
trans-diasteromers (19.3 mg, 16%), as a yellow oil: R (TLC) 0.29
f
(
CHCl −MeOH, 90:10); the spectroscopic data were in agreement
3
32
with previous values reported in the literature.
washed with water, dried over Na
dryness. The crude mixture was purified by silica gel flash
chromatography eluted with CHCl
2 4
SO , filtered, and evaporated to
2
-(4-Hydroxy-3-methoxyphenyl)-7-methoxy-N-(4-methylbenzyl)-
5
-[(E)-3-(4-methylbenzyl)amino-3-oxoprop-1-en-1-yl]-2,3-dihydro-
3
−MeOH (100:0 → 90:10), giving
benzofuran-3-carboxamide [(±)-21]. Amide 13 (122.6 mg, 0.41
mmol) was solubilized in EtOAc (61.3 mL), and the organic solution
was stirred with the enzyme solution (118.2 mg of TvL in 59.1 mL of
acetate buffer) at rt for 4 h. The two phases were separated, and the
aqueous phase was partitioned with EtOAc (2 × 25 mL). The total
the neolignanamide (±)-25 (24.8 mg, 24%) as a yellow oil: R (TLC)
f
0.55 (CHCl −MeOH, 92:8); UV (MeOH) λ (log ε) 284 (4.44),
3
max
1
13
316 (4.26) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/
z 715.2 [M − H] (calcd for C H N O , 715.25); anal. C 63.65, H
5.60, N 3.87%, calcd for C H N O , C 63.68, H 5,63, N 3.91%.
−
3
8
39
2
12
38
40
2
12
K
DOI: 10.1021/acs.jnatprod.6b00577
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
N-(4-Hydroxyphenethyl)-5-[(E)-3-(4-hydroxyphenethyl)amino-3-
oxoprop-1-en-1-yl]-2-(4-hydroxyphenyl)-2,3-dihydrobenzofuran-3-
carboxamide [(±)-26]. Coumaroyl amide 18 (105.3 mg, 0.37 mmol)
was dissolved in DMSO−EtOAc (1:99; 51.0 mL). The mixture was
stirred with a solution of TvL (102.0 mg in 51.3 mL of acetate buffer)
at rt for 24 h. The two phases were separated, the aqueous layer was
partitioned with EtOAc (2 × 20 mL), and the combined organic
phases were washed with water, dried, and evaporated. Purification by
flash column chromatography on silica gel, eluted with CH Cl −
[α]20 +108 (c 0.1, CH OH); ECD (MeOH) λ (Δε) 276 (+5.25),
D
3
max
2
92 (+4.32). Enantiomer 27b (2.7 mg): (2R,3R)-27; white,
20
amorphous powder; tR = 11.42 min; [α]D −105 (c 0.1, CH OH);
3
ECD (MeOH) λmax (Δε) 276 (−5.88), 292 (−4.95) nm.
Antiproliferative Activity Assay. Human Cell Cultures. Human
colorectal adenocarcinoma cell line Caco-2 (ATCC number: HTB-
3
7), human mammary adenocarcinoma MCF-7 (ATCC number:
HTB22), and human prostate cancer PC-3 (ATCC number: CLR-
2
2
1
435) have been used in the present work and have been cultured as
MeOH (98:2 → 90:10), afforded the racemate (±)-26 (16.8 mg, 16%)
as a white, amorphous powder: R (TLC) 0.35 (CH Cl −MeOH,
4
5
previously reported.
f
2
2
1
9
2:8); UV (MeOH) λmax (log ε) 286 (4.42), 313 (4.38) nm; H and
C NMR data, see Tables 1 and 2; ESIMS m/z 563.3 [M − H]
calcd for C H N O , 563.22); anal. C 72.27, H 5.75, N 4.93%, calcd
Treatment with Neolignanamides (±)-4 and (±)-21−(±)-28 and
3
1
3
−
MTT Colorimetric Assay. Human cancer cell lines ((2.5−3.0) × 10
2
cells/0.33 cm ) were plated in Nunclon Microwell (Nunc) 96-well
(
34
31
2
6
for C H N O , C 72.32, H 5.71, N 4.96%.
plates and were incubated at 37 °C. After 24 h, cells were treated with
the compounds under study (final concentration 0.01−100 μM). Cells
treated with 0.5−1% DMSO were used as controls. Microplates were
34
32
2
6
2
-(4-Hydroxyphenyl)-N-(4-methylbenzyl)-5-[(E)-3-(4-methyl-
benzyl)amino-3-oxoprop-1-en-1-yl]-2,3-dihydrobenzofuran-3-car-
boxamide [(±)-27]. Amide 19 (130.8 mg, 0.49 mmol) was dissolved
in DMSO−EtOAc (1:99; 65.1 mL), and the mixture was stirred with
TvL solution (115.2 mg in 57.8 mL of acetate buffer) at rt for 24 h.
After the separation of the two phases, the aqueous layer was
partitioned with EtOAc (2 × 25 mL) and the total organic phase was
washed with water, dried over anhydrous Na SO , filtered, and
incubated at 37 °C in a humidified atmosphere of 5% CO and 95% air
2
for 3 days, and then cell viability was measured with a colorimetric
assay based on the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
3
4
tetrazolium bromide. The results were read on a multiwell scanning
spectrophotometer (AF2200 plate reader, Eppendorf, Milan, Italy),
using a wavelength of 570 nm. Each value was the average of 6−8
2
4
evaporated in vacuo. The crude mixture was purified by flash column
chromatography on silica gel with CH Cl −MeOH (100:0 → 95:5),
wells. The 50% grown inhibition (GI ) value was calculated according
50
2
2
4
6
and the neolignanamide (±)-27 was obtained after crystallization
to the NCI (Bethesda, MD, USA): thus, GI50 is the concentration of
) = 50 (where T is the
(
acetone; 38.3 mg, 29%) as a white, amorphous powder: R (TLC)
test compound where 100(T − T
0
)/(C − T
0
f
0
3
.25 (CH Cl −MeOH, 96:4); UV (MeOH) λ (log ε) 297 (4.11),
optical density of the test well after a 72 h period of exposure to test
2
2
max
1
13
10 (4.09) nm; H and C NMR data, see Tables 1 and 2; ESIMS m/
compound, T is the optical density at time zero, and C is the DMSO
0
−
z 531.3 [M − H] (calcd for C H N O , 531.23); anal. C 76.64, H
34
31
2
4
control optical density). The cytotoxicity effect was calculated
according to the NCI when the optical density of treated cells was
lower than the T value using the following formula: 100(T − T )/T .
6
.03, N 5.28%, calcd for C H N O , C 76.67, H 6.06, N 5.26%.
34 32 2 4
2
-(4-Hydroxyphenyl)-5-[(E)-3-oxo-3-(3,4,5-trimethoxybenzyl)-
0
0
0
aminoprop-1-en-1-yl]-N-(3,4,5-trimethoxybenzyl)-2,3-dihydro-
benzofuran-3-carboxamide [(±)-28]. Coumaroyl amide 20 (117.6
mg, 0.34 mmol) was solubilized in DMSO−EtOAc (1:99; 58.6 mL),
and this solution was stirred together with the enzyme solution (108.2
mg of TvL in 54.5 mL of acetate buffer, pH = 4.5) at rt for 24 h. After
separation, the aqueous layer was partitioned with EtOAc (2 × 25
mL), and the combined organic layers were washed with water, dried,
and evaporated in vacuo. Silica gel column chromatography, eluted
with CH Cl −acetone (96:4 → 80:20), gave the dimer (±)-28 (39.4
Cell Cycle Analysis. Cell cycle progression was evaluated by DNA
flow cytometry, with PI used to stain cells. Caco-2 cells were cultured
in six-well plates and treated with compound (±)-21 and 5-FU. Cells
were harvested by trypsinization after 24−36 h of treatment, washed in
PBS, and cooled in 70% ethanol at 4 °C for at least 30 min. Cells were
washed in PBS to remove the ethanol and incubated for 1 h at 37 °C
in a PBS solution containing RNase A (final concentration 100 μm/
mL; Bio Basic Inc., Markham, Ontario, Canada). Finally, PI was added
(final concentration 50 μg/mL; Agros Organics, Geel, Belgium), and
the cellular suspension was incubated for 1 h at 37 °C. The cellular
suspension was analyzed on a flow cytometer (Amnis FlowSight
Millipore, Merck KgaA, Darmstadt, Germany), and results were
analyzed using Image Data Exploration and Analysis (IDEAS) software
2
2
mg, 34%) as a white, amorphous powder: R (TLC) 0.35 (CH Cl −
f
2
2
acetone, 84:16); UV (MeOH) λ (log ε) 276 (4.59), 322 (4.21) nm;
max
1
13
H and C NMR data, see Tables 1 and 2; ESIMS m/z 683.4 [M −
H] (calcd for C H N O 683.26); anal. C 66.61, H 5.92, N 4.04%,
−
38
39
2
10
calcd for C H N O , C 66.65, H 5.89, N 4.09%.
38
40
2
10
Measurement of the Capacity Factor (K). Measurements of the
capacity factor of neolignanamides (±)-4 and (±)-21−(±)-28 were
performed using a Luna-C column, and as eluent a gradient of H O/
(Amnis part of EMD Millipore, Seattle, WA, USA).
Apoptosis Analysis. Apoptotic cell death was measured by flow
1
8
2
+
+
cytometry (Amnis FlowSight Millipore) using Alexa Fluor 488 annexin
V/dead cell apoptosis kit with Alexa Fluor 488 annexin V and PI for
flow cytometry (V13241, Invitrogen, Monza MB, Italy) according to
the manufacturer’s instructions. The numbers of live (annexin
negative/PI negative), early apoptotic (annexin positive/PI negative),
and late apoptotic/necrotic (annexin and PI positive) cells were
determined using IDEAS software (Amnis part of EMD Millipore).
H (99/1; A)−CH CN/H (99/1; B) was used at 1 mL/min with the
following eluting profile: t0
3
B = 20%, t
B = 80%. The capacity
min
30 min
factor K was calculated using the following expression: K = (t − t )/
R
0
t , where t is the retention time of the test substance and t is the
0
R
0
retention time of AA used as standard. Calculated log P values were
obtained with the ACD/Labs log P program version 11.
Chiral Resolution of Racemic Mixtures (± )-21 and (± )-27.
Racemic neolignanamide 21 (5.7 mg) was dissolved in EtOH (1.5
mL) and resolved by HPLC-UV using a Lux Cellulose-2 chiral column
ASSOCIATED CONTENT
(
eluting solvent: n-hexane−EtOH, 30:70; flow rate 0.5 mL/min; 30
■
injections; 50 μL each) to yield two enantiomers. Enantiomer 21a (2.5
*
S
Supporting Information
20
mg): (2S,3S)-21; white, amorphous powder; t 11.59 min; [α] +117
R
D
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00577.
(
c 0.1, CH OH); ECD (MeOH) λ (Δε) 250 (+2.36), 310 (+2.32)
3
max
nm. Enantiomer 21b (2.6 mg): (2R,3R)-21; white, amorphous
powder; t 13.63 min; [α] −115 (c 0.1, CH OH); ECD (MeOH)
λmax (Δε) 250 (−3.69), 310 (−2.96) nm.
20
R
D
3
1
13
ESIMS and H and C NMR spectra for compounds 13,
1
Racemic neolignanamide 27 (6.1 mg) was dissolved in EtOH (1.5
mL) and was resolved by HPLC-UV using a Lux Cellulose-2 chiral
column (eluting solvent: n-hexane−EtOH, 35:65; flow rate 0.5 mL/
min; 30 injections; 50 μL each) to yield two enantiomers. Enantiomer
1
13
4, 16, 17, 19, 20; ESIMS, H, C COSY, HSQC, and
HMBC spectra for neolignanamides (±)-21−(±)-28;
stacked plot of RP-18-HPLC-UV of (±)-4 and (±)-21−
(±)-28 (PDF)
2
7a (2.8 mg): (2S,3S)-27; white, amorphous powder; t = 8.09 min;
R
L
DOI: 10.1021/acs.jnatprod.6b00577
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(
27) Basini, G.; Spatafora, C.; Tringali, C.; Bussolati, S.; Grasselli, F. J.
AUTHOR INFORMATION
■
Biomol. Screening 2014, 19, 1282−1289.
(
Corresponding Author
28) Mogharabi, M.; Faramarzi, M. A. Adv. Synth. Catal. 2014, 356,
*
Tel (C. Spatafora): +39 095 7385015. Fax: +39 095 580138.
E-mail: cspatafo@unict.it.
897−927.
(29) Spatafora, C.; Tringali, C. In Targets in Heterocyclic Systems.
Chemistry and Properties; Attanasi, O. A., Spinelli, D., Eds.; Societa
Chimica Italiana: Rome, 2007; Vol. 11, pp 284−312.
̀
Notes
The authors declare no competing financial interest.
(30) Bruschi, M.; Orlandi, M.; Rindone, B.; Rummakko, P.; Zoia, L. J.
Phys. Org. Chem. 2006, 19, 592−596.
ACKNOWLEDGMENTS
(31) Orlandi, M.; Rindone, B.; Molteni, G.; Rummakko, P.; Brunow,
G. Tetrahedron 2001, 57, 371−378.
■
This research was supported by a grant of the University of
Catania (FIR−“Finanziamento della Ricerca” 2014).
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DOI: 10.1021/acs.jnatprod.6b00577
J. Nat. Prod. XXXX, XXX, XXX−XXX