Resveratrol-Related Polymethoxystilbene Glycosides: Synthesis, Antiproliferative Activity, and Glycosidase Inhibition

Article abstract of DOI:10.1021/acs.jnatprod.5b00619

A small library of polymethoxystilbene glycosides (20-25) related to the natural polyphenol resveratrol have been synthesized and subjected, together with their aglycones 17-19, to an antiproliferative activity bioassay toward Caco-2 and SH-SY5Y cancer cells. Six of the compounds exhibit antiproliferative activity against at least one cell line. In particular, compounds 17 and 18 proved highly active on at least one of the two cell cultures. Compound 18 showed a GI₅₀ value of 3 ΜM against Caco-2 cells, a value comparable to that of the anticancer drug 5-fluorouracil. The closely related compound 19 proved inactive, and its conjugates 22 and 25 showed weak cell growth inhibition. The results indicate that minimal differences in the structure of both polymethoxystilbenes and their glycosides can substantially affect the antiproliferative activity. The possible hydrolytic release of the aglycones 17-19 by β-glucosidase or β-galactosidase was also evaluated. Compounds 20-25 were also tested as potential β-glucosidase, β-galactosidase, and α-glucosidase inhibitors. A promising inhibitory activity toward α-glucosidase was observed for 21 (IC₅₀ = 78 ΜM) and 25 (IC₅₀ = 70 ΜM), which might be indicative of their potential as lead compounds for development of antidiabetic or antiobesity agents.

Full text of DOI:10.1021/acs.jnatprod.5b00619

Article
pubs.acs.org/jnp
Resveratrol-Related Polymethoxystilbene Glycosides: Synthesis,
Antiproliferative Activity, and Glycosidase Inhibition
Nunzio Cardullo,† Carmela Spatafora,*,† Nicolo Musso,‡ Vincenza Barresi,‡ Daniele Condorelli,‡
̀
and Corrado Tringali†
‡
†Dipartimento di Scienze Chimiche and Dipartimento di Scienze Bio-Mediche, Sezione di Biochimica, Universita
̀
degli Studi di
Catania, Viale A. Doria 6, I-95125 Catania, Italy
S
* Supporting Information
ABSTRACT: A small library of polymethoxystilbene glycosides (20−25) related to the natural polyphenol resveratrol have been
synthesized and subjected, together with their aglycones 17−19, to an antiproliferative activity bioassay toward Caco-2 and SH-
SY5Y cancer cells. Six of the compounds exhibit antiproliferative activity against at least one cell line. In particular, compounds 17
and 18 proved highly active on at least one of the two cell cultures. Compound 18 showed a GI50 value of 3 μM against Caco-2
cells, a value comparable to that of the anticancer drug 5-fluorouracil. The closely related compound 19 proved inactive, and its
conjugates 22 and 25 showed weak cell growth inhibition. The results indicate that minimal differences in the structure of both
polymethoxystilbenes and their glycosides can substantially affect the antiproliferative activity. The possible hydrolytic release of
the aglycones 17−19 by β-glucosidase or β-galactosidase was also evaluated. Compounds 20−25 were also tested as potential β-
glucosidase, β-galactosidase, and α-glucosidase inhibitors. A promising inhibitory activity toward α-glucosidase was observed for
21 (IC50 = 78 μM) and 25 (IC50 = 70 μM), which might be indicative of their potential as lead compounds for development of
antidiabetic or antiobesity agents.
tilbenoids are an important family of natural products
better oral bioavailability, longer half-life, greater plasma
Ssharing with flavonoids shikimate as common biosynthetic exposure, and lower clearance than resveratrol.16 Polymethox-
precursor.1 Probably the most popular polyphenolic stilbenoid
ystilbenes are frequently much more antiproliferative than
resveratrol14,17 and show other promising properties such as
is resveratrol (1, Figure 1). This compound, originally identified
as a phytoalexin produced by Vitis vinifera, has subsequently
antiangiogenic activity and VEGF inhibition,9,15,18 inhibition of
NF-kB activation,19 and inhibition of multidrug resistance
been found in red wine and in other plants and became an
(MDR) of tumor cells.20 Further studies indicate that 2 and
outstanding natural product for the high number of studies
other polymethoxystilbenes are able to interact with biomem-
brane models21−23 and have a higher bioavailability than
resveratrol.24−27 Among the methoxylated stilbenes and related
compounds, some cis-isomers have shown higher antiprolifer-
ative28 or antimetastatic14 activity than their trans-isomers, but
they are prone to isomerization during storage and
administration and during metabolism in liver microsomes.29
We expanded our studies on polymethoxystilbenes by
exploring the antiproliferative properties of some of their
glycosides, due to the fact that biological data on these
glycosides have not been reported, although many natural
indicating 1 as possessing cardioprotective, cancer chemo-
preventive, antioxidative, and many other beneficial proper-
ties.2,3 As a continuation of our long-standing interest toward
bioactive natural products and especially polyphenols,4,5 we
have devoted part of our research activities to analogues and
derivatives of resveratrol.6−9 Various in vivo studies have shown
that 1 is impaired by a short half-life, rapid clearance, and low
bioavailability, being rapidly metabolized.10,11 Thus, efforts have
been made to obtain synthetic analogues with an increased
metabolic stability and potentially enhanced antitumor activity.
A simple resveratrol analogue, 3,5,4′-trimethoxystilbene (2), a
natural product isolated from Virola elongatae,12 is significantly
more potent than 1 in inhibiting growth or motility of human
cancer cells7,13,14 and as an antiangiogenic15 agent and showed
Received: July 15, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 2. Chemical structures of compounds 6 and 7.
subsequent mild regioselective hydroxylation at C-2 by m-
CPBA afforded the corresponding 2-hydroxypolymethoxystil-
benes 17−19. We previously reported their synthesis, where
especially 17 showed antiangiogenic activity.9,18 Compound 17
also proved to be an inhibitor of VEGF production in a
granulosa cell model; compounds 17−19 were also able to
inhibit the proliferation of SW480 colorectal tumor cells,6 and
19 proved more potent than both 17 and 18.
Figure 1. Chemical structures of compounds 1−5 and 26.
glycosides display antitumor activity or other promising
biological properties.30,31 Bioactive glycostilbenoids have been
found in many families of higher plants.30 Among these,
resveratroloside (3, Figure 1) showed cytotoxic activity against
human tumor cell lines; piceid32 (4, Figure 1) and 3,5,4′-
trihydroxystilbene-2-O-β-D-glucopyranoside (5, Figure 1) in-
hibited tumor growth and lung metastasis in mice bearing
highly metastatic Lewis lung carcinoma and showed antiangio-
genic activity.33 Angiogenesis inhibitors are intensively pursued
as a promising approach to cancer therapy.34 Antiangiogenic
activity through fibroblast growth factor-2 (FGF-2) inhibition
has also been reported for a resveratrol-related natural glycoside
bearing a disaccharide moiety.35
The growing attention to glycoconjugates as target-specific
antiproliferative agents or effective prodrugs36 boosted our
interest further. Indeed, glycoconjugates might target specific
saccharide transporters and primarily glucose transporters;
however, these cells exhibit an altered glucose metabolism,
known as the “Warburg effect”, which has recently been
indicated as an emerging hallmark of cancer.37 O-Glycosides
should accumulate selectively in tumor cells, thus allowing dose
reduction of the drug and consequently its general toxicity.38 As
prodrugs, they might be hydrolyzed by specific glycosidases,
thereby allowing the release of the active aglycone. In this
regard it is worth citing a report on target-specific
glycoconjugates evaluated for cytotoxic properties:39 the β-D-
galactoside of the synthetic drug diethylstilbestrol (6, Figure 2)
showed an IC50 value of 75 μM on Caco-2 cells; the 4-O-β-D-
glucoside of curcumin (7, Figure 2), the pigment found in
Curcuma longa,40 proved the most potent, with an IC50 value of
10.3 μM on Caco-2 cells. Further glycosylated resveratrol
prodrugs have been reported as anti-inflammatory agents, of
which two were effective prodrugs in mice.41
The final step was the conjugation of compounds 17−19 via
a nucleophilic substitution to the anomeric carbon of an
activated sugar. The glycosylation can be troublesome with
phenols because of the low reactivity of the phenolic group as a
glycosyl acceptor. Glycosyl halides (especially bromides) are
the most frequently used carbohydrate donors for aromatic O-
glycosylation. Unlike other types of donors, glycosyl halides
selectively give the O-glycosylation product with the inversion
of anomeric configuration (SN2 mechanism) and with accept-
able yields.42 Thus, to obtain, respectively, the 2-β-D-O-gluco-
and galactopyranosides, tetra-O-acetyl-α-D-glucopyranosyl bro-
mide and tetra-O-acetyl-α-D-galactopyranosyl bromide were
employed. In a first attempt, the glycosylation was carried out
in dry ethanol at room temperature using basic conditions, but
slow reactions, accompanied by the formation of a complex
mixture of products, were observed. Better results were
obtained in a biphasic system and in the presence of a phase
transfer catalyst (tetrabutylammonium chloride), in basic
medium. The procedure was further optimized by temperature
modulation, and glycosides 20−25 were obtained in 30−46%
yield through RP-18 reversed-phase PLC (see Experimental
Section). No detectable byproduct was obtained, and the
recovered unreacted substrate, when necessary, was recycled.
Indeed, under these reaction conditions both glycosylation and
acetate hydrolysis were achieved in one step. Compounds 20−
25 were subjected to full structural characterization, and NMR
methods (COSY, HSQC, HMBC) allowed unambiguous
assignment of all their 1H and 13C NMR signals (see
Experimental Section).
This prompted the syntheses of gluco- and galactopyrano-
sides of three polymethoxystilbenes bearing a 2-OH group, to
be exploited as glycosyl acceptors.
The glycoconjugates 20−25 were evaluated for antiprolifer-
ative activity toward Caco-2 and SH-SY5Y cell lines with the
Mosmann bioassay, using 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT). The 2-hydroxypolyme-
thoxystilbenes 17−19 were included in the panel, both for
comparison with the related conjugates and for a further
evaluation of their previously observed antiproliferative activity
on SW480 colon carcinoma cells.6 5-Fluorouracil (5-FU) was
used as positive control. In Table 1 only results for compounds
showing a GI50 value lower than at least 10 μM are included.
RESULTS AND DISCUSSION
■
The synthetic procedure for the glycoconjugate polymethox-
ystilbenes is shown in Scheme 1. The polymethoxystilbenes 11,
15, and 16 were synthesized via Arbuzov rearrangement
followed by Horner−Emmons−Wadsworth olefination to
afford the trans-isomer with high diastereoselectivity. A
B
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a
Scheme 1. Synthetic Procedures for Compounds 20−25
a
(a) P(OCH2CH3)3, 130 °C, 5 h; (b) CH3ONa, DMF, 0 °C, 30 min, rt, 1 h, 100 °C, 1 h, rt, 24 h; (c) m-CPBA, CH2Cl2, rt, 35 min; (d) K2CO3,
TBACl, MeOH/H2O/CHCl3, tetra-O-acetyl-α-D-glucopyranosyl bromide, 24 h; (e) K2CO3, TBACl, MeOH/H2O/CHCl3, tetra-O-acetyl-α-D-
galactopyranosyl bromide.
a
Table 1. Antiproliferative Activity of Compounds 17−25
cytotoxic effect up to the maximum tested concentration of 100
μM) is rather surprising due to its close structural similarity to
18. Hence, the substitution pattern of the ring devoid of the
hydroxy group seems to be critical for the activity of these
unconjugated stilbenoids.
The interpretation of the biological data of the glycosides
20−25 is not straightforward. A reduction of the potency is
observed due to conjugation of 17 and 18 with both glucose
and galactose: their glycosides 20, 21 and 23, 24 are inactive
toward Caco-2 cells, whereas a weak growth inhibition is
observed only for 20 and 23 toward SH-SY5Y cells. This
suggests that, at least in Caco-2 cells, these glycosides are not
effectively taken up or are not adequately hydrolyzed and do
not interact with the biological target. Something different is
observed for the inactive aglycone 19: its galactoside (25)
showed a weak antiproliferative effect toward Caco-2 cells and
SH-SY5Y cells.
We therefore subjected the glycosides 20−25 to hydrolytic
reactions carried out in the presence of β-glucosidase or β-
galactosidase, as an in vitro experimental model for a possible
hydrolysis by intracellular enzymes (see Experimental Section).
We hypothesized that the lack of antiproliferative activity of
glucosides and galactosides of the active aglycones 17 and 18
could be due to poor hydrolysis by intracellular enzymes.
Figure 3a shows the kinetic profile of compounds 20−25 in the
b
GI50 (μM) SD
c
d
compound
Caco-2
SH-SY5Y
17
5.8 0.9
3.0 0.4
2.2 0.8
4.5 0.5
14.3 3.5
1.5 0.1
18
5-FU
a
Compounds 19−25 were inactive against both tested cancer cell lines
or showed GI50 > 10 μM. GI50 calculated after 72 h of continuous
exposure relative to untreated controls; values are the mean ( SD) of
four experiments. Caco-2: human colon carcinoma. SH-SY5Y:
neuroblastoma.
b
c
d
Six of the tested compounds exhibit growth inhibition against
at least one cell line, although the biological response shows
significant variations for the different cell lines. In particular,
compound 17 proved highly active on both Caco-2 and SH-
SY5Y cells, and compound 18 was potently active toward Caco-
2 (GI50 = 3.0 μM), with a GI50 value comparable to that of the
anticancer drug 5-FU. Compounds 22 and 25 showed a weak
growth inhibition of Caco-2 cells (GI50 = 44.2 and 16.0 μM,
respectively), and a similar weak inhibition was observed for
compounds 20, 23, and 25 toward SH-SY5Y cells (GI50 = 21.9,
48.4, and 22.5 μM, respectively). Compounds 19, 21, and 24
were inactive on both cell lines. The inactivity of 19 (no
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The glucosides 20 and 21 were progressively hydrolyzed by
almond glucosidase after a few hours; a slower hydrolysis was
observed for 22, with a half-life of 70 h. No significant
hydrolysis was observed for the three galactosides 23−25 up to
96 h. Figure 3b shows that compounds 21−25 were not
hydrolyzed by β-galactosidase up to 96 h. Compound 20
underwent slow hydrolysis, with a half-life of 77 h. The amount
of 17 released from 20 after 72 h of β-galactosidase treatment is
less than 50%. In summary, only 20 and 21 were significantly
hydrolyzed by β-glucosidase, with a half-life of 11 and 20 h,
respectively, much shorter than the 72 h duration of the
biological assays. Nevertheless, these compounds are probably
not adequately absorbed, with the exception of compound 20
by SH-SY5Y cells. In this latter case even a low uptake if
coupled with a high hydrolytic rate by intracellular β-
glucosidases, affording the active aglycone 17, might justify
the observed activity.
The lack of hydrolytic effect by β-glucosidase and especially
by β-galactosidase for most of the glycosides prompted us to
investigate if these compounds might act as β-glucosidase or β-
galactosidase inhibitors. This led to enzymatic inhibition assays
for both enzymes in the presence of compounds 20−25, as
detailed in the Experimental Section. In the analysis of β-
glycosidase activities p-nitrophenyl-β-glucopyranoside and o-
nitrophenyl-β-galactopyranoside were employed as substrates.
The results are summarized in Table 2, where the μM
concentration required for 50% inhibition of the glycosidase
activity (IC50) is reported.
Figure 3. Hydrolysis of 20−25 at 37 °C (pH = 7.2) in the presence of
enzymes: (a) β-glucosidase from almonds, (b) β-galactosidase from A.
oryzae. S = substrate.
Table 2. β-Glucosidase, β-Galactosidase, and α-Glucosidase
Inhibitory Activities of Compounds 20−25
a
IC50 (μM) SD
presence of β-glucosidase from almonds, whereas Figure 3b
gives the results of the same experiment in the presence of β-
galactosidase from Aspergillus oryzae. These results were
confirmed by the quantification of the released aglycone, as
reported in Figure 4.
β-glucosidase
β-galactosidase
α-glucosidase
20
208 47
192 50
272 16
n.d.
91 17
78 21
21
258 42
297 68
298 68
299 53
296 12
22
133 19
201 29
23
350
6
24
170 60
n.i.
70
332 70
65
3
25
acarbose
9
a
Values are the mean ( SD) of three experiments; n.d.: not
determined, n.i.: no inhibition.
Most of the compounds showed moderate inhibitory activity
toward both enzymes, with IC50 values in the approximate
range 170−350 μM. All the determined IC50 values are
substantially similar, and no significant difference was observed
between glucosides and galactosides, except for the lack of β-
glucosidase inhibition by 25 up to 1 × 10−4 M. However, the
data are noteworthy, if compared with previously reported IC50
values of other glycosidase inhibitors.43 This prompted the
evaluation of these stilbenoid glycosides as α-glucosidase
inhibitors, taking into account the growing interest toward
new potential antidiabetic agents. We carried out an enzymatic
inhibition assay employing p-nitrophenyl-α-glucopyranoside as
the substrate. The results are reported in Table 2 as IC50 values
(μM). The glycoconjugates 20−24 showed IC50 values in the
approximate range 70−200 μM, whereas 25 showed an IC50
value higher than 300 μM. Compounds 20, 21, and 24 (IC50
=
91, 78, 70 μM, respectively) showed a promising inhibitory
activity when compared with the antidiabetic drug acarbose
(IC50 = 65 μM)44,45 or with many previously reported α-
Figure 4. (a) Percentage of aglycone released by β-glucosidase from
20−25; (b) percentage of aglycone released by β-galactosidase from
20−25.
D
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staining with a solution of cerium sulfate and phosphomolybdic acid
followed by heating.
glucosidase inhibitors, whose IC50 values are higher than 100
μM.43 Resveratrol (1) and other related stilbenoids have
previously been reported as α-glucosidase inhibitors46,47 or
antidiabetic or antiobesity agents.48 In addition, the stilbenoid
glucoside desoxyrhaponticin (5-hydroxy-4′-methoxystilbene-3-
O-β-D-glucopyranoside, 26), isolated from Rheum emodi49 and
closely related to compounds 20−25, was reported as a yeast α-
glucosidase inhibitor.
The enzymatic reactions were incubated at 37 °C under shaking at
400 rpm; control reactions without enzyme were carried out under the
same conditions. The progress of the reactions was monitored at
regular time intervals by HPLC using an analytic reversed-phase
column (Luna C18 column, 5 μm; 4.6 × 250 mm; Phenomenex) and
the following solvent system at 1 mL/min: eluent A: H2O and
HCOOH, 99:1 (v/v); eluent B: CH3CN and HCOOH, 99:1 (v/v).
The elution gradient had the following profile: t0 min B = 45%, t7 min
= 80%, t10 min B = 90%, t12 min B = 100%.
B
In conclusion the polymethoxystilbene glycosides 20−25
were synthesized and, together with the related 2-hydroxy-
polymethoxystilbenes 17−19, were subjected to an antiprolifer-
ative activity bioassay toward Caco-2 (human colon) and SH-
SY5Y (neuroblastoma) cancer cells. Compounds 17 and 18
proved highly active on both cells (Table 1); 18 showed a GI50
value of 3 μM (Caco-2 cells), a value comparable to that of 5-
FU. Compound 19 was inactive, although its glucoside 22 and
galactoside 25 showed a weak growth inhibition of Caco-2 and
SH-SY5Y cells. The possible hydrolytic release of the aglycones
17−19 by β-glucosidase or β-galactosidase was also evaluated;
only 20 and 21 were significantly hydrolyzed by β-glucosidase.
Collectively, the data show that minimal differences in the
structure of both stilbenoids and conjugates may substantially
affect their antiproliferative activity. An evaluation of the β-
glucosidase or β-galactosidase activity in the presence of
compounds 20−25 revealed that all are moderate inhibitors at
least toward one of these enzymes. Finally, compounds 21 and
24 showed a promising α-glucosidase inhibition. Thus, these
resveratrol-related conjugates may be considered as lead
compounds for future development of antidiabetic or
antiobesity agents.
All chemicals were of reagent grade and were used without further
purification. All chemicals, β-glucosidase from almonds, β-galactosi-
dase from Aspergillus oryzae, α-glucosidase from Saccharomyces
cerevisiae, p-nitrophenyl-β-D-glucopyranoside (pNP-β-Glc), o-nitro-
phenyl β-D-galactopyranoside (oNP-β-Gal), and p-nitrophenyl-α-D-
glucopyranoside (pNP-α-Glc), were purchased from Sigma.
Compounds 11 and 15−19 were synthesized as previously
described by Spatafora et al.9 The stilbenoid 11 was obtained by
reacting the diethyl (4-methoxybenzyl)phosphonate 9 (prepared from
4-methoxybenzyl chloride 8) with the aldehyde 10; analogously, 15
was synthesized by reacting the diethyl (3,5-dimethoxybenzyl)-
phosphonate 13 (prepared from 3,5-dimethoxybenzyl bromide 12)
with the aldehyde 14; analogously, 16 was obtained by reaction of 13
with 10. Finally, compounds 11, 15, and 16 were subjected to a mild
hydroxylation in the presence of m-chloroperbenzoic acid to give the
2-hydroxystilbenes 17−19.
Synthesis of Compounds 20−25. Preliminary Experiments.
Reaction conditions were optimized by subjecting compound 19 to
preliminary experiments (below indicated as a−c).
a. Synthesis in Homogeneous Phase. Compound 19 (0.0160 g,
0.017 mmol) was dissolved in KOH solution (1 mL of 1.25 M) in dry
EtOH and stirred for 15 min. Tetra-O-acetyl-α-D-glucopyranosyl
bromide (0.0102 g, 0.024 mmol) was added, and the mixture was
stirred at room temperature for 3 days. The reaction was monitored by
TLC (5% CH3OH/CHCl3); two further aliquots of tetra-O-acetyl-α-D-
glucopyranosyl bromide (0.0050 g, 0.012 mmol) were added after 12
and 36 h.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured with a Rudolph Research Analytical (Autopol I) polarimeter
at 25 °C and 589 nm. UV spectra were recorded on a Jasco V630
spectrometer. NMR spectra were run on a Varian Unity Inova
spectrometer operating at 499.86 (1H) and 125.70 MHz (13C) and
equipped with a gradient-enhanced, reverse-detection probe. Chemical
shifts (δ) are indirectly referred to TMS using solvent signals. The 2D
g-HSQCAD experiments were performed with matched adiabatic
sweeps for coherence transfer, corresponding to a central 13C−1H J-
value of 146 Hz. G-HMBCAD experiments were optimized for a long-
range 13C−1H 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 manufacturers and
acquired at constant temperature (300 K). Methanol-d4 or pyridine-d5
were used as solvents. Mass spectra were acquired with an Agilent
6410 triple quadrupole (1200 Series) mass spectrometer 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) with MeOH/H2O/
HCOOH (98:2:0.1). The following parameters were used for sample
ionization: gas temperature 300 °C; vaporizer temperature 250 °C; gas
flow 10 L/min; nebulizer 60 psi; fragmentator 135 V; capillary voltage
3500 V; charging 2000 V. Elemental analyses were performed on a
PerkinElmer 240B microanalyzer. High-performance liquid chroma-
tography (HPLC) was carried out using an Agilent Series G1354A
pump and an Agilent UV G1315D as diode array detector (DAD). An
Agilent Series 1100 G1313A autosampler was used for sample
injection. PLC was performed on LiChroprep Si 60 (0.025−0.040
mm; Merck) or on silica gel RP-18 (0.025−0.040 mm; Merck) using
different solvent systems, as reported for each compound. Thin-layer
chromatography (TLC) was carried out using precoated silica gel
F254 plates (Merck); visualization of reaction components was
achieved under UV light at a wavelength of 254 and 366 nm or by
b. Synthesis in Heterogeneous Phase at Room Temperature.
Compound 19 (0.0044 g, 0.014 mmol) was dissolved in MeOH/H2O
(55:45, 2 mL) and stirred together with K2CO3 (0.0200 g, 0.145
mmol) for 15 min at room temperature. A mixture of tetrabuty-
lammonium chloride (TBACl, 0.0042 g, 0.014 mmol) and tetra-O-
acetyl-α-D-glucopyranosyl bromide (0.0145 g, 0.035 mmol) in CHCl3
(2 mL) were added. The biphasic system was stirred at room
temperature for 20 h, and the reaction was monitored by TLC (5%
CH3OH/CHCl3).
c. Synthesis in Heterogeneous Phase under Reflux. Compound 19
(0.0057 g, 0.020 mmol) was dissolved in MeOH/H2O (45:55, 2.2
mL), and the solution was stirred with K2CO3 (0.0248 g, 0.179 mmol)
at room temperature for 15 min. To the solution were added 1 mL of
CHCl3, TBACl (0.0055 g, 0.018 mmol), and a chloroformic solution
of tetra-O-acetyl-α-D-glucopyranosyl bromide (0.0185 g, 0.045 mmol,
dissolved in 1.2 mL of CHCl3). The heterogeneous mixture was stirred
at 60 °C for 24 h. After 10 h, a solution of tetra-O-acetyl-α-D-
glucopyranosyl bromide (0.0098 g, 0.024 mmol/2 mL of CHCl3) was
added. The reaction was monitored by TLC (5% CH3OH/CHCl3).
The procedure c gave a higher yield of compound 25 and was also
employed to synthesize compounds 21, 22, and 24. Under these
conditions 20 and 23 were obtained in low yields; higher yields were
obtained carrying out the reaction at room temperature (procedure b).
(2R,3S,4S,5S)-2-[2-(4-Methoxystyryl)-4,6-dimethoxyphenoxy]-
tetrahydro-6-hydroxymethyl-2H-pyran-3,4,5-triol (20). Compound
17 (0.0184 g, 0.064 mmol) was stirred with K2CO3 (0.0880 g, 0.637
mmol) in MeOH/H2O (45:55, 8.3 mL) for 15 min at room
temperature. The catalyst TBACl (0.0178 g, 0.064 mmol), 3.6 mL of
CHCl3, and a solution of tetra-O-acetyl-α-D-glucopyranosyl bromide
(0.0396 g, 0.096 mmol in 4 mL of CHCl3) were added. The mixture
was stirred at room temperature for 24 h. The reaction was monitored
by TLC (5% CH3OH/CHCl3), and a solution of tetra-O-acetyl-α-D-
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glucopyranosyl bromide (0.0175 g, 0.042 mmol/2 mL of CHCl3) was
added after 10 h. The reaction was quenched by phase separation. The
organic phase was dried over anhydrous Na2SO4, and evaporated to
dryness, and the crude residue was purified by flash chromatography
on RP-18 silica gel using a gradient of CH3CN/H2O (from 10% to
70%). Amorphous powder (0.0056 g, 33% yield): Rf (TLC) = 0.25
(5% CH3OH/CHCl3); [α]25D = +73 (c 0.1, CH3OH); UV (CH3OH)
λmax (log ε) 306 (4.32) nm; 1H NMR (500 MHz, methanol-d4, 300 K)
δ 7.67 (d, J = 16.5 Hz, 1 H, 7-H), 7.54 (d, J = 8.7 Hz, 2 H, 2′-H and 6′-
H), 7.04 (d, J = 16.5 Hz, 1 H, 8-H), 6.92 (d, J = 8.7 Hz, 2 H, 3′-H and
5′-H), 6.82 (d, J = 2.5 Hz, 1 H, 6-H), 6.53 (d, J = 2.5 Hz, 1 H, 4-H),
4.72 (d, J = 8.0 Hz, 1 H, 1″-H), 3.86 (s, 3 H, 3-OCH3), 3.84 (s, 3 H, 5-
OCH3), 3.82 (s, 3 H, 4′-OCH3), 3.77 (dd, J = 12.0, 3.0 Hz, 1 H, 6a″-
H), 3.67 (dd, J = 12.0, J = 5.0 Hz, 1 H, 6b″-H), 3.56 (dd, J = 9.0, 8.0
Hz, 1 H, 2″-H), 3.48−3.42 (m, 2 H, 3″-H and 4″-H), 3.20 (ddd, J =
6.5, 5.0, 3.0 Hz, 1H, 5″-H); 13C NMR (125 MHz, methanol-d4, 300 K)
δ 160.9 (C-4′), 158.4 (C-5), 154.5 (C-3), 139.1 (C-2), 134.1 (C-1),
131.9 (C-1′), 130.1 (C-8), 129.1 (C-2′ and C-6′), 123.1 (C-7), 115.1
(C-3′ and C-5′), 106.4 (C-1″), 101.5 (C-6), 100.5 (C-4), 78.1 (C-5″),
77.9 (C-3″), 75.9 (C-2″), 71.5 (C-4″), 62.7 (C-6″), 56.7 (C-3-OCH3),
56.1 (C-5-OCH3), 55.7 (C4′-OCH3); ESIMS calcd for C23H28O9Na
[M + Na]+ 471.16; found 471.2; anal. calcd for C23H28O9 C, 61.60; H,
6.29; found C, 61.24; H, 6.27.
The aqueous phase was extracted with CHCl3 (3 × 6 mL), and the
combined organic phases were washed with H2O (3 × 15 mL). The
CHCl3 phase was dried over Na2SO4, filtered, and dried in vacuo. The
residue was purified by liquid chromatography using RP-18 silica gel
with a gradient of CH3CN in H2O (from 40% to 80%). Product 22
was obtained (0.0063 g, 38%): amorphous powder; Rf (TLC) = 0.37
(4% CH3OH/CHCl3); [α]25D = +55 (c 0.1, CH3OH); UV (CH3OH)
λmax (log ε) 303 (4.30) nm. 1H NMR (500 MHz, methanol-d4, 300 K)
δ 7.77 (d, J = 16.2 Hz, 1 H, 7-H), 7.02 (d, J = 16.2 Hz, 1 H, 8-H), 6.83
(d, J = 2.7 Hz, 1 H, 6-H), 6.77 (d, J = 2.0 Hz, 2 H, 2′-H and 6′-H),
6.55 (d, J = 2.7 Hz, 1 H, 4-H), 6.40 (t, J = 2.0 Hz, 1 H, 4′-H), 4.74 (d,
J = 8.0 Hz, 1 H, 1″-H), 3.86 (s, 3 H, 3-OCH3), 3.84 (s, 3 H, 5-OCH3),
3.83 (s, 6 H, 3′-OCH3 and 5′-OCH3), 3.77 (dd, J = 11.0, J = 2.5 Hz,1
H, 6a″-H), 3.65 (dd, J = 11.0, J = 5.5 Hz, 1 H, 6b″-H), 3.56 (dd, J =
8.5, 8.0 Hz,1 H, 2″-H), 3.46−342 (m, 2 H, 3″-H and 4″-H), 3.18 (m, 1
H, 5″-H); 13C NMR (125 MHz, methanol-d4, 300 K) δ 162.5 (C-3′
and C-5′), 158.4 (C-5), 154.7 (C-3), 141.1 (C-1), 139.2 (C-2),133.5
(C-1′), 130.5 (C-8), 125.8 (C-7), 106.2 (C-1″), 105.7 (C-2′ and C-
6′), 101.11 (C-4), 101.66 (C-6), 101.08 (C-4′), 78.02 (C-5″), 77.92
(C-3″), 75.9 (C-2″), 68.7 (C-4″), 63.2 (C-6″), 56.7 (C-3-OCH3), 55.9
(C-3′-OCH3 and C-5′-OCH3), 56.1 (C-5-OCH3); ESIMS calcd for
C24H30O10Na [M + Na]+ 501.17; found 501.2; anal. calcd for
C24H30O10 C, 60.24; H, 6.32; found C, 60.30; H, 6.21.
(2R,3S,4S,5S)-2-[2-(3,4-Dimethoxystyryl)-4,6-dimethoxyphenoxy]-
tetrahydro-6-hydroxymethyl-2H-pyran-3,4,5-triol (21). Compound
18 (0.0212 g, 0.067 mmol) was dissolved in MeOH/H2O (45:55, 8.5
mL), and the solution was stirred with K2CO3 (0.0926 g, 0.670 mmol)
at room temperature for 15 min. To the aqueous solution was added
TBACl (0.0182 g, 0.067 mmol) in CHCl3 (4 mL), and subsequently, a
solution of tetra-O-acetyl-α-D-glucopyranosyl bromide (0.0688 g,
0.168 mmol in 4.5 mL of CHCl3) was added. The heterogeneous
mixture was heated at 60 °C for 24 h. The reaction was monitored by
TLC (4% CH3OH/CHCl3), and after 10 h tetra-O-acetyl-α-D-
glucopyranosyl bromide in CHCl3 (0.0301 g/2 mL) was added.
The aqueous phase was extracted with CHCl3 (3 × 8 mL); the
combined organic phases were washed with H2O (3 × 16 mL), dried
over Na2SO4, filtered, and evaporated to dryness. The crude extract
was purified by column chromatography with RP-18 silica gel with a
gradient of CH3CN in H2O (from 10% to 50%), affording 21 (0.0087
g, 43%): amorphous powder; Rf (TLC) = 0.16 (4% CH3OH/CHCl3);
(2R,3S,4S,5R)-2-[2-(4-Methoxystyryl)-4,6-dimethoxyphenoxy]-
tetrahydro-6-hydroxymethyl-2H-pyran-3,4,5-triol (23). Compound
17 (0.0232 g, 0.081 mmol) was stirred with K2CO3 (0.1123 g, 0.813
mmol) in MeOH/H2O (45:55, 10.2 mL) for 15 min at room
temperature. To the aqueous solution were added CHCl3 (4.3 mL),
TBACl (0.0226 g, 0.049 mmol), and a solution containing tetra-O-
acetyl-α-D-galactopyranosyl bromide (0.0666 g, 0.162 mmol) in
CHCl3 (4.8 mL). The heterogeneous mixture was stirred at room
temperature for 24 h, and the reaction was monitored by TLC (5%
CH3OH/CHCl3). Another addition of sugar was done after 10 h
(0.0293 g/2.0 mL). The reaction was quenched by phase separation.
The CHCl3 phase was dried over anhydrous Na2SO4, filtered, and
evaporated in vacuo. The organic extract was purified by column
chromatography with RP-18 silica gel with a gradient of CH3CN in
H2O (from 10% to 70%). Amorphous powder (0.0069 g, 30%); Rf
(TLC) = 0.23 (5% CH3OH/CHCl3); UV (CH3OH) λmax (log ε) 306
1
(4.01) nm; H NMR (500 MHz, pyridine-d5, 300 K) δ 8.60 (d, J =
[α]25 = 22 (c 0.2, CH3OH); UV (CH3OH) λmax (log ε) 320 (3.94)
16.5 Hz, 1 H, 7-H), 7.86 (d, J = 8.5 Hz, 2 H, 2′-H and 6′-H),7.49 (d, J
= 16.5 Hz, 1 H, 8-H), 7.23 (d, J = 3.0 Hz, 1 H, 6-H), 6.99 (d, J = 8.5
Hz, 2 H, 3′-H and 5′-H), 6.76 (d, J = 3.0 Hz, 1 H, 4-H), 5.46 (d, J =
8.0 Hz, 1 H, 1″-H), 4.93 (dd, J = 9.0, 8.0 Hz, 1 H, 2″-H), 4.75 (bd, J =
3.5 Hz, 1 H, 4″-H), 4.63 (dd, J = 10.5, J = 6.5 Hz, 1 H, 6a″-H), 4.36
(dd, J = 10.5, J = 6.5, 1 H, 6b″-H), 4.32 (dd, J = 9.0, 3.5 Hz, 1 H, 3″-
H), 4.08 (bt, J = 6.5 Hz, 5″-H) 3.84 (s, 3 H, 5-OCH3), 3.83 (s, 3 H, 3-
OCH3), 3.70 (s, 3 H, 4′-OCH3). 13C NMR (125 MHz, pyridine-d5,
300 K) δ 159.9 (C-4′), 157.4 (C-5), 154.6 (C-3), 139.5 (C-2), 133.6
(C-1), 131.4 (C-1′), 129.3 (C-8), 128.7 (C-2′ and C-6′), 123.2* (C-
7), 114.7 (C-3′ and C-5′), 107.6 (C-1″), 101.2 (C-6), 100.7 (C-4),
77.0 (C-5″), 75.4 (C-3″), 73.6 (C-2″), 69.9 (C-4″), 61.9 (C-6″), 56.4
(C-3-OCH3), 55.6 (C-5-OCH3), 55.3 (C4′-OCH3); value with
superscript (*) was assigned through HMBC correlations; the signal
was partially overlapped with that of the residual undeuterated solvent;
ESIMS calcd for C23H28O9Na [M + Na]+ 471.16; found 471.2; anal.
calcd for C23H28O9 C, 61.60; H, 6.29; found C, 61.42; H, 6.23.
( 2 R , 3 S , 4 S , 5 R ) - 2 - [ 2 - ( 3 , 4 - D i m e t h o x y s t y r y l ) - 4 , 6 -
dimethoxyphenoxy]tetrahydro-6-hydroxymethyl-2H-pyran-3,4,5-
triol (24). Compound 18 (0.0190 g, 0.060 mmol) was stirred with
K2CO3 (0.0838 g, 0.606 mmol) in MeOH/H2O (45:55, 7.7 mL) at
room temperature for 15 min. CHCl3 (4 mL), TBACl (0.0163 g, 0.060
mmol), and an organic solution of tetra-O-acetyl-α-D-galactopyranosyl
bromide (0.0617 g, 0.150 mmol in 4 mL of CHCl3) were added to the
aqueous phase. The mixture was heated at 60 °C for 24 h, and the
reaction was monitored by TLC. After 10 h tetra-O-acetyl-α-D-
galactopyranosyl bromide (0.0301 g) in 2 mL of CHCl3 was added to
the reaction mixture.
D
nm; 1H NMR (500 MHz, methanol-d4, 300 K) δ 7.68 (d, J = 16.5 Hz,
1 H, 7-H), 7.27 (d, J = 1.5 Hz, 1 H, 2′-H), 7.13 (dd, J = 8.0 Hz, J = 1.5
Hz, 1 H, 6′-H), 7.03 (d, J = 16.5 Hz, 1 H, 8-H), 6.94 (d, J = 8.0 Hz, 1
H, 5′-H), 6.82 (d, J = 3.0 Hz, 1 H, 6-H), 6.53 (d, J = 3.0 Hz, 1 H, 4-
H), 4.72 (d, J = 8.5 Hz, 1 H, 1″-H), 3.91 (s, 3 H, 3′-OCH3), 3.86 (s, 3
H, 3-OCH3), 3.85 (s, 3 H, 4′-OCH3), 3.84 (s, 3 H, 5-OCH3), 3.76
3
(dd, J = 12.0, J = 3.5 Hz, 1 H, 6a″-H), 3.66 (dd, J = 12.0, JH,H = 5.0
Hz, 1 H, 6b″-H), 3.57 (dd, J = 8.5, 7.0 Hz, 1 H, 2″-H), 3.43* (dd, J =
7.5, 7.0 Hz, 1 H, 3″-H), 3.45* (dd, J = 7.0, 4.0, 1 H, 4″-H), 3.18 (m, 1
H, 5″-H); the signals with identical superscript (*) are partially
overlapped; 13C NMR (125 MHz, methanol-d4, 300 K) δ 158.4 (C-5),
154.7 (C-3), 150.7 (C-3′), 150.5 (C-4′), 139.1 (C-2), 133.9 (C-1),
132.6 (C-1′), 130.3 (C-8), 123.4 (C-7), 121.5 (C-6′), 112.9 (C-5′),
110.8 (C-2′), 106.4 (C-1″), 101.5 (C-6), 100.7 (C-4), 78.01 (C-5″),
77.9 (C-3″), 75.9 (C-2″), 71.6 (C-4″), 62.7 (C-6″), 56.72 (C-4′-
OCH3), 56.63 (C-3-OCH3), 56.49 (C-3′-OCH3), 56.07 (C-5-OCH3);
ESIMS calcd for C24H30O10Na [M + Na]+ 501.17; found 501.2; anal
calcd for C24H30O10 C, 60.24; H, 6.32; found C, 60.25; H, 6.19.
(2R,3S,4S,5S)-2-[2-(3,5-Dimethoxystyryl)-4,6-dimethoxyphenoxy]-
tetrahydro-6-hydroxymethyl-2H-pyran-3,4,5-triol (22). Compound
19 (0.0162 g, 0.051 mmol) was dissolved in MeOH/H2O (45:55, 7.5
mL), and the solution was stirred with K2CO3 (0.0714 g, 0.517 mmol)
at room temperature for 15 min. CHCl3 (4 mL), TBACl (0.0142 g,
0.051 mmol), and the solution of tetra-O-acetyl-α-D-glucopyranosyl
bromide previously prepared (0.0524 g, 0.127 mmol in 3 mL of
CHCl3) were added to the aqueous phase. The reaction mixture was
stirred at 60 °C for 24 h, and tetra-O-acetyl-α-D-glucopyranosyl
bromide (0.025 g) in CHCl3 (1.5 mL) was added after 10 h. The
reaction was monitored by TLC (4% CH3OH/CHCl3).
The aqueous phase was extracted with CHCl3 (3 × 6 mL), and the
combined organic phases were washed with H2O (3 × 15 mL), dried
F
DOI: 10.1021/acs.jnatprod.5b00619
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
with anhydrous Na2SO4, filtered, and evaporated to dryness in vacuo.
The crude extract was purified by column chromatography with RP-18
silica gel eluting with a gradient of CH3CN in H2O (from 10% to
30%). By PLC 0.0115 g of galactoside 24 was recovered (46%): Rf
fetal bovine serum, 2 mM L-alanyl-L-glutamine, and penicillin−
streptomycin (50 units−50 mg per mL), and cell cultures were
incubated at 37 °C under a humidified atmosphere of 5% CO2/95%
air. The culture media were changed twice a week.
(TLC) = 0.23 (4% CH3OH/CHCl3); [α]25 = +80 (c 0.2, CH3OH);
D
Treatment with Cytotoxic Compounds and MTT Colorimetric
Assay. Human cancer cell lines ((2.5−3.0) × 103 cells/0.33 cm2) were
plated in Nunclon Microwell 96-well plates (Nunc) and were
incubated at 37 °C. After 24 h, cells were treated with the compounds
(final concentration 0.01−100 μM). Cells treated with 0.5−1% of
DMSO were used as controls. Microplates were incubated at 37 °C in
humidified atmosphere of 5% CO2/95% air for 3 days, and then
cytotoxicity was measured with a colorimetric assay based on the use
of a tetrazolium salt [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide].50 The results were read on a multiwall scanning
spectrophotometer (Multiscan reader), using a wavelength of 570 nm.
Each value was the average of 6−8 wells. The GI50 value was calculated
according to the NCI:51 thus, GI50 is the concentration of test
compound where 100(T − T0)/(C − T0) = 50 (where T is the optical
density of the test well after a 72 h period of exposure to test
compound, T0 is the optical density at time zero, C is the DMSO
control optical density). The cytotoxicity effect was calculated
according to NCI when the optical density of treated cells was
lower than the T0 value using the following formula: 100(T − T0)/T0
< 0.
Enzymatic Cleavage Assay. Compounds 20−25 were dissolved in
DMSO and diluted with phosphate buffer at pH = 7.2 (Na2HPO4/
KH2PO4 0.050 M) to a final concentration of 5.0 × 10−5 M (the final
concentration of DMSO did not exceeded 1.0%).
β-Glucosidase from almonds (1.2 U/mL) or β-galactosidase from A.
oryzae (0.7 U/mL) was added, and the mixtures were incubated at 37
°C. Samples (200 μL) were taken at regular time intervals up to 96 h,
and the enzymatic hydrolysis was monitored by HPLC on an RP-18
column at 305 nm (see General Experimental Procedures).
1
UV (CH3OH) λmax (log ε) 322 (4.28) nm; H NMR (500 MHz,
pyridine-d5, 300 K) δ 8.64 (d, J = 16.5 Hz, 1 H, 7-H), 7.72 (d, J = 2.0
Hz, 1 H, 2′-H), 7.52 (d, J = 16.5 Hz, 1 H, 8-H), 7.40 (dd, J = 8.5, J =
2.0 Hz, 1 H, 6′-H), 7.26 (d, J = 3.0 Hz, 1 H, 6-H), 6.94 (d, J = 8.5 Hz,
1 H, 5′-H), 6.77 (d, J = 3.0 Hz, 1 H, 4-H), 5.49 (d, J = 7.5 Hz, 1 H, 1″-
H), 4.92 (bdd, J = 7.5 Hz, 1H, 2″-H), 4.72 (bs, 1 H, 4″-H), 4.59 (bdd,
J = 10.5, J = 7.0 Hz, 1H, 6a″-H), 4.36* (dd, J = 10.5, J = 5.5 Hz, 1 H,
6b″-H), 4.32* (dd, J = 9.3, 5.5 Hz, 1 H, 3″-H), 4.09 (bdd, J = 7.0, 5.5
Hz, 1 H, 5″-H), 3.94 (s, 3 H, 3′-OCH3), 3.84 (s, 6 H, 3-OCH3 and 5-
OCH3), 3.79 (s, 3 H, 4′-OCH3); the signals with identical superscript
(*) are partially overlapped; 13C NMR (125 MHz, pyridine-d5, 300 K)
δ 157.2 (C-5), 154.5 (C-3), 150.1# (C-3′), 149.6# (C-4′), 139.2 (C-2),
133.4 (C-1), 131.7 (C-1′), 129.4 (C-8), 123.8 (C-7), 120.8 (C-6′),
112.3 (C-5′), 110.0 (C-2′), 107.2 (C-1″), 100.9 (C-6), 100.6 (C-4),
76.7 (C-5″), 75.2 (C-3″), 73.3 (C-2″), 69.6 (C-4″), 61.7 (C-6″), 56.2
(C-3-OCH3), 55.8 (C-3′-OCH3), 55.7 (C-4′-OCH3), 55.4 (C-5-
OCH3); values with superscript (#) were attributed through HMBC
correlations; the signals were partially overlapped with that of the
residual undeuterated solvent; ESI-MS calcd for C24H30O10Na [M +
Na]+ 501.17; found 501.2; anal. calcd for C24H30O10 C, 60.24; H, 6.32;
found C, 60.17; H, 6.31.
( 2 R , 3 S , 4 S , 5 R ) - 2 - [ 2 - ( 3 , 5 - D i m e t h o x y s t y r y l ) - 4 , 6 -
dimethoxyphenoxy]tetrahydro-6-hydroxymethyl-2H-pyran-3,4,5-
triol (25). Compound 19 (0.0280 g, 0.088 mmol) was stirred in
MeOH/H2O (45:55, 11.3 mL) with K2CO3 (0.1226 g, 0.887 mmol) at
room temperature for 15 min. After this time, CHCl3 (5 mL), TBACl
(0.0244 g, 0.088 mmol), and a solution of tetra-O-acetyl-α-D-
galactopyranosyl bromide (0.0904 g, 0.220 mmol) in CHCl3 (6.5
mL) were added to the reaction mixture. The heterogeneous mixture
was stirred at 60 °C for 24 h, and at 10 h, 0.0396 g/3 mL of sugar in
CHCl3 was added. The aqueous phase was extracted with CHCl3 (3 ×
10 mL), and the combined CHCl3 phases were washed with H2O (3 ×
20 mL), dried over anhydrous Na2SO4, filtered, and evaporated to
dryness. The crude extraction was purified by column chromatography
with RP-18 silica gel with a gradient of CH3CN in H2O (from 40% to
70%). From PLC 25 was recovered (0.0109 g, 40%): Rf (TLC) = 0.38
The activity of the enzymes does not change when the solution
contains 1.0% DMSO. All assays were performed in triplicate.
In order to quantify the released aglycone by the cleavage assay,
calibration curves were created for compounds 17−19: four solutions
of the aglycones prepared within the concentration range of the test
(10−5−10−6 M) were eluted by HPLC on an RP-18 column under the
same conditions of the assay.
(4% CH3OH/CHCl3); [α]25 = +70 (c 0.03, CH3OH); UV
D
(CH3OH) λmax (log ε) 305 (4.46) nm; 1H NMR (500 MHz,
pyridine-d5, 300 K) δ 8.75 (d, JH,H = 16.4 Hz, 1 H, 7-H), 7.54 (d, J =
16.4 Hz, 1 H, 8-H), 7.27 (d, J = 2.3 Hz, 2 H, 2′-H and 6′-H), 7.25 (d, J
= 2.5 Hz, 1 H, 6-H), 6.78 (d, J = 2.5 Hz, 1 H, 4-H), 6.71 (t, J = 2,3 Hz,
1 H, 4′-H), 5.48 (d, J = 8.0 Hz, 1 H, 1″-H), 4.91 (dd, J = 8.3, 8.0 Hz, 1
Glycosidase Enzyme Inhibition Assay. A slight modification of the
method of Tsujii and co-workers52 was used to evaluate the inhibition
of β-glucosidase from almonds, β-galactosidase from A. oryzae, and α-
glucosidase from S. cerevisiae by compounds 20−25. The enzymatic
activity was determined spectrophotometrically at 400 nm by
monitoring the release of p-nitrophenol and o-nitrophenol from the
substrates pNP-β-Glc or pNP-α-Glc (for glucosidases) and oNP-β-Gal
(for galactosidase), respectively. Each substrate was dissolved in
DMSO/phosphate buffer (Na2HPO4/KH2PO4 0.050 M, pH = 7.2) in
order to have a final concentration of 6.5 × 10−5 M. In each assay, 3
mL of substrate solution were incubated with 0.1, 0.2, 0.4, 0.6, or 1.0
mL of the stock solution of each glycoside (2.6−4.7 × 10−4 M; for
details see the Supporting Information) in the presence of the
corresponding enzyme (2.9 U/mL for β-glucosidase, 4.0 U/mL for β-
galactosidase, and 2.0 U/mL for α-glucosidase) at 25 °C. The final
concentration of DMSO of the solutions did not exceed 1.0%. In each
set of experiments, the assay was performed in triplicate with five
different concentrations. Preliminary kinetic studies of the three
enzymes in the presence of pertinent substrate have provided the
following incubation times: 30 min for β-glucosidase and β-
galactosidase and 2 h for α-glucosidase. The percentage inhibition
was determined for each compound from the residual activity by
comparing the enzyme activity with and without compounds. The
concentration of inhibition required for 50% of glycosidase activity
under the assay conditions was defined as the IC50 value.
H, 2″-H), 4.70 (bd, J = 3.3 Hz, 1 H, 4″-H), 4.59 (dd, J = 10.5, 3JH,H
=
7.0 Hz, 1 H, 6a″-H), 4.35 (dd, J = 10.5, J = 5.5 Hz, 1 H, 6b″-H), 4.32
(dd, J = 8.3, 3.3 Hz, 1 H, 3″-H), 4.08 (bdd, J 7.0, 5.5 Hz, 1 H, 5″-H),
3.84 (s, 3 H, 3-OCH3), 3.83 (s, 3 H, 5-OCH3), 3.82 (s, 6 H, 3′-OCH3
and 5′-OCH3); 13C NMR (125 MHz, pyridine-d5, 300 K) δ 161.6 (C-
3′ and C-5′), 157.3 (C-5), 154.6 (C-3), 140.6 (C-1), 139.4 (C-2),
133.0 (C-1′), 129.6 (C-8), 123.0* (C-7), 107.2 (C-1″), 105.1 (C-2′
and C-6′), 101.13 (C-4 and C-4′), 101.06 (C-6), 76.7 (C-5″), 75.1 (C-
3″), 73.2 (C-2″), 69.5 (C-4″), 61.5 (C-6″), 56.2 (C-3-OCH3), 55.4
(C-5-OCH3), 55.3 (C-3′-OCH3 and C-5′-OCH3); value with
superscript (*) was assigned through HMBC correlations; the signal
was partially overlapped with that of the residual undeuterated solvent;
ESIMS calcd for C24H30O10Na [M + Na]+ 501.17; found 501.2; anal.
calcd for C24H30O10 C, 60.24; H, 6.32; found C, 60.19; H, 6.29.
Antiproliferative Activity Assay. Human Cell Cultures. The
colorectal adenocarcinoma Caco-2 cells (ATCC number: HTB-37)
were obtained from the American Type Culture Collection (ATCC,
Teddington, UK). The Caco-2 cell line was grown in RPMI 1640
medium (cat. 61870-010, Gibco by Life Technologies, Italy). The SH-
SY5Y (ATCC CRL-2266) was kindly provided by Prof. Maria Angela
Sortino (University of Catania, Italy). The SH-SY5Y cells were grown
in DMEM-F12 (cat. no. 21331-046, Gibco by Life Technologies,
Italy). All media were supplemented with 10% (v/v) heat-inactivated
G
DOI: 10.1021/acs.jnatprod.5b00619
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Journal of Natural Products
Article
(19) Heynekamp, J. J.; Weber, W. M.; Hunsaker, L. A.; Gonzales, A.
M.; Orlando, R. A.; Deck, L. M.; Vander Jagt, D. L. J. Med. Chem.
2006, 49, 7182−7189.
(20) Valdameri, G.; Rangel, L. P.; Spatafora, C.; Guitton, J.; Gauthier,
C.; Arnaud, O.; Ferreira-Pereira, A.; Falson, P.; Winnischofer, S. M. B.;
Rocha, M. E. M.; Tringali, C.; Di Pietro, A. ACS Chem. Biol. 2012, 7,
322−330.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00619.
■
S
ESI mass, 1D and 2D NMR, and UV spectra of
compounds 20−25; α-glycosidase inhibition assay
(PDF)
(21) Sarpietro, M. G.; Ottimo, S.; Giuffrida, M. C.; Spatafora, C.;
Tringali, C.; Castelli, F. Int. J. Pharm. 2010, 388, 144−150.
(22) Sarpietro, M. G.; Spatafora, C.; Accolla, M. L.; Cascio, O.;
Tringali, C.; Castelli, F. J. Nat. Prod. 2013, 76, 1424−1431.
(23) Sarpietro, M. G.; Spatafora, C.; Tringali, C.; Micieli, D.; Castelli,
F. J. Agric. Food Chem. 2007, 55, 3720−3728.
AUTHOR INFORMATION
Corresponding Author
*Tel: +39 095 7385015. Fax: +39 095 580138. E-mail:
cspatafo@unict.it.
■
(24) Lin, H.-S.; Spatafora, C.; Tringali, C.; Ho, P. C. J. Pharm.
Biomed. Anal. 2012, 57, 94−98.
(25) Lin, H. S.; Tringali, C.; Spatafora, C.; Choo, Q. Y.; Ho, P. C.
Chromatographia 2010, 72, 827−832.
Notes
The authors declare no competing financial interest.
(26) Lin, H. S.; Tringali, C.; Spatafora, C.; Wu, C.; Ho, P. C. J.
Pharm. Biomed. Anal. 2010, 51, 679−684.
ACKNOWLEDGMENTS
(27) Lin, H. S.; Zhang, W.; Go, M. L.; Tringali, C.; Spatafora, C.; Ho,
P. C. J. Agric. Food Chem. 2011, 59, 1072−1077.
(28) Cardile, V.; Chillemi, R.; Lombardo, L.; Sciuto, S.; Spatafora, C.;
Tringali, C. Z. Naturforsch., C: J. Biosci. 2007, 62, 189−195.
(29) Lee, J.; Kim, S. J.; Choi, H.; Kim, Y. H.; Lim, I. T.; Yang, H. M.;
Lee, C. S.; Kang, H. R.; Ahn, S. K.; Moon, S. K.; Kim, D. H.; Lee, S.;
Choi, N. S.; Lee, K. J. J. Med. Chem. 2010, 53, 6337−6354.
(30) Dembitsky, V. M. Lipids 2005, 40, 869−900.
(31) La Ferla, B.; Airoldi, C.; Zona, C.; Orsato, A.; Cardona, F.;
Merlo, S.; Sironi, E.; D’Orazio, G.; Nicotra, F. Nat. Prod. Rep. 2011, 28,
630−648.
■
This research was supported by grants of Regione Siciliana
(FERS 4.1.2.A 2007−2013 “Piattaforma regionale di ricerca
traslazionale per la salute”) and of the University of Catania
(FIR, “Finanziamento della Ricerca” 2014).
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