Benzofurans from styrax agrestis as acetylcholinesterase inhibitors: Structure-activity relationships and molecular modeling studies

Article abstract of DOI:10.1021/np200308j

An extract of Styrax agrestis fruits, collected in Vietnam, significantly inhibited acetylcholinesterase (AChE) in vitro. Bioassay-guided fractionation revealed three new egonol-type benzofurans: egonol-9(Z),12(Z) linoleate (1), 7-demethoxyegonol-9(Z),12(Z) linoleate (2), and 7-demethoxyegonol oleate (4). Ten known egonol-type benzofurans were also isolated (3, 5, 6-13). In order to better understand structure-activity relationships in this series, egonol derivatives 14-19 were prepared by chemical modifications and evaluated for their inhibition of AChE, butyrylcholinesterase (BChE), and AChE-induced Aβ aggregation. Compounds 1-4 were the most potent inhibitors of the series, which exhibited inhibitory activity against AChE (IC₅₀ 1.4-3.1 μM) and, for 1, Aβ aggregation (77.6%). Molecular docking studies were also performed to investigate interaction of these compounds with the active site of AChE.

Full text of DOI:10.1021/np200308j

ARTICLE
pubs.acs.org/jnp
Benzofurans from Styrax agrestis As Acetylcholinesterase Inhibitors:
StructureꢀActivity Relationships and Molecular Modeling Studies
Jiawei Liu,†,‡ Vincent Dumontet,*,‡ Anne-Laure Simonin,‡ Bogdan I. Iorga,‡ Vincent Guerineau,‡
Marc Litaudon,‡ Van Hung Nguyen,§ and Franc-oise Gueritte‡
†Research Center of Medicinal Plants Resource Science and Engineering, Guangzhou University of Chinese Medicine,
232 Waihuandong Road, Higher Education Mega Center, Guangzhou 510006, People's Republic of China
‡Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles, CNRS, B^atiment 27, Avenue de la Terrasse, CNRS,
91198 Gif-sur-Yvette, France
§Institute of Chemistry, VAST, 18 Hoang Quoc Viet Street, Cau Giay District, Hanoi, Vietnam
S Supporting Information
b
ABSTRACT: An extract of Styrax agrestis fruits, collected in
Vietnam, significantly inhibited acetylcholinesterase (AChE) in
vitro. Bioassay-guided fractionation revealed three new egonol-
type benzofurans: egonol-9(Z),12(Z) linoleate (1), 7-demethoxy-
egonol-9(Z),12(Z) linoleate (2), and 7-demethoxyegonol oleate
(4). Ten known egonol-type benzofurans were also isolated
(3, 5, 6ꢀ13). In order to better understand structureꢀactivity
relationships in this series, egonol derivatives 14ꢀ19 were
prepared by chemical modifications and evaluated for their
inhibition of AChE, butyrylcholinesterase (BChE), and AChE-induced Aβ aggregation. Compounds 1ꢀ4 were the most potent
inhibitors of the series, which exhibited inhibitory activity against AChE (IC50 1.4ꢀ3.1 μM) and, for 1, Aβ aggregation (77.6%).
Molecular docking studies were also performed to investigate interaction of these compounds with the active site of AChE.
lzheimer’s disease (AD), a progressive neurodegenerative
disorder of the central nervous system associated with
significantly inhibited AChE activity. Styrax is the largest genus
of the Styracaceae family, which contains approximately 130
species occurring in tropical and subtropical regions.12 Styrax
species have been traditionally used in herbal medicine to
treat various diseases.13 Styrax species contain egonol, a natural
benzofuran having anticomplement, antifungal, and antibacterial
activities.13ꢀ15 A literature survey revealed that no phytochem-
ical study has been carried out on S. agrestis. In the present study
we report a bioassay-guided isolation and structure elucidation of
three new and 10 known egonol-type benzofurans from an
EtOAc extract of fruits of S. agrestis, as well as their inhibitory
activities on AChE, BChE, and AChE-induced Aβ aggregation.
Several egonol analogues were also prepared to study structureꢀ
activity relationships in this series. The binding mode of some of
the molecules to AChE binding sites was also investigated by
molecular docking simulations.
A
cognitive and memory loss, is becoming one of the most serious
threats to life expectancy for elderly people.1,2 The main patho-
logical changes in the AD brain include extracellular deposition
of beta amyloid (Aβ) in senile plaques, the appearance of intra-
cellular neurofibrillary tangles, and extensive neuronal loss.3,4 AD
is also characterized by a pronounced alteration of the cholinergic
neurotransmitter system, a consistent cholinergic deficit, and
synaptic changes in the brain resulting in memory impairments.5
Acetylcholinesterase (AChE) is the key enzyme responsible for
the metabolic and catalytic hydrolysis of acetylcholine and
promotes the aggregation and deposition of Aβ peptide.6ꢀ8 Aβ
deposition leads to neuronal death through oxidative stress,
excitotoxicity, energy depletion, inflammation, and apoptosis.4
One promising therapeutic strategy for AD may consist of
concomitantly restoring native ACh levels and reducing the
formation of toxic Aβ fibrils.4,9 Such a strategy may be realized
by dual-function molecules possessing the ability to inhibit AChE
activity and prevent Aβ peptide aggregation.
’ RESULTS AND DISCUSSION
The EtOAc extract of fruits of S. agrestis significantly inhibited
recombinant human AChE (hAChE) and Electrophorus electricus
AChE (EeAChE) activity (100% inhibitory effect at 10 μg/mL).
The MeOH extract was inactive. One hundred milligrams of the
EtOAc extract was filtered on polyamide to remove tannins, and
In our ongoing efforts to find new AChE inhibitors from
natural sources, part of our plant extract library (9327 extracts)
was screened by high-throughput assay. In preceding studies we
isolated acylphenols from Myristica crassa (Myristicaceae) and
turrianes from Kermadecia rotundifolia (Proteaceae) as new
AChE inhibitors.10,11 We have since found that an EtOAc extract
obtained from the fruits of Styrax agrestis A. Chev. (Styracaceae)
Received: April 12, 2011
Published: September 22, 2011
r
Copyright
2011 American Chemical Society and
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15 mg of the filtered extract was fractionated on a semipre-
parative C18 column to give nine subfractions according to a
standardized method.16 The AChE bioassay yielded two active
subfractions (100% inhibitory effect at 10 μg/mL), which were
used as references for further fractionation. The EtOAc extract
(3.5 g) was then subjected to silica gel column chromatography
(CC), and preparative HPLC led to the isolation of three new
(1, 2, and 4) and 10 known 2-arylbenzofurans (3, 5, 6ꢀ13). The
known compounds were identified by comparing their physical
constants with those reported in the literature: egonol oleate
(3),17 egonol-2-methylbutanoate (5),18 7-demethoxylegonol-2-
methylbutanoate (6),18 7-demethoxylegonol acetate (7),19 ego-
nol propanoate (8),20 egonol-2-methylpropanoate (9),21 egonol
butanoate (10),19 egonol acetate (11),22 7-demethoxyegonol
(12),23 and egonol (13).15 To the best of our knowledge, 3 has
not been described before as a natural compound but has been
obtained by semisynthesis from egonol.17 7-Demethoxyegonol
(12) and egonol (13) were also obtained after alkaline hydrolysis
of the EtOAc extract.
Compound 1 was a pale yellow oil, and its sodiated molecular
formula was determined to be C37H48O6Na by HRESIMS. The
13C NMR signals of the alcohol part of compound 1 were
superimposable to those of 13, while the 13C NMR signals of
the unsaturated fatty acid moiety were similar to those of linoleic
acid.24 The 1H NMR and 13C NMR spectra, HSQC, and HMBC
of 1 showed signals assignable to a cis,cis-1,4-pentadiene unit of a
long-chain unsaturated fatty acid moiety.25 Comparison of the
13C NMR data of 1 with those of 13 indicated an esterification
shift at the C-300 position of 1, and a long-range correlation was
observed between H-300 and C-1000 in the HMBC spectra.
Ozonolysis of 1 gave aldehyde 14. This indicated that the cis,
cis-1,4-pentadiene unit was at C-9000 of the unsaturated fatty acid.
Alkaline hydrolysis of 1 with 10% NaOHꢀEtOH yielded linoleic
acid and 13. Compound 1 was thus assigned the structure
egonol-9(Z),12(Z)-linoleate.
The sodiated molecular formula of compound 4 (C36H48-
O5Na) was deduced from HRESIMS measurements, and the
1H and 13C NMR spectra of 4 were similar to those of 2. The
carbon signals of the alcohol moiety were nearly superimposable
with those of 12, while the signals of the fatty acid moiety
were similar to those of oleic acid.26 Comparison of the 13C NMR
data of 4 with those of 12 indicated an esterification shift at C-300,
and the HMBC experiment exhibited a long-range correlation
between H-300 and C-1000. Alkaline hydrolysis of 4 yielded oleic
acid and 12, and ozonolysis of 4 yielded aldehyde 15. Consequently,
compound 4 was characterized as 7-demethoxyegonol-9(Z)-
oleate.
The HRESIMS of compound 5 revealed a sodiated molecular
formula of C24H26O6Na. Examination of the 1D and 2D NMR
spectra suggested that 5 was egonol-2000-methyl butanoate, pre-
viously isolated from Styrax obassia.18 As the C-2 configuration
was unknown, the (S)-2000-methyl butyrate ester was prepared
from egonol (Figure 1). Spectral and analytical data were similar
to those of 5, and the sign of optical rotation of the synthesized
compound and of 5 was the same. Thus, the absolute configura-
tion of compound 5 was deduced as S at C-2000.
Compound 6 was obtained as an optically active oil, and the
HRESIMS indicated a sodiated molecular formula of C23H24-
O5Na. Examination of the 1D and 2D NMR spectra suggested
that 6 was 7-demethoxylegonol-2-methylbutanoate, previously
isolated from S. obassia.18 The positive sign of the optical rotation
for 6 was the same as that of 5, suggesting the S absolute
configuration at C-2000.
In order to examine structureꢀactivity relationships in this
benzofuran series, other derivatives were prepared from 13. The
synthetic pathway employed for the preparation of egonol
derivatives is outlined in Figure 1.
Compound 16 was obtained by oxidation of 13 using Dess-
Martin periodinane in CH2Cl2. The 1H and 13C NMR spectra of
16 showed an aldehyde proton signal at δH 9.74 (H-300)
corresponding to the carbon signal at δC 202.9 (C-300) instead
of signals for a primary alcohol as in egonol. The structure of 16
was established as 3-[2-(1,3-benzodioxol-5-yl)-7-methoxy-1-
benzofuran-5-yl]propanal.17
Compound 17 was prepared from 13 by oxidation using CrO3
1
in CH2Cl2ꢀpyridine. The main differences in the H and 13C
NMR spectra of 17 and 16 were the absence of signals at δH 9.74
(H-300 in 16) and δC 202.9 (C-300 in 16) and the presence of a
signal at δC 177.0, which confirmed the carboxylic function.
Therefore, 17 was established as the known 3-[2-(1,3-benzo-
dioxol-5-yl)-7-methoxy-1-benzofuran-5-yl]propionic acid
(egonoic acid).27
Compound 18 was obtained from the reaction of 13 with tert-
butyl isocyanate in dry CH3CN and gave an ion peak at m/z
448.1733 [M + Na]+ (C24H27NO6Na). The 1H NMR spectrum
of 18 showed that the resonance of H-300 was shifted downfield
compared to that of egonol. The 13C NMR spectrum displayed
24 carbons signals. A HMBC correlation was observed be-
tween the C-1000 carbonyl of a carbamate group and H-300 in
the egonol moiety. Thus, compound 18 was identified as N-tert-
butyl egonolcarbamate.
Compound 2 gave a sodiated molecular formula of
C36H46O5Na (HRESIMS). The 13C NMR signals of the 2-aryl-
benzofuran part were superimposable with those of 12, and the
signals of the fatty acid moiety were similar to those of linoleic
acid. Ozonolysis of 2 yielded aldehyde 15, and alkaline hydrolysis
of 2 with 10% NaOHꢀEtOH liberated linoleic acid and 12.
Thus, 2 was determined to be 7-demethoxyegonol-9(Z),12(Z)-
linoleate.
Compound 19 was prepared by treatment of 13 with
1
phenyl isocyanate in dry CH3CN. The H NMR spectrum of
19 showed that the resonance of H-300 was also shifted down-
field compared to that of egonol. The 13C NMR spectrum of 19
exhibited 26 carbon signals, and a HMBC correlation was
observed between the carbonyl carbon C-1000 of the carbamate
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Figure 1. Preparation of egonol derivatives.
Table 1. Inhibition of EeAChE and hAChE by Benzofuran
Table 2. Inhibition of AChE-Induced Aβ Aggregation by
Benzofuran Derivatives
Derivativesa,b
compound
EeAChE
hAChE
compound
inhibition (%) at 100 μM
1
1.4 ( 0.3
2.0 ( 0.9
1.4 ( 0.5
2.2 ( 0.8
4.8 ( 0.6
5.5 ( 0.7
3.6 ( 0.6
3.3 ( 0.6
50 ( 3 nM
1.7 ( 0.2
2.7 ( 0.4
1
77.6
70.5
64.6
61.6
86.5
54.1
2
5
3
1.8 ( 0.3
3.1 ( 0.6
11
4
13
5
5.9 ( 0.7
19
6
6.1 ( 0.8
propidium
9 + 10
19
tacrinec
5.2 ( 0.5
4.5 ( 0.6
AChE. None of the compounds (1ꢀ19) inhibited BChE activity
(IC50 > 20 μM).
1026 ( 72 nM
a Results are expressed as means of IC50 values (the concentration that
inhibits AChE by 50%) in μM, and data were obtained from duplicate
experiments. b For the enzymes used, see Experimental Section.
c Tacrine was used as reference.
Five compounds (1, 5, 11, 13, and 19) were selected to
evaluate their ability to inhibit AChE-induced Aβ aggregation
using a thioflavin T fluorimetric assay.29 Aβ1ꢀ40 (230 μM) was
incubated at 25 °C for 48 h with AChE (2.3 μM) in the presence
of test compounds at 100 μM, and propidium iodine was used as
a reference. The Aβ aggregation inhibitory potency of the
compounds is reported in Table 2. Compound 1, a selective
AChE inhibitor, significantly inhibited AChE-induced Aβ aggre-
gation by 77.6%. Compounds 11 and 13 did not show any AChE
inhibition, whereas their aggregation inhibitory potency was
64.6% and 61.6%, respectively, at 100 μM concentrations. These
data suggest that the inhibitory effect of benzofurans on Aβ
aggregation is probably independent of AChE inhibition and may
be associated with their direct binding to Aβ peptide. In
accordance with our results, Howlett et al. revealed that the
benzofuran analogue SKF-64346 prevented aggregation by a
process involving binding of the benzofuran to Aβ peptide,
suggesting that a specific recognition site might exist for benzo-
furans on Aβ peptide.30 These findings are also consistent with
the observations that benzofuran-based hybrid compounds sig-
nificantly inhibited AChE/BChE activities and exhibited signifi-
cant inhibition of Aβ aggregation.31
group and H-300. Thus, compound 19 was identified as N-phenyl
egonolcarbamate.
The inhibitory potency and selectivity of benzofurans 1ꢀ19
were evaluated on EeAChE, hAChE, and BChE by a modified
Ellman’s colorimetric method.28 The IC50 values of active
compounds are compared in Table 1. The egonol esters 1ꢀ6,
9 + 10, and 19 significantly inhibited both EeAChE and hAChE
activity, whereas 7, 8, 11ꢀ13, 16, and 17 were inactive. The data
indicate that the degree of inhibition of AChE is related to the
length and bulkiness of the alkyl ester group. Alkyl chains with
more than three carbon units led to compounds with antic-
holinesterase activity. Nevertheless, further increase in chain
length did not significantly enhance AChE inhibition, as illu-
strated by the close IC50 values of 1 and 3 compared to 5 or 2 and
4 compared to 6. In addition, it was found that a methoxy group
at C-7 had no effect on activity since the linoleate and oleate
esters of egonol (1 and 3, respectively) have IC50 values very
close to their 7-demethoxylate analogues (2 and 4). Further-
more, oxidation and conversion of the double bond of the furan
ring of 1ꢀ4 into the corresponding aldehyde-ester derivatives
(14 and 15) resulted in loss of anti-AChE activity. Thus, both the
furan and ester groups play important roles in the inhibition of
Molecular modeling studies were performed in order to
investigate how these compounds might interact with the active
site of AChE. During the last two decades, a large number of
molecular modeling studies focused on AChE have been pub-
lished using different approaches (pharmacophore-based virtual
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Among these, three main hydrogen bonds were evident
during the molecular dynamics simulations, which anchor the
ligand in the AChE binding site: the carbonyl O atom from the
ligand ester group interacts with the backbone NH from Phe288
and O atoms from the ligand 1,2-(methylenedioxy)benzene
group interact with the backbone NH from Ser124 and OH
group from Tyr130. Particularly interesting are the hydrophobic
residues situated at the peripheral site (Figure 2), which are
involved in ligand stabilization. These interactions are essential
for AChE inhibition since 7 and 11ꢀ13, which do not contain a
reasonably voluminous hydrophobic ester substituent, are not
active. However, very long aliphatic chains do not increase the
inhibitory activity, as the terminal atoms fall outside the binding
site. Finally the methoxy substituent of the benzofuran moiety does
not seem to be essential for inhibition, this substituent making only
limited hydrophobic contacts with Asp72 and Tyr334.
In conclusion, we have identified a novel series of benzofuran
AChE inhibitors with significant inhibitory effects on Aβ aggre-
gation. The structureꢀactivity relationships suggest that the
furan ring, the ester group, and the length of the hydrocarbon
side chain are the key structural features contributing to the
inhibitory potency of the benzofuran derivatives against AChE.
Molecular modeling calculations propose a binding mode for this
class of compounds on AChE and have identified several residues
at the peripheral site that are important for the inhibitory effect.
Our data suggest that these benzofuran derivative dual-function
molecules are capable not only of inhibiting AChE activity but
also of preventing Aβ aggregation and provide a chemical
template for further optimization.
Figure 2. Superposition of 3 (green) and 5 (yellow) in the binding site
of acetylcholinesterase, at the termination of the molecular dynamics
simulation (10 ns). Hydrophobic residues at the peripheral site involved
in ligand stabilization are shown in magenta. This mode of binding is
representative for compounds 1ꢀ5 isolated from S. agrestis.
screening, different types of molecular docking and structure-
based virtual screening, molecular dynamics) or a combination of
these (see Supporting Information for references). All of these
studies involved AChE inhibitors having at least one positively
charged nitrogen atom or a nitrogen-containing polar group,
which generally drives the overall binding mode in the AChE site.
Several nitrogen-free AChE inhibitors have been reported re-
cently, mainly isolated from natural sources.32 Three-dimen-
sional structures of compounds 1ꢀ7 and 11ꢀ13 were built and
used for molecular docking in the binding site of AChE. In
agreement with the experimental data for anticholinesterase
activity, only compounds 1ꢀ6 could be successfully docked,
and the resulting ligand conformations showed interactions with
both catalytic and peripheral sites, the more rigid cyclic system
being positioned inside the catalytic site of the protein (Figure 2).
During the docking process, the ligands were fully flexible and the
protein was rigid, the binding site being defined as a sphere with a
radius of 20 Å around the OH group of SER200. For compounds
presenting ambiguity in the assignment of a chiral atom config-
uration (e.g., 6), both stereoisomers were considered.
Using the docking results as starting conformations, molecular
dynamics simulations were carried out over 10 ns in order to
introduce protein flexibility and to evaluate the possibility of an
induced fit that could take place upon ligand binding. In most
cases a slight rearrangement of the ligand in the binding site was
observed, concomitant with some changes in the protein side-
chain conformations in order to accommodate the presence of
the ligand. Root-mean-square deviation (rmsd) values of 2.5 Å
were typically obtained for the protein. For the ligand, the rmsd
values were more variable, with an important contribution from
the movement of the long aliphatic chains in the case of 1ꢀ4 (see
Supporting Information). Very few proteinꢀligand hydrogen
bonds were identified, the interactions being mainly hydrophobic.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
recorded in CH2Cl2 at 25 °C with a Perkin-Elmer 341 polarimeter. IR
spectra were recorded on a Nicolet FT-IR 205 spectrophotometer. UV
spectra were recorded on a Varian Cary 100 UVꢀvisible spectro-
photometer. 1H and 13C spectra were recorded in CDCl3 on an Avance
300 Bruker spectrometer operating at 300.17 and 75.48 MHz, respec-
tively, or on an Avance 500 Bruker (1H, 500.13; 13C, 125.77 MHz). 1H
chemical shifts were referenced relative to CDCl3 at 7.24 ppm and 13
C
chemical shifts to the central peak of CDCl3 at 77.23 ppm. High-
resolution mass spectra (HRMS) were recorded with either a MALDI-
TOF mass spectrometer (Voyager-De STR; Perspective Biosystems) or
an ESI-TOF mass spectrometer (LCT Premier; Waters). TLC was
performed on Si gel 60 F254. Column chromatography was carried out
on Merck Si gel 60. Analytical and preparative HPLC were performed on
a Waters autopurification HPLC system (Waters 2525) using a Kromasil
C18 column (250 ꢁ 4.5 mm i.d. for analytical and a 250 ꢁ 21.2 mm i.d.
column for preparative, stainless steel, 5 μm Thermo). The gradient
mobile phase was MeCNꢀH2O (3:1) to 100% MeCN. The flow rate
was 17 mL/min, and the elution was monitored with a UVꢀvis diode
array detector (190ꢀ600 nm, Waters 2996) and a PL-ELS 1000 ELSD
Polymer Laboratory detector. CombiFlash Companion chromatogra-
phy (CCC) was carried out on silica gel colums (Redisep 4 g).
Plant Material. Ripe fruits of S. agrestis were collected in Vietnam by
D. D. Cuong (Institute of Chemistry, VAST, Hanoi) in May 2005 at Thua
Thien, Hue district, and identified by Q. B. Nguyen from the Institute of
Botany, VAST, Hanoi, where a voucher specimen VN-1512 is deposited.
Extraction and Isolation. Dried, powdered fruits of S. agrestis
(1060 g) were extracted with EtOAc (250 mL ꢁ 3) and MeOH
(250 mL ꢁ 3) at 40 °C. The filtrates were evaporated under vacuum
to give the EtOAc (26.5 g) and MeOH (100.4 g) extracts. These extracts
were evaluated using the AChE assay. The MeOH extract had no activity
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at 10 μg/mL. The EtOAc extract exhibited 100% inhibition at 10 μg/mL.
A 100 mg aliquot of the crude EtOAC extract was dissolved in a 1:1
mixture of EtOAcꢀMeOH and filtered on a polyamide cartridge. The
filtered extract was then fractionated on a semipreparative C18 column,
according to a previous method.16 Subfractions 6 and 7 (Fr.6, Fr.7) gave
100% inhibition of AChE activity at 10 mg/mL and were used as positive
references. The EtOAc extract (3.5 g) was then subjected to silica gel
CCC eluted with a heptaneꢀEtOAcꢀMeOH gradient. Fractions 3, 4,
and 5 (Fr.3ꢀ5) ofthe EtOAc extractwere the most active. Fr.3 (165.8 mg)
was further separated by CC on a Sunfire RP-18 column (290 ꢁ 150 mm
i.d., Waters) using MeCN. Four compounds were obtained: 1 (7.4 mg),
2 (5.0 mg), 3 (11.9 mg), and 4 (4.1 mg). Fr.4 (126.2 mg) was further
separated by HPLC (H2OꢀMeCN, 5:95) to give compounds 5 (31.0 mg)
and 6 (6.8 mg). Fr.5 (155.6 mg) was subjected to RP-HPLC eluted with
H2OꢀMeCN (10:90) at 17 mL/min to yield compounds 7 (2.3 mg),
8 (1.3 mg), a 66:44 mixture of 9 and 10 (8.1 mg), 11 (7.1 mg), 12
(12.4 mg), and 13 (276 mg).
Compound 1: oil; UV (CH2Cl2) λmax (log ε) 211 (4.03), 228 (4.21),
318 (4.23) nm; IR (CH2Cl2) νmax 2925, 1731, 1600, 1475, 1366, 1232,
1146, 1040, 930 cmꢀ1; 1H NMR (CDCl3, 300 MHz) δ 7.38 (1H, dd, J =
8.2, 1.7 Hz, H-60), 7.30 (1H, d, J = 1.7 Hz, H-20), 6.93 (1H, d, J = 1.3 Hz,
H-4), 6.85 (1H, d, J = 8.2 Hz, H-50), 6.77 (1H, s, H-3), 6.58 (1H, d, J =
1.3 Hz, H-6), 5.99 (2H, s, OCH2O), 5.36 (1H, dt, J = 10.7, 7.0 Hz,
H-12000), 5.34 (1H, dt, J = 10.8, 7.0 Hz, H-10000), 5.32 (1H, dt, J = 10.8,
7.1 Hz, H-9000), 5.30 (1H, dt, J = 10.7, 7.0 Hz, H-13000), 4.10 (2H, t, J =
6.5 Hz, H-300), 4.01 (3H, s, OCH3), 2.75(2H, brt, J =5.8 Hz, H-11000), 2.73
(2H, brt, J = 7.5 Hz, H-100), 2.29 (2H, t, J = 7.5 Hz, H-2000), 2.03 (2H, dd,
J = 8.7, 6.4 Hz, H-800), 2.03 (2H, dd, J = 13.1, 6.6 Hz, H-8000), 1.98 (2H,
dd, J = 7.5, 6.5 Hz, H-200), 1.61 (2H, m, H-3000), 1.30 (14H, m, H-4000-H-
7000, H-15000-H-17000), 0.87 (3H, t, J = 6.8 Hz, H-18000); 13C NMR data
(CDCl3, 75 MHz) δ 174.1 (C, C-1000), 156.4 (C, C-2), 148.3 (C, C-30),
148.2 (C, C-40), 145.0 (C, C-7), 142.8 (C, C-8), 137.2 (C, C-5), 131.3 (C,
C-9), 130.4 (CH, C-13000), 130.2 (CH, C-9000), 128.3(CH, C-12000), 128.1
(CH, C-10000), 124.9 (C, C-10), 119.4 (CH, C-60), 112.6 (CH, C-4), 108.8
(CH, C-50), 107.7 (CH, C-6), 105.8 (CH, C-20), 101.3 (CH2, OCH2O),
100.6 (CH, C-3), 63.8 (CH2, C-300), 56.4 (CH3, OCH3), 34.6 (CH2,
C-2000), 32.8 (CH2, C-100), 31.7 (CH2, C-16000), 31.0 (CH2, C-200), 29.8
(CH2, C-7000), 29.6 (CH2, C-6000), 29.4 (CH2, C5000), 29.4 (CH2, C-15000),
29.3 (CH2, C-4000), 27.4 (CH2,C-8000), 27.4 (CH2, C-14000), 25.8 (CH2,
C-11000), 22.8 (CH2, C-17000), 14.3 (CH3, C-18000); MALDI-TOF-HRMS
m/z 611.3359 [M + Na]+ (calcd for C37H48O6Na, 611.3349).
Compound 2: oil; UV (CH2Cl2) λmax (log ε) 228 (4.04), 320
(4.23) nm; IR (CH2Cl2) νmax 2925, 2854, 1731, 1503, 1488, 1472,
1234, 1175, 1039, 931 cmꢀ1; 1H NMR (CDCl3, 500 MHz) δ 7.37 (1H,
d, J = 8.4 Hz, H-7), 7.36 (1H, dd, J = 8.2, 1.4 Hz, H-60), 7.32 (1H, brs,
H-4), 7.28 (1H, d, J = 1.4 Hz, H-20), 7.05 (1H, dd, J = 8.4, 1.3 Hz, H-6),
6.86 (1H, d, J = 8.2 Hz, H-50), 6.79 (H, s, H-3), 5.99 (2H, s, OCH2O),
5.37 (1H, dd, J = 10.8, 5.2 Hz, H-12000), 5.34 (1H, dd, J = 10.8, 7.4 Hz,
H-10000), 5.32 (1H, dd, J = 10.8, 8.0 Hz, H-9000), 5.30 (1H, dd, J = 10.1, 7.0
Hz, H-13000), 4.09 (2H, t, J = 6.5 Hz, H-300), 2.75 (2H, d, J = 6.4 Hz,
H-11000), 2.75 (2H, t, J = 7.6 HZ, H-100), 2.28 (H, t, J = 7.5 Hz, H-2000),
2.03 (2H, dd, J = 13.7, 6.8 Hz, H-14000), 1.99 (2H, dd, J = 9.7, 6.4 Hz,
H-8000), 1.96 (2H, dd, J = 7.5, 6.5 Hz, H-200), 1.61 (2H, m, H-3000), 1.30
(14H, m, H-4000-H-7000, H-15000-H-17000), 0.87 (3H; t, J = 6.7 Hz H-18000);
13C NMR data (CDCl3, 125 MHz) δ 174.1 (C, C-1000), 156.3 (C, C-2),
153.6 (C, C-8), 148.3 (C, C-30), 148.2 (C, C-40), 136.1 (C, C-5), 130.5
(CH, C-13000), 130.3 (CH, C-9000), 129.8 (C, C-9), 128.3 (CH, C-12000),
128.1 (CH, C-10000), 125.1 (C, C-10), 124.7 (C, C-6), 120.3 (CH, C-4),
119.3 (CH, C-60), 111.0 (CH, C-7), 108.9 (CH, C-50), 105.7 (CH,
C-20), 101.5 (CH2, OCH2O), 100.2 (CH, C-3), 63.8 (CH2, C-300), 34.6
(CH2, C-2000), 32.4 (CH2, C-100), 31.8 (CH2, C-16000), 31.1 (CH2, C-200),
29.8 (CH2, C-7000), 29.6 (CH2,C-6000), 29.4 (CH2,C-4000), 29.4 (CH2,C-
5000), 29.4 (CH2,C-15000), 27.4 (CH2, C-8000), 27.4 (CH2, C-14000), 25.8
(CH2, C-11000), 25.2 (CH2, C-3000), 22.8 (CH2, C-17000), 14.3 (CH3,
C-18000); MALDI-TOF-HRESIMS m/z 581.3284 [M + Na]+ (calcd for
C36H46O5Na, 581.3243).
Compound 3: oil; UV (CH2Cl2) λmax (log ε) 210 (4.16), 229 (4.04),
319 (4.23) nm; IR (CH2Cl2) νmax 2922, 2851, 1734, 1600, 1503, 1475,
1
1364, 1232, 1146, 1039, 930 cmꢀ1; H and 13C NMR spectra, see
Ozturk et al.;17 HRESIMS m/z 613.3506 [M + Na]+ (calcd for C37H50-
O6Na, 613.3505).
Compound 4: oil; UV (CH2Cl2) λmax (log ε) 210 (4.14), 229 (4.05),
319 (4.23) nm; IR (CH2Cl2) νmax 2923, 1732, 1600, 1503, 1467, 1364,
1233, 1146, 1039, 930 cmꢀ1; 1H NMR (CDCl3, 500 MHz) δ 7.37 (1H,
d, J = 8.4 Hz, H-7), 7.36 (1H, dd, J = 8.2, 1.4 Hz, H-60), 7.32 (1H, d, J =
1.3 Hz, H-4), 7.28 (1H, d, J = 1.4 Hz, H-20), 7.05 (1H, brd, J = 8.4 Hz,
H-6), 6.86 (1H, d, J = 8.2 Hz, H-50), 6.79 (H, s, H-3), 5.99 (2H, s,
OCH2O), 5.32 (1H, ddt, J = 11.1, 10.7, 6.0 Hz, H-9000), 5.32 (1H, dd, J =
11.1, 5.9 Hz, H-10000), 4.09 (2H, t, J = 6.5 Hz, H-300), 2.75 (2H, t, J = 7.6
HZ, H-100), 2.28 (H, t, J = 7.5 Hz, H-2000), 1.98 (2H, m, H-8000), 1.98 (2H,
m, H-11000), 1.98 (2H, m, H-200), 1.60 (2H, m, H-3000), 1.29 (20H, m,
H4000-H-7000, H-12000ꢀH-17000), 0.86 (3H; t, J = 6.7 Hz, H-18000); 13C
NMR data (CDCl3, 125 MHz) δ 174.1 (C, C-1000), 156.3 (C, C-2), 153.6
(C, C-8), 148.3 (C, C-30), 148.2 (C, C-40), 136.1 (C, C-5), 130.2 (CH,
C-9000), 129.9 (CH, C-10000), 129.8 (C, C-9), 125.1 (C, C-10), 124.8 (C,
C-6), 120.2 (CH, C-4), 119.3 (CH, C-60), 111.0 (CH, C-7), 108.9 (CH,
C-50), 105.7 (CH, C-20), 101.5 (CH2, OCH2O), 100.2 (CH, C-3), 63.8
(CH2, C-300), 34.6 (CH2, C-2000), 32.4 (CH2, C-100), 31.1 (CH2, C-200),
29.9 (CH2, C-7000), 29.9 (CH2, C-12000), 29.7 (CH2, C-6000), 29.5 (CH2,
C-5000), 29.5 (CH2, C-13000), 29.5 (CH2, C-14000), 29.4 (CH2, C-4000), 29.4
(CH2, C-15000), 27.4 (CH2,C-8000), 27.4 (CH2, C-11000), 25.2 (CH2,
C-3000), 22.9 (CH2, C-17000), 14.3 (CH3, C-18000); MALDI-TOF-HRE-
SIMS m/z 583.3436 [M + Na]+ (calcd for C36H48O5Na, 583.3399).
Compound 5: oil; [α]25D +5.2 (c 1.0, CH2Cl2); UV (CH2Cl2) λmax
(log ε) 211 (4.05), 228 (4.09), 318 (4.22) nm; IR (CH2Cl2) νmax 2922,
1
1729, 1600, 1475, 1365, 1231, 1146, 1040, 930 cmꢀ1; H and 13C
spectra of 5, see Takanashi et al.;18 MALDI-TOF-HRESIMS, m/z
433.1682 [M + Na]+ (calcd for C24H26O6Na, 433.1627).
Synthesis of Compound 5. To a stirred solution of 13 (20.0 mg,
0.061 mmol) in pyridine (0.5 mL) were added (S)-(+)-2-methylbutyric
acid (12.5 mg, 0.061 mmol) and p-toluenesulfonyl chloride (2.2 mg,
0.003 mmol) in CH2Cl2 (1 mL). The reaction mixture was refluxed for
8 h and stirred for a further 72 h at rt. The reaction mixture was diluted
with H2O (10 mL) and extracted with CH2Cl2 (15 mL ꢁ 3). The com-
bined organic layers were dried over anhydrous Na2SO4, filtered
through Celite, and evaporated to dryness. The residue was purified
by CCC (heptaneꢀEtOAc, 4:1) to afford a product identical to isolated
compound 5 (13.4 mg, 54%); [α]25D +4.4 (c 1.0, CH2Cl2).
Compound 6: oil; [α]25D +8.5 (c 0.5, CH2Cl2). UV (CH2Cl2) λmax
(log ε) 228 (3.93), 320 (4.23) nm; IR (CH2Cl2) νmax 2926, 1727, 1613,
1503, 1488, 1446, 1468, 1360, 1231, 1182, 1149, 1037, 930 cmꢀ1
;
1H and 13C spectra, see Takanashi et al.;18 HRESIMS m/z 403.1566
[M + Na]+ (calcd for C23H24O5Na, 403.1580).
Alkaline Hydrolysis. The crude EtOAc extract (16.5 g) of
S. agrestis was hydrolyzed with 15% NaOH in 50% EtOH at 100 °C for
4 h under argon. The reaction mixture was cooled to rt. The resulting
suspension, after removal of alcoholic solvent under reduced pressure,
was diluted with H2O (500 mL) and then extracted with CH2Cl2
(150 mL ꢁ 3). The combined organic layers were dried over anhydrous
Na2SO4, filtered trough Celite, and evaporated to give the crude
hydrolysate (5.9 g). The latter was purified by CCC (heptaneꢀEtOAc;
linear gradient from 1:0 to 1:1) to afford 12 (58.6 mg, demethoxyegonol23)
and 13 (947.3 mg, egonol15).
Ozonolysis of Compounds 1, 2, 3, and 4. Ozonolysis33 was
carried out in a long-necked, 50 mL standard flask equipped with a
magnetic stirring bar and with an insert containing a dropping funnel and
a gas inlet tube that reached the bottom of the reactor. A solution of 1ꢀ4
(3ꢀ5 mg each) in CH2Cl2ꢀMeOH (1:1, 5 mL) was added to the
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Journal of Natural Products
ARTICLE
reactor, which was cooled to ꢀ78 °C with a dry iceꢀacetone bath. The
reactor content was mixed with a slow stream of argon. Ozone was
generated and passed through the mixture. Excess ozone was removed
by flushing the reactor with argon. Dimethyl sulfide (0.5 mL) was added,
and the mixture was stirred for 12 h at rt. The volatile components were
removed on a rotary evaporator. Ozonolysis of 1 and 3 yielded aldehyde
14, and ozonolysis of 2 and 4 gave aldehyde 15.
Na2SO4 and filtered, and the solvent was evaporated in vacuo. The residue
was purified by CCC (heptaneꢀEtOAc; linear gradient from 1:0 to 1:1)
to give 18 (10.4 mg, 75%). Under similar conditions, 13 (20.0 mg,
0.061 mmol) was treated with phenyl isocyanate (7.4 mg, 0.06 mmol) in
dry MeCN (1 mL) to yield 19 (27.3 mg, 98%).
Compound 18: amorphous solid; UV (CH2Cl2) λmax (log ε) 229
(4.24), 318 (4.34) nm; IR νmax CH2Cl2 2921, 2852, 1709, 1473, 363,
1222, 1033, 927 cmꢀ1; 1H NMR (CDCl3, 300 MHz) δ 7.37 (1H, dd, J =
8.2, 1.8 Hz, H-60), 7.29 (1H, d, J = 1.7 Hz, H-20), 6.93 (1H, d, J = 1.3 Hz,
H-4), 6.84 (1H, d, J = 8.2 Hz, H-50), 6.76 (1H, s, H-3), 6.58 (1H, d, J =
1.3 Hz, H-6), 5.97 (2H, s, OCH2O), 4.62 (1H, brs, NH), 4.10 (2H, t, J =
6.5 Hz, H-300), 4.00 (3H, s, OCH3), 2.72 (2H, t, J = 7.5 Hz, H-100), 1.94
(2H, dd, J = 7.5, 6.5 Hz, H-200), 1.30 (3H, s, H-4000), 1.30 (3H, s, H-6000),
1.29 (3H, s, H-5000); 13C NMR data (CDCl3, 75 MHz) δ 157.0 (C, C-1000),
156.3 (C, C-2), 148.2 (C, C-30), 148.1 (C, C-40), 144.9 (C, C-7), 142.7
(C, C-8), 137.4 (C, C-5), 131.2 (C, C-9), 124.9 (C, C-10), 119.4 (CH,
C-60), 112.5 (CH, C-4), 108.8 (CH, C-50), 107.6 (CH, C-6), 105.7 (CH,
C-20), 101.5 (CH2, OCH2O), 100.6 (CH, C-3), 63.8 (CH2, C-300), 56.4
(CH3, OCH3), 50.4 (C, C-3000), 32.8 (CH2, C-100), 31.4 (CH2, C-200),
29.67 (CH3, C-5000), 29.2 (CH3, C-4000), 29.2 (CH3, C-6000); MALDI-
TOF-HRESIMS m/z 448.1733 [M + Na]+ (calcd for C24H27NO6Na,
448.1736).
Compound 19: amorphous solid; UV (CH2Cl2) λmax (log ε) 219
(4.30), 233 (4.20), 318 (3.04) nm; IR νmax CH2Cl2 3320, 2938, 1726,
1597, 1537, 1501, 1475, 1444, 1364, 1313, 929 cmꢀ1; 1H NMR (CDCl3,
300 MHz) of the major rotamer δ 10.81 (1H, brs, NH), 7.53 (2H, ddd,
J = 8.2, 2.5, 1.5 Hz, H-4000 and H-8000), 7.37 (1H, dd, J = 8.2, 1.7 Hz, H-60),
7.29 (1H, d, J = 1.7 Hz, H-20), 7.26 (2H, ddd, J = 8.2, 7.4, 1.5 Hz, H-5000
and H-7000), 7.04 (1H, tdd, J = 7.4, 1.8, 1.5 Hz, H-6000), 6.94 (1H, d, J = 1.3
Hz, H-4), 6.84 (1H, d, J = 8.2 Hz, H-50), 6.76 (1H, s, H-3), 6.59 (1H, d,
J = 1.3 Hz, H-6), 5.98 (2H, s, OCH2O), 4.20 (2H, t, J = 6.5 Hz, H-300),
4.00 (3H, s, OCH3), 2.76 (2H, dd, J = 9.3, 6.5 Hz, H-100), 2.03 (2H, ddd,
J = 12.7, 9.3, 6.5 Hz, H-200); 13C NMR data (CDCl3, 125 MHz) of the
major rotamer δ 156.4 (C, C-2), 153.8 (C, C-1000), 148.3 (C, C-30), 148.2
(C, C-40), 145.0 (C, C-7), 142.7 (C, C-8), 138.1 (C, C-3000) 137.4 (C, C-5),
131.3 (C, C-9), 129.3 (CH, C-5000), 129.3(CH, C-7000), 124.9 (C, C-10),
123.6 (CH, C-6000) 120.2 (CH, C-8000), 120.2 (CH, C-4000) 119.4 (CH,
C-60), 112.6 (CH, C-4), 108.8 (CH, C-50), 107.6 (CH, C-6), 105.8 (CH,
C-20), 101.5 (CH2, OCH2O), 100.6 (CH, C-3), 64.9 (CH2, C-300), 56.4
(CH3, OCH3), 32.8 (CH2, C-100), 31.2 (CH2, C-200); MALDI-TOF-
HRESIMS m/z 468.1419 [M + Na]+ (calcdfor C26H23NO6Na, 468.1423).
In Vitro AChE and BChE Inhibition. Enzymes EeAChE from
Electrophorus electricus (ref C2888), recombinant hAChE from hu-
man (reference C1682), and hBChE from human serum (reference
B4186) were purchased from Sigma. Inhibition of AChE activity was
determined by the spectroscopic method of Ellman et al.,28 using acetyl-
thiocholine iodide as substrate, in 96-well microtiter plates. All solutions
were brought to room temperature prior to use. Aliquots of 200 μL of a
solution containing 640 μL of 10 mM 5,50-dithiobis-2-nitrobenzoic acid
(DTNB) in 0.1 M sodium phosphate, pH 8.0, 19.2 mL of the same
buffer, and 13 μL of a solution of AChE (100 U/mL) in water were
added to each well, followed by 2 μL of a DMSO solution of the inhibitor
(0.9% final volume). The reaction was initiated by adding 20 μL of
acetylthiocholine iodide (7.5 mM) to each well and was followed by
monitoring the appearance of the thiolate dianion produced by reduc-
tion of DTNB at 412 nm every 13 s for 120 s at 25 °C in a Molecular
Devices Max 384 Plus plate reader. Each inhibitor was evaluated at 10
concentrations (from 1 mg to 0.05 mg/mL by diluting by a factor 3).
Percentage inhibition was calculatedrelative toa control sample ofDMSO
(% inhibition = [1 ꢀ (slope cpd/slope DMSO)] ꢁ 100) with SoftMax
Pro software. IC50 values displayed represent the means ( SD of three
individual determinations each performed in triplicate assays. Tacrine
hydrochloride (Sigma) was used as the reference compound.
1
Compound 14: oil; H NMR (CDCl3, 500 MHz) δ 10,14 (1H, s,
H-1), 9.74 (1H, t, J = 1.5 Hz, H-9000), 7.84 (1H, dd, J = 8.2, 1.7 Hz, H-70),
7.61 (1H, d, J = 1.7 Hz, H-30), 7.32 (1H, d, J = 1.8 Hz, H-3), 7.04 (1H, d,
J = 1.6 Hz, H-5), 6.91 (1H, d, J = 8.2 Hz, H-60), 6.07 (2H, s, OCH2O),
4.11 (2H, brt, J = 6.4 Hz, H-300), 3.82 (3H, s, OCH3), 2.73 (2H, dd, J =
9.0, 6.7 Hz, H-100), 2.40 (2H, td, J = 7.3, 1.7 Hz, H-8000), 2.30 (2H, t J =
7.5 Hz, H-2000), 1.98 (2H, dd, J = 9.0, 6.4 Hz H-200), 1.60 (2H, m, H-3000),
1.60 (2H, m, H-7000), 1.30 (2H, m, H-4000), 1.30 (2H, m, H-5000), 1.30 (2H,
m, H-6000); 13C NMR data (CDCl3, 125 MHz) δ 202.9 (C, C-9000), 189.2
(CH, C-1), 174.1 (C, C-1000), 164.3 (C, C-10), 152.9 (C, C-50), 152.0 (C,
C-40), 129.3 (C, C-2), 126.9 (CH, C-70), 124.9 (C, C-20), 119.8 (CH,
C-3), 118.3 (CH, C-5), 110.4 (CH, C-30), 108.5 (CH, C-60), 102.5
(CH2, OCH2O), 63.6 (CH2, C-300), 56.6 (CH3, OCH3), 44.5 (CH2,
C-8000), 34.7 (CH2, C-2000), 32.4 (CH2, C-100), 30.3 (CH2, C-200), 29.3
(CH2, C-6000), 29.2 (CH2, C-5000), 29.2 (CH2, C-4000), 28.4 (CH2, C-7000),
25.1 (CH2, C-3000); MALDI-TOF-MS 519.23 [M + Li]+, 535.22
[M + Na]+; MALDI-TOF-HRMS m/z 535.1938 [M + Na]+ (calcd
for C28H32O9Na, 535.1944).
1
Compound 15: oil; H NMR (CDCl3, 500 MHz) δ 10,16 (1H, s,
H-1), 9.74 (1H, t, J = 1.8 Hz, H-9000), 7.83 (1H, dd, J = 8.2, 1.7 Hz, H-70),
7.74, (1H, d, J = 2.0 Hz, H-3), 7.60 (1H, d, J = 1.7 Hz, H-30), 7.47 (1H,
dd, J = 8.2, 2.0 Hz, H-5), 7.21 (1H, d, J = 8.2 Hz, H-6), 6.91 (1H, d, J =
8.2 Hz, H-60), 6.08 (2H, s, OCH2O), 4.10 (2H, brt, J = 6.4 Hz, H-300),
2.75 (2H, brt, J = 7.9 Hz, H-100), 2.40 (2H, td, J = 7.3, 1.7 Hz, H-8000), 2.29
(2H, t J = 7.5 Hz, H-2000), 1.98 (2H, dd, J = 7.9, 6.4 Hz, H-200), 1.61 (2H,
m, H-3000), 1.61 (2H, m, H-7000), 1.32 (2H, m, H-4000), 1.32 (2H, m Hz
H-5000), 1.32 (2H, m, H-6000); 13C NMR data (CDCl3, 125 MHz) δ 202.9
(C, C-9000), 188.9 (CH, C-1), 173.8 (C, C-1000), 164.7 (C, C-10), 152.8
(C, C-50), 148.2 (C, C-40), 139.8 (C, C-4), 135.5 (CH, C-5), 135.2 (C,
C-7), 129.5 (CH, C-3), 129.3 (C, C-2), 126.8 (CH, C-70), 123.8 (CH,
C-6), 122.7 (C, C-20), 110.1 (CH, C-30), 108.5 (CH, C-60), 102.2 (CH2,
OCH2O), 63.5 (CH2, C-300), 43.9 (CH2, C-8000), 34.3 (CH2, C-2000), 31.6
(CH2, C-100), 30.1 (CH2, C-200), 28.9 (CH2, C-4000), 28.9 (CH2, C-5000),
28,9 (CH2, C-6000), 24.9 (CH2, C-3000), 22.9 (CH2, C-7000); MALDI-
TOF-MS 489.21 [M + Li]+, 505.18 [M + Na]+; MALDI-TOF-HRMS
m/z 505.1833 [M + Na]+ (calcd for C27H30O8Na, 505.1838).
Preparation of 16. Dess-Martin periodinane (79.6 mg, 0.19 mmol)
was added to a solution of 13 (61.2 mg, 0.19 mmol) in CH2Cl2 (2 mL),
and the mixture was stirred at rt for 12 h. The reaction mixture was
washed successively with water (50 mL ꢁ 3) and brine (50 mL ꢁ 2),
dried over anhydrous Na2SO4, and filtered, and the solvent was
evaporated in vacuo. The residue was purified by PTLC using heptaneꢀ
EtOAc (1:1) to give 1617 (54.7 mg, 88%).
Preparation of 17. Chromium trioxide (500 mg, 5.0 mmol) was
added gradually to pyridine (1 mL). The solution was mixed with egonol
(40 mg, 0.12 mmol) dissolved in pyridine (1 mL). The reaction mixture
was stirred at rt overnight. Subsequently, the reaction mixture was
poured into water and extracted with ether, the organic phase was dried
over anhydrous Na2SO4 and filtered, and the solvent was evaporated in
vacuo. The residue was purified by CCC (petroleum etherꢀEtOAc;
linear gradient from 1:0 to 2:3) to give 17 (35.0 mg, 84%).27
Preparation of 18 and 19. To a solution of 13 (20.0 mg,
0.061 mmol) in dry MeCN (2 mL) was added tert-butyl isocyanate
(6.1 mg, 0.061 mmol) under argon. The reaction mixture was stirred and
refluxed for 4 h. Subsequently, the solvent was evaporated in vacuo, the
reaction residue was quenched with water (10 mL) and extracted with
CH2Cl2 (15 mL ꢁ 3), the organic phase was dried over anhydrous
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Journal of Natural Products
ARTICLE
Inhibition of BChE was measured as described above, substituting
100 U/mL of hBChE and 75 mM butyrylthiocholine iodide for enzyme
and substrate, respectively.
compounds 1ꢀ6. This material is available free of charge via the
Internet at http://pubs.acs.org.
Inhibition of AChE-Induced Aβ-Aggregation. To quantify
amyloid fibril formation, the thioflavin T fluorescence method was
applied.29 Aliquots of 2 μL of β-amyloid1ꢀ40 (Aβ1ꢀ40) peptide
(Butendorf, Switzland), lyophilized from 2 mg/mL HFIP solution and
dissolved in DMSO at a final concentration of 230 μM, were incubated
for 24 h at rt (25 °C) in 0.215 M sodium phosphate buffer (pH 8.0).
For co-incubation experiments, aliquots (16 μL) of EeAChE from
Electrophorus electricus (Sigma-Aldrich) (final concentration of 2.3 μM,
Aβ/AChE molar ratio of 100:1) and EeAChE in the presence of 2 μL of
the tested inhibitors (final inhibitor concentration 100 μM) were added.
Blanks containing Aβ, EeAChE, Aβ plus inhibitors in 0.215 M sodium
phosphate buffer (pH 8.0), and EeAChE plus inhibitors in 0.215 M
sodium phosphate buffer (pH 8.0) were prepared. The final volume of
each vial was 20 μL. Each assay was run in duplicate. After incubation,
the samples containing Aβ, Aβ plus AChE, or Aβ plus AChE in the
presence of inhibitors were diluted with 50 mM glycineꢀNaOH buffer
(pH 8.5) containing 1.5 μM thioflavin T (Sigma-Aldrich) to a final
volume of 2.0 mL. For the thioflavin T-based fluorometric assay, analyses
were performed with a Jasco spectrofluorimeter FP-750 using a 2 mL
’ AUTHOR INFORMATION
Corresponding Author
*Tel: +33 1 69 82 30 38. Fax: +33 1 69 07 72 47. E-mail: vincent.
dumontet@icsn.cnrs-gif.fr.
’ ACKNOWLEDGMENT
This study was carried out in the framework of a collaboration
agreement between the Centre National de la Recherche Scien-
tifique (France) and the Vietnamese Academy of Sciences and
Technology (Vietnam) and was funded by a grant from ICSN-
CNRS to one of us (J.L.). The authors are very grateful to M.-T.
Martin for her assistance in the structural determinations, and to
J. Rouleau for helpful advice in the thioflavin T assay. We thank
J.-F. Betzer and C. Apel for technical assistance with the
ozonolysis reaction, and O. Thoison, F. Pelissier, and B. Morleo
for HPLC analyses. Wealso thankM. Xu, Q. Wang, and C. Guillou
for helpful discussions.
quartz cell. Fluorescence was monitored with excitation at 446 nm (λexc
)
and emission at 490 nm (λem). A time scan of fluorescence was
performed, and the intensity values reached at the plateau (around
300 s) were averaged after subtracting the background fluorescence from
1.5 μM thioflavin T and AChE. The fluorescence intensities were
compared, and the percent inhibition due to the presence of the test
compounds was calculated. The percent inhibition of the AChE-induced
aggregation due to the presence of the tested compound was calculated
by the following expression: 100 ꢀ [IFi ꢀ IFc)/IFo ꢁ 100], where IFi,
IFo, and IFc are the fluorescence intensities obtained for Aβ plus AChE
in the presence and in the absence of inhibitor and the tested compound,
respectively, after subtracting the fluorescence of respective blanks. In
our testing procedure of thioflavin T, tested benzofuran analogues were
found to be strong innately fluorescent compounds with two excitation
wavelengths, at 336 and 470 nm (λexc), and two emission wavelengths, at
370 and 490 nm (λem). So the percent inhibition was calculated by
subtracting their own fluorescence intensity of tested compounds.
Molecular Modeling. Molecular docking was carried out using
GOLD 4.034 with standard parameters. The published crystal structure
of EeAChE (pdb code 1EEA) was used, with a binding site defined as a
20 Å radius sphere around the OH group of Ser200. The 3D structures of
ligands were constructed with CORINA 3.44.35 Molecular dynamics
simulations were carried out with GROMACS version 4.0.236 using the
OPLS-AA37 force field. Each system was energy-minimized until con-
vergence using a steepest descents algorithm. Molecular dynamics with
position restraints for 200 ps was performed followed by the production
run of 10 ns. During the position restraints and production runs, the
ParinelloꢀRahman method38 was used for pressure coupling, and the
temperature was coupled using the NosꢀeꢀHoover method39 at 300 K.
Electrostatics were calculated with the particle mesh Ewald method.40
The P-LINCS algorithm41 was used to constrain bond lengths, and a
time step of 2 fs was used throughout. Ligand topologies for the OPLS-
AA force field were obtained in a semiautomated manner using an in-
house developed script. All calculations were performed using the high-
performance computing (HPC) facilities at the ICSN. Images were
generated with Chimera42 and raytraced with the POV-ray module.
’ REFERENCES
(1) Walsh, D. M.; Selkoe, D. J. Neuron 2004, 44, 181–193.
(2) Ferri, C. P.; Prince, M.; Brayne, C.; Brodaty, H.; Fratiglioni, L.;
Ganguli, M.; Hall, K.; Hasegawa, K.; Hendrie, H.; Huang, Y.; Jorm, A.;
Mathers, C.; Menezes, P. R.; Rimmer, E.; Scazufca, M. Lancet 2005,
336, 2112–2117.
(3) DeKosky, S. T. J. Am. Geriatr. Soc. 2003, 51, 314–320.
(4) Parihar, M. S.; Hemnani, T. J. Clin. Neurosci. 2004, 11, 456–467.
(5) Bartus, R. T.; Dean, R. L.; Beer, B.; Lippa, A. S. Science 1982,
217, 408–414.
(6) Alvarez, A.; Bronfman, F.; Perez, C. A.; Vicente, M.; Garrido, J.;
Inestrosa, N. C. Neurosci. Lett. 1995, 49–52.
(7) Alvarez, A.; Opazo, C.; Alarcon, R.; Garrido, J.; Inestrosa, N. C.
J. Mol. Biol. 1997, 272, 348–361.
(8) Inestrosa, N. C.; Alvarez, A.; Perez, C. A.; Moreno, R. D.;
Vicente, M.; Linker, C.; Casanueva, O. I.; Soto, C.; Garrido, J. Neuron
1996, 16, 881–891.
(9) Jacobsen, J. S. Curr. Top. Med. Chem. 2002, 2, 343–352.
(10) Maia, A.; Schmitz-Alfonso, I.; Martin, M.-T.; Awang, K.;
Laprꢀevote, O.; Guꢀeritte, F.; Litaudon, M. Planta Med. 2008, 74, 1–6.
(11) Beniddir, M. A.; Simonin, A.-L.; Martin, M.-T.; Dumontet, V.;
Poullain, C.; Guꢀeritte, F.; Litaudon, M. Phytochem. Lett. 2010, 3, 75–78.
(12) Fritsch, P. W. Mol. Phylogenet. Evol. 2001, 19, 387–408.
^
(13) Pauletti, P. M.; Teles, H. L.; Silva, D. H. S.; Araꢀujo, A. R.;
Bolzani, V. S. Braz. J. Pharmacogn. 2006, 16, 576–590.
(14) Min, B. S.; Oh, S. R.; Ahn, K. S.; Kim, J. H.; Lee, J.; Kim, D. Y.;
Kim, E. H.; Lee, H. K. Planta Med. 2004, 70, 1210–1215.
(15) Pauletti, P. M.; Araujo, A. R.; Young, M. C.; Giesbrecht, A. M.;
Bolzani, V. D. Phytochemistry 2000, 55, 597–601.
(16) Bousserouel, H.; Litaudon, M.; Morleo, B.; Martin, M. T.;
Thoison, O.; Nosjean, O.; Boutin, J. A.; Renard, P.; Sevenet, T.
Tetrahedron 2005, 61, 845–851.
(17) Ozturk, S. E.; Akgul, Y.; Anil, H. Bioorg. Med. Chem. 2008,
16, 4431–4437.
(18) Takanashi, M.; Takisawa, Y. Phytochemistry 1988, 27, 1224–1226.
(19) Li, Q. L.; Li, B. G.; Qi, H. Y.; Gao, X. P.; Zhang, G. L. Planta
Med. 2005, 71, 847–851.
(20) Park, S. Y.; Lee, H.-J.; Lee, O.-K.; Kang, H.-Y.; Choi, D.-H.;
Paik, K.-H.; Khan, M. Bull. Korean Chem. Soc. 2007, 28, 1874–1876.
(21) Takanashi, M.; Takizawa, Y. J. Oleo Sci. 2002, 51, 151–155.
(22) Akgul, Y. Y.; Anil, H. Phytochemistry 2003, 63, 939–943.
’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra of compounds 1,
b
2, 4, 14, 15, 18, and 19 and molecular dynamics simulations of
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Journal of Natural Products
ARTICLE
(23) Takanashi, M.; Takizawa., Y.; Mitsuhashi, T. Chem. Lett. 1974,
8, 869–871.
(24) Aursand, M.; Grasdalen, H. Chem. Phys. Lipids 1992, 62,
239–251.
(25) Gunstone, F. D.; Jacobsberg, F. R. Chem. Phys. Lipids 1972, 9,
112–122.
(26) Gunstone, F. D. In Advances in Lipid MethodologyꢀTwo;
Christie, W. W., Ed.; Oily Press: Dundee, 1993; Chapter 1, pp 1ꢀ68.
(27) Puentes de Diaz, A. Phytochemistry 1997, 44, 345–346.
(28) Ellman, G. L.; Courtney, K. D.; Andres, V., Jr.; Feather-Stone,
R. M. Biochem. Pharmacol. 1961, 7, 88–95.
(29) Bartolini, M.; Bertucci, C.; Cavrini, V.; Andrisano, V. Biochem.
Pharmacol. 2003, 65, 407–416.
(30) Howlett, D. R.; Perry, A. E.; Godfrey, F.; Swatton, J. E.;
Jennings, K. H.; Spitzfaden, C.; Wadsworth, H.; Wood, S. J.; Markwell,
R. E. Biochem. J. 1999, 340, 283–289.
(31) Rizzo, S.; Riviere, C.; Piazzi, L.; Bisi, A.; Gobbi, S.; Bartolini, M.;
Andrisano, V.; Morroni, F.; Tarozzi, A.; Monti, J. P.; Rampa, A. J. Med.
Chem. 2008, 51, 2883–2886.
(32) William, P.; Sorribas, A.; Howes, M.-J. Nat. Prod. Rep. 2011,
28, 48–77.
(33) Ackman, R. G.; Reston, M. E.; Gallay, L. R.; Vandenheuvel, F. A.
Can. J. Chem. 1961, 39, 1956–1963.
(34) Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.;
Taylor, R. D. Proteins 2003, 52, 609–623.
(35) http://www.molecular-networks.com/products/corina (accessed
on Sept 6, 2011).
(36) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. J. Chem.
Theory Comput. 2008, 4, 435–447.
(37) Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. J. Am. Chem.
Soc. 1996, 118, 11225–11236.
(38) Parinello, M.; Rahman, A. J. Appl. Phys. 1981, 52, 7182–7190.
(39) Nosꢀe, S. Mol. Phys. 1984, 52, 255–268.
(40) Essman, U.; Perela, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;
Pedersen, L. G. J. Chem. Phys. 1995, 103, 8577–8592.
(41) Hess, B. J. Chem. Theory Comput. 2007, 4, 116–122.
(42) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.;
Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. J. Comput. Chem. 2004,
25, 1605–1612.
2088
dx.doi.org/10.1021/np200308j |J. Nat. Prod. 2011, 74, 2081–2088