Natural β-Dihydroagarofuran-Type Sesquiterpenoids as Cognition-Enhancing and Neuroprotective Agents from Medicinal Plants of the Genus Celastrus

Article abstract of DOI:10.1021/acs.jnatprod.5b00234

Alzheimers disease (AD) is an irreversible, multifaceted, and progressive neurodegenerative disorder. Over the past 30 years, the search for anti-AD drugs has been primarily based on the cholinergic deficiency hypothesis and/or the β-amyloid (Aβ) cascade hypothesis. In this study, we report the identification of 16 new and 38 known β-dihydroagarofuran-type sesquiterpenoids from Celastrus flagellaris and Celastrus angulatus. The β-dihydroagarofuran-type sesquiterpenoids 58, 59, 61, and 63 significantly attenuated scopolamine-induced prolonged escape latency and increased number of errors compared with the control group. At 10 μM, 21 of the 62 tested β-dihydroagarofuran-type sesquiterpenoids rescued Aβ_25-35-induced SH-SY5Y cells from viability reduction, which increased the cell viability from 64.6% for the model to more than 74.0%. The majority of the β-dihydroagarofuran-type sesquiterpenoids with ester groups exhibited stronger activity than those with free hydroxy groups or without substituents at the same positions. These results identified a new chemical skeleton as drug lead for the investigation of novel therapeutic agents against AD.

Full text of DOI:10.1021/acs.jnatprod.5b00234

Article
pubs.acs.org/jnp
Natural β‑Dihydroagarofuran-Type Sesquiterpenoids as Cognition-
Enhancing and Neuroprotective Agents from Medicinal Plants of the
Genus Celastrus
Ruonan Ning,†,§ Yun Lei,‡,§ Shuangzhu Liu,† Huan Wang,‡ Rujun Zhang,† Wei Wang,‡ Yingdong Zhu,†
Haiyan Zhang,*,‡ and Weimin Zhao*,†
†State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
People’s Republic of China
‡CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
People’s Republic of China
S
* Supporting Information
ABSTRACT: Alzheimer’s disease (AD) is an irreversible,
multifaceted, and progressive neurodegenerative disorder.
Over the past 30 years, the search for anti-AD drugs has
been primarily based on the cholinergic deficiency hypothesis
and/or the β-amyloid (Aβ) cascade hypothesis. In this study,
we report the identification of 16 new and 38 known β-
dihydroagarofuran-type sesquiterpenoids from Celastrus flag-
ellaris and Celastrus angulatus. The β-dihydroagarofuran-type
sesquiterpenoids 58, 59, 61, and 63 significantly attenuated
scopolamine-induced prolonged escape latency and increased
number of errors compared with the control group. At 10 μM,
21 of the 62 tested β-dihydroagarofuran-type sesquiterpenoids
rescued Aβ25−35-induced SH-SY5Y cells from viability
reduction, which increased the cell viability from 64.6% for the model to more than 74.0%. The majority of the β-
dihydroagarofuran-type sesquiterpenoids with ester groups exhibited stronger activity than those with free hydroxy groups or
without substituents at the same positions. These results identified a new chemical skeleton as drug lead for the investigation of
novel therapeutic agents against AD.
galanthamine from Leucojum aestivum,8 and betulinic acid
lzheimer’s disease (AD), characterized by the loss of
from Bacopa monnieria.9
The family Celastraceae is widespread in the tropical and
Acognitive functions, is the most common cause of
dementia, which seriously affects the life quality of patients.1
subtropical regions of the world, including North Africa, South
America, and East Asia, and is composed of approximately 88
genera, including trees, shrubs, and liana plants.10 β-
Dihydroagarofuran-type sesquiterpenoids are characteristic
natural products of the family Celastraceae and are regarded
as privileged structures due to their chemical diversity and
attractive biological activities, including their ability to exert
multidrug-resistant,11 antitumor-promoting,12 antiplasmodial,13
antitubercular,14 anti-inflammatory,15 anti-HIV,16 cytotoxic,17
immunosuppressant,18 and insecticidal effects.19 The fruits of
Celastrus orbiculatus Thunb. var. cancatus (Rehd. et Wils.) have
been used in China for their activity against amnesia.20 The
seeds of Celastrus paniculatus Willd. have been used in China to
refresh mental activity and have a long history of use in India to
alleviate cognitive disturbances, and are revered as “Elixir of
Life” in the latter country.20 The methanol extract of the seeds
The cholinergic pathway plays an important role in the
regulation of cognition, learning, and memory processes.2 A
large body of evidence emphasizes the abnormal accumulation
of β-amyloid (Aβ) in the pathology of AD.3 Therefore, the
research on AD drugs over the past 30 years has been primarily
based on the cholinergic deficiency and Aβ cascade hypotheses.
However, the currently approved AD drugs do not prevent the
progression of the disease and show only marginal benefits in
moderately to severely affected patients,4 which emphasizes the
necessity of discovering new effective therapeutic agents.2b,5
Natural products have been shown to be reliable resources in
drug discovery and development processes. In the 2012
Newman and Cragg’s review, of the 1355 new drugs approved
from 1980 to 2010, 26.8% are either natural products or
therapeutic agents directly derived from natural products.6 In
the area of dementia research, natural products have played an
important role in providing potentially valuable compounds for
drug discovery, such as curcumin from Curcuma longa,7
Received: March 17, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
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Figure 1. β-Dihydroagarofuran-type sesquiterpenoids isolated from C. flagellaris, C. angulatus, and C. orbiculatus.
of C. paniculatus was found to attenuate H2O2-induced injury in
embryonic rat forebrain neuronal cells.21 We recently reported
the identification of dimeric trinorditerpenoids with neuro-
protective activity from the root bark of C. orbiculatus.22 Herein,
we report the identification of new β-dihydroagarofuran-type
sesquiterpenoids from Celastrus plants and their neuro-
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Figure 2. ORTEP drawing of compound 1.
protective effects against Aβ25−35 toxicity, and their nootropic
effects against scopolamine-induced memory impairment in
mice, as measured using a water maze.
NMR spectrum. Single-crystal X-ray diffraction analysis with
Cu Kα radiation was used to establish the structure and
absolute configuration of 1 [Flack parameter: 0.14 (14)]
(Figure 2). Thus, the structure of 1 was unequivocally defined
as (1R,2S,4R,5S,7R,9S,10S)-1,15-diacetoxy-2-benzoyloxy-9-cin-
namoyloxy-β-dihydroagarofuran.
RESULTS AND DISCUSSION
■
Ten new (1−10) and 10 known (11−20) β-dihydroagarofuran-
type sesquiterpenoids were isolated from the seeds of Celastrus
flagellaris Rupr. Six new (21−26) and 28 known analogues
(27−54) were obtained from the seeds and root bark of
Celastrus angulatus Maxim. by using various chromatographic
methods (Figure 1).
Compound 2 had the molecular formula C37H42O9, as
determined by HRTOFMS (m/z 653.2739 [M + Na]+, calcd
653.2727) and its 13C NMR data. A β-dihydroagarofuran
1
skeleton was established from the H−1H COSY cross signals
for the H-1/H-2/H-3/H-4/H3-14 and H-6/H-7/H-8/H-9 spin
systems and the HMBC correlations between H2-15 and C-1,
C-9, between H-9, H3-14 and C-5, and between H-4, H-6 and
C-10. The 1H and 13C NMR data revealed 2 to be a
tetrasubstituted β-dihydroagarofuran-type sesquiterpenoid with
two cinnamoyloxy and two acetoxy groups. In the HMBC
spectrum, cross peaks between H-1 (δH 5.75) and the AcO-1
carbonyl carbon (δC 170.0), between H-2 (δH 5.66) and the
CinO-2 carbonyl carbon (δC 166.2), between H-9 (δH 5.24)
and the CinO-9 carbonyl carbon (δC 166.2), and between H2-
15 (δH 5.03, 4.49) and the AcO-15 carbonyl carbon (δC 170.8)
indicated the locations of the four substituents of 2 (Figure 3).
The relative configuration of 2 was established by the coupling
constants of the key proton signals and the NOESY data.
Signals at δH 5.75 (1H, d, J1,2 = 3.4 Hz, H-1), δH 5.66 (1H, dd,
J1,2 and 2,3 = 3.4, 7.5 Hz, H-2), and δH 5.24 (1H, d, J8,9 = 6.8 Hz,
Compound 1 showed 35 signals in its 13C NMR spectrum
and an accurate [M + Na]+ ion at m/z 627.2579 in the
HRTOFMS, corresponding to the molecular formula
C35H40O9Na (m/z 627.2570) and indicative of 16 indices of
1
hydrogen deficiency. Its H NMR spectrum indicated signals
for 12 protons in the aromatic region for the benzoyl and
cinnamoyl groups at δH 8.13 (d, 2H, J = 7.0 Hz), 7.69 (d, 1H, J
= 16.0 Hz), 7.57 (m, 3H), 7.50 (t, 2H, J = 7.5 Hz), 7.39 (m,
3H), and 6.39 (d, 1H, J = 16.0 Hz), two acetyl groups at δH
1.77 and 2.17 (s, each 3H), three acylated oxymethine protons
at δH 5.80 (m, 2H) and 5.23 (d, 1H, J = 6.9 Hz), and a pair of
acylated oxymethylene protons at δH 5.12 and 4.48 (d, each 1H,
J = 12.1 Hz). The 13C NMR spectrum exhibited 35 carbon
signals, including a benzoyl, a cinnamoyl, and two acetyl
moieties, as well as 15 sp3 carbon signals, comprising an
oxygenated primary carbon (C-15), three oxygenated secon-
dary carbons (C-1, C-2, C-9), two oxygenated tertiary carbons
(C-5, C-11), three primary carbons (C-12, C-13, C-14), three
secondary carbons (C-3, C-6, C-8), two tertiary carbons (C-4,
C-7), and a quaternary (C-10) carbon, hence representing
three indices of hydrogen deficiency and reflecting the tricyclic
molecular backbone of 1. These data suggested that compound
1 is a tetrasubstituted β-dihydroagarofuran-type sesquiterpe-
noid.
The locations of two of the four ester groups were defined
based on the HMBC correlations between H-9 (δH 5.23) and
the CinO-9 carbonyl carbon (δC 166.3) and between H2-15 (δH
5.12, 4.48) and the AcO-15 carbonyl carbon (δC 170.9).
However, the locations of the remaining ester groups could not
be deduced via NMR analysis due to the overlapping of the two
Figure 3. 1H−1H COSY (bold) and selected HMBC (arrows)
correlations of compound 2.
1
acylated oxymethine protons at δH 5.80 (m, 2H) in the H
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Figure 4. Key NOESY correlations, ECD exciton coupling, and ECD spectrum of compound 2.
H-9) (Table 1) were compatible with equatorial orientations of
H-1, H-2, and H-9, which are supported by the NOE
associations between H-1/H-9 and H2-15 and between H-1
and the CinO-2 olefinic protons. Its electronic circular
dichroism (ECD) spectrum showed a Davidoff-type split
curve with a positive first Cotton effect at 291 nm (Δε =
29.74) and a negative second Cotton effect at 260 nm (Δε =
−4.32) (Figure 4),23 due to the coupling of the two
cinnamoyloxy chromophores at C-2β and C-9α. Therefore,
the structure of 2 was characterized as (1S,2S,4R,5S,7R,9S,10S)-
1,15-diacetoxy-2,9-dicinnamoyloxy-β-dihydroagarofuran.
diacetoxy-2-cinnamoyloxy-9-benzoyloxy-β-dihydroagarofuran,
respectively.
The structure of 5 was similar to that of 3, except for an
additional acetoxy group at C-15, whereas the structure of 6
was similar to that of 4, except for replacement of a
cinnamoyloxy by a benzoyloxy group at C-2. The relative
configurations of 5 and 6 were deduced to be different from
those of 3 and 4 at C-1 based on the NOE correlations between
H-4 and H-1/H-2 in 5 and between H-1 and H-3α in 6.
Furthermore, the Davidoff-type split curve of 5 with a positive
first Cotton effect at 292 nm (Δε = 27.36) and a negative
second Cotton effect at 262 nm (Δε = −6.75) (Figure S49,
Supporting Information), and the Davidoff-type split curve of 6
with a positive first Cotton effect at 235 nm (Δε = 22.88) and a
negative second Cotton effect at 213 nm (Δε = 3.07) in the
ECD spectrum23 (Figure S59, Supporting Information)
indicated their absolute configurations. Thus, the structures of
5 and 6 were defined as (1R,2S,4R,5S,6R,7R,9S,10S)-1,6,15-
triacetoxy-2,9-dicinnamoyloxy-β-dihydroagarofuran and
(1R,2S,4R,5S,6R,7R,9S,10S)-1,6-diacetoxy-2,9-dibenzoyloxy-β-
dihydroagarofuran, respectively.
Compounds 3 and 4 were assigned the molecular formulas
C37H42O9 and C35H40O9, respectively, according to their
HRTOFMS (m/z 653.2742 [M + Na]+, calcd 653.2727; m/z
627.2588 [M + Na]+, calcd 627.2570) and 13C NMR
spectroscopic data. The NMR data showed that 3 and 4 are
both tetrasubstituted β-dihydroagarofuran-type sesquiterpe-
noids. In the HMBC spectrum of 3, cross peaks between H-1
(δH 5.65) and the AcO-1 carbonyl carbon (δC 170.2), between
H-2 (δH 5.70) and the CinO-2 carbonyl carbon (δC 166.2),
between H-6 (δH 5.42) and the AcO-6 carbonyl carbon (δC
170.3), and between H-9 (δH 4.77) and the CinO-9 carbonyl
carbon (δC 166.3) suggested that an acetoxy group is located at
C-6 in 3 instead of the acetoxy group at C-15 in 2. The
substitution of an acetoxy group at C-6 in 4 instead of the
acetoxy group at C-15 in 2, as well as a benzoyloxy group at C-
9 in 4 instead of the cinnamoyloxy group in 2, was verified by
the HMBC correlations between H-6 (δH 5.46) and the AcO-6
carbonyl carbon (δC 170.3) and between H-9 (δH 4.99) and the
BzO-9 carbonyl carbon (δC 165.7). Their relative config-
urations were deduced by analyses of key coupling constants
(J1,2 = 3.9 Hz, J2,3 = 7.8 Hz, J8,9 = 7.0 Hz in 3 and J1,2 = 3.2 Hz,
J2,3 = 7.5 Hz, J8,9 = 6.9 Hz in 4) and were confirmed by the
NOESY experiment, in which NOE correlations were observed
between H-1 and H-9/H3-15; between H-6 and H3-14 in 3;
and between H3-15 and H-1/H-6/H-9 in 4. The absolute
configurations of 3 and 4 were established by analyses of their
ECD spectra, which showed Davidoff-type split curves with a
positive first Cotton effect at 292 nm (Δε = 38.52) and a
negative second Cotton effect at 260 nm (Δε = −9.36) due to
the coupling of the two cinnamoyloxy chromophores at C-2β
and C-9α in 3 (Figure S29, Supporting Information), and with
a positive first Cotton effect at 277 nm (Δε = 9.87) and a
negative second Cotton effect at 238 nm (Δε = −1.65) due to
the coupling of the cinnamoyloxy and benzoyloxy chromo-
phores at C-2β and C-9α in 4 (Figure S39, Supporting
Information).23 Thus, the structures of 3 and 4 were assigned
as (1S,2S,4R,5S,6R,7R,9S,10S)-1,6-diacetoxy-2,9-dicinnamoy-
loxy-β-dihydroagarofuran and (1S,2S,4R,5S,6R,7R,9S,10S)-1,6-
The 1H and 13C NMR data indicated that 7 is a trisubstituted
β-dihydroagarofuran-type sesquiterpenoid with a cinnamoyloxy,
an acetoxy, and a hexanoyloxy moiety, and 8 is a
tetrasubstituted sesquiterpenoid with a cinnamoyloxy, a
1
butanoyloxy, and two acetoxy groups. Analyses of the H−1H
COSY, HSQC, and HMBC spectra of 7 and 8 enabled the
establishment of their 2D structures. The relative configurations
of 7 and 8 were determined from the coupling constants (J1,2
3.4 Hz, J2,3 = 7.4 Hz, J8,9 = 6.8 Hz in 7, and J1,2 = 3.6 Hz, J2,3
=
=
7.5 Hz, J8,9 = 6.9 Hz in 8) of key adjacent protons and from the
NOE correlations between H-1 and H-3α, between H-9 and
H3-15 in 7, and between H3-15 and H-1/H-6/H-9 in 8. Thus,
the structures of 7 and 8 were defined as 1β-acetoxy-2β-
hexanoyloxy-9α-cinnamoyloxy-β-dihydroagarofuran and 1α,6α-
diacetoxy-2β-butanoyloxy-9α-cinnamoyloxy-β-dihydroagarofur-
an, respectively.
Compound 9 possessed an identical molecular formula and
similar 1D NMR data to those of 8, except for the presence of a
pair of acylated oxymethylene proton signals and the absence of
an acylated oxymethine singlet proton signal. The NMR data of
10 were similar to those of 5, except for the replacement of a
cinnamoyloxy group by an acetoxy group at C-2 in 10. Analyses
1
of H−1H COSY, HSQC, and HMBC spectra enabled the
deduction of the 2D structures of 9 and 10. Their relative
configurations were established using the methods described
above and were defined as 1α,15-diacetoxy-2β-butanoyloxy-9α-
cinnamoyloxy-β-dihydroagarofuran and 1β,2β,6α,15-tetraace-
toxy-9α-cinnamoyloxy-β-dihydroagarofuran, respectively.
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1
Table 2. H NMR Data of the Carbon Framework of Compounds 21−26 (δ ppm, J in Hz in Parentheses) in CDCl3
a
no.
21
22
23
24
25
26
1
4.28 d (3.4)
4.10 brs
5.57 d (3.2)
6.17 d (2.7)
5.44 dd (11.9, 4.3)
1.82 m
5.57 dd (10.4, 5.8)
1.82 m
5.50 dd (12.2, 4.2)
1.87 m
2α
2β
3α
3β
4
5.42 dd (6.4, 3.2)
5.82 dd (5.7, 2.7)
1.58 m
1.79 m
1.48 m
1.86 m
2.25 m
2.29 m
1.76 m
1.79 m
1.48 m
1.52 m
1.47 m
2.47 m
2.49 m
2.27 m
2.24 m
2.28 m
1.94 m
2.00 m
1.88 m
2.31 m
1.92 m
6α
6β
7
2.04 dd (13.0, 4.0)
2.92 d (13.0)
2.55 brt (3.6)
5.71 dd (5.6, 3.2)
5.76 d (5.6)
1.59 s
2.17 dd (13.4, 3.2)
2.98 d (13.4)
2.53 brt (3.7)
6.13 dd (5.4, 3.4)
6.20 d (5.4)
1.50 s
2.13 dd (12.8, 4.8)
2.65 d (12.8)
2.39 brt (3.9)
5.58 dd (9.8, 3.3)
6.08 d (9.8)
1.59 s
2.20 dd (12.6, 4.8)
2.30 d (12.6)
2.26 m
6.49 s
6.15 s
2.66 d (4.1)
5.81 dd (5.8, 4.1)
5.91 d (5.8)
1.61 s
2.63 d (4.2)
5.82 dd (5.2, 4.2)
5.78 d (5.2)
1.65 s
8
5.57 brs
9
5.57 brs
12
13
14
15
1.52 s
1.45 s
1.26 s
1.24 s
1.21 s
1.47 s
1.22 s
1.25 d (7.6)
5.24 d (12.5)
4.89 d (12.5)
1.32 d (8.0)
5.27 d (12.6)
4.87 d (12.6)
1.41 d (8.0)
5.97 d (12.0)
5.47 d (12.0)
1.16 d (7.8)
5.10 d (12.0)
4.90 d (12.0)
1.21 d (7.4)
1.66 s
1.10 d (7.8)
4.56 d (12.1)
4.42 d (12.1)
a
Recorded in pyridine-d5.
Analyses of the NMR and IR spectra of 21 revealed it to be a
The locations of the five ester functions were established using
1H−1H COSY, HSQC, and HMBC experiments. The relative
configuration of 23 was determined as 1β,2β,8β,9β by analyses
of the coupling constants (J1,2 = 2.7 Hz, J2,3 = 5.7 Hz, J8,9 = 5.4
Hz) of key adjacent protons and NOE correlations between H-
1 and H-9, and between H-2 and H-4. Thus, the structure of 23
was defined as 1β,2β-diacetoxy-8β,9β-dibenzoyloxy-15-nicoti-
noyloxy-β-dihydroagarofuran.
β-dihydroagarofuran-type sesquiterpenoid carrying two benzoy-
loxy, two acetoxy, and two secondary hydroxy groups. The
HMBC correlations between H-8 (δH 5.81) and the BzO-8
carbonyl carbon (δC 166.3), between H-9 (δH 5.91) and the
BzO-9 carbonyl carbon (δC 166.0), between H-6 (δH 6.49) and
the AcO-6 carbonyl carbon (δC 170.0), and between H2-15 (δH
4.89, 5.24) and the AcO-15 carbonyl carbon (δC 170.3)
established the locations of the four substituents. The
remaining two hydroxy groups were assigned to C-1 and C-2
according to the chemical shifts of H-1/C-1 at δH 4.28/δC 78.5,
and H-2/C-2 at δH 4.10/δC 71.5, confirmed by the HSQC and
HMBC data. Comparison of the NMR spectroscopic data of 22
with those of 21 indicated that the two hydroxy groups at C-1
and C-2 are acetylated, and the acetoxy group at C-6 in 21 is
absent in 22. Their relative configurations were established
using the methods described above. In their ECD spectra, the
observation of a negative first Cotton effect at 240 nm (Δε =
−19.22) and a positive second Cotton effect at 222 nm (Δε =
15.82) in 21 (Figure S104, Supporting Information), as well as
a negative first Cotton effect at 238 nm (Δε = −51.14) and a
positive second Cotton effect at 219 nm (Δε = 4.17) in 22
(Figure S113, Supporting Information),23 allowed the deter-
mination of their absolute configurations. Thus, the structures
of 21 and 22 were defined as (1R,2S,4R,5S,6R,7R,8R,9S,10S)-
6,15-diacetoxy-8,9-dibenzoyloxy-1,2-dihydroxy-β-dihydroagaro-
furan and (1R,2S,4R,5S,7R,8R,9S,10S)-1,2,15-triacetoxy-8,9-di-
benzoyloxy-β-dihydroagarofuran, respectively.
The 2D structures of 24 and 25 were established by analyses
1
of their H−1H COSY, HSQC, and HMBC spectra. They are
also nicotinates of β-dihydroagarofuran-type sesquiterpenoids.
The large coupling constant of H-8 with H-9 (J8,9 = 9.8 Hz) in
24, and the smaller coupling constant between H-8 and H-9
(J8,9 = 5.2 Hz) in 25 revealed their trans and cis orientations,
respectively. In their NOESY spectra, NOE cross peaks were
observed between H-1 and H-4/H-9 in 24, and between H-1
and H-6/H-9, between H-4 and H-6 in 25. Thus, the structures
of 24 and 25 were defined as 1β-acetoxy-9β-benzoyloxy-8α-
hexanoyloxy-15-nicotinoyloxy-β-dihydroagarofuran and 6α-ace-
toxy-1β,9β-dibenzoyloxy-8β-nicotinoyloxy-β-dihydroagarofur-
an, respectively.
The spectroscopic data of 26 were similar to those of 42, a β-
dihydroagarofuran-type sesquiterpenoid previously identified
from C. paniculatus.24 Compound 26 lacks the C-2 acetoxy
group of 42, as is evidenced by the signals at δH 1.48 and 1.87
(H-2) and δC 22.4 (C-2) (Tables 2 and 3). The correlations
between H-1 and H-4 and between H-8 and H-9/H-15a in the
NOESY spectrum permitted the establishment of its relative
configuration. Thus, the structure of 26 was defined as
1β,8α,15-triacetoxy-9α-benzoyloxy-β-dihydroagarofuran.
The structures of the known compounds 11,25 12,26 13,27
14,28 15,29 16,27 17,30 18,27 19,25 20,31 32,32 33,33 34,33 35,34
36,34 37,35 38,36 39,37 40,37 41,38 42,24 43,39 44,39 45,39 46,40
47,41 48,19 49,19 50,38 51,32 52,38 53,33 and 5438 were
characterized by comparison of their observed and reported
NMR data.
The fruits and seeds of several Celastrus plants have been
used in China and India to enhance memory. According to our
experimental results, the seeds of C. flagellaris, C. angulatus, and
C. orbiculatus mainly contain fatty acids and β-dihydroagar-
ofuran-type sesquiterpenoids. Linolenic acid, one of the fatty
acids from the seeds of Celastrus plants, has been well-known to
Compound 23 gave the molecular formula C39H41NO11, as
deduced from the HRESIMS (m/z 722.2588 [M + Na]+, calcd
722.2577) and 13C NMR data. The presence of a nicotinoyl
function was revealed by the resonances at δH 9.14 (brs), 8.60
(brd, J = 4.8 Hz), 8.13 (brd, J = 8.0 Hz), and 7.07 (dd, J = 8.0,
1
4.8 Hz) in the H NMR spectrum, and at δC 123.0 (d), 126.0
(s), 137.0 (d), 151.2 (d), 153.6 (d), 165.6 (s) in its 13C NMR
spectrum. The presence of two acetoxy and two benzoyloxy
1
groups could also be deduced from the H and 13C NMR data
(Tables 2 and 3). A three-proton multiplet centered at δH
1
5.75−5.80 in the H NMR spectrum (CDCl3) of 23 was
resolved into two doublets at δH 6.17 (1H, J = 2.7 Hz) and 6.20
(1H, J = 5.4 Hz), and a doublet of doublets at δH 6.13 (1H, J =
5.4, 3.4 Hz) when the spectrum was recorded in pyridine-d5.
F
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Article
improve memory.42 However, the biological activities relating
to the cognition-enhancing and neuroprotective effects of β-
dihydroagarofuran-type sesquiterpenoids of the genus Celastrus
have not been reported. Scopolamine, a nonselective
muscarinic antagonist, can block the cholinergic pathway.43
On the basis of the blockade effects, the use of scopolamine on
animals provides a simple and rapid method for testing the
cognition-enhancing properties of new drugs.44 Thus, the
cognition-enhancing effects of compounds 58, 59, and 61−63,
which are available in sufficient quantities for animal testing,
were evaluated on scopolamine-treated mice using a passage-
way water maze. As shown in Figure 5, mice injected with
Figure 5. Effects of compounds 58, 59, and 61−63 on scopolamine-
induced cognitive deficits of mice. (A) The escape latency of mice in
passageway water maze. (B) The number of errors of mice in
passageway water maze. Data are shown as mean SEM n = 12. ***p
#
< 0.001 vs control group, p < 0.05, ##p < 0.01 vs scopolamine group.
Control and huperzine A are, respectively, abbreviated as Con and
Hup A.
scopolamine (4.5 mg/kg) exhibited longer escape latency and
increased number of errors compared with the saline-injected
control group. Compounds 58, 59, 61, and 63, as well as
huperzine A (positive control), rescued the escape latency and
significantly reduced the number of errors. Compound 58
showed cognition-enhancing effects in a dose-dependent
manner, whereas compounds 59, 61, and 63 showed better
cognition-enhancing effects at 20 mg/kg. Compound 62
showed no statistical difference compared with the scopol-
amine-treated group.
Aβ has generally been adopted as an inducer of neuronal
injury to analyze the protective effects and mechanism of action
of new pharmacotherapies for AD.45 Aβ25−35, a synthetic
peptide that possesses the same β-sheet structure and exhibits
large fibrils, possesses most of the physical and biological
properties of full length-Aβ46 and is often used to study the
neuroprotective effects of various compounds predicted to
modulate Aβ toxicity in vitro.47 The 3-(4,5-dimethylthiazol-2-
G
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Journal of Natural Products
Article
yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to
evaluate the neuroprotective effects of 62 β-dihydroagarofuran-
type sesquiterpenoids, including the 47 sesquiterpenoids
described above, 11 sesquiterpenoids isolated previously from
C. orbiculatus,28 and four synthetic sesquiterpenoid derivatives,
against Aβ25−35-induced SH-SY5Y cell injury. As shown in
Table 4, 21 tested compounds increased the cell viability from
neuroprotective effect, i.e., 1 (BzO-2) > 17 (AcO-2); 11
(BzO-2) > 14 (AcO-2); 7 (HexO-2)/12 (CinO-2) > 13 (BzO-
2); and 7 (HexO-2)/12 (CinO-2) > 16 (AcO-2). Therefore,
the bulk of the substituents at C-2 may also play an important
role in the neuroprotective effects of this kind of
sesquiterpenoids. No unambiguous SAR could be deduced
concerning the substituents at other positions.
In summary, we report the identification of 16 new and 38
known β-dihydroagarofuran-type sesquiterpenoids from the
seed of C. flagellaris and the seed and root bark of C. angulatus.
Twenty-one of the 62 tested compounds were found to possess
neuroprotective effects against Aβ25−35-induced toxicity on SH-
SY5Y cells. According to animal behavior test results, four
compounds rescued scopolamine-induced memory impairment
in mice. These results revealed that certain β-dihydroagarofur-
an-type sesquiterpenoids may contribute to the memory-
improving effect of several Celastrus plants used in Chinese
and Indian folk medicines, and identified a new chemical
skeleton as drug lead for the research of novel therapeutic
agents against AD. Further in vitro and in vivo investigations on
the neuroprotective and memory-enhancing effects, as well as
their pharmacological mechanism, are required.
Table 4. Neuroprotective Effects of Selected Compounds
Isolated from Celastrus Plants against Aβ25−35 Induced
Neurotoxicity in SH-SY5Y Cells
a
a
cell viability (%)
cell viability (%)
compd
1 μM
10 μM
compd
1 μM
10 μM
b
1
−
86.9
82.3
84.5
87.3
−
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
64
65
66
67
68
69
70
EGCG
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
74.3
76.2
82.3
−
−
−
2
−
−
−
−
−
−
−
−
−
76.3
−
79.6
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
3
4
5
6
−
7
85.0
85.0
−
77.7
87.4
73.9
86.2
−
9
10
11
12
13
14
15
16
17
18
19
20
21
23
28
29
30
32
33
34
35
36
37
38
EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were
■
measured using a Buchi 510 melting point apparatus (Buchi, Flawil,
̈
̈
Switzerland). Optical rotations were measured on a PerkinElmer 341
polarimeter, and IR spectra were recorded using KBr disks on a
PerkinElmer 577 spectrometer (PerkinElmer, Waltham, MA). ECD
spectra were obtained on a JASCO J-810 spectrometer (JASCO
Corporation, Tokyo, Japan). UV spectra were obtained using a Varian
Cary 50-vis spectrophotometer (Varian, Melbourne, Australia). NMR
experiments were performed on Bruker AM-400, Bruker Advance III
500, Bruker Advance III 600 (Bruker, Ettlingen, Germany), or Varian-
MERCURY Plus-400 (Varian, Palo Alto, CA) using TMS as an
internal standard. ESIMS analyses were performed on a Shimadzu LC-
MS-2020 spectrometer with a Shimadzu SPD-M20 (Shimadzu, Kyoto,
Japan) diode array detector using CNW C18 (2.1 × 50 mm, 3.5 μm) or
CNW C18 (2.1 × 100 mm, 3.5 μm) columns (Anpel Scientific
Instrument Co., Ltd., Shanghai, People’s Republic of China).
HRESIMS analyses were performed on a Waters-Micromass QTOF
Ultima Global mass spectrometer (Waters, Milford, MA). Semi-
preparative HPLC was performed on a Unimicro EasySep-1010 binary
pump system with a Unimicro EasySep-1010 detector (Unimicro,
Shanghai, People’s Republic of China) using YMC-Pack ODS-A (250
× 20 mm, 5 μm) or YMC-Pack ODS-A (250 × 10 mm, 5 μm)
columns (YMC Co., Ltd., Kyoto, Japan). Precoated silica gel GF254
plates, silica gel (300−400 mesh) (Qingdao Haiyang Chemical Co.,
Ltd., Qingdao, People’s Republic of China), C18 reversed-phase (RP-
18) silica gel (150−200 mesh) (Merck, Whitehouse Station, NJ), and
CHP20P MCI gel (75−150 μm) (Mitsubishi Chemical Industries,
Ltd., Tokyo, Japan) were used for column chromatography (CC) and
TLC detection.
76.7
−
74.4
−
−
−
−
−
−
−
77.8
−
77.7
79.0
81.7
78.7
−
−
−
−
−
−
−
−
−
−
79.7
−
−
−
−
c
N.T.
80.8
a
The neuroprotective effect of these compounds on Aβ25−35-induced
neurotoxicity in SH-SY5Y cells. The cell viability in control was taken
as 100%, and the average value of cell viability under Aβ25−35 exposure
was 64.6
1.4%. The positive control is epigallocatechin gallate
b
c
(EGCG). The − means not active. N.T. means not tested.
Plant Material, Extraction, and Isolation. The seeds of C.
flagellaris were collected in October 2013 in the suburb of Jilin, Jilin
Province, People’s Republic of China, and were identified by Prof.
Zhixin Ju of Jilin Agricultural University. A voucher specimen was
deposited in Shanghai Institute of Materia Medica, Chinese Academy
of Sciences (SIMM20131016-CB). The seeds of C. flagellaris (98 g)
were powdered and ultrasonically extracted with CH2Cl2 at room
temperature (1 L × 3) to give a crude extract (36 g). The crude extract
was fractionated on a silica gel (300−400 mesh) column eluted with a
petroleum ether/EtOAc gradient (from 40:1 to 10:1, v/v) to afford six
fractions (Fr.1−Fr.6). Fr.2−5 were further separated using column
chromatography over silica gel (petroleum ether/acetone of increasing
polarity) and semipreparative HPLC to give the new compounds 1
(25 mg), 2 (6 mg), 3 (15 mg), 4 (4 mg), 5 (8 mg), 6 (5 mg), 7 (5
64.6% for the model to more than 74.0%, significantly
improving the cell viability at 10 μM. Among them, compounds
12 and 14 also showed neuroprotective effects at 1 μM.
Comparison of the activities of 7, 12, 13, 16, 58 and 11, 14, 15
revealed that compounds containing a C-2 ester group are
more potent than those without a substituent at the same
position. Two derivatives obtained by hydrolysis, 67 and 70,
displayed weak activities, suggesting that β-dihydroagarofuran-
type sesquiterpenoids with only two or three hydroxy groups
provide no neuroprotective effect. Sesquiterpenoids with
different ester groups at C-2 exhibited variations in the
H
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1
mg), 8 (2 mg), 9 (4 mg), and 10 (5 mg), as well as the known
compounds 11−20.
1745, 1711, 1637, 1450, 1367, 1234, 1165, 1016, 767, 710 cm−1; H
NMR (CDCl3) δ 1.81 (3H, s, AcO-1), 2.13 (3H, s, AcO-6), CinO-2
and CinO-9 [7.67 (d, 1H, J = 16.5 Hz), 7.63 (d, 1H, J = 16.5 Hz), 7.53
(m, 4H), 7.38 (m, 6H), 6.38 (d, 1H, J = 16.5 Hz), 6.37 (d, 1H, J = 16.5
Hz)], for other signals, see Table 1; 13C NMR (CDCl3) δ AcO-1 [20.9
(q), 170.2 (s)], AcO-6 [21.6 (q), 170.3 (s)], CinO-2 and CinO-9
[118.1 (d), 118.4 (d), 128.2 × 2 (d), 128.4 × 2 (d), 129.0 × 2 (d),
129.1 × 2 (d), 130.5 × 2 (d), 134.4 (s), 134.5 (s), 145.1 (d), 145.7
(d), 166.2 (s), 166.3 (s)], for other signals, see Table 3; HRTOFMS
m/z 653.2742 [M + Na]+ (calcd for C37H42O9Na, 653.2727).
The seeds of C. angulatus were collected in October 2010 in
Xinxiang, Henan Province, People’s Republic of China, and were
identified by Prof. Jingui Shen of Shanghai Institute of Materia Medica.
A voucher specimen was deposited in Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (SIMM20111010). The seeds
of C. angulatus (1.8 kg) were powdered and ultrasonically extracted
with EtOH at room temperature (10 L × 3) to yield a crude extract
(300 g). The extract was fractionated over a silica gel (300−400 mesh)
column and eluted with a petroleum ether/acetone gradient (from
20:1 to 1:1, v/v) to afford six fractions (Fr.1−Fr.6). Crude crystals
(200 mg) precipitated from Fr.2 were chromatographed over silica gel
(petroleum ether/EtOAc of increasing polarity) to give 26 (8 mg) and
40. A portion (24 g) of the filtrate from Fr.2 was subjected to
chromatography over C18 reversed-phase silica gel and semipreparative
HPLC to give 24 (16 mg), 37, and 50. Fr.3 (18 g) and Fr.4 (16 g)
were subjected to chromatography over silica gel (petroleum ether/
acetone of increasing polarity) and semipreparative HPLC to afford
the new compounds 22 (10 mg) and 25 (2 mg), as well as the known
compounds 27, 28, 31−36, 39, 41, 42, 51, 52, and 54. New
compounds 21 (23 mg) and 23 (6 mg) and known compounds 29,
30, 38, and 53 were obtained from Fr.5 (15 g) using isolation methods
similar to those mentioned above.
(1S,2S,4R,5S,6R,7R,9S,10S)-1,6-Diacetoxy-2-cinnamoyloxy-9-ben-
zoyloxy-β-dihydroagarofuran (4): white amorphous powder; [α]21
D
+ 87 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 218 (4.20), 223 (4.11),
276 (4.23) nm; ECD (MeOH) λmax (Δε) 238 (−1.65), 277 (9.87)
nm; IR (KBr) νmax 1742, 1713, 1639, 1392, 1366, 1280, 1227, 1100,
1015, 768, 709 cm−1; H NMR (CDCl3) δ 1.62 (3H, s, AcO-1), 2.13
1
(3H, s, AcO-6), CinO-2 and BzO-9 [8.06 (d, 2H, J = 7.4 Hz), 7.63 (d,
1H, J = 16.1 Hz), 7.56 (t, 1H, J = 7.0 Hz), 7.51 (m, 2H), 7.44 (t, 2H, J
= 7.5 Hz), 7.39 (m, 3H), 6.37 (d, 1H, J = 16.1 Hz)], for other signals,
see Table 1; 13C NMR (CDCl3) δ AcO-1 [20.6 (q), 169.8 (s)], AcO-6
[21.6 (q), 170.3 (s)], CinO-2 and BzO-9 [118.3 (d), 128.3 × 2 (d),
128.4 × 2 (d), 129.1 × 2 (d), 129.6 (s), 130.2 × 2 (d), 130.6 (d),
133.4 (d), 134.4 (s), 145.1 (d), 165.7 (s), 166.3 (s)], for other signals,
see Table 3; HRTOFMS m/z 627.2588 [M + Na]+ (calcd for
C35H40O9Na, 627.2570).
The root bark of C. angulatus was collected in May 2010 in
Xinxiang, Henan Province, People’s Republic of China, and was
identified by Prof. Jingui Shen of Shanghai Institute of Materia Medica.
A voucher specimen was deposited in Shanghai Institute of Materia
Medica, Chinese Academy of Sciences (SIMM20111011). The root
bark of C. angulatus (1.8 kg) was powdered and ultrasonically extracted
with EtOH at room temperature (10 L × 3) to give a crude extract
(240 g). The crude extract was fractionated using silica gel (300−400
mesh) eluted with a petroleum ether/acetone gradient (from 20:1 to
1:1, v/v) to afford five fractions (Fr.1 ∼ Fr.5). Fr.2 (26 g) and Fr.4 (30
g) were further chromatographed over silica gel (petroleum ether/
acetone of increasing polarity) and subjected to semipreparative
HPLC to yield the known compounds 43−49.
(1R,2S,4R,5S,6R,7R,9S,10S)-1,6,15-Triacetoxy-2,9-dicinnamoy-
loxy-β-dihydroagarofuran (5): white amorphous powder; [α]21
+
D
211 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 218 (4.33), 279 (4.43)
nm; ECD (MeOH) λmax (Δε) 262 (−6.75), 292 (27.36) nm; IR (KBr)
νmax 1743, 1711, 1639, 1388, 1370, 1280, 1225, 1159, 1093, 1016, 768,
712 cm−1; H NMR (CDCl3) δ 1.79 (3H, s, AcO-1), 2.10 (3H, s,
1
AcO-6), 2.27 (3H, s, AcO-15), CinO-2 and CinO-9 [7.76 (d, 1H, J =
16.1 Hz), 7.69 (d, 1H, J = 16.0 Hz), 7.64 (m, 2H), 7.55 (m, 2H), 7.39
(m, 6H), 6.44 (d, 1H, J = 16.1 Hz), 6.34 (d, 1H, J = 16.0 Hz)], for
other signals, see Table 1; 13C NMR (CDCl3) δ AcO-1 [20.9 (q),
169.8 (s)], AcO-6 [21.4 (q), 170.2 (s)], AcO-15 [21.5 (q), 170.8 (s)],
CinO-2 and CinO-9 [117.8 (d), 118.0 (d), 128.4 × 4 (d), 129.0 × 4
(d), 130.6 × 2 (d), 134.4 × 2 (s), 145.4 (d), 146.0 (d), 165.9 (s),
166.1 (s)], for other signals, see Table 3; HRTOFMS m/z 711.2783
[M + Na]+ (calcd for C39H44O11Na, 711.2781).
(1R,2S,4R,5S,7R,9S,10S)-1,15-Diacetoxy-2-benzoyloxy-9-cinna-
moyloxy-β-dihydroagarofuran (1): colorless orthorhombic crystals
(acetone); mp 218−220 °C; [α]22 + 143 (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 219 (4.40), 223 (4.41), 279 (4.42) nm; ECD
(MeOH) λmax (Δε) 238 (0.84), 275 (4.17) nm; IR (KBr) νmax 1742,
(1R,2S,4R,5S,6R,7R,9S,10S)-1,6-Diacetoxy-2,9-dibenzoyloxy-β-di-
hydroagarofuran (6): white amorphous powder; [α]21 + 92 (c 0.03,
D
1
1713, 1636, 1452, 1367, 1274, 1227, 1165, 776, 720 cm−1; H NMR
CHCl3); UV (MeOH) λmax (log ε) 231 (4.33) nm; ECD (MeOH)
λmax (Δε) 213 (3.07), 235 (22.88) nm; IR (KBr) νmax 1747, 1730,
(CDCl3) δ 1.77 (3H, s, AcO-1), 2.17 (3H, s, AcO-15), BzO-2 and
CinO-9 [8.13 (d, 2H, J = 7.0 Hz), 7.69 (d, 1H, J = 16.0 Hz), 7.57 (m,
3H), 7.50 (t, 2H, J = 7.5 Hz), 7.39 (m, 3H), 6.39 (d, 1H, J = 16.0
Hz)], for other signals, see Table 1; 13C NMR (CDCl3) δ AcO-1 [20.9
(q), 170.0 (s)], AcO-15 [21.5 (q), 170.9 (s)], BzO-2 and CinO-9
[118.2 (d), 128.4 × 2 (d), 128.8 × 2 (d), 129.0 × 2 (d), 130.0 (s),
130.0 × 2 (d), 130.5 (d), 133.3 (d), 134.5 (s), 145.6 (d), 166.2 (s),
166.3 (s)], for other signals, see Table 3; HRTOFMS m/z 627.2579
[M + Na]+ (calcd for C35H40O9Na, 627.2570).
(1S,2S,4R,5S,7R,9S,10S)-1,15-Diacetoxy-2,9-dicinnamoyloxy-β-di-
hydroagarofuran (2): white amorphous powder; [α]22D + 150 (c 0.1,
CHCl3); UV (MeOH) λmax (log ε) 218 (4.02), 279 (4.13) nm; ECD
(MeOH) λmax (Δε) 260 (−4.32), 291 (29.74) nm; IR (KBr) νmax
1742, 1712, 1639, 1450, 1367, 1261, 1226, 1162, 1013, 769, 715 cm−1;
1H NMR (CDCl3) δ 1.80 (3H, s, AcO-1), 2.18 (3H, s, AcO-15),
1718, 1456, 1367, 1280, 1230, 1111, 1096, 1009, 709 cm−1; H NMR
1
(CDCl3) δ 1.59 (3H, s, AcO-1), 2.13 (3H, s, AcO-6), BzO-2 and BzO-
9 [8.06 (dd, 2H, J = 7.0, 1.3 Hz), 7.98 (dd, 2H, J = 7.0, 1.3 Hz), 7.57
(t, 2H, J = 7.5 Hz), 7.45 (t, 4H, J = 7.0 Hz)], for other signals, see
Table 1; 13C NMR (CDCl3) δ AcO-1 [20.5 (q), 169.7 (s)], AcO-6
[21.4 (q), 170.2 (s)], BzO-2 and BzO-9 [128.3 × 2 (d), 128.5 × 2 (d),
129.4 (d), 129.5 × 2 (d), 130.1 × 2 (d), 130.3 (s), 132.9 (d), 133.3
(d), 165.5 (s), 166.1 (s)], for other signals, see Table 3; HRTOFMS
m/z 601.2421 [M + Na]+ (calcd for C33H38O9Na, 601.2414).
1β-Acetoxy-2β-hexanoyloxy-9α-cinnamoyloxy-β-dihydroagaro-
furan (7): white amorphous powder; [α]21 + 72 (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 218 (4.13), 223 (4.13), 277 (4.22) nm; IR
(KBr) νmax 2925, 1747, 1718, 1641, 1456, 1362, 1233, 1133, 1013, 878,
770, 713 cm−1; H NMR (CDCl3) δ 7.66 (d, 1H, J = 16.0 Hz), 7.53
1
(m, 2H), 7.37 (m, 3H), 6.38 (d, 1H, J = 16.0 Hz), 5.55 (m, 2H), 4.75
(d, 1H, J = 6.5 Hz), 2.42 (ddd, 1H, J = 15.0, 6.5, 3.3 Hz), 2.28 (t, 2H, J
= 7.4 Hz), 2.17 (m, 1H), 2.00−2.10 (m, 4H), 1.90 (t, 1H, J = 7.8 Hz),
1.80 (s, 3H), 1.73 (d, 1H, J = 15.4 Hz), 1.37 (s, 3H), 1.34 (s, 3H), 1.25
CinO-2 and CinO-9 [7.76 (d, 1H, J = 16.2 Hz), 7.69 (d, 1H, J = 16.0
Hz), 7.64 (m, 2H), 7.55 (m, 2H), 7.39 (m, 6H), 6.43 (d, 1H, J = 16.2
Hz), 6.39 (d, 1H, J = 16.2 Hz)], for other signals, see Table 1; 13C
NMR (CDCl3) δ AcO-1 [20.9 (q), 170.0 (s)], AcO-15 [21.6 (q),
170.8 (s)], CinO-2 and CinO-9 [118.2 × 2 (d), 128.4 × 4 (d), 129.0 ×
4 (d), 130.5 × 2 (d), 134.5 × 2 (s), 145.3 (d), 145.6 (d), 166.2 × 2
(s)], for other signals, see Table 3; HRTOFMS m/z 653.2739 [M +
Na]+ (calcd for C37H42O9Na, 653.2727).
1
(d, 3H, J = 7.0 Hz), 1.20 (s, 3H), 0.88 (t, 3H, J = 6.5 Hz); H NMR
(pyridine-d5) δ 1.97 (3H, s, AcO-1), HexO-2 [2.36 (t, 2H, J = 7.2 Hz),
2.11 (m, 2H), 1.66 (m, 2H), 1.27 (m, 2H), 0.85 (t, 3H, J = 7.1 Hz)],
CinO-9 [8.03 (d, 1H, J = 16.0 Hz), 7.56 (m, 2H), 7.32 (m, 3H), 6.78
(d, 1H, J = 16.0 Hz)], for other signals, see Table 1; 13C NMR
(pyridine-d5) δ AcO-1 [21.3 (q), 170.7 (s)], HexO-2 [14.5 (q), 23.0
(t), 25.4 (t), 32.0 (t), 35.2 (t), 173.4 (s)], CinO-9 [119.7 (d), 129.1 ×
2 (d), 129.7 × 2 (d), 131.1 (d), 136.3 (s), 145.6 (d), 166.6 (s)], for
(1S,2S,4R,5S,6R,7R,9S,10S)-1,6-Diacetoxy-2,9-dicinnamoyloxy-β-
dihydroagarofuran (3): white amorphous powder; [α]23 + 210 (c
D
0.1, CHCl3); UV (MeOH) λmax (log ε) 218 (3.91), 279 (4.03) nm;
ECD (MeOH) λmax (Δε) 260 (−9.36), 292 (38.52) nm; IR (KBr) νmax
I
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Journal of Natural Products
Article
other signals, see Table 3; HRTOFMS m/z 563.2996 [M + Na]+
(calcd for C32H44O7Na, 563.2985).
signals, see Table 2; 13C NMR (CDCl3) δ AcO-1 [20.8 (q), 170.1 (s)],
AcO-2 [21.5 (q), 170.3 (s)], AcO-15 [21.5 (q), 171.0 (s)], BzO-8 and
BzO-9 [128.5 × 2 (d), 128.7 × 2 (d), 129.8 × 2 (d), 129.9 × 2 (d),
130.3 × 2 (s), 133.4 × 2 (d), 165.1 (s), 166.2 (s)], for other signals,
see Table 3; HRESIMS m/z 659.2479 [M + Na]+ (calcd for
C35H40O11Na, 659.2468).
1α,6α-Diacetoxy-2β-butanoyloxy-9α-cinnamoyloxy-β-dihydroa-
garofuran (8): white amorphous powder; [α]21D + 96 (c 0.1, CHCl3);
UV (MeOH) λmax (log ε) 206 (4.01), 218 (4.03), 277 (4.21) nm; IR
(KBr) νmax 2925, 1744, 1709, 1637, 1388, 1367, 1234, 1165, 1014,
1
768,712 cm−1; H NMR (CDCl3) δ 7.66 (d, 1H, J = 16.0 Hz), 7.53
1β,2β-Diacetoxy-8β,9β-dibenzoyloxy-15-nicotinoyloxy-β-dihy-
droagarofuran (23): white amorphous powder; [α]22 −164 (c 0.1,
(m, 2H), 7.38 (m, 3H), 6.38 (d, 1H, J = 16.0 Hz), 5.59 (m, 2H), 5.38
(s, 1H), 4.74 (d, 1H, J = 6.5 Hz), 2.41 (ddd, 1H, J = 15.0, 6.3, 3.0 Hz),
2.32 (t, 1H, J = 7.0 Hz), 2.26 (t, 2H, J = 7.4 Hz), 2.22 (m, 1H), 2.12
(s, 3H), 1.79 (s, 3H), 1.76 (m, 1H), 1.44 (s, 3H), 1.40 (s, 3H), 1.39 (s,
D
MeOH); UV (MeOH) λmax (log ε) 227 (4.48), 264 (3.63) nm; IR
(KBr) νmax 1743, 1720, 1603, 1591, 1450, 1367, 1284, 1292, 1271,
1
1244, 1115, 1084, 1026, 854, 712 cm−1; H NMR (CDCl3) δ 9.14
1
(brs, 1H), 8.60 (brd, 1H, J = 4.8 Hz), 8.13 (brd, 1H, J = 8.0 Hz), 8.05
(d, 2H, J = 7.6 Hz), 7.65 (d, 2H, J = 7.6 Hz), 7.56 (t, 1H, J = 7.6 Hz),
7.43 (t, 2H, J = 7.6 Hz), 7.22 (t, 1H, J = 7.6 Hz), 7.07 (dd, 1H, J = 8.0,
4.8 Hz), 6.89 (t, 2H, J = 7.6 Hz), 5.80 (d, 1H, J = 5.8 Hz), 5.76 (d, 1H,
J = 3.1 Hz), 5.75 (dd, 1H, J = 5.8, 3.3 Hz), 5.58 (d, 1H, J = 12.1 Hz),
5.47 (dd, 1H, J = 6.1, 3.1 Hz), 5.17 (d, 1H, J = 12.1 Hz), 2.77 (d, 1H, J
= 13.2 Hz), 2.60 (brt, 1H, J = 3.7 Hz), 2.51 (m), 2.14 (dd, 1H, J =
13.2, 4.1 Hz), 2.03 (m), 1.82 (m),1.73 (3H, s, AcO-1), 1.72 (3H, s,
AcO-2), 1.42 (d, 1H, J = 8.0 Hz), 1.61 (s, 1H), 1.29 (s, 1H); 1H NMR
(pyridine-d5) δ 1.92 (AcO-1), 1.81 (AcO-2), BzO-8 and BzO-9 [8.34
(d, 2H, J = 7.6 Hz), 7.97 (d, 2H, J = 7.6 Hz), 7.56 (t, 1H, J = 7.6 Hz),
7.48 (t, 2H, J = 7.6 Hz), 7.23 (overlapped, 1H), 6.95 (t, 2H, J = 7.6
Hz)], NicO-15 [9.54 (brs, 1H), 8.77 (brd, 1H, J = 4.8 Hz), 8.39 (brd,
1H, J = 8.0 Hz), 7.23 (overlapped, 1H)], for other signals, see Table 2;
13C NMR (CDCl3) δ AcO-1 [21.5 (q), 170.6 (s)], AcO-2 [21.2 (q),
170.2 (s)], BzO-8 and BzO-9 [128.2 × 2 (d), 128.7 × 2 (d), 129.7 × 2
(d), 129.8 × 2 (d), 129.2 (s), 129.7 (s), 133.2 (d), 133.5 (d), 165.0
(s), 166.0 (s)], NicO-15 [123.0 (d), 126.0 (s), 137.0 (d), 151.2 (d),
153.6 (d), 165.6 (s)]; 13C NMR (pyridine-d5) δ AcO-1 [21.5 (q),
171.2 (s)], AcO-2 [21.5 (q), 170.5 (s)], BzO-8 and BzO-9 [128.9 × 2
(d), 129.4 × 2 (d), 130.2 × 2 (d), 130.6 × 2 (d), 130.1 (s), 130.7 (s),
133.8 (d), 134.2 (d), 165.9 (s), 166.5 (s)], NicO-15 [123.5 (d), 126.8
(s), 137.5 (d), 151.9 (d), 154.4 (d), 166.2 (s)], for other signals, see
Table 3 ; HRESIMS m/z 722.2588 [M + Na]+ (calcd for
C39H41NO11Na, 722.2577)
3H), 1.20 (d, 3H, J = 7.0 Hz), 0.94 (t, 3H, J = 6.5 Hz); H NMR
(pyridine-d5) δ 1.94 (3H, s, AcO-1), 2.18 (3H, s, AcO-6), ButO-2
[2.31 (m, 2H), 1.65 (m, 2H), 0.92 (t, 3H, J = 7.0 Hz)], CinO-9 [8.03
(d, 1H, J = 16.0 Hz), 7.61 (m, 2H), 7.34 (m, 3H), 6.76 (d, 1H, J = 16.0
Hz)], for other signals, see Table 1; 13C NMR (pyridine-d5) δ AcO-1
[21.3 (q), 170.6 (s)], AcO-6 [21.7 (q), 170.5 (s)], ButO-2 [14.2 (q),
19.2 (t), 37.1 (t), 173.2 (s)], CinO-9 [119.3 (d), 129.1 × 2 (d), 129.8
× 2 (d), 131.2 (d), 135.3 (s), 146.0 (d), 166.3 (s)], for other signals,
see Table 3; HRTOFMS m/z 593.2748 [M + Na]+ (calcd for
C32H42O9Na, 593.2727).
1α,15-Diacetoxy-2β-butanoyloxy-9α-cinnamoyloxy-β-dihydroa-
garofuran (9): white amorphous powder; [α]22 + 100 (c 0.1,
D
CHCl3); UV (MeOH) λmax (log ε) 206 (4.40), 218 (4.40), 279 (4.64)
nm; IR (KBr) νmax 1746, 1719, 1643, 1452, 1365, 1223, 1162, 1134,
1
1012, 879, 767, 710 cm−1; H NMR (CDCl3) δ 1.78 (3H, s, AcO-1),
2.16 (3H, s, AcO-15), ButO-2 [2.33 (m, 2H), 1.69 (m, 2H), 0.96 (t,
3H, J = 7.0 Hz)], CinO-9 [7.67 (d, 1H, J = 16.0 Hz), 7.54 (m, 2H),
7.38 (m, 3H), 6.36 (d, 1H, J = 16.0 Hz)], for other signals, see Table
1; 13C NMR (CDCl3) δ AcO-1 [20.9 (q), 169.8 (s)], AcO-15 [21.6
(q), 170.8 (s)], ButO-2 [13.8 (q), 18.5 (t), 36.8 (t), 172.9 (s)], CinO-
9 [118.2 (d), 128.4 × 2 (d), 129.0 × 2 (d), 130.5 (d), 134.5 (s), 145.5
(d), 166.2 (s)], for other signals, see Table 3; HRTOFMS m/z
593.2734 [M + Na]+ (calcd for C32H42O9Na, 593.2727).
1β,2β,6α,15-Tetraacetoxy-9α-cinnamoyloxy-β-dihydroagarofur-
an (10): white amorphous powder; [α]22 + 77 (c 0.1, CHCl3); UV
D
(MeOH) λmax (log ε) 207 (4.03), 218 (4.05), 279 (4.23) nm; IR
(KBr) νmax 1745, 1710, 1637, 1368, 1237, 1158, 1091, 878, 768 cm−1;
1H NMR (CDCl3) δ 1.78 (3H, s, AcO-1), 2.10 (3H, s, AcO-2), 2.09
1β-Acetoxy-9β-benzoyloxy-8α-hexanoyloxy-15-nicotinoyloxy-β-
dihydroagarofuran (24): white amorphous powder; [α]22 + 36 (c
D
0.1, MeOH); UV (MeOH) λmax (log ε) 225 (3.78), 263 (3.12) nm; IR
(KBr) νmax 1726, 1589, 1452, 1369, 1282, 1269, 1228, 1109, 1025, 977,
714 cm−1; 1H NMR (CDCl3) δ 1.57 (3H, s, AcO-1), HexO-8 [2.12 (t,
2H, J = 7.6 Hz), 1.36 (m, 2H), 1.03 (m, 2H), 1.07 (m, 2H), 0.69 (t,
3H, J = 7.0 Hz)], BzO-9 [7.85 (d, 2H, J = 7.6 Hz), 7.48 (t, 1H, J = 7.6
Hz), 7.32 (t, 2H, J = 7.6 Hz)], NicO-15 [9.45 (brs, 1H), 8.86 (brs,
1H), 8.51 (brd, 1H, J = 7.8 Hz), 7.49 (dd, 1H, J = 7.8, 4.8 Hz)], for
other signals, see Table 2; 13C NMR (CDCl3) δ AcO-1 [21.3 (q),
170.2 (s)], HexO-8 [13.9 (q), 22.3 (t), 31.3 (t), 21.7 (t), 34.7 (t),
173.4 (s)], BzO-9 [128.7 × 2 (d), 129.6 × 2 (d), 129.9 (s), 133.2 (d),
165.7 (s)], NicO-15 [123.8 (d), 124.0 (s), 137.4 (d), 151.4 (d), 154.2
(d), 165.7 (s)], for other signals, see Table 3; HRESIMS m/z
636.3177 [M + H]+ (calcd for C36H46NO9, 636.3173).
(3H, s, AcO-6), 2.23 (3H, s, AcO-15), CinO-9 [7.69 (d, 1H, J = 16.0
Hz), 7.55 (dd, 2H, J = 6.8, 2.3 Hz), 7.39 (m, 3H), 6.36 (d, 1H, J = 16.0
Hz)], for other signals, see Table 1; 13C NMR (CDCl3) δ AcO-1 [20.9
(q), 169.8 (s)], AcO-2 [21.4 (q), 170.1 (s)], AcO-6 [21.5 (q), 170.2
(s)], AcO-15 [21.5 (q), 170.7 (s)], CinO-9 [117.7 (d), 128.4 × 2 (d),
129.1 × 2 (d), 130.7 (d), 134.4 (s), 146.0 (d), 165.9 (s)], for other
signals, see Table 3; HRTOFMS m/z 623.2458 [M + Na]+ (calcd for
C32H40O11Na, 623.2468).
(1R,2S,4R,5S,6R,7R,8R,9S,10S)-6,15-Diacetoxy-8,9-dibenzoyloxy-
1,2-dihydroxy-β-dihydroagarofuran (21): white amorphous powder;
[α]22D −39 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 228 (4.34), 273
(3.22) nm; ECD (MeOH) λmax (Δε) 222 (15.82), 240 (−19.22) nm;
IR (KBr) νmax 3514, 2929, 1726, 1603, 1452, 1371, 1284, 1263, 1234,
1101, 1041, 860, 712 cm−1; 1H NMR (CDCl3) δ 2.12 (3H, s, AcO-6),
1.89 (3H, s, AcO-15), BzO-8 and BzO-9 [8.04 (d, 2H, J = 7.2 Hz),
7.87 (d, 2H, J = 7.2 Hz), 7.56 (t, 1H, J = 7.2 Hz), 7.50 (t, 1H, J = 7.2
Hz), 7.44 (t, 2H, J = 7.2 Hz), 7.32 (t, 2H, J = 7.2 Hz)], for other
signals, see Table 2; 13C NMR (CDCl3) δ AcO-6 [21.5 (q), 170.0 (s)],
AcO-15 [21.5 (q), 170.3 (s)], BzO-8 and BzO-9 [128.6 × 2 (d), 128.7
× 2 (d), 129.9 × 2 (d), 130.1 × 2 (d), 130.1 × 2 (s), 133.5 × 2 (d),
166.0 (s), 166.3 (s)], for other signals, see Table 3; HRESIMS m/z
633.2322 [M + Na]+ (calcd for C33H38O11Na, 633.2312).
6α-Acetoxy-1β,9β-dibenzoyloxy-8β-nicotinoyloxy-β-dihydroa-
garofuran (25): white amorphous powder; [α]22 −67 (c 0.1,
D
MeOH); UV (MeOH) λmax (log ε) 226 (4.34), 263 (3.51) nm; IR
(KBr) νmax 1730, 1591, 1452, 1337, 1286, 1228, 1113, 1093, 1026, 872,
706 cm−1; 1H NMR (CDCl3) δ 2.13 (3H, s, AcO-6), BzO-1 [7.58 (d,
2H, J = 7.6 Hz), 7.27 (t, 1H, J = 7.6 Hz), 7.01 (t, 2H, J = 7.6 Hz)],
NicO-8 [9.24 (brs, 1H), 8.80 (brd, 1H, J = 4.8 Hz), 8.32 (brd, 1H, J =
7.8 Hz), 7.43 (dd, 1H, J = 7.8, 4.8 Hz)], BzO-9 [7.48 (d, 2H, J = 7.6
Hz), 7.15 (t, 1H, J = 7.6 Hz), 6.88 (t, 2H, J = 7.6 Hz)], for other
signals, see Table 2; 13C NMR (CDCl3) δ AcO-1 [21.5 (q), 170.3 (s)],
BzO-1 [128.1 × 2 (d), 129.4 × 2 (d), 129.9 (s), 132.7 (d), 165.7 (s)],
NicO-8 [123.7 (d), 124.9 (s), 137.4 (d), 151.2 (d), 153.8 (d), 164.4
(s)], BzO-9 [127.8 × 2 (d), 129.4 × 2 (d), 129.6 (s), 132.4 (d), 165.1
(s)], for other signals, see Table 3; HRESIMS m/z 642.2708 [M + H]+
(calcd for C37H40NO9, 642.2703).
(1R,2S,4R,5S,7R,8R,9S,10S)-1,2,15-Triacetoxy-8,9-dibenzoyloxy-β-
dihydroagarofuran (22): white amorphous powder; [α]22 −126 (c
D
0.2, MeOH); UV (MeOH) λmax (log ε) 229 (4.39), 274 (3.34) nm;
ECD (MeOH) λmax (Δε) 219 (4.17), 238 (−51.14) nm; IR (KBr) νmax
1739, 1603, 1452, 1369, 1284, 1255, 1234, 1115, 1088, 1026, 711
cm−1; 1H NMR (CDCl3) δ 1.53 (3H, s, AcO-1), 2.01 (3H, s, AcO-2),
1.89 (3H, s, AcO-15), BzO-8 and BzO-9 [8.00 (d, 2H, J = 7.6 Hz),
7.90 (d, 2H, J = 7.6 Hz), 7.58 (t, 1H, J = 7.6 Hz), 7.49 (t, 1H, J = 7.6
Hz), 7.45 (t, 2H, J = 7.6 Hz), 7.31 (t, 2H, J = 7.6 Hz)], for other
1β,8α,15-Triacetoxy-9α-benzoyloxy-β-dihydroagarofuran (26):
white amorphous powder; [α]22 −4 (c 0.1, MeOH); UV (MeOH)
D
λmax (log ε) 231 (4.05), 274 (2.95) nm; IR (KBr) νmax 1743, 1722,
1363, 1281, 1228, 1095, 1072, 1045, 887, 715 cm−1; 1H NMR
J
DOI: 10.1021/acs.jnatprod.5b00234
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(CDCl3) δ 1.62 (3H, s, AcO-1), 1.89 (3H, s, AcO-8), 2.25 (3H, s,
AcO-15), BzO-9 [8.09 (d, 2H, J = 7.2 Hz), 7.45 (t, 1H, J = 7.2 Hz),
7.57 (t, 2H, J = 7.2 Hz)], for other signals, see Table 2; 13C NMR
(CDCl3) δ AcO-1 [20.9 (q), 169.9 (s)], AcO-8 [21.1 (q), 170.2 (s)],
AcO-15 [21.5 (q), 170.3 (s)], BzO-9 [128.5 × 2 (d), 130.4 × 2 (d),
129.6 (s), 133.5 (d), 166.0 (s)], for other signals, see Table 3;
HRESIMS m/z 539.2244 [M + Na]+ (calcd for C28H36O9Na,
539.2257).
Statistical Analysis. The detailed operation is described in
Supporting Information.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00234.
Preparation of 67 and 68. An excess amount of NaH (10 mg)
was added to a solution of compound 59 (30 mg, 0.066 mmol) in
MeOH (25 mL), and the mixture was refluxed for 24 h. The reaction
mixture was cooled to room temperature, and the solution was
acidified to pH 7 with 3 N HCl. After evaporation of the solvent, the
residue was extracted with EtOAc, and the organic layer was washed
with H2O and brine and dried over anhydrous Na2SO4. The solvent
was evaporated, and the residue was purified by column chromatog-
raphy over silica gel (petroleum ether/acetone, 5:1) to give 67 (5 mg,
0.019 mmol) and 68 (3 mg, 0.0072 mmol). 67: white amorphous
UV, IR, MS, HRMS, 1H NMR, 13C NMR, 1H−1H
COSY, NOESY, HSQC, and HMBC spectra of all new
compounds described and the ECD spectra of
compounds 1−6, 21, and 22; 1H NMR spectra of
compounds 67−70; detailed operation of portions of the
biological assays, such as animals, behavioral test, MTT
assay, and statistical analysis (PDF)
Crystallographic data for compound 1 (CIF)
48
1
powder; H NMR (CDCl3) data are identical to reported data. 68:
white amorphous powder; 1H NMR (CDCl3) δ 5.46 (dd, 1H, J = 12.0,
4.0 Hz, H-1), 4.41 (1H, s, H-6), 4.96 (d, 1H, J = 7.0 Hz, H-9), 1.51
(3H, s, H-12); 1.39 (3H, s, H-13); 1.30 (3H, s, H-15); 1.60 (3H, s,
AcO-9), BzO-1 [8.08 (d, 2H, J = 7.2 Hz), 7.45 (t, 1H, J = 7.2 Hz), 7.57
(t, 2H, J = 7.2 Hz)].
AUTHOR INFORMATION
Corresponding Authors
*Tel/Fax: 86-21-50806052. E-mail: wmzhao@simm.ac.cn
(W.Z.).
■
*Tel/Fax: 86-21-50806710. E-mail: hzhang@simm.ac.cn
Preparation of 69 and 70. Compounds 69 (3 mg, 0.0068 mmol)
and 70 (5 mg, 0.020 mmol) were prepared from 15 (30 mg, 0.062
mmol) and 58 (30 mg, 0.070 mmol), respectively, using the same
chemical transformation method described above. 68 and 69: white
(H.Z.).
Author Contributions
§These authors contributed equally.
1
amorphous powder; H NMR (CDCl3) data are identical to reported
data.49
Notes
The authors declare no competing financial interest.
X-ray Crystallographic Analysis of 1. All data were collected
using a Bruker APEX-II CCD diffractometer with graphite-
monochromated Cu Kα radiation (1.54178 Å). The structure was
solved by direct methods using SHELXL-97 and was refined using full-
matrix least-squares calculation on F2 using SHELXL-97. The
hydrogen atom positions were geometrically idealized and were
allowed to ride on their parent atoms. Non-hydrogen atoms were
refined anisotropically. Crystallographic data for 1 has been deposited
at the Cambridge Crystallographic Data Centre under the deposition
number CCDC 1401060. Copies of these data can be obtained free of
charge via the Internet at www.ccdc.cam.ac.uk/conts/retrieving.html
or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. [Tel:
(+44) 1223−336−408. Fax: (+44) 1223-336-033. E-mail: deposit@
ccdc.cam.ac.uk].
Crystal Data of 1: C35H40O9, Mr = 604.67, orthorhombic, P212121,
a = 9.1983 (2) Å, b = 18.6272 (4) Å, c = 18.7098 (4) Å, V = 3205.71
(12) Å3, Z = 4, Dcalc = 1.253 mg/m3. Crystal dimensions: 0.2 × 0.1 ×
0.05 mm3, μ = 0.74 mm−1, F(000) = 1288, T = 296 K. Independent
reflections: 5599 (Rint = 0.0063). The final R1 values were 0.035, wR2 =
0.087 [I > 2σ(I)]. Flack parameter: 0.14 (14).
Biological Assay. Animals. Imprinting control region (ICR) mice
(18−20 g) were obtained from Shanghai Laboratory Animal Center,
Chinese Academy of Sciences. The detailed operation is described in
the Supporting Information.
Behavioral Test. Animal behavior was determined by a passageway
water maze, as described above.50 The detailed operation is described
in the Supporting Information.
Cell Culture and Compound Treatment. Human neuroblastoma
SH-SY5Y cells were purchased from ATCC and were maintained in
MEM/F12 medium supplemented with 10% fetal bovine serum, 100
U/mL penicillin and 100 U/mL streptomycin in a humid atmosphere
of 5% CO2 at 37 °C. Cells were pretreated with each compound (1
and 10 μM) and epigallocatechin gallate (EGCG, 10 μM) for 2 h,
followed by exposure to 10 μM of Aβ25−35 (Sigma) in the presence of
compounds for another 24 h.
MTT Assay. Cell viability was determined using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sangon
Biotech) reduction assay, as previously described.22b The detailed
operation is described in Supporting Information.
ACKNOWLEDGMENTS
■
This study was supported by the National Natural Science
Foundation of China (81473113) and the State Key Laboratory
of Drug Research (SIMM1403KF-04). We are grateful to Prof.
Zhixin Ju of Jilin Agricultural University for collection and
identification of the plant material of C. flagellaris.
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DOI: 10.1021/acs.jnatprod.5b00234
J. Nat. Prod. XXXX, XXX, XXX−XXX