Isolation, Structure Elucidation, Semi-Synthesis, and Structural Modification of C19-Diterpenoid Alkaloids fromAconitum apetalumand Their Neuroprotective Activities

Article abstract of DOI:10.1021/acs.jnatprod.0c01111

Five new aconitine-type C₁₉-diterpenoid alkaloids, apetalrines A-E (1-5), were isolated fromAconitum apetalum. Their structures were determined by analysis of 1D and 2D NMR, IR, and HRESIMS data. Semisynthesis of apetalrine B (2) from its parent compound aconorine was achieved to confirm the structure proposed. Twenty derivatives of2(11a-11l12a12b12d12e12j12k12m12n) were synthesized via a unified approach relying on simple coupling reactions. The evaluation of neuroprotective effects of compounds (1-511b11c11f-11i12a12b12d12e12k12m12n) with low cytotoxicity revealed compound2to exhibit good neuroprotective effects in H₂O₂-treated SH-SY5Y cells at a concentration of 50 μM. A series of studies using flow cytometry, staining, and Western blotting on2indicated that its neuroprotective effects may arise from inhibiting cell apoptosis.

Full text of DOI:10.1021/acs.jnatprod.0c01111

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Isolation, Structure Elucidation, Semi-Synthesis, and Structural
Modification of C19-Diterpenoid Alkaloids from Aconitum apetalum
and Their Neuroprotective Activities
Lin-Xi Wan, Ji-Fa Zhang, Yong-Qi Zhen, Lan Zhang, Xiaohuan Li, Feng Gao,* and Xian-Li Zhou*
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ABSTRACT: Five new aconitine-type C19-diterpenoid alkaloids,
apetalrines A−E (1−5), were isolated from Aconitum apetalum.
Their structures were determined by analysis of 1D and 2D NMR,
IR, and HRESIMS data. Semisynthesis of apetalrine B (2) from its
parent compound aconorine was achieved to confirm the structure
proposed. Twenty derivatives of 2 (11a−11l, 12a, 12b, 12d, 12e,
12j, 12k, 12m, 12n) were synthesized via a unified approach relying
on simple coupling reactions. The evaluation of neuroprotective
effects of compounds (1−5, 11b, 11c, 11f−11i, 12a, 12b, 12d, 12e,
12k, 12m, 12n) with low cytotoxicity revealed compound 2 to
exhibit good neuroprotective effects in H2O2-treated SH-SY5Y cells
at a concentration of 50 μM. A series of studies using flow cytometry, staining, and Western blotting on 2 indicated that its
neuroprotective effects may arise from inhibiting cell apoptosis.
MLA must be considered. Many efforts have been made to
n recent years, natural products have become an important
Isource of precursors or drugs for the treatment of decrease the toxicity of MLA, while maintaining its neuro-
protective activity.7
Alzheimer’s disease (AD). The approved acetylcholinesterase
As part of an ongoing interest in the chemistry of
diterpenoid alkaloids, in preliminary work an extract of
Aconitum apaletum (Huth) B. Fedtsch. (Ranunculaceae)
showed a promising neuroprotective effect.8 Herein, we report
the isolation of five new aconitine-type C19-diterpenoid
alkaloids apetalrines A−E (1−5) from Aconitum apetalum,
the semisynthesis of apetalrine B (2), and the synthesis of 20
new derivatives of the apetalrines, taking advantage of simple
esterification. The neuroprotective activities of several of the
new compounds obtained were evaluated against H2O2-
induced damage in human neuroblastoma SH-SY5Y cells,
and the mechanism of the neuroprotection of the most potent
compound, apetalrine B (2), was investigated.
inhibitor, galanthamine, which modulates nicotinic receptors in
the brain, belongs to the Amaryllidaceae alkaloid family.1
Huperzine A, a sesquiterpene alkaloid group found in Huperzia
serrata, is currently marketed in the People’s Republic of China
for the indications of AD and is recommended as a standard
therapy for memory disorders such as dementia.2 The
antidementia drug candidate, (−)-Clausenamide, originally
isolated from Clausena lansium, exhibits multitarget actions of
improving cognition and anti-AD pathogenesis and has entered
Phase II clinical trials in China.3 These successfully developed
phytochemicals inspired our group to find anti-AD lead
compounds from plant-derived natural products.
Diterpenoid alkaloids, as the predominant components from
the plant genera Aconitum and Delphinium, have been found to
exhibit a wide spectrum of pharmacological properties.4 In
addition to analgesic, anti-inflammatory, and cardiotonic
effects, some diterpenoid alkaloids exhibit neuroprotection.
Talatisamine has been identified as a specific potassium ion
(K+) channel blocker based on virtual screening and
electrophysiological characterization, thus representing a
potential lead compound for neuroprotection.5 Also, methyl-
lycaconitine (MLA) is an active nonprotein competitive
neuronal nicotinic acetylcholine receptor (nAChR) antagonist,
which may improve the symptoms of AD by regulating
neurons to affect cognitive, learning, memory, and other brain
functions.6 However, as a diterpenoid alkaloid, the toxicity of
RESULTS AND DISCUSSION
■
Isolation and Structure Elucidation of Apetalrines A−
E (1−5). The aerial parts of Aconitum apetalum were extracted
as described in the Experimental Section to give four major
Received: October 14, 2020
Published: March 5, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
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fractions, A−D. Chromatographic separation of each fraction
on silica gel columns yielded five new C19-diterpenoid alkaloids
designated as apetalrines A−E (1−5, Figure 1). These were all
obtained as amorphous powders, and their structures were
elucidated using a combination of spectroscopic methods (IR,
NMR, and HRESIMS).
C-7′ (δC 168.6), and CH3-9″/10″ (δH1.57, overlapped each
other) and both C-7″ (δC175.9) and C-8″ (δC73.7), indicating
the presence of a fragment of vaginatunine A.10 The fragment
of vaginatunine A was located at C-18 via an ester group on the
basis of the HMBC correlation between H-18 and the ester
carbonyl of anthranoyl group (Figure 2). The relative
1
Figure 2. Key H−1H COSY, HMBC, and NOESY of compound 1.
configuration of compound 1 was deduced from the NOESY
and 1H−1H COSY connections and vicinal coupling constants.
A H-13 and H-16 coupling correlation was not observed in the
1H−1H COSY spectrum of 1, giving evidence that ring D is in
a boat conformation, and consequently, OCH3-16 is in a β-
position,11 which was confirmed by a NOESY cross-peak
between H-16 and H-17. The cross-peak between H-5 and H-
10 indicated that H-10 is β-oriented. Both H-1 and H-14 were
assigned as β-oriented based on the characteristic coupling
constants of H-1 (δH 3.16, 1H, dd, J = 10.8, 6.6 Hz) and H-14
(δH 4.82, 1H, t, J = 4.8 Hz).12 These observations led to the
assignment of the structure of apetalrine A as 1 (Figure 2).
Apetalrines B−E (2−5) were obtained as amorphous
Figure 1. Structures of compounds 1−5.
Compound 1 was obtained as amorphous powder. Its
molecular formula was deduced as C43H55N3O10 on the basis
of a protonated molecular ion peak at m/z 774.3962 [M + H]+
in the HRESIMS (calcd 774.3966). The IR spectrum displayed
absorption bands characterized as hydroxy (3362 cm−1),
phenyl (1581 and 1519 cm−1), and ester carbonyl groups
1
(1689 and 1665 cm−1). The H and 13C NMR spectra of
1
apetalrine A (1) exhibited signals at δH 1.08 (3H, t, J = 7.2 Hz)
and δC 49.4 (t) and 13.7 (q), for an N-ethyl group, δH 2.04
(3H, s) and δC170.9 (s) and 21.5 (q), for an acetyl group, δH
3.22, and 3.28 (each 3H, s) and δC 56.3 (q) and 56.5 (q), for
two aliphatic methoxy groups, δH 12.04 and 11.94, each 1H, s,
for protons of two monosubstituted acylamino groups, and two
ortho-substituted phenyl groups [8.72 (1H, d, J = 7.8 Hz), 8.83
(1H, d, J = 7.8 Hz), 8.03 (1H, dd, J = 7.8, 1.2 Hz), 7.86 (1H, d,
J = 7.8 Hz), 7.62 (1H, dt, J = 7.8, 1.2 Hz), 7.54 (1H, t, J = 7.8
Hz), 7.22 (1H, t, J = 7.8 Hz), 7.17 (1H, d, J = 7.8 Hz)]. Along
with the above-mentioned signals, its 13C NMR spectrum
displayed six oxygenated carbon signals (δC 85.4 d, 73.7 s, 77.0
d, 81.8 d, 71.2 t, 73.7 s), suggesting that compound 1 possesses
two hydroxy groups in addition to one acetyl group, two
methoxy groups, and one anthranoyl ester group. Moreover,
the signals at δH 1.57 (6H, s) and δC 28.1 q, 28.1 q, and 73.7 s
indicated the occurrence of a 2-hydroxy-2-methylpropanoyl
group. All the above evidence along with careful inspection of
powders and exhibited nearly identical H and 13C NMR
resonances to those of 1 (Tables 1 and 2). Their molecular
formulas, C41H53N3O9, C45H57N3O11, C43H57N3O9, and
C44H57N3O10, respectively, were inferred from their HRESIMS
and 13C NMR spectra. The most obvious difference between
compounds 1 and 2 in their NMR spectra was the absence of
an acetyl group in 2, thus validating the loss of 42 mass units in
2 as found by mass spectrometry. The proton signal of H-14
was shifted upfield from δH 4.82 in 1 to δH 4.05 in 2,
suggesting that the acetyl group at H-14 in 1 was replaced by
hydroxy group in 2. In contrast, the spectroscopic data of
compound 3 revealed it to have an additional acetyl group
when compared with that of 1. This additional acetyl group
was assigned to C-8 from the evidence that the carbon signal of
C-8 at δC 73.7 in 1 was shifted downfield to δC 86.0. On
comparing the HRESIMS and NMR spectra of 2 and 4, an
additional ethyl group was found in 4. The carbon signal of C-
8 in 4 was deshielded from δC 72.9 in 2 to δC 78.1 along with
the long-range correlations between protons of an ethoxy
group at δH 3.36/3.38 and C-8 at δC 73.7, suggesting that the
the NMR data (1H, 13C, DEPT, HMQC, HMBC, H−1H
1
COSY, and NOESY) revealed apetalrine A (1) to be an
aconitine-type C19-diterpenoid alkaloid.9 The two methoxy
groups could be assigned at C-1 and C-16 due to the
correlations between OCH3-1 (δH 3.28) and C-1 (δC 85.4) and
between OCH3-16 (δH 3.22) and C-16 (δC 81.8), respectively,
in the HMBC spectrum. Similarly, the correlation between H-
14 (δH 4.82) and the carbonyl carbon (δC 170.9) in the
HMBC spectrum implied that the acetoxy group is located at
C-14. Furthermore, the HMBC spectrum showed correlations
of NH-2′ (δH 12.04) and C-8′ (δC167.7), H-6′ (δH 8.03) and
1
ethoxy group is located at C-8. Both the H and 13C NMR
signals of the skeleton of 5 were the same as those of 1. The
difference between 5 and 1 was in the side chain at C-18,
which had one more ethylene signal at δH 1.78 and 1.94 and δC
33.7 in the NMR spectra. The HMBC spectrum of 5 displayed
correlations between H-10″ (δH 1.78) and C-11″ (δC 33.7)
and between H-11″ (δH 0.96) and C-10″ (δC 8.1)/C-8″ (δC
76.2), indicating that a methyl group at C-8″ in 1 was replaced
by an ethyl group. Thus, the structures of apetalrines B−E (2−
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1
Table 1. H NMR Data of Compounds 1−5 (600 MHz, CDCl3, δ in ppm, J in Hz)
position
1
2
3
4
5
1
2a
3.16, dd (10.8, 6.6)
1.86, m
3.04, dd (10.8, 6.6)
1.93, m
3.14, dd (10.2, 6.6)
1.82, m
3.14, dd (10.8, 6.6)
2.06, m
3.16, dd (11.2, 6.6)
1.87, m
2b
3a
3b
2.33, t (5.4)
1.45, br t (12.6)
1.88, m
2.21, m
1.35, br t (12.6)
1.75, m
2.11, m
1.48, br t (12.6)
1.86, m
2.21, m
1.46, br t (12.6)
1.88, m
2.32, t (6.0)
1.49, br t (12.0)
1.89, m
5
6a
6b
7
1.70, d (7.2)
1.56, (overlapped)
2.04, (overlapped)
2.14, m
1.61, d (7.2)
1.43, dd (15.0, 7.8)
1.77, m
1.74, d (7.2)
1.46, dd (15.0, 7.8)
1.88, m
1.72, d (7.2)
1.41, dd (15.0, 7.8)
1.99, m
1.71, d (7.8)
1.56, (overlapped)
2.13, (overlapped)
2.15, m
2.19, m
3.21, m
2.43, m
9
1.83, m
1.66, m
1.73, m
1.86, m
1.72, m
10
2.62, t (6.6)
2.16, m
2.43, m
2.27, t (6.0)
2.06, m
2.45, m
2.35, t (6.6)
2.12, m
2.44, m
2.35, t (6.0)
2.08, m
2.41, m
2.65, t (6.0)
2.16, m
2.44, m
12a
12b
13
2.34, m
2.41, m
2.61, m
2.22, m
2.35, m
14
4.82, t (4.8)
1.90, m
2.42, m
3.20, m
3.05, s
4.05, (overlapped)
1.95, m
2.35, m
3.34, d (7.8)
3.13, s
4.79, t (4.8)
2.10, m
2.81, m
3.24, m
2.93, s
4.02, dd (5.4, 10.8)
2.06, m
2.42, m
3.35, m
3.02, s
4.83, t (4.8)
1.88, m
2.43, m
3.2, dd (9.6, 4.2)
3.06, s
15a
15b
16
17
18a
18b
19a
19b
21a
21b
22
OMe-1
OMe-16
Ac-8
Ac-14
OEt-8
4.01, ABq (10.8)
4.12, ABq (10.8)
2.15, ABq (11.4)
2.65, ABq (11.4)
2.42, m
2.54, m
1.08, t (7.2)
3.28, s
3.93, ABq (12.6)
4.02, ABq (12.6)
2.11, ABq (10.8)
2.54, ABq (10.8)
2.33, m
2.46, m
0.99, t (7.2)
3.25, s
4.02, ABq (10.8)
4.06, ABq (10.8)
2.12, m
2.60, m
2.36, m
2.56, m
1.09, t (7.2)
3.31, s
3.28, s
1.92, s
4.06, ABq(11.4)
4.08, ABq (11.4)
2.16, m
2.65, ABq (11.4)
2.28, m
2.54, m
1.08, t (7.2)
3.36, s
4.03, ABq (10.8)
4.13, ABq (10.8)
2.14, m
2.65, m
2.43, m
2.55, m
1.09, t (7.2)
3.29, s
3.22, s
3.18, s
3.28, s
3.23, s
2.04, s
2.02, s
2.05, s
3.36, m,
1.12, t (7.2)
8.84, d (8.4)
7.63, dt (7.8, 1.2)
7.19, t (7.8)
8.04, dt (7.8, 1.2)
12.05, s
7.87, d (7.8)
7.56, t (7.8)
7.23, t (7.8)
8.72, d (8.4)
11.94, s
3′
4′
5′
6′
NH-2′
3″
4″
5″
6″
8.83, d (7.8)
7.62, dt (7.8, 1.2)
7.17, t (7.2)
8.03, dd (7.8, 1.2)
12.04, s
7.86, d (7.2)
7.54, t (7.8)
7.22, t (7.8)
8.72, d (7.8)
11.94, s
8.74, d (8.4)
7.53, t (7.8)
7.08, t (7.8)
7.94, d (7.8)
11.97, s
7.78, d (7.8)
7.45, t (7.8)
7.14, t (7.8)
8.63, d (8.4)
11.85, s
8.84, d (8.4)
7.63, dt (7.8, 1.2)
7.19, dt (7.8, 1.2)
8.03, dd (7.8, 1.2)
12.03, s
7.87, dd (7.8, 1.2)
7.55, dt (7.8, 1.2)
7.23, dt (7.8, 1.2)
8.72, dd (7.8, 1.2)
11.93, s
8.84, d (8.4)
7.64, dt (7.8, 1.2)
7.19, dt (7.8, 1.2)
8.04, dd (7.8, 1.2)
12.06, s
7.88, dd (7.8, 1.2)
7.55, dt (7.2, 1.2)
7.24, dt (7.2, 1.2)
8.73, dd (8.4, 1.2)
11.89, s
NH-2″
9″
1.57, s
1.47, s
1.57, s
1.58, s
1.54, s
10″
1.57, s
1.47, s
1.57, s
1.58, s
1.78, dd (14.4, 7.2)
1.94, m
11″
0.96, t (7.2)
5) were determined as shown in Figure 1, which were
confirmed by the analysis of their 2D NMR data (Supporting
Information).
SY5Y cell damage with low cytotoxicity (Table 3 and Table S1,
Supporting Information), it was decided to conduct the
synthesis of 2 and its derivatives.
Synthesis of Apetalrine B (2) and Its Derivatives. The
observation of good neuroprotective activity and low
cytotoxicity for apetalrines A−E (1−5, Table 3) was
considered promising. This prompted a further investigation
of this series of C19-diterpenoid alkaloids, but only milligram
amounts of 1−5 were obtained by isolation. Accordingly, this
was not considered a sustainable source to provide starting
material for the synthesis of analogues and systematic
evaluation of their bioactivities. Because compound 2 showed
protective effects on H2O2-induced human neuroblastoma SH-
Apetalrine B (2) appeared synthetically approachable by
semisynthesis from the naturally enriched C19-diterpenoid
a l k a l o i d a c o n o r i n e a n d 2 - ( 2 - ( 2 - h y d r o x y - 2 -
methylpropanamido)benzamido)-benzoic acid. As shown in
Scheme 1, the removal of the 2-acetamido benzoate ester at C-
18 of aconorine afforded compound 6, which has the same
skeleton as that of 2. Meanwhile, 2-(2-(2-hydroxy-2-
methylpropanamido)benzamido)-benzoic acid 7 was obtained
by deprotection of 8 using Na2CO3/MeOH, which resulted
from the coupling reaction of the known compound 913 with
1069
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Article
Table 2. 13C NMR Data of Compounds 1−5 (150 MHz,
CDCl3, δ in ppm)
Table 3. Neuroprotective Effects of Selected Compounds
against H2O2-Induced Neurotoxicity in SH-SY5Y Cells
position
1
2
3
4
5
neuroprotective
neuroprotective
a
a
compound
rate (%)
compound
rate (%)
1
2
3
4
5
6
7
8
85.4, CH
86.1, CH
85.1, CH
85.6, CH
85.5, CH
1
2
3
4
48.6 0.82
77.4 0.76
54.2 0.54
58.6 0.79
62.1 0.65
11.7 0.24
22.5 1.27
6.5 0.15
11h
11i
2.2 0.06
14.5 0.24
21.3 0.90
33.0 0.58
5.5 0.18
13.0 0.72
10.8 0.67
19.9 0.48
12.4 0.57
26.2, CH2 25.9, CH2 26.4, CH2 26.2, CH2 26.2, CH2
32.9, CH2 33.0, CH2 32.8, CH2 32.9, CH2 32.9, CH2
38.3, C
12a
12b
12d
12e
12k
12m
12n
38.5, C
46.3, CH
38.1, C
46.1, CH
38.7, C
46.4, CH
38.3, C
46.2, CH
46.2, CH
5
25.4, CH2 25.2, CH2 25.2, CH2 24.3, CH2 25.4, CH2
46.4, CH
73.7, C
45.0, CH
35.5, CH
49.0, C
28.6, CH2 27.8, CH2 28.8, CH2 28.9, CH2 28.6, CH2
45.5, CH
77.0, CH
41.0, CH2 38.5, CH2 37.7, CH2 34.8, CH2 41.1, CH2
81.8, CH
62.0, CH
71.2, CH2 71.2, CH2 71.1, CH2 71.4, CH2 71.2, CH2
52.9, CH2 53.0, CH2 52.6, CH2 52.8, CH2 52.9, CH2
49.4, CH2 49.6, CH2 49.3, CH2 49.4, CH2 49.5, CH2
13.7, CH3 13.8, CH3 13.6, CH3 13.7, CH3 13.7, CH3
56.5, CH3 56.7, CH3 56.7, CH3 56.5, CH3 56.5, CH3
11b
11c
11f
11g
47.1, CH
72.9, C
45.9, CH
37.7, CH
49.0, C
41.7, CH
86.0, C
45.0, CH
38.6, CH
49.0, C
41.0, CH
78.1, C
46.0, CH
38.2, CH
49.1, C
46.5, CH
73.7, C
45.1, CH
35.6, CH
49.0, C
9
15.6 1.01
10
11
12
13
14
15
16
17
18
19
21
22
OMe-1
a
Neuroprotective effects of compounds (50 μM) on H2O2-induced
(500 μM) neurotoxicity in SH-SY5Y cells. Neuroprotective rate % =
(ODtest group − ODmodel group)/(ODcontrol group − ODmodel group) × 100%.
The cell viability in the control was taken as 100%, and the average
value of cell viability under H2O2 exposure was 68.2 1.34% (n = 5).
46.2, CH
75.7, CH
42.5, CH
75.3, CH
45.6, CH
75.2, CH
45.6, CH
76.9, CH
82.3, CH
62.7, CH
82.9, CH
61.7, CH
82.6, CH
62.5, CH
81.8, CH
62.1, CH
to determine structure−activity relationship information. It
was found that compounds 1−5 showed little cytotoxicity
(Table S1, Supporting Information). Because the aconitine-
type C19-diterpenoid alkaloids usually exhibit toxicity to cells,14
it was speculated that the vaginatunine A fragment at C-18
position of apetalrines might affect this toxicity. Thus,
following the above synthetic route, 20 derivatives of
apetalrines were synthesized by condensation of the C19-
diterpenoid alkaloid 6 with different carboxylic acids that came
from the reaction of the known compound 9 with different acyl
chlorides, 10a−10m (Scheme 2). To confirm the effect of the
vaginatunine A fragment on toxicity, not only the monoesters
(11a−11l) were synthesized but also the C-18 and C-14
diesters (12a, 12b, 12d, 12e, 12j, 12k, 12m, 12n) were
obtained. A difference in the synthesis of monoesters and
diesters was the amount of the carboxylate acids. Adding 1.1
equiv of the carboxylate acids yielded only monoesters in 41−
46% yields, while 2.2 equiv gave the diesters in 62−69% yields.
Biological Activity of Apetalrines A−E (1−5) and the
Derivatives of the Apetalrines. After we screened the
cytotoxicity of all diterpenoid alkaloids mentioned above
(Table S1, Supporting Information), the low-cytotoxic
compounds 1−5, 11 (b, c, f−i) and 12 (a, b, d, e, k, m, n)
were examined for their potential neuroprotective effects
toward H2O2-induced human neuroblastoma SH-SY5Y cell
damage (Table 3), using an MTT method. The percentage of
surviving SH-SY5Y cells decreased to 68.2% after treatment
with H2O2 (500 μM) but increased to 87.5% following
treatment with 50 μM 2. The neuroprotective rate of 2 was
77.4% thus indicating good neuroprotective activity.
Because apetalrine B (2) exhibited demonstrable neuro-
protective effects, further studies were performed to investigate
whether the effect of 2 resulted from the inhibition of
apoptosis. The production of intracellular ROS was detected
by flow cytometry. The results showed that ROS production
was increased significantly in H2O2-treated cells, relative to the
control group. Compared with model cells, a reduced level of
intracellular ROS was observed in cells treated with apetalrine
B (2) (Figure 4A). In addition, H2O2 treatment induced
obvious cell death via Hoechst 33258 staining and flow
cytometry analysis with Annexin-V/PI, while apetalrine B (2)
markedly reversed the cellular apoptosis (Figure 4B,C). The
expression of apoptosis-related proteins such as PARP, Bcl-2,
Bax, and caspase-3 was also evaluated by Western blotting
OMe-16 56.3, CH3 56.5, CH3 56.4, CH3 56.5, CH3 56.4, CH3
1′
2′
3′
4′
5′
6′
7′
115.8, C
141.5, C
115.9, C
141.5, C
115.8, C
141.5, C
115.8, C
141.4, C
115.9, C
141.5, C
121.0, CH 121.0, CH 121.0, CH 121.1, CH 121.0, CH
135.0, CH 134.9, CH 135.0, CH 135.0, CH 135.0, CH
123.3, CH 123.3, CH 123.4, CH 123.4, CH 123.3, CH
130.8, CH 130.9, CH 130.8, CH 130.9, CH 130.9, CH
168.6, C
167.7, C
121.3, C
140.0, C
121.5, CH 121.5, CH 121.5, CH 121.5, CH 121.5, CH
133.2, CH 133.3, CH 133.3, CH 133.3, CH 133.3, CH
127.4, CH 127.4, CH 127.4, CH 127.4, CH 127.4, CH
123.6, CH 123.7, CH 123.6, CH 123.6, CH 123.7, CH
175.9, C
73.7, C
168.6, C
167.8, C
121.2, C
140.1, C
168.6, C
167.8, C
121.3, C
140.1, C
168.7, C
167.8, C
121.5, C
140.0, C
168.6, C
167.8, C
121.2, C
140.1, C
8′
1″
2″
3″
4″
5″
6″
7″
8″
9″
10″
11″
Ac-8
175.9, C
73.7, C
175.9, C
73.7, C
175.8, C
73.7, C
175.4, C
76.2, C
28.1, CH3 28.1, CH3 28.1, CH3 28.1, CH3 26.5, CH3
28.1, CH3 28.1, CH3 28.1, CH3 28.1, CH3 33.7, CH2
8.1, CH3
169.7, C
22.6, CH3
56.1, CH2
16.3, CH3
171.2, C
21.5, CH3
OEt-8
Ac-14
170.9, C
21.5, CH3
170.9, C
21.5, CH3
1-chloro-2-methyl-1-oxopropan-2-yl acetate 10 in 75% yield.
Then, the semisynthesis of apetalrine B (2) was achieved in
40% yield by crossed ester condensation of C-18-OH in C19-
diterpenoid alkaloid 6 (1.0 equiv) with carboxylic acid 7 (1.1
equiv) when reacted with EDCI and DMAP. The NMR
spectrum of the synthesized compound was consistent with
that of isolated 2 (Figure 3), which confirmed the absolute
structure of this new natural product.
Synthesis of derivatives of the apetalrines provided greater
amounts of these compounds to evaluate their bioactivities and
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Scheme 1. Semisynthesis of Apetalrine B (2)
1
Figure 3. Comparison of H NMR and 13C NMR spectra of the natural and synthetic apetalrine B (2).
instrument (600 MHz). Chemical shifts were recorded in parts per
million (ppm) relative to tetramethylsilane as the internal standard.
13C NMR data were collected on a Bruker AV 400 nuclear magnetic
resonance instrument (100 MHz) or a Bruker AV 600 nuclear
magnetic resonance instrument (150 MHz) with complete proton
decoupling. Chemical shifts are reported in parts per million (ppm)
with tetramethylsilane as the internal standard. HRESIMS were
determined using a Waters Acquity UPLC/Xevo G2-S QTof mass
spectrometer. Thin-layer chromatography silica gel GF254 plates and
silica gel G and H (200−400 mesh) for column chromatography were
purchased from Qingdao Ocean Chemical Plant (Qingdao, People’s
Republic of China). Unless otherwise specified, the reagents and
solvents used in this work were all commercially available analytical or
chemical grades and used directly without any purification. The TLC
analysis. As expected, apetalrine B (2) decreased the rate of
apoptosis in H2O2-treated SH-SY5Y cells (Figure 4D). Taken
together, the results suggested that apetalrine B (2) inhibited
ROS production in SH-SY5Y cells and thereby inhibited
H2O2-induced cell apoptosis.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
determined using a PerkinElmer polarimeter with a sodium lamp
operating at 598 nm and 20 °C. IR spectra were obtained using a
1
Thermo Fisher Nicolet 6700 spectrometer. H NMR spectra were
recorded on a Bruker AV 400 nuclear magnetic resonance instrument
(400 MHz) or a Bruker AV 600 nuclear magnetic resonance
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Scheme 2. Synthesis of Derivatives of Apetalrines
plates were visualized under a UV lamp at 254 nm or by spraying the
Dragendorff’s reagent or by iodine.
(600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3)
data, see Table 2; HRESIMS m/z 774.3962 [M + H]+ (calcd for
C43H56N3O10, 774.3966).
Apetalrine B (2). This compound is a white amorphous powder;
[α]20D −4.0 (c 0.25, CHCl3); IR (KBr) vmax 3408, 3305, 2924, 2852,
1688, 1697, 1666, 1581, 1519, 1264, 1093, 759 cm−1; 1H NMR (600
MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data,
see Table 2; HRESIMS m/z 732.3857 [M + H]+ (calcd for
C41H54N3O9, 732.3860).
Plant Material. Aconitum apetalum was collected from Yili,
Xinjiang, People’s Republic of China, in August 2014. The plant was
authenticated by Professor Qing-Er Yang of the Institute of Botany,
Chinese Academy of Sciences. A voucher specimen (C. Ren & L.
Wang 748) was deposited at the School of Life Science and
Engineering, Southwest Jiaotong University, Sichuan, People’s
Republic of China.
Extraction and Isolation. The powdered roots of Aconitum
apetalum (1.5 kg) were extracted with 95% EtOH at room
temperature five times (5 L 95% EtOH each time for 2 days each).
The combined EtOH extract was concentrated under reduced
pressure to give a crude extract (92 g). Then, the extract was
suspended in H2O (1 L). The pH was adjusted to 3.0 using 10% HCl
solution, and the suspension was extracted with light petroleum ether
(1 L × 3) and EtOAc (1 L × 3), successively. The acid aqueous
solution was basified with 10% aqueous NH4OH to pH 10 and then
extracted with CH2Cl2 (1 L × 3). Removal of the solvent under
reduced pressure afforded the total crude alkaloids (11 g), which were
chromatographed over a silica gel column, eluting with a CH2Cl2−
MeOH (50:1 to 3:1) gradient system, to give fractions A (2.1 g), B
(1.7 g), C (2.5 g), and D (2.4 g). Fraction A (2.1 g) was further
chromatographed on a silica gel column employing petroleum ether−
acetone (40:1 to 10:1) as eluent to afford compounds 3 (6 mg) and 4
(4 mg). Purification of fraction B by silica gel column chromatog-
raphy, eluting with petroleum ether−acetone (10:1 to 3:1), yielded
compound 5 (5 mg). Column chromatography of fraction C over a
silica gel column using CH2Cl2−MeOH (30:1 to 1:1) as eluent
afforded compound 1 (19 mg). Separation of fraction D by silica gel
column chromatography using petroleum ether−acetone (6:1 to 3:1)
as eluent provided compound 2 (9 mg).
Apetalrine C (3). This compound is a white amorphous powder;
D
1727, 1689, 1666, 1582, 1521, 1438, 1264, 1093, 759 cm−1; 1H NMR
(600 MHz, CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3)
data, see Table 2; HRESIMS m/z 816.4072 [M + H]+ (calcd for
C45H58N3O11, 816.4071).
[α]20 +5.2 (c 0.50, CHCl3); IR (KBr) vmax 3446, 3305, 2969, 2928,
Apetalrine D (4). This compound is a white amorphous powder;
[α]20 +4.8 (c 0.50, CHCl3); IR (KBr) vmax 3435, 3302, 2969, 2926,
D
1689, 1666, 1521, 1438, 1263, 1093, 758 cm−1; 1H NMR (600 MHz,
CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see
Table 2; HRESIMS m/z 760.4173 [M + H]+ (calcd for C43H58N3O9,
760.4173).
Apetalrine E (5). This compound is a white amorphous powder;
[α]20D +11.8 (c 0.50, CHCl3); IR (KBr) vmax 3446, 3300, 2924, 2854,
1741, 1689, 1581, 1439, 1263, 1093, 757 cm−1; 1H NMR (600 MHz,
CDCl3) data, see Table 1; 13C NMR (150 MHz, CDCl3) data, see
Table 2; HRESIMS m/z 788.4127 [M + H]+ (calcd for C44H58N3O10,
788.4122).
Compound 6. This was prepared using NaOH (4.22 g, 105.50
mmol) added to a solution of aconorine (10.00 g, 17.58 mmol) in
95% EtOH (50 mL). The reaction mixture was refluxed for 3 h,
following by removal of solvent under reduced pressure. The crude
mixture was subjected to silica gel column chromatography, eluting
with CH2Cl2−MeOH (25:1), to yield compound 6 as a white
Apetalrine A (1). This compound is a white amorphous powder;
[α]20D +11.6 (c 0.50, CHCl3); IR (KBr) vmax 3445, 3300, 2968, 2929,
1740, 1689, 1665, 1581, 1519, 1439, 1262, 1093, 758 cm−1; 1H NMR
1
amorphous powder (6.6 g, 92.2%): H NMR (400 MHz, CDCl3): δ
4.84 (1H, d, J = 4.0 Hz), 4.12 (1H, s), 3.64 (1H, s), 3.48−3.35 (2H,
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Figure 4. Apetalrine B (2) inhibited ROS production and protected H2O2-induced apoptosis in SH-SY5Y cells. (A) SH-SY5Y cells were pretreated
with 50 μM apetalrine B (2) for 1 h, followed by treatment with 500 μM H2O2 for 4 h, and then the ROS levels of SH-SY5Y cells were detected by
flow cytometry. Representative images and quantification of mean ROS FITC are shown. (B) SH-SY5Y cells were cotreated with H2O2 and
apetalrine B (2), as described above, and then stained with Hoechst 33258 and observed by fluorescence microscope. Scale bar: 50 μm. The
apoptotic cells are indicated by red arrows. (C) SH-SY5Y cells were cotreated with H2O2 and apetalrine B (2), as described above, and the
apoptosis ratios were determined by flow cytometry analysis of Annexin-V/PI double staining. Representative images and quantification of
apoptosis are shown. (D) Immunoblotting analysis of apoptosis-related proteins (PARP, Bcl-2, Bax, caspase-3). β-actin was used as a loading
control. Quantification of immunoblotting were shown. Data are expressed as means
SEM. All data were representative of at least three
independent experiments. ###p < 0.001, vs control group. **p < 0.01 and ***p < 0.001, vs H2O2-treated group.
m), 3.33 (3H, d, J = 2.0 Hz), 3.26 (3H, d, J = 1.6 Hz), 3.16 (1H, s),
3.12−3.02 (1H, m), 2.50 (2H, d, J = 10.4 Hz), 2.46−2.31 (3H, m),
2.31−2.18 (2H, m), 2.14−2.04 (2H, m), 2.05−1.90 (5H, m), 1.88−
1.79 (2H, m), 1.78−1.66 (3H, m), 1.63 (1H, d, J = 7.2 Hz), 1.49−
1.18 (2H, m), 1.11−1.00 (3H, m); 13C NMR (100 MHz, CDCl3): δ
86.5, 82.4, 75.8, 73.1, 69.0, 63.2, 56.7, 56.6, 53.2, 49.7, 49.0, 47.1,
46.1, 46.0, 45.8, 39.3, 38.5, 37.7, 32.4, 27.9, 26.0, 24.8, 13.9;
HRESIMS m/z 408.2763 [M + H]+ (calcd for C23H38NO5,
408.2750).
General Procedure for the Synthesis of Compounds 8 and 7a−
7m. Pyridine (3.00 mmol, 3.0 equiv) and acyl chloride (1.50 mmol,
1.5 equiv) were added to a solution of the known compound 9 (1.00
mmol, 1.0 equiv) in dry dichloromethane (5 mL) dropwise at 0 °C.
Then, the reaction solution was stirred at ambient temperature for 10
h. The solvent was removed under reduced pressure to obtain a crude
yellow mixture. Purification of the crude mixture by column
chromatography over silica gel (petroleum ether−EtOAc 10:1 to
4:1) gave compounds 8 and 7a−7m. The NMR and HRESIMS data
of compounds 8 and 7a−7m are listed in the Supporting Information.
Compound 7. A suspension of compound 8 (0.80 mmol, 1.0
equiv) and Na2CO3 (8.00 mmol, 10.0 equiv) in MeOH (5 mL) was
stirred at room temperature for 5 h. Then, the mixture was acidized to
pH 4−5 by 1 M HCl at 0 °C. After concentration under reduced
pressure, the crude mixture was loaded onto a silica gel column, which
was eluted with petroleum ether−EtOAc (3:1 to 1:1), and gave
compound 7 as a white solid (274 mg, 89.0%): 1H NMR (400 MHz,
DMSO-d6): δ 12.02 (1H, s), 11.63 (1H, s), 8.61 (1H, d, J = 8.4 Hz),
8.55 (1H, d, J = 8.0 Hz), 8.05 (1H, dd, J = 8.0, 1.6 Hz), 7.87 (1H, dd,
J = 8.0, 1.2 Hz), 7.70−7.65 (1H, m), 7.61−7.56 (1H, m), 7.28−7.22
(2H, m), 1.33 (6H, s); 13C NMR (100 MHz, DMSO-d6): δ 176.1,
169.8, 166.5, 140.4, 138.9, 134.1, 132.7, 131.3, 127.7, 123.5, 123.2,
122.1, 120.6, 120.4, 117.7, 72.6, 27.7, 27.7; HRESIMS m/z 343.1290
[M + H]+ (calcd for C18H19N2O5, 343.1294).
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General Procedure for the Synthesis of Apetalrine B (2) and
Derivatives of the Apetalrines. EDCI (0.60 mmol, 3.0 equiv) and
DMAP (0.10 mmol, 0.5 equiv) were added to a solution of compound
6 (0.20 mmol, 1.0 equiv) and different carboxylate acids 8 and 7a−
7m (0.22 mmol, 1.1 equiv for the synthesis of monoesters; 0.44
mmol, 2.2 equiv for the synthesis of diesters) in CH2Cl2 (5 mL). Each
reaction mixture was stirred at room temperature for 10 h, followed
by concentration under reduced pressure. Each crude residue was
separated by silica gel column chromatography, using CH2Cl2−
MeOH (40:1 to 20:1) or petroleum ether−EtOAc (3:1 to 1:1) as
eluent to yield the apetalrines (2, 11a−11l, 12a, 12b, 12d, 12e, 12j,
12k, 12m, 12n) in 40−69% yields.
= 7.1 Hz), 1.03 (3H, s), 1.02 (3H, s); 13C NMR (150 MHz, CDCl3):
δ 171.7, 168.6, 168.1, 141.5, 140.7, 134.9, 133.4, 131.0, 127.3, 123.4,
123.2, 121.7, 120.9, 120.5, 116.0, 86.1, 82.3, 75.7, 72.9, 71.3, 62.8,
56.7, 56.6, 52.9, 49.6, 49.0, 48.1, 47.1, 46.3, 46.2, 45.9, 38.5, 38.4,
37.7, 33.0, 29.9, 27.8, 26.3, 25.8, 25.2, 22.7, 13.8; HRESIMS m/z
730.4050 [M + H]+ (calcd for C42H56N3O8, 730.4067).
Compound 11f. This compound is a white amorphous powder;
yield 45%; 1H NMR (400 MHz, CDCl3): δ 12.05 (1H, s), 11.23 (1H,
s), 8.82 (1H, d, J = 8.4 Hz), 8.74 (1H, d, J = 8.3 Hz), 8.05 (1H, dd, J
= 8.0, 1.3 Hz), 7.86 (1H, d, J = 7.9 Hz), 7.64 (1H, dd, J = 11.5, 4.3
Hz), 7.52 (1H, d, J = 7.3 Hz), 7.19 (2H, td, J = 7.2, 3.1 Hz), 4.15
(1H, d, J = 4.5 Hz), 4.08 (2H, dd, J = 31.8, 8.5 Hz), 3.34 (6H, d, J =
2.6 Hz), 3.27 (6H, d, J = 3.8 Hz), 0.96 (3H, t, J = 7.4 Hz); 13C NMR
(100 MHz, CDCl3): δ 175.4, 168.7, 168.1, 141.6, 140.7, 135.0, 133.4,
131.0, 127.3, 123.4, 123.3, 121.8, 120.9, 120.6, 116.0, 86.3, 82.4, 75.7,
73.0, 70.0, 63.0, 56.7, 56.6, 53.0, 51.6, 49.7, 49.0, 47.1, 46.4, 46.2,
46.0, 38.5, 38.1, 37.7, 32.7, 32.0, 29.9, 27.9, 25.9, 25.7, 23.0, 22.8,
13.9, 12.3, 12.1; HRESIMS m/z 772.4518 [M + H]+ (calcd for
C45H62N3O8, 772.4537).
Compound 11g. This compound is a white amorphous powder;
yield 42%; 1H NMR (400 MHz, CDCl3): δ 12.07 (1H, s), 11.28 (1H,
s), 8.79 (1H, d, J = 8.1 Hz), 8.67 (1H, d, J = 8.1 Hz), 8.05 (1H, dd, J
= 8.0, 1.4 Hz), 7.91−7.83 (1H, m), 7.70−7.61 (1H, m), 7.54 (1H, dd,
J = 11.4, 4.0 Hz), 7.24−7.16 (2H, m), 4.15 (1H, d, J = 7.0 Hz), 4.13−
4.01 (2H, m), 3.34 (3H, d, J = 2.3 Hz), 3.27 (3H, d, J = 5.2 Hz), 1.08
(3H, t, J = 7.1 Hz); 13C NMR (100 MHz, CDCl3): δ 170.8, 168.7,
168.1, 141.4, 140.5, 135.0, 133.4, 131.0, 127.4, 123.5, 121.7, 121.0,
120.6, 116.1, 86.1, 82.4, 77.4, 75.7, 72.9, 71.3, 62.8, 56.7, 56.6, 53.0,
49.6, 49.1, 47.2, 46.3, 46.2, 45.9, 44.6, 38.5, 37.7, 35.3, 33.0, 31.6,
30.4, 28.2, 27.9, 25.9, 25.2, 13.9; HRESIMS m/z 750.3511 [M + H]+
(calcd for C41H53ClN3O8, 750.3521).
Compound 11h. This compound is a white amorphous powder;
yield 43%; 1H NMR (600 MHz, CDCl3): δ 12.09 (1H, s), 11.95 (1H,
s), 8.86 (1H, d, J = 8.5 Hz), 8.67 (1H, d, J = 8.3 Hz), 8.05 (1H, dd, J
= 8.0, 1.3 Hz), 7.89 (1H, d, J = 7.1 Hz), 7.72−7.69 (2H, m), 7.65
(1H, t, J = 7.2 Hz), 7.20 (1H, t, J = 7.3 Hz), 4.16−4.14 (1H, m),
4.14−4.02 (2H, m), 3.35 (3H, s), 3.28 (3H, s), 1.09 (3H, t, J = 7.1
Hz), 0.96 (3H, s); 13C NMR (150 MHz, CDCl3): δ 168.7, 167.9,
167.7, 141.5, 139.7, 135.1, 133.3, 129.1, 129.0, 127.4, 124.2, 123.5,
121.8, 121.1, 116.0, 86.2, 82.4, 75.7, 72.9, 68.4, 62.8, 56.8, 56.6, 56.3,
53.0, 49.7, 49.1, 47.2, 46.4, 46.2, 45.9, 38.9, 38.5, 37.7, 32.2, 27.9,
25.9, 24.0, 22.8, 14.0; HRESIMS m/z 736.3349 [M + H]+ (calcd for
C40H51ClN3O8, 736.3365).
Compound 11i. This compound is a white amorphous powder;
yield 45%; 1H NMR (600 MHz, CDCl3): δ 12.83 (1H, s), 12.23 (1H,
s), 8.84 (1H, d, J = 7.8 Hz), 8.69−8.65 (1H, m), 8.06 (1H, dd, J = 8.0,
1.5 Hz), 7.95 (1H, dd, J = 8.0, 1.3 Hz), 7.67−7.61 (2H, m), 7.37−
7.34 (1H, m), 7.23−7.20 (1H, m), 4.82 (1H, d, J = 4.2 Hz), 4.15 (2H,
dd, J = 10.3, 4.7 Hz), 3.35 (2H, s), 3.29 (3H, s), 1.09 (3H, t, J = 7.1
Hz); 13C NMR (150 MHz, CDCl3): δ 168.8, 167.6, 160.4, 141.3,
139.2, 135.2, 133.66, 131.0, 127.6, 125.2, 123.7, 121.7, 121.6, 121.2,
116.1, 93.5, 86.1, 82.4, 75.7, 72.9, 71.4, 63.3, 56.8, 56.7, 53.0, 49.7,
49.1, 47.2, 46.4, 46.2, 46.0, 38.6, 37.7, 33.1, 27.9, 25.9, 25.3, 13.9;
HRESIMS m/z 790.2420 [M + H]+ (calcd for C39H47Cl3N3O8,
790.2429).
Compound 11j. This compound is a white amorphous powder;
yield 44%; 1H NMR (600 MHz, CDCl3): δ 12.09 (1H, s), 11.82 (1H,
s), 8.82 (1H, d, J = 8.0 Hz), 8.70−8.67 (1H, m), 8.04 (1H, dd, J = 8.0,
1.5 Hz), 7.84 (1H, dd, J = 8.0, 1.2 Hz), 7.61−7.58 (1H, m), 7.53−
7.50 (1H, m), 7.20 (1H, dd, J = 11.1, 4.2 Hz), 7.18−7.15 (1H, m),
4.16−4.13 (1H, m), 4.07 (2H, dd, J = 49.2, 10.9 Hz), 3.33 (3H, s),
3.26 (3H, s), 2.19 (3H, s), 1.71 (6H, s), 1.07 (3H, t, J = 7.1 Hz); 13C
NMR (150 MHz, CDCl3): δ 172.2, 170.1, 168.7, 168.1, 141.7, 140.4,
134.9, 133.5, 131.0, 127.3, 123.6, 123.4, 121.7, 121.0, 120.7, 116.0,
86.2, 82.4, 81.7, 75.7, 72.9, 71.3, 62.9, 56.8, 56.6, 53.0, 49.7, 49.1,
47.2, 46.4, 46.2, 45.9, 38.6, 38.5, 37.7, 33.1, 27.9, 25.9, 25.3, 24.8,
24.7, 22.2, 13.9; HRESIMS m/z 774.3955 [M + H]+ (calcd for
C43H56N3O10, 774.3966).
Compound 2. The NMR data of synthesized compound 2 were
consistent with those of the isolated compound. The compared
spectra are shown in Figure 3.
Compound 11a. This compound is a white amorphous powder;
yield 47%; 1H NMR (600 MHz, CDCl3): δ 12.04 (1H, s), 11.19 (1H,
s), 8.79 (1H, d, J = 8.3 Hz), 8.71 (1H, d, J = 8.3 Hz), 8.05 (1H, dd, J
= 8.0, 1.4 Hz), 7.85 (1H, dd, J = 7.9, 0.9 Hz), 7.66−7.61 (1H, m),
7.52 (1H, t, J = 7.3 Hz), 7.19 (2H, t, J = 7.6 Hz), 4.14 (1H, d, J = 2.8
Hz), 4.07 (2H, dd, J = 52.3, 10.9 Hz), 3.34 (3H, s), 3.28 (3H, s), 1.08
(3H, t, J = 7.1 Hz), 1.01 (3H, t, J = 7.4 Hz); 13C NMR (150 MHz,
CDCl3): δ 172.2, 168.6, 168.1, 141.5, 140.7, 135.0, 133.4, 131.0,
127.3, 123.4, 123.3, 121.7, 120.9, 120.5, 116.1, 86.1, 82.3, 75.7, 72.9,
71.3, 62.8, 56.7, 56.6, 53.0, 49.6, 49.0, 47.1, 46.3, 46.2, 45.9, 40.7,
38.5, 38.5, 37.7, 33.0, 27.9, 25.9, 25.2, 19.2, 14.0, 13.9; HRESIMS m/z
716.3900 [M + H]+ (calcd for C41H54N3O8, 716.3911).
Compound 11b. This compound is a white amorphous powder;
yield 46%; 1H NMR (400 MHz, CDCl3): δ 12.04 (1H, s), 11.18 (1H,
s), 8.80 (1H, d, J = 8.4 Hz), 8.71 (1H, d, J = 8.4 Hz), 8.06 (1H, d, J =
8.0 Hz), 7.85 (1H, d, J = 7.9 Hz), 7.65 (1H, t, J = 7.9 Hz), 7.53 (1H,
dd, J = 10.0, 5.5 Hz), 7.20 (2H, t, J = 7.6 Hz), 4.14 (1H, s), 4.13−3.98
(2H, m), 0.94 (3H, d, J = 7.4 Hz), 0.88 (3H, t, J = 6.7 Hz); 13C NMR
(100 MHz, CDCl3): δ 172.4, 168.7, 168.2, 141.5, 140.7, 135.0, 133.4,
131.0, 127.3, 123.3, 121.8, 121.0, 120.5, 116.1, 114.3, 86.1, 82.4, 77.4,
75.7, 73.0, 62.8, 56.8, 56.6, 53.0, 49.7, 49.1, 47.2, 46.4, 46.2, 45.9,
38.6, 38.5, 32.1, 29.9, 29.6, 27.9, 27.8, 25.2, 22.9, 22.6, 14.3, 14.1;
HRESIMS m/z 730.4055 [M + H]+ (calcd for C42H56N3O8,
730.4067).
Compound 11c. This compound is a white amorphous powder;
yield 45%; 1H NMR (400 MHz, CDCl3): δ 12.04 (1H, s), 11.18 (1H,
s), 8.79 (1H, d, J = 8.4 Hz), 8.69 (1H, d, J = 8.3 Hz), 8.04 (1H, dd, J
= 8.0, 1.3 Hz), 7.84 (1H, d, J = 7.1 Hz), 7.68−7.60 (1H, m), 7.52
(1H, t, J = 7.9 Hz), 7.18 (2H, t, J = 7.6 Hz), 4.13 (1H, s), 4.12−3.95
(2H, m), 3.34 (3H, s), 3.27 (3H, s), 1.08 (3H, t, J = 7.1 Hz), 0.89
(3H, t, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3): δ 172.4, 168.6,
168.1, 141. 5, 140.7, 134.9, 133.3, 131.0, 127.3, 123.4, 123.2, 121.7,
120.9, 120.5, 116.0, 86.1, 82.3, 77.4, 75.6, 72.9, 71.3, 62.8, 56.7, 56.6,
52.9, 49.6, 49.0, 47.1, 46.3, 46.2, 45.9, 38.7, 38.5, 38.4, 37.7, 33.0,
31.6, 27.8, 25.4, 25.2, 22.6, 14.1, 13.8; HRESIMS m/z 744.4210 [M +
H]+ (calcd for C43H58N3O8, 744.4224).
Compound 11d. This compound is a white amorphous powder;
yield 41%; 1H NMR (400 MHz, CDCl3): δ 12.05 (1H, s), 11.28 (1H,
s), 8.80 (1H, d, J = 8.5 Hz), 8.71 (1H, d, J = 8.4 Hz), 8.04 (1H, dd, J
= 8.0, 1.2 Hz), 7.84 (1H, d, J = 7.9 Hz), 7.69−7.58 (1H, m), 7.51
(1H, dd, J = 11.6, 4.1 Hz), 7.18 (2H, t, J = 7.6 Hz), 4.13 (1H, d, J =
4.3 Hz), 4.07 (2H, dd, J = 32.6, 8.2 Hz), 3.34 (3H, s), 3.27 (3H, s),
1.29 (3H, s), 1.27 (3H, s), 1.08 (3H, t, J = 7.1 Hz); 13C NMR (100
MHz, CDCl3): δ 176.2, 168.6, 168.1, 141.5, 140.9, 134.9, 133.3,
131.0, 127.3, 123.4, 123.2, 121.7, 120.9, 120.5, 116.0, 86.1, 82.3, 77.4,
75.6, 72.9, 71.3, 62.8, 56.7, 56.6, 52.9, 49.6, 49.0, 47.1, 46.3, 46.2,
45.9, 38.5, 38.4, 37.7, 37.6, 33.0, 27.8, 25.8, 25.2, 19.8, 13.8;
HRESIMS m/z 716.3901 [M + H]+ (calcd for C41H54N3O8,
716.3911).
Compound 11e. This compound is a white amorphous powder;
yield 48%; 1H NMR (600 MHz, CDCl3): δ 12.05 (1H, s), 11.18 (1H,
s), 8.80 (1H, d, J = 8.0 Hz), 8.72 (1H, d, J = 7.9 Hz), 8.05 (1H, dd, J
= 8.0, 1.5 Hz), 7.85 (1H, dd, J = 8.0, 1.1 Hz), 7.66−7.63 (1H, m),
7.55−7.51 (1H, m), 7.21−7.18 (2H, m), 4.17−4.14 (1H, m), 4.08
(2H, dd, J = 52.0, 10.9 Hz), 3.35 (3H, s), 3.28 (3H, s), 1.09 (3H, t, J
Compound 11k. This compound is a white amorphous powder;
yield 41%; 1H NMR (600 MHz, CDCl3): δ 12.25 (1H, s), 12.14 (1H,
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s), 8.90 (1H, dd, J = 8.4, 0.9 Hz), 8.87 (1H, dd, J = 8.4, 0.7 Hz),
8.09−8.05 (3H, m), 7.93 (1H, dd, J = 8.0, 1.3 Hz), 7.68−7.64 (1H,
m), 7.63−7.59 (1H, m), 7.56−7.50 (3H, m), 7.28 (1H, d, J = 1.1 Hz),
7.22−7.19 (1H, m), 4.19−4.14 (1H, m), 4.09 (2H, dd, J = 48.9, 8.4
Hz), 3.35 (3H, s), 3.28 (3H, s), 1.09 (3H, t, J = 7.2 Hz); 13C NMR
(150 MHz, CDCl3): δ 168.7, 168.4, 165.9, 141.5, 141.0, 135.2, 135.0,
133.6, 132.0, 131.0, 129.0, 127.7, 127.5, 123.6, 123.5, 121.9, 121.1,
120.8, 116.1, 86.2, 82.4, 75.7, 72.9, 71.3, 62.9, 56.8, 56.6, 53.0, 49.7,
49.1, 47.2, 46.4, 46.2, 45.9, 38.5, 37.7, 33.1, 27.9, 25.9, 25.3, 13.9;
HRESIMS m/z 750.3740 [M + H]+ (calcd for C44H52N3O8,
750.3754).
Compound 11l. This compound is a white amorphous powder;
yield 46%; 1H NMR (600 MHz, CDCl3): δ 11.81 (1H, d, J = 9.4 Hz),
8.83 (1H, d, J = 8.4 Hz), 8.04−8.01 (1H, m), 7.71 (1H, dd, J = 8.0,
1.1 Hz), 7.61−7.58 (1H, m), 7.25−7.24 (1H, m), 7.14−7.11 (1H, m),
6.79−6.76 (1H, m), 6.72−6.70 (1H, m), 4.16−4.14 (1H, m), 4.12−
4.02 (2H, m), 3.35 (3H, s), 3.28 (3H, s), 1.09 (3H, t, J = 7.1 Hz); 13C
NMR (150 MHz, CDCl3): δ 168.7, 168.4, 150.0, 142.3, 134.8, 133.1,
130.9, 127.9, 122.6, 120.8, 117.7, 117.3, 115.9, 115.6, 86.2, 82.4, 75.7,
73.0, 71.1, 62.9, 56.8, 56.6, 53.0, 49.7, 49.1, 47.2, 46.4, 46.2, 45.9,
38.5, 37.7, 33.1, 27.9, 25.9, 25.2, 13.9; HRESIMS m/z 646.3469 [M +
H]+ (calcd for C37H48N3O7, 646.3487).
Compound 12a. This compound is a white amorphous powder;
yield 63%; 1H NMR (600 MHz, CDCl3): δ 12.01 (1H, s), 11.88 (1H,
s), 11.15 (2H, d, J = 3.0 Hz), 8.80 (1H, d, J = 8.3 Hz), 8.72 (1H, d, J
= 8.2 Hz), 8.69 (2H, d, J = 8.4 Hz), 8.08 (1H, dd, J = 8.0, 1.4 Hz),
8.05 (1H, dd, J = 8.0, 1.4 Hz), 7.83 (2H, d, J = 7.9 Hz), 7.66−7.63
(1H, m), 7.62−7.59 (1H, m), 7.51 (2H, t, J = 7.9 Hz), 7.21−7.15
(4H, m), 5.15 (1H, t, J = 4.9 Hz), 4.08 (2H, dd, J = 54.9, 11.0 Hz),
3.31 (3H, s), 3.20 (3H, s), 1.10 (3H, t, J = 7.1 Hz), 1.01 (6H, td, J =
7.3, 0.9 Hz); 13C NMR (150 MHz, CDCl3): δ 172.2, 172.2, 168.7,
168.6, 168.1, 168.0, 141.5, 141.0, 140.7, 140.6, 135.0, 134.6, 133.3,
133.2, 131.4, 130.9, 127.3, 127.2, 123.7, 123.4, 123.2, 123.1, 121.7,
121.7, 120.9, 120.9, 120.7, 120.5, 116.8, 116.0, 85.4, 81.6, 77.3, 73.8,
71.2, 62.2, 56.5, 56.3, 52.8, 49.5, 49.1, 46.1, 45.3, 45.2, 40.7, 40.7,
40.6, 38.3, 36.8, 32.9, 29.9, 28.4, 26.2, 25.4, 19.2, 19.2, 13.9, 13.9,
13.7; HRESIMS m/z 1024.5065 [M + H]+ (calcd for C59H70N5O11,
1024.5072).
Compound 12b. This compound is a white amorphous powder;
yield 62%; 1H NMR (600 MHz, CDCl3): δ 12.01 (1H, s), 11.88 (1H,
s), 11.15 (2H, d, J = 4.7 Hz), 8.80 (1H, d, J = 8.3 Hz), 8.73 (1H, d, J
= 8.0 Hz), 8.69 (2H, d, J = 8.4 Hz), 8.07 (2H, ddd, J = 16.7, 7.9, 1.5
Hz), 7.83 (2H, d, J = 8.0 Hz), 7.67−7.63 (1H, m), 7.61 (1H, d, J =
7.2 Hz), 7.54−7.49 (2H, m), 7.43 (1H, s), 7.19−7.16 (2H, m), 7.08
(1H, s), 5.15 (1H, t, J = 5.0 Hz), 4.15−4.04 (2H, m), 3.31 (3H, s),
3.20 (3H, s), 2.45−2.41 (3H, m), 1.11 (3H, t, J = 7.1 Hz), 0.95 (6H,
t, J = 7.4 Hz); 13C NMR (100 MHz, CDCl3): δ 172.4, 172.4, 168.7,
168.6, 168.1, 168.0, 141.5, 141.0, 140.7, 140.7, 140.6, 139.5, 135.0,
134.6, 133.4, 133.2, 131.4, 131.0, 127.3, 123.8, 123.4, 123.2, 123.1,
121.8, 121.0, 120.9, 120.8, 120.5, 116.0, 114.2, 85.2, 81.7, 77.4, 73.8,
71.2, 62.3, 56.5, 56.4, 49.5, 49.1, 47.3, 45.3, 45.2, 40.4, 38.5, 38.3,
34.1, 32.1, 31.7, 31.7, 31.1, 29.9, 29.5, 28.4, 27.8, 25.4, 22.9, 22.5,
14.3, 14.3, 14.0; HRESIMS m/z 1052.5369 [M + H]+ (calcd for
C61H74N5O11, 1052.5385).
Compound 12e. This compound is a white amorphous powder;
yield 62%; 1H NMR (600 MHz, CDCl3): δ 12.01 (1H, s), 11.88 (1H,
s), 11.14 (2H, d, J = 3.8 Hz), 8.80 (1H, d, J = 8.4 Hz), 8.73 (1H, d, J
= 8.5 Hz), 8.70 (2H, d, J = 8.4 Hz), 8.08 (1H, dd, J = 8.0, 1.5 Hz),
8.05 (1H, dd, J = 8.0, 1.6 Hz), 7.84 (2H, d, J = 8.0 Hz), 7.65 (1H, dd,
J = 11.5, 4.3 Hz), 7.61 (1H, dd, J = 11.4, 4.3 Hz), 7.53−7.50 (2H, m),
7.22−7.17 (4H, m), 5.15 (1H, t, J = 4.9 Hz), 4.09 (2H, dd, J = 53.6,
11.0 Hz), 3.31 (3H, s), 3.20 (3H, s), 1.10 (3H, t, J = 7.1 Hz), 1.02
(6H, s), 1.01 (6H, s); 13C NMR (150 MHz, CDCl3): δ 171.8, 171.7,
168.7, 168.6, 168.1, 168.0, 141.5, 141.0, 140.6, 140.6, 135.0, 134.6,
133.4, 133.2, 131.4, 130.9, 127.3, 127.2, 123.7, 123.4, 123.2, 123.1,
121.7, 121.7, 121.0, 120.9, 120.7, 120.5, 116.8, 116.0, 85.5, 81.7, 77.4,
73.8, 71.2, 62.2, 56.5, 56.4, 52.8, 49.5, 49.1, 48.0, 48.0, 47.3, 46.2,
45.3, 45.2, 40.6, 38.3, 36.8, 32.9, 29.9, 28.5, 26.3, 26.3, 26.2, 25.4,
22.7, 13.7; HRESIMS m/z 1052.5369 [M + H]+ (calcd for
C61H74N5O11, 1052.5385).
Compound 12j. This compound is a white amorphous powder;
yield 67%; 1H NMR (600 MHz, CDCl3): δ 12.08 (1H, s), 11.94 (1H,
s), 11.80 (2H, s), 8.83 (1H, d, J = 8.2 Hz), 8.76 (1H, d, J = 8.1 Hz),
8.69 (2H, dd, J = 7.8, 3.7 Hz), 8.08 (1H, dd, J = 8.0, 1.5 Hz), 8.05
(1H, dd, J = 8.0, 1.5 Hz), 7.85 (2H, d, J = 7.9 Hz), 7.64−7.61 (1H,
m), 7.60−7.56 (1H, m), 7.52 (2H, t, J = 7.4 Hz), 7.22−7.16 (4H, m),
5.16 (1H, t, J = 4.9 Hz), 4.09 (2H, dd, J = 51.7, 11.0 Hz), 3.31 (3H,
s), 3.20 (3H, s), 2.19 (3H, s), 2.19 (3H, s), 1.72 (6H, s), 1.59 (6H, s),
1.11 (3H, t, J = 7.1 Hz); 13C NMR (150 MHz, CDCl3): δ 172.2,
172.2, 170.1, 170.1, 168.7, 168.6, 168.0, 167.9, 141.6, 141.1, 140.3,
140.3, 134.9, 134.5, 133.4, 133.2, 131.4, 131.0, 127.3, 127.2, 123.7,
123.5, 123.4, 123.3, 121.7, 121.7, 121.1, 121.0, 120.7, 120.6, 116.7,
115.9, 85.5, 81.7, 81.6, 81.1, 77.4, 73.8, 71.2, 62.2, 56.6, 56.4, 52.8,
49.5, 49.1, 47.3, 46.2, 45.3, 45.2, 40.5, 38.3, 36.8, 32.9, 29.9, 28.5,
26.2, 25.4, 24.7, 24.7, 24.7, 24.7, 22.0, 22.0, 13.7; HRESIMS m/z
1140.5170 [M + H]+ (calcd for C63H74N5O15, 1140.5181).
Compound 12k. This compound is a white amorphous powder;
yield 65%; 1H NMR (600 MHz, CDCl3): δ 12.21 (2H, d, J = 3.9 Hz),
12.10 (1H, s), 11.98 (1H, s), 8.90−8.85 (3H, m), 8.79 (1H, dd, J =
8.4, 0.8 Hz), 8.08 (1H, dd, J = 8.0, 1.5 Hz), 8.07−8.04 (5H, m), 7.91
(2H, dd, J = 8.0, 1.2 Hz), 7.68−7.64 (1H, m), 7.64−7.60 (1H, m),
7.58 (2H, dd, J = 12.1, 4.8 Hz), 7.56−7.50 (6H, m), 7.25−7.17 (4H,
m), 5.16 (1H, t, J = 5.0 Hz), 4.10 (2H, dd, J = 56.8, 11.0 Hz), 3.31
(3H, s), 3.19 (3H, s), 1.10 (3H, t, J = 7.1 Hz); 13C NMR (150 MHz,
CDCl3): δ 168.7, 168.6, 168.3, 168.2, 165.9, 165.8, 141.4, 141.0,
141.0, 140.9, 135.1, 135.1, 135.0, 134.6, 133.5, 133.4, 132.0, 132.0,
131.4, 130.9, 128.9, 127.6, 127.4, 127.4, 123.8, 123.5, 123.5, 123.4,
121.9, 121.9, 121.1, 121.0, 121.0, 120.8, 116.8, 116.0, 85.5, 81.6, 77.4,
73.8, 71.2, 62.2, 56.6, 56.4, 53.4, 49.5, 49.1, 47.3, 46.2, 45.3, 45.2,
40.6, 38.3, 36.8, 33.1, 28.4, 26.3, 24.4, 13.7; HRESIMS m/z
1091.4740 [M + H]+ (calcd for C65H66N5O11, 1091.4759).
Compound 12m. This compound is a white amorphous powder;
yield 62%; 1H NMR (600 MHz, CDCl3): δ 12.02 (1H, s), 11.88 (1H,
s), 11.13 (2H, s), 8.79 (1H, d, J = 8.5 Hz), 8.71 (1H, d, J = 8.4 Hz),
8.65 (2H, d, J = 8.4 Hz), 8.09 (1H, dd, J = 8.0, 1.5 Hz), 8.05 (1H, dd,
J = 8.0, 1.6 Hz), 7.83 (2H, d, J = 8.0 Hz), 7.67−7.63 (1H, m), 7.61
(1H, dd, J = 11.4, 4.3 Hz), 7.54−7.50 (2H, m), 7.24−7.15 (4H, m),
5.15 (1H, t, J = 4.9 Hz), 4.09 (2H, dd, J = 60.3, 11.0 Hz), 3.31 (3H,
s), 3.20 (3H, s), 1.25 (6H, s), 1.11 (3H, t, J = 7.1 Hz); 13C NMR
(150 MHz, CDCl3): δ 169.2, 169.2, 168.7, 168.6, 168.1, 168.0, 141.4,
140.9, 140.5, 140.5, 135.0, 134.6, 133.4, 133.2, 131.4, 131.0, 129.8,
127.3, 127.2, 123.8, 123.5, 123.3, 123.2, 121.8, 121.0, 120.9, 120.8,
120.6, 116.8, 116.0, 85.5, 81.7, 73.8, 71.2, 62.2, 56.5, 56.4, 52.8, 49.5,
49.1, 47.3, 46.1, 45.3, 45.2, 40.6, 38.3, 36.8, 32.9, 29.9, 28.4, 27.4,
26.2, 25.6, 13.7; HRESIMS m/z 968.4431 [M + H]+ (calcd for
C55H62N5O11, 968.4446).
Compound 12d. This compound is a white amorphous powder;
yield 69%; 1H NMR (600 MHz, CDCl3): δ 12.02 (1H, s), 11.89 (1H,
s), 11.24 (2H, s), 8.81 (1H, dd, J = 8.5, 0.8 Hz), 8.74 (1H, dd, J = 8.4,
0.8 Hz), 8.71 (2H, d, J = 8.5 Hz), 8.07 (2H, ddd, J = 16.7, 8.0, 1.5
Hz), 7.84 (2H, d, J = 7.9 Hz), 7.67−7.63 (1H, m), 7.62−7.59 (1H,
m), 7.53−7.50 (2H, m), 7.21−7.15 (4H, m), 5.15 (1H, t, J = 5.0 Hz),
4.09 (2H, dd, J = 54.8, 11.0 Hz), 3.31 (3H, s), 3.20 (3H, s), 1.29 (6H,
d, J = 2.1 Hz), 1.28 (6H, d, J = 2.1 Hz), 1.11 (3H, t, J = 7.1 Hz); 13C
NMR (150 MHz, CDCl3): δ 176.3, 176.2, 168.7, 168.6, 168.1, 168.0,
141.5, 141.0, 140.9, 140.8, 135.0, 134.6, 133.4, 133.2, 131.4, 130.9,
127.3, 127.2, 123.7, 123.4, 123.2, 123.0, 121.8, 121.8, 121.0, 120.9,
120.7, 120.5, 116.7, 115.9, 85.5, 81.7, 77.4, 73.8, 71.2, 62.2, 56.6, 56.4,
52.8, 49.5, 49.1, 47.3, 46.2, 45.3, 45.2, 40.6, 38.3, 37.6, 36.8, 32.9,
28.5, 26.2, 25.4, 19.8, 13.7; HRESIMS m/z 1024.5061 [M + H]+
(calcd for C59H70N5O11, 1024.5072).
Compound 12n. This compound is a white amorphous powder;
1
yield 63%; H NMR (400 MHz, CDCl3): δ 12.01 (1H, s), 11.96−
11.79 (3H, m), 8.83 (1H, d, J = 8.4 Hz), 8.75 (1H, d, J = 8.3 Hz),
8.70 (2H, d, J = 8.4 Hz), 8.05 (2H, m), 7.85 (2H, d, J = 8.0 Hz),
7.66−7.45 (4H, m), 7.18 (4H, m, J = 14.4, 13.2, 7.4 Hz), 5.13 (1H, t,
J = 4.9 Hz), 4.07 (2H, dd, J = 44.6, 11.0 Hz), 3.31 (3H, s), 3.19 (3H,
s), 1.56 (12H, s), 1.10 (3H, t, J = 7.1 Hz); 13C NMR (100 MHz,
CDCl3): δ 176.0, 175.9, 168.7, 168.6, 167.8, 167.7, 141.5, 141.0,
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140.0, 140.0, 135.0, 134.6, 133.3, 133.1, 131.3, 130.9, 127.5, 127.4,
123.7, 123.7, 123.6, 123.3, 121.6, 121.5, 121.5, 121.3, 121.1, 121.0,
116.6, 115.8, 85.4, 81.7, 77.3, 77.4, 73.8, 73.8, 71.1, 62.2, 56.5, 56.3,
52.8, 49.5, 49.1, 47.3, 46.1, 45.3, 45.1, 40.5, 38.3, 36.9, 28.4, 28.1,
27.1, 26.2, 25.4, 18.8, 13.7; HRESIMS m/z 1056.4960 [M + H]+
(calcd for C59H70N5O13, 1056.4970).
Authors
Lin-Xi Wan − School of Life Science and Engineering,
Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China
Ji-Fa Zhang − School of Life Science and Engineering,
Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China
Yong-Qi Zhen − School of Life Science and Engineering,
Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China
Lan Zhang − School of Life Science and Engineering,
Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China
Cell Culture and Treatment. The percentage neuroprotective
rates were assessed using an MTT assay. Human SH-SY5Y
neuroblastoma cells were obtained from ATCC (Manassas, VA,
USA) and were cultured at 37 °C in a humidified atmosphere of 5%
CO2, in GibcoTM Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum. MTT
was purchased from Sigma-Aldrich (St. Louis, MO, USA). Briefly,
SH-SY5Y cells were seeded in 96-well plates and then pretreated with
50 μM of each test compound. After 1 h of incubation, hydrogen
peroxide solution (H2O2, 500 μM) was added into cells and incubated
for 4 h. Then, MTT (5 mg/mL) was added into each well. After a 4 h
treatment, the supernatant was removed, and DMSO (150 μL/well)
was added to dissolve the insoluble formazan crystals. Then, the plate
was vibrated, and the absorbance was measured at 490 nm using a
microplate reader.
Xiaohuan Li − School of Life Science and Engineering,
Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01111
Reactive Oxygen Species (ROS) for Flow Cytometric
Analysis. The H2O2-induced SH-SY5Y cells were collected and
centrifuged. After centrifugation, the cells were washed with PBS
three times. Then, DCFH-DA was added to stain the reactive oxygen
species in cells at 37 °C for 15 min. Stained cells were collected and
analyzed using a FACScalibur instrument (Becton Dickinson,
Mountain View, CA, USA).
Immunofluorescence Analysis. For the apoptosis assay, the
cells were stained with Hoechst 33258 in the dark at 37 °C for 30 min
and then observed under a fluorescence microscope.
Annexin-V/PI Dual Staining. The apoptotic ratio was measured
by flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA) after
employing an Annexin-V-FLUOS Staining Kit (Roche, Germany).
The detailed procedures were performed according to the
manufacturer’s instructions.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was finically supported by the NSF of China
(81773605) and the Science and Technology Program of
Sichuan, China (2018JY0077).
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ASSOCIATED CONTENT
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sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01111.
HRESIMS, IR, and NMR spectra for compounds and
cytotoxicity of tested compounds (PDF)
AUTHOR INFORMATION
■
Corresponding Authors
Feng Gao − School of Life Science and Engineering, Southwest
Jiaotong University, Chengdu 610031, People’s Republic of
China; orcid.org/0000-0001-9436-681X; Phone: +86-
28-87603202; Email: gaof@swjtu.edu.cn
Xian-Li Zhou − School of Life Science and Engineering,
Southwest Jiaotong University, Chengdu 610031, People’s
Republic of China; orcid.org/0000-0002-1690-0578;
Phone: +86-28-87603202; Email: zhouxl@swjtu.edu.cn
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