Diverse Chemosensitizing 8,9-Secolindenane-Type Sesquiterpenoid Oligomers and Monomers from Sarcandra glabra

Article abstract of DOI:10.1021/acs.joc.9b00986

The theoretical analysis and biomimetic conversion confirmed that the oxidative cleavage of the Δ^8,9 double bond of chloranthalactone A (I), an abundant lindenane-type sesquiterpenoid in Chloranthaceae plants, generates 8,9-secolindenane (II) with active aldehyde and maleic anhydride fragments that can capture other fragments and produce oligomeric molecules. A careful phytochemical investigation of the leaves of Sarcandra glabra led to the discovery of eight novel 8,9-secolindenane-type sesquiterpenoid oligomers (sarglalactones A-H, compounds 1-8), including three unprecedented trimers (1-3), five unusual dimers (4-8), and five 8,9-secolindenane monomers (sarglalactones I-M, 9-13). Their structures were determined by comprehensive HRMS, NMR, circular dichroism, and X-ray diffraction analyses. Bioassay results showed that these oligomers significantly reversed the multidrug resistance of MCF-7/doxorubicin (DOX) cells and increased the sensitivity of U2 OS cells to DOX by downregulating the HMGB1 expression.

Full text of DOI:10.1021/acs.joc.9b00986

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Article
Diverse chemosensitizing 8,9-secolindenane-type
sesquiterpenoid oligomers and monomers from Sarcandra glabra
Jun Chi, Shanshan Wei, Hongliang Gao, Dingqiao Xu, Lina
Zhang, Lei Yang, Wenjun Xu, Jun Luo, and Ling-yi Kong
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00986 • Publication Date (Web): 13 Jun 2019
Downloaded from http://pubs.acs.org on June 15, 2019
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Diverse chemosensitizing 8,9-secolindenane-type sesquiterpenoid
oligomers and monomers from Sarcandra glabra
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Jun Chi, Shanshan Wei, Hongliang Gao, Dingqiao Xu, Lina Zhang, Lei Yang,
Wenjun Xu, Jun Luo*, and Lingyi Kong*
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural
Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing
2
*
10009, P. R. China
Corresponding authors
E-mail: cpu_lykong@126.com
luojun@cpu.edu.cn
O
H
O
O
8''
H
9
O
O
H
H
O
O
9'
H
O
O
O
12'
O
1
2
9
4
O
8
8
HO
9''
O
O
H
O
oxidative
cleavage
H
9
1
Sarcandra glabra
H
O
9
O
9
O
H
8
O
O
O
H
O
O
H
8
9'
H
O
O
O
O
9
H
12
8
O 4 9'S
5 9'R
chloranthalactone A (I)
II
H
Abstract: The theoretical analysis and biomimetic conversion confirmed that the oxidative cleavage of
the ∆8,9 double bond of chloranthalactone A (I), an abundant lindenane-type sesquiterpenoid in
Chloranthaceae plants, generates 8,9-secolindenane (II) with active aldehyde and maleic anhydride
fragments that can capture other fragments and produce oligomeric molecules. A careful
phytochemical investigation of the leaves of Sarcandra glabra led to the discovery of eight novel 8,9-
secolindenane-type sesquiterpenoid oligomers (sarglalactones A-H, compounds 1-8), including three
unprecedented trimers (1-3) and five unusual dimers (4-8), and five 8,9-secolindenane monomers
(sarglalactones I-M, 9-13). Their structures were determined by comprehensive HRMS, NMR, CD and
X-ray diffraction analyses. Bioassay results showed that these oligomers significantly reversed the
MDR of MCF-7/DOX cells and increased the sensitivity of U2 OS cells to doxorubicin by
downregulating HMGB1 expression.
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INTRODUCTION
Chloranthalactone A (I), also named shizukanolide B, was the first and most abundant lindenane-type
1
sesquiterpenoid discovered in plants of the Chloranthaceae family. Structurally, chloranthalactone A
0
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possesses a highly conjugated α,β,γ,δ-unsaturated lactone unit (∆ /∆7,11/CO-12) and a conjugation-
preferred moiety (∆4,15/C-5,6/∆7,11/CO-12) that readily undergo oxidation, Diels-Alder, [2 + 2]
cyclization and Michael addition reactions to construct complicated lindenane conjugates (Scheme
8,9
2
S1). Most of these molecules exhibit important bioactivities, including cytotoxic, anti-HIV, anti-
3
inflammatory and antimalarial actions. Their highly complex structures and potential bioactivities
have attracted increasing interest from natural product and synthetic chemists, and the biomimetic
4
synthesis of some structures has continuously been achieved in recent years. In our previous study, a
number of novel and anti-inflammatory lindenane monomers and oligomers based on monomer I were
5
discovered from relevant Chloranthaceae plants. By analyzing the changes in the C-C bonds of the
reported structures, in addition to the [4 + 2] /[2 + 2] cycloaddition and Michael addition reactions, the
oxidative cleavage of the ∆8,9 double bond in precursor I became our particular focus. The oxidative
reaction formed highly active 9-aldehyde and maleic anhydride moieties that function like the two arms
of a person, which can catch other fragments to construct unprecedented molecules.
6
2
In this paper, the biomimetic oxidative cleavage of chloranthalactone A under O and metal-free
conditions generated 8,9-secolindenane (II), and a subsequent acetal reaction successfully produced the
dimeric chlojapolactone A (III), the only 8,9-secolindenane-based oligomer reported from Chloranthus
7
japonicus (Scheme 1). The results of the biomimetic synthesis suggested that more rare and diverse
oligomers based on 8,9-secolindenane (II) might be created in plants under physiological conditions.
Sarcandra glabra (Thunb.) Nakai, which is widely distributed in southern Asia, is one of the renowned
traditional Chinese medicines used for anti-inflammatory and adjuvant treatment for cancer.8
According to our previous study, chloranthalactone A (I) and several of its derivatives exist in this
plant,1b,5b and thus a detailed isolation of the leaves of S. glabra led to the discovery of eight 8,9-
secolindenane-based oligomers (sarglalactones A-H, compounds 1-8), including three unprecedented
trimers and five unusual dimers, along with five 8,9-secolindenane monomers (sarglalactones I-M, 9-
1
3). Here, we report the characterization of these novel 8,9-secolindenane oligomers and their
bioactivities by enhancing the sensitivity of MCF-7/DOX cells and U2 OS cells to doxorubicin (DOX).
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RESULTS AND DISCUSSION
As mentioned above, the highly conjugated α, β, γ, δ-unsaturated lactone moiety enabled the ∆8,9
9
double bond of chloranthalactone A (I) to display a high reactivity, and this compound was largely
1
1
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obtained from the seeds of S. glabra in our previous research.5b Thus, the biomimetic oxidation of
precursor I was performed under atmosphere and visible light irradiation (Table S1-1 and Figure S1-
2
8
).1b,10 As a result, the 8,9-epoxide chloranthalactone B (IV) was spontaneously formed with a yield of
0% over 30 days, and the trace C8-C9 cleavage product 8,9-secolindenane (II), which was previous
semisynthesized from I through three step oxidations,6a was also measured using H-NMR within 2
days (Figure S1-1). Furthermore, a variety of photocatalysts were employed to improve the yield of
II.11 Among these photocatalysts, 9-mesityl-10-methylacridinium perchlorate (Mes-Acr ) showed the
best transformation to product II, with a yield of 25%, and the reaction time was limited to 5 h.12
With 8,9-secolindenane (II) in hand, the biomimetic acetalization and alcoholysis reactions of its
two active arms were designed and conducted (Scheme 1). Treatment of compound II with 10% p-
1
+
TsOH and MeOH-H
2
O
(1:1, V:V) smoothly opened the maleic anhydride to yield
chloranerectuslactone V (IIa), which was also reported in Chloranthus erectus.6b The epoxide IV was
treated with 10% p-TsOH and DCM to produce a 91% yield of 8,9-diol chloranthalactone E (IVa),13
which was unstable and existed as an epimeric mixture at C-8 due to the fast ketone-hemiacetal
tautomerization.14 After screening and optimization of the reaction conditions (Figure S1-3),
chlojapolactone A (III) and 9′R-III were obtained as the acetalization dimers of 9-aldehyde IIa and
8,9-diol IVa at 30% and 4% yields following the application of 10% p-TsOH as an acid catalyst and
molecular sieve in toluene to remove water at 100℃. Another trace product IIIa, was identified as an
acetalized dimer of compounds II and IVa through an HPLC-HRMS analysis (Figures S1-4 and -5).
Overall, the successful biomimetic syntheses of 8,9-secolindenanes and its oligomers confirmed our
aforementioned hypothesis, and more diverse and unprecedented 8,9-secolindenane-based oligomers
might be created by plants with abundant chloranthalactone A (I).
Scheme 1. Biomimetic conversion of chloranthalactone A (I).
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OH
O
p-TsOH
H2O, DCM
OH
solventless
in air
O
O
O
O
H
H
9
8
O
chloranthalactone B (IV)
chloranthalactone E (IVa)
O
H
O
H
Mes-Ars+
O2, MeCN
blue LED
H
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chloranthalactone A (I)
H
p-TsOH, H2O
MeOH, 50℃
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H
O
O
O
O
O
O
II
chloranerectuslactone V (IIa)
IVa,10% p-TsOH
toluene,100℃
molecular sieve
IVa,10% p-TsOH
toluene,100℃
molecular sieve
O
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O
O
O
H
O
H
H
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H
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H
H
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H
H
H
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O
H
H
H
IIIa (trace)
C30H32O7
chlojapolactone A (Ⅲ)
9'R-III
(4%)
+
m/z 522.2481 [M+NH4]
(30%)
The air-dried and powdered leaves of S. glabra (4.6 kg) were exhaustively extracted with 95% EtOH
under reflux. After removing the solvent, the residue (757.8 g) was successively extracted with
petroleum ether and dichloromethane to obtain two extracts (150.1 g and 80.5 g, respectively). A
detailed isolation of the petroleum ether fraction and small polar section of the dichloromethane
fraction was conducted and led to the discovery of eight 8,9-secolindenane-type oligomers
(
sarglalactones A-H, 1-8), including three unprecedented trimers and five unusual dimers, along with
five 8,9-secolindenane monomers (sarglalactones I-M, 9-13) (Figure 1).
2
'
15'
3
'
Unit B
O
O
O
O
O
O
O
1'
4'
5'
O
4'
H
8'
H
8'
H
8'
O
O
1
0'
O
O
12''
13''
HO
O
O
O
9'
7'
12'
O
H
O
9'
O
9'
O
1
4 H
H
6'
11''
7''
H
O
H
H
O
12'
8''
HO
2
1
O
O 12
14'
11'
O
8''
O
O
O
O
O
12'
9
9''
9
9
8''
10
13'
8
8
8
9''
10''
6''
9''
3
5
HO
H
O
H
4
7
11
14''
5''
6
H
H
4''
H
H
1
''
1
5
15''
1
3
Unit A
Unit A'
3''
1
2''
2
3
Unit B
O
O
O
O
O
4'
4
O
4'
H
H
8'
8'
O
14
10
9R1
8
8
'
O
O
O
O
O
9'
O
9'
H
H
H
O
H
H
O
9'
H
1
2'
12
O
O
O
O
O
O
12'
OR2
9
9
O
O
H
R1
R2
Et
8
8
OH
O
O
9
8
9
CHO
O
H
H
1
0 COOH
Me
Et
Unit A
1
0,14
H
11
4
9'S
5 9'R
6 9'S
7 9'R
1
2 CH(OMe)2 Me
8
1
3 CH(OMe)2 Et
Figure 1. Structures of sarglalactones A-M (1-13).
Sarglalactone A (1), a white amorphous powder, was assigned the molecular formula of C45
50 11
H O
based on the HRMS data. Three pairs of characteristic upfield shifted methylene protons attributed to
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the cyclopropane ring of lindenanes identified by evaluating H NMR (Table 1) and 2D NMR (Figures
15
2
and 3) data indicated that compound 1 was an unprecedented lindenane trimer. A careful analysis of
its 1D and 2D NMR data indicated the presence of two 8,9-diol lindenanes [chloranthalactone E (IVa),
units A and A′]13 and one 8,9-secolindenane [chloranerectuslactone V (IIa), unit B] . The key HMBC
6b
1
1
1
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1
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1
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correlations from the characteristic acetal methine H-9′ [δ
H
5.15 (s) and δ
109.7) indicated that the aldehyde arm of unit B captured 8,9-diol unit A
105.6 and C-9′′ at δ 4.18 (s)
C C
109.7] to C-8 (δ 109.6) and
H-9 at δ
H
4.31 (s) to C-9′ (δ
C
7
by acetalization. The good agreement of chemical shifts of C-8′′ at δ
C
H
and δ
C
75.2 in unit A′′ with those of C-8 in unit A and C-9 in diol IVa, as well as the “loose end” ester
166.8) of unit B, suggested that unit B caught unit A′ by esterification between
carbonyl of C-12′ (δ
C
OH-8′′ and COOH-12′. The result was also supported by the lack of an HMBC correlation (Figure 2) of
H-9′′ /C-12′ and the tautomerism of 8,9-diol IVa. The key ROESY correlation (Figure 3) of H-9 /H-9′
of 1 revealed that these protons were located co-facially on the 1,3-dioxolane ring. The obvious
differences in the chemical shifts of CH-5 [δ
H
3.42 (m) and δ
C
50.5] and CH
3
-14 [δ
H
0.52 (s) and δ
C
C
1
1
7.3] in unit A and CH-5′′ [δ
H
2.95 (dd, J = 13.3, 2.8 Hz) and δ
C
57.2] and CH
3
-14′′ [δ
H
0.78 (s) and δ
6.7] in unit A′ that were attributed to the anisotropy of the α, β-unsaturated lactone indicated that C-8-
O-C-12 and H-14 were located co-facially and C-8′′-O-C-12′′ were located in the opposite direction to
H-14′′.5d The relative structure of 1 was further verified by the ROESY data (Figure 3). Therefore, the
structure of 1 was determined as an unprecedented lindenane trimer arising from one 8,9-secolindenane
core with two active arms to capture two 8,9-diol lindenanes by acetalization and esterification,
respectively.
17'
16'
1
5'
15'
3'
Unit B
3'
3'
15'
O
1'
O 8'
1'
O
O
1'
4'
O
O
4'
O
4'
5'
H
H
10'
H
10'
O
O
10'
O
O
8'
5'
7'
11'
12''
13''
7'
O
8''
7'
O
9
9
'
12'
O
11''
5''
H
O
9
9'
5'
12'
HO
H
O
O
O
O
8''
1
4
H
5
H 6'
H
H
14'
12'
14
10
9''
14
10
11'
HO
9''
1
O 14'
O12
8''
14'
11'
9
O
O
O
O
O
7
''
O
1
0
8
13'
9''
8
8
13'
3
13'
14''
O
14''
4
7
O
HO 10''
14''
O
10''
H
H
1
1
10''
H
H
H
H
1''
15
13
Unit A'
15''
Unit A
3''
2
1
3
15'
15'
3'
3
'
3'
1
5'
1'
O
O
O
4'
5'
1
'
4
'
1'
4'
O
O
10'
9'
10'
10'
5'
O
14'
7'
O
8
'
O
12'
7'
5'
7'
9'
O
12'
O
9'
6'
H
O
12'
H
H
H
14'
16'
H
14
16'
14
14'
9
O
O
9
O
11'
3'
O
O
H
O
17'
14
OH
O
17'
10
8
O 12
10
8
O
13'
9
13'
1
O
Unit B
O
10
8
O
H
H
5
Unit A
H
4/5
6/7
8
Figure 2. Key HMBC correlations of sarglalactones A-H (1-8).
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9
0
Figure 3. Key ROESY correlations of sarglalactones A-H (1-8).
50 54
The molecular formulas C45H O11 and C47H O11 of sarglalactones B (2) and C (3) obtained by
HRMS spectra and the characteristic NMR data indicated that 2 and 3 were also two lindenane trimers.
After a careful comparison, the NMR (Table S1-2) data of 2 was closely related to the data from 1,
reflecting the similarity of their structures. Two sets of NMR signals with a certain ratio were observed
in Figures S2-31~35, and key HMBC correlations (Figure 2) from H-9′′ [δ
H
5.18/5.56 (s)] of unit A′ to
CO-12′ (δ 168.1/170.6) of unit B were present. These facts supported the connection of units B and A′
C
in 2 through esterification between COOH-12′ and OH-9′′, as well as the existence of the
tautomerization at C-8′′ as monomer IVa. The NMR spectra of 3 was extremely similar to those of 2,
revealing that 3 was also a tautomeric 8,9-secolindenane trimer. One set of additional terminal double
bond signals from C-15′ at δ
H
4.87/4.80 (s) and 4.63/4.54 (s) and δ
C
107.8 (Table S1-2), and HMBC
-17′ [δ 1.20 (t, J
-16′ to CO-8′ (δ 168.2)
correlations (Figure 2) from H
2
-16′ [δ 4.20/4.10 (m)] to C-17′ (δ 13.8/13.9) and H
H
C
3
H
=
7.1 Hz) ] to C-16′ (δ
C
62.4/61.5) belonging to an ethoxy side chain, from H
2
C
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and from H-9′′ [δ
H C
5.16/5.50 (s)] to C-12′ (δ 168.7/168.8), indicated that the maleic anhydride arm of
8
,9-secolindenane core (unit B) was esterified with ethanol and 8,9-diol lindenane moieties,
respectively. The relative configurations of 2 and 3 were verified by the ROESY correlations and are
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
shown in Figure 3.
1
13
1
3
Table 1. H (600 MHz) and C{ H} (150 MHz) NMR data for 1 and 4 (δ in ppm, J in Hz, CDCl )
1
4
no.a
Unit A
Unit B
Unit A′
Unit A
Unit B
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
δ
H
δ
C
1
2
1.83 (m)
0.87 (m)
23.6
15.8
1.70 (m)
27.4
8.0
1.86 (m)
0.90 (m)
0.78 (m)
1.86 (m)
24.1
15.8
1.99 (m)
0.83 (m)
0.67 (m)
1.84 (m)
23.5
15.7
1.86 (m)
0.82 (m)
0.71 (m)
1.90 (m)
28.1
0.78 (m)
11.2
25.0
0
.70 (m)
0.57 (q, 4.8)
1.63 (m)
3
4
5
1.97 (m)
23.7
151.3
50.5
29.5
93.2
47.1
23.0
150.4
57.2
23.7
151.8
50.4
153.5
44.4
3.42 (m)
2.31 (m)
2.42 (m)
2.07 (m)
2.95 (dd, 13.3, 2.8)
2.62 (dd, 13.3, 2.8)
2.38 (m)
3.42 (m)
2.75 (dd, 12.4, 5.4)
2.54 (dd, 13.5, 5.6)
2.27 (dd, 13.5, 13.5)
2
2
.51 (m)
.27 (m)
2.48 (dd, 17.8, 5.9)
2.23 (dd, 14.0,12.6)
6
22.3
26.3
23.1
22.3
28.2
7
8
9
154.2
109.6
85.4
125.1
166.3
109.7
49.0
155.8
105.6
75.2
154.6
109.7
85.3
143.5
165.8
109.4
48.1
4.31 (s)
5.15 (s)
4.18 (s)
4.26 (s)
5.08 (s)
1
1
1
0
1
2
42.8
42.2
42.8
128.0
171.2
8.9
139.5
166.8
17.0
126.6
171.7
8.9
125.8
171.4
8.8
142.0
166.2
10.3
13
14
1.87 (s)
0.52 (s)
5.00 (s)
4.74 (s)
1.95 (s)
1.12 (s)
1.91 (s)
0.78 (s)
5.00 (s)
4.76 (s)
1.84 (s)
0.48 (s)
5.04 (s)
4.71 (s)
2.01 (s)
1.26 (s)
4.90 (s)
4.33 (s)
17.3
16.1
16.7
17.3
15.5
1
5
106.8
1.58 (s)
36.1
106.7
106.5
107.7
a
Numbers of carbon atoms in units A/B/A′ are represented as 1-15, 1′-15′, and 1′′-15′′, respectively.
The presence of 30-32 carbon atoms in the molecular formulas of sarglalactones D-H (4-8) by the
HRMS spectra and the characteristic NMR data indicated that these compounds were lindenane dimers.
A cyclic acetal linkage between units A and B in 4-7 was established by 9′-acetal methine at δ
and δ 109.4 (Tables 1 and S1-3, only data for 4 are listed here) and the HMBC (Figure 2) correlations
from H-9′ to C-8 (δ
set of maleic anhydride signals at δ
66.2/166.3 (C-12′) and 10.3/10.2 (CH
s) and 4.33/4.39 (s), and δ 107.7/107.3 in their 1D and 2D NMR spectra identified that 4/5 were the
H
5.08 (s)
C
C
109.7) and H-9 [δ
H
4.26 (s)] to C-9′. For compounds 4 and 5, the presence of one
143.5/143.8 (C-7′), 165.8/165.9 (C-8′), 142.0/141.9 (C-11′),
C
1
3 H
-13′) and terminal double bond signals of C-15′ at δ 4.90/4.92
(
C
ones that conserved maleic anhydride units for 8,9-secolindenane core (unit B). Additionally, a
comparative HPLC analyses of IIIa with the isolated compounds 4 and 5 (Figure S1-4) confirmed that
the structure of trace product IIIa in biomimetic conversion section (Scheme 1) was sarglalactones D
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
4). For compounds 6/7, the observations of methyl signals from CH
3
-15′ at δ
92.6/93.1, and the key HMBC
4.21/4.22 (m)] to the ester carbon C-12′ (δ
C
H C
1.64/1.68 (s) and δ
3
1.3/30.8, an upfield shift of an oxyquaternary carbon of C-4′ at δ
correlation from the ethoxy side chain H -16′ [δ
C
2
H
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
169.7/169.9) indicated that their 8,9-secolindenane cores existed as a lactone ring type that was similar
to 1/2. The identical HMBC correlations and obvious difference in the chemical shifts of C8/9/9′
suggested that 4/5 and 6/7 were two pair of epimers of an acetal carbon (C-9′), which was also
confirmed by key ROESY correlations (Figure 3) of H-9/H-9′ in 4/6, and H-5/H-9′ in 5/7. For
compound 8, the absence of the characteristic acetal methine and obvious HMBC correlations between
carbonyl carbon C-9′ (δ
common aldehyde arm was oxidized to a carboxyl group that captured one 8,9-diol-lindenane moiety
unit A) through esterification. The characteristic protons H-5 at δ 2.92 (m) and CH -14 at δ 1.07 (s)
C H 3 H
176.0) and H-9 [δ 5.49 (s)] and H -14′ [δ 1.21 (s)] indicated that the
(
H
3
H
of 8 confirmed that the configuration of C-8 was consistent with the configuration of C-8′′ in 1. The
relative structures of 4-8 were further verified by their ROESY data (Figure 3).
4
6
Figure 4. X-ray structures of sarglalactones D (4) and F (6).
The absolute configurations of compounds 1-7 were determined by combination of X-ray diffraction
and circular dichroism analyses. Suitable crystals of 4 and 6 were finally obtained in methanol-water
and acetone systems, respectively, and subjected to X-ray diffraction experiments. The Flack
parameters of 0.00 (11) and 0.15 (11) for 4 and 6 (CCDC no 1893947 and 1893946) allowed the
unambiguous assignments of their absolute configurations as 1R, 3S, 5S, 8S, 9S, 10S, 1′R, 3′S, 5′S, 9′S,
10′S and 1R, 3S, 5S, 8S, 9S, 10S, 1′R, 3′S, 4′S, 5′R, 9′S, 10′S, respectively (Figure 4). Furthermore, the
CD spectra of 1-7 were detected in MeOH (Figure 5) and applied to resolve the stereochemistry of
these oligomers. For epimers 6 and 7, their curves displayed opposite exciton chirality in the 220-280
nm region dominated by the configurations of C-9′. An obvious negative Cotton effect at 253 nm (Δε -
7
.3) and a positive Cotton effect at 230 nm (Δε +6.4) were observed for 7, indicating a anticlockwise
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mode of two coupling α,β-unsaturated lactone chromophores and the 9′R configuration in 7 (Figure 1).
The assignment of 9′S-configuration was also confirmed in 6 by its positive exciton chirality in the
2
20-280 nm region, as determined by X-ray diffraction. Accordingly, the absolute configurations of C-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
9′ in compounds 1-3 were determined to be S based on the similar Cotton effects in range of 220-280
nm as that in 6 (Figure 5). For compound 5, its CD curve was in good agreement with that of 7 (Figure
S1-8), although the absorption value at 231 nm of 5 (Δε -0.31) was not positive as that of 7 (Δε +6.4),
which indicated the 9′R configuration in 5 as that in 7. Thus, the absolute configurations of these
oligomers were determined and shown in Figure 1.
1
2
3
4
5
6
7
7
6
Figure 5. CD spectra of sarglalactones A-G (1-7) in MeOH.
According to the HRMS and 13C{ H}-NMR data for sarglalactones I-M (9-13), these compounds
contained 16-19 carbon atoms. Their 1D NMR (Table S1-4) data were similar to that of
chloranerectuslactone V (IIa), indicating that they were also 8,9-secolindenanes. Structurally,
compounds 10-13 were the oxidation, decarbonylation and acetalization derivatives of aldehyde IIa or
1
9
, as supported by the HMBC (Figure S1-9) correlations from H-1, H-5 and H-14 to the carboxyl group
of C-9 (δ 181.4) of 10, terminal double bond protons CH -14 at δ 5.08 (dd, J = 2.1 Hz) and 4.85 (s) to
C-1, C-5 and C-10 (δ 153.2) of 11, and two methoxy protons at δ 3.56 (s) to C-9 (δ 114.0) of 12 and
3. In addition, a methoxy or ethoxyl group was attached to CO-12 of 9-13, which was easily
C
2
H
C
H
C
1
confirmed by the HMBC spectra. The relative structures of 9-13 were further verified by their ROESY
data (Figure S1-9). Based on the CD spectra of 9 and 11 (Figure S1-10), the negative exciton chirality
in 200-280 nm indicated a clockwise mode of two coupling chromophores and the absolute
configurations of 9 and 11 were assigned as shown in Figure 1. In addition, monomer 9 with 9-
aldehyde had a same 8,9-secolindenane unit of dimer 6 that have been identified by X-ray diffraction,
and therefore the chemical conversion of 9 to 6 was performed using biomimetic acetalization
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9
1
1
1
1
1
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1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
conditions described in Scheme 1. The HPLC and HPLC-HRMS analyses (Figures S1-11 and -12)
indicated the formation of 6 from 9, which confirmed the absolute configurations of 9 as 1′R, 3′S, 4′S,
5
′R, 10′S (Figure 1). Limited by the amount of isolation, monomers 10 and 12-13 were confirmed to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
possess the identical absolute configurations as that of 9 with the biosynthesis consideration.
After isolation and identification, the bioactivities of these novel and diverse lindenane oligomers
were screened. In an in vitro cytotoxicity assay, almost all the tested compounds were not cytotoxic
toward MCF-7/DOX and U2OS cells (Table S1-5). However, compounds 1 and 4-8 potentially
reversed the multidrug resistance of MCF-7/DOX cells, and the reversal fold values are shown in Table
2
. Meanwhile, 1-8 showed significant synergistic cytotoxic effects in combination with DOX on U2
OS cells, and the combined indexes (CIs) were all less than 1 (Table 2). Based on the results, these
oligomers increased the chemotherapeutic sensitivity of human tumor cells to doxorubicin. As reported,
anticancer agents such as doxorubicin upregulate HMGB1 expression in U2 OS cells, and
downregulation of HMGB1 expression restores the chemosensitivity of U2 OS cells.16
Immunofluorescence staining revealed a reversal of HMGB1 expression in U2 OS cells following
treatment with most of the compounds, as shown in Figure 6A. Western blots further revealed a
concentration-dependent effect of 4 on decreasing HMGB1 levels in these cells to render them more
sensitive to doxorubicin (Figure 6B).
Table 2. The in vitro MDR reversal activity and synergistic cytotoxicity of compounds 1-9.
MDR reversal effects on MCF-7/DOX cells
Synergistic effects with doxorubicin
Compounds
c
on U2 OS cells (CI )
IC50 (μM)a
156.0 ± 29.9
2.7 ± 0.2
-
RF valueb
Doxorubicin
1
2
3
4
5
6
7
8
55.8
-
0.64 ± 0.08
0.92 ± 0.06
0.67 ± 0.07
0.68 ± 0.06
0.97 ± 0.06
0.80 ± 0.06
0.65 ± 0.08
0.65 ± 0.05
1.24 ± 0.09
-
-
6.9 ± 1.1
13.2 ± 1.6
3.4 ± 0.9
1.2 ± 0.9
8.1 ± 2.3
22.3
11.8
45.2
129.2
19.3
9
> 50
-
Verapamild
13.7 ± 2.7
11.3
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9
a
b
c
IC50: 50% inhibitory concentration of DOX in the presence of 1 and 8 (10 μM), 4-7 (30 μM), and 9 (50 μM).
The reversal fold (RF) value was calculated as follows: IC50 (DOX cells) /IC50 (DOX cells in the presence of isolates).
17
The combined index (CI) value at 50% inhibition. According to the method described by Soriano et al., 1.1 ≤ CI ≤ 1.3
was considered as moderate antagonism, 0.9 ≤ CI < 1.1 as an additive effect, 0.8 ≤ CI < 0.9 as slight synergism, 0.6 ≤ CI
<
0.8 as moderate synergism, 0.4 ≤ CI < 0.6 as synergism, and 0.2 ≤ CI <0.4 as strong synergism.
Verapamil was used as a positive control (10 μM).
d
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. Inhibition of HMGB1 expression increased the sensitivity of U2 OS cells to doxorubicin in
vitro. A Cells were treated with 50 μM isolates and 10 μM doxorubicin, and the expression levels of
HMGB1 of compounds 1-9 were analyzed under high content microscope. B Cells were treated with
different concentrations of 4 in combination with 10 μM doxorubicin, and HMGB1 expression was
analyzed by Western blot assay.
CONCLUSIONS
In summary, a series of novel 8,9-secolindenanes and 8,9-secolindenane-based oligomers
(
compounds 1-13) with effects on increasing chemosensitivity were successfully discovered in our
current project guided by the strategy of theoretical prediction, biomimetic reaction verification, and
detailed phytochemical investigations. These results also proved the effectiveness and practicality of
this strategy for the discovery of valuable natural molecules from the plant kingdom. We believe that
more interesting and valuable molecules are created by plants and are waiting to be discovered.
EXPERIMENTAL SECTION
General Experimental Details. Optical rotations were measured using a JASCO P-1020
polarimeter. The UV spectra were recorded using a UV-2450 UV/vis spectrophotometer. The CD
spectra were recorded with a JASCO J-810 spectrometer. The IR measurements were performed using
a Bruker Tensor 27 spectrometer. X-ray crystallographic data were collected with a Bruker Smart 1000
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1
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CCD diffractometer equipped with a graphite monochromator. The 1D and 2D NMR spectra were
1
recorded using a Bruker Avance III NMR spectrometer with standard pulse sequences ( H: 500 or 600
MHz, 13C{ H} NMR: 125 or 150 MHz) and TMS as an internal standard. High-resolution (HR)-ESI
mass data were required with an Agilent 6520B UPLC-Q-TOF instrument. Column chromatography
(CC) was performed with silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), MCI gel
(Mitsubishi Chemical Industries Ltd., Japan), ODS (40-63 μm, Fuji, Japan), Sephadex LH-20
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Pharmacia, Sweden), and Toyoperal HW-40C (Tosoh Corporation, Japan). Preparative HPLC was
performed using a Shimadzu LC-6AD series instrument with a Shim-park RP-C18 column (20 × 200
mm) and a Shimadzu SPD-20A detector.
Procedure for the Biomimetic Conversion of Chloranthalactone A (I).
Chloranthalactone A (I). Compound I (310.9 mg, 0.03 ‰) was provided as colorless oil from the
fresh seeds of S. glabra (10 kg) in our previous study.5b 1H NMR (600 MHz, CDCl
.5 Hz, H-1), 0.92 (m H-2), 1.98 (m, H-3), 2.97 (dq, J = 13.3, 3.0 Hz, H-5), 2.70 (dd, J = 16.8, 3.0 Hz,
H-6a), 2.27 (ddd, J = 16.8, 13.3, 2.5 Hz, H-6b), 6.25 (s, H-9), 1.90 (s, H-13), 0.79 (s, H-14), 5.06 (s, H-
) δ 1.66 (td, J = 7.6,
3
3
13
1
1
3
5a), 4.79 (s, H-15b); C{ H} NMR (150 MHz, CDCl ) δ 26.6 (C-1), 17.1 (C-2), 22.6 (C-3), 150.2 (C-
4), 62.2 (C-5), 21.5 (C-6), 148.2 (C-7), 149.7 (C-8), 120.0 (C-9), 40.2 (C-10), 122.6 (C-11), 171.3 (C-
+
12), 8.8 (C-13), 22.3 (C-14), 106.7 (C-15). HRMS (ESI-TOF) m/z: [M + H] Calcd for C15
17 2
H O
229.1224; Found 229.1224.
8
,9-Seco-chloranthalactone A (II) and Chloranthalactone B (IV). A solution of compound I (10.0
+
mg, 0.04 mmol) and Mes-Ars (0.2 mg, 0.005 mmol) in dry ACN (2 ml) was irradiated with a 7 W blue
LED under an oxygen atmosphere for 5 h. The resulting products were concentrated under reduced
pressure. The residues were purified by using preparative TLC to afford II (2.3 mg, 23%) and IV (4.1
mg, 50%) as colorless oil. The details of the oxidation reaction of I to produce compounds II and IV
under different conditions are shown in Table S1-1 and Figure S1-2.
1
Compound II: H NMR (600 MHz, CDCl
3
) δ 1.84 (ddd, J = 8.6, 5.5, 3.8 Hz, H-1), 0.97 (td, J = 8.6,
5
5
.5 Hz, H-2a), 0.84 (dt, J = 5.5, 3.8 Hz, H-2b), 2.01 (ddd, J = 8.6, 5.5, 3.8 Hz, H-3), 2.94 (dd, J = 12.1,
.3 Hz, H-5), 2.57 (dd, J = 13.3, 5.3 Hz, H-6a), 2.34 (dd, J = 13.3, 12.1 Hz, H-6b), 9.50 (s, H-9), 2.06
1
3
1
3
(s, H-13), 1.27 (s, H-14), 5.01 (s, H-15a), 4.46 (s, H-15b); C{ H} NMR (150 MHz, CDCl ) δ 27.9 (C-
1), 11.3 (C-2), 24.8 (C-3), 152.0 (C-4), 42.6 (C-5), 27.4 (C-6), 142.8 (C-7), 165.6 (C-8), 202.05 (C-9),
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5.9 (C-10), 142.2 (C-11), 165.8 (C-12), 10.1 (C-13), 14.9 (C-14), 108.8 (C-15). HRMS (ESI-TOF)
+
m/z: [M + Na] Calcd for C15
H
16
O
4
Na 283.0941; Found 283.0943.
1
Compound IV: H NMR (500 MHz, CDCl ) δ 1.71 (td, J = 8.0, 3.6 Hz, H-1), 0.97 (ddd, J = 9.1, 8.0,
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5.5 Hz, H-2a), 0.84 (dt, J = 5.5, 3.6 Hz, H-2b), 1.99 (m, H-3), 3.38 (ddt, J = 13.0, 5.1, 2.0 Hz, H-5),
2.55 (ddq, J = 19.0, 5.1, 2.0 Hz, H-6a), 2.11 (ddq, J = 19.0, 13.0, 2.0 Hz, H-6b), 4.18 (s, H-9), 1.89 (s,
1
3
1
H-13), 0.64 (s, H-14), 5.03 (s, H-15a), 4.70 (s, H-15b); C{ H} NMR (125 MHz, CDCl
3
) δ 24.0 (C-1),
1
6.9 (C-2), 23.1 (C-3), 150.0 (C-4), 50.7 (C-5), 21.4 (C-6), 152.5 (C-7), 88.0 (C-8), 64.6 (C-9), 41.4
(C-10), 129.2 (C-11), 170.5 (C-12), 9.1 (C-13), 17.1 (C-14), 106.9 (C-15). HRMS (ESI-TOF) m/z: [M
+
+
Na] Calcd for C15
H
16
O
3
Na 267.0992; Found 267.0991.
Chloranerectuslactone V (IIa). To a solution of II (50.2 mg, 0.19 mmol) in methanol (1 ml) was
added dropwise an aqueous solution of p-TsOH (3.3 mg, 1 ml, 0.02 mmol). The solution was stirred in
an oil bath at 50℃ using heating for 24 h. The resulting products were concentrated under reduced
pressure. The residues were purified by using preparative TLC to afford IIa (45.9 mg, 83%) as a
1
colorless oil. H NMR (600 MHz, CDCl
3
) δ 0.79 (m, H-2a), 0.73 (m, H-2b), 2.01 (ddd, J = 8.6, 5.5, 3.8
Hz, H-3), 2.65 (dd, J = 13.5, 6.4 Hz, H-5), 2.47 (dd, J = 13.5, 6.5 Hz, H-6a), 2.00 (m, H-6b), 9.60 (s,
1
3
1
3
H-9), 1.99 (s, H-13), 1.21 (s, H-14), 1.54 (s, H-15), 3.78 (s, H-16). C{ H} NMR (150 MHz, CDCl ) δ
27.4 (C-1), 7.5 (C-2), 29.1 (C-3), 91.8 (C-4), 44.1 (C-5), 25.6 (C-6), 127.2 (C-7), 166.5 (C-8), 202.2
(C-9), 56.9 (C-10), 139.2 (C-11), 170.4 (C-12), 15.8 (C-13), 16.5 (C-14), 31.8 (C-15), 52.9 (C-16).
+
HRMS (ESI-TOF) m/z: [M + Na] Calcd for C16
H
20
O
5
Na 315.1203; Found 315.1204.
Chloranthalactone E (IVa). Compound IV (11.0 mg, 0.04 mmol) was dissolved in 1 ml of
acetonitrile/water (5:1). The solution was stirred at room temperature for 48 h. The resulting product
was concentrated under reduced pressure. The residue was purified by using preparative TLC to afford
IVa (2.1 mg, 20%) as crystalline solid. To IV (10.5 mg, 0.04 mmol) in 1 ml of DCM was added
aqueous solution of p-TsOH (8.7 mg, 0.2 ml, 0.05 mmol) dropwise manner. The solution was stirred at
room temperature for 2 h. The resulting product was concentrated under reduced pressure. The residue
was purified using preparative TLC to afford IVa (9.6 mg, 91%) as crystalline solid. 8α-OH /8β-OH-
1
3
IVa: H NMR (500 MHz, CDCl ) δ 1.99/1.90 (m, H-1), 0.81 (td, J = 8.6, 5.1 Hz, H-2a)/0.92 (q, J = 4.2
Hz, H-2a), 0.84 (q, J = 5.1 Hz, H-2b)/0.79 (m, H-2b), 1.99 /1.90 (m, H-3), 3.38 (ddt, J = 12.2, 6.1, 2.8
Hz, H-5) /2.96 (dq, J = 13.2, 2.8 Hz, H-5), 2.51 (m H-6a), 2.22 (dd, J = 17.8, 12.2 Hz, H-6b)/2.12 (t, J
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=
13.2 Hz, H-6b), 3.94 (s, H-9)/4.12 (s, H-9), 1.80/1.81 (s, H-13), 0.53/0.95 (s, H-14), 4.99 (s, H-15a),
.70/4.72 (s, H-15b); 13C{ H} NMR (125 MHz, CDCl
1
4
3
) δ 23.1/24.3 (C-1), 15.8/16.1 (C-2), 23.9/23.0
(C-3), 152.1/150.9 (C-4), 51.6/57.1 (C-5), 22.4 (C-6), 159.7/159.3 (C-7), 104.8/105.9 (C-8), 76.3 (C-9),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
43.5/42.2 (C-10), 124.9/124.3 (C-11), 173.9/173.6 (C-12), 8.4/8.5 (C-13), 20.1/16.5 (C-14),
+
106.4/106.1 (C-15). HRMS (ESI-TOF) m/z: [M + Na] Calcd for C15
18 4
H O Na 285.1097; Found
285.1099.
Chlojapolactone A (III) and 9′R-III. To a solution of IIa (27.80 mg, 0.095 mmol) and IVa (50.12
mg, 0.191 mmol) in 2 ml toluene were added with 10% p-TsOH solution (8.22 mg, 0.03 mmol) and
molecular sieve. The solutions were stirred in an oil bath at 100℃ or room temperature. The resulting
2
products were reverse extracted with H O, and mother liquors were retained and concentrated under
reduced pressure. The residue was purified by using preparative TLC to afford III (15.33 mg, 30%)
and 9′R-III (1.82 mg, 4%) as amorphous solids. The details for the acetal reaction of compounds II and
IV under different conditions are shown in Figure S1-4.
1
Compound III: H NMR (500 MHz, CDCl
3
) δ 1.81 (m, H-1), 0.84 (m, H-2a), 0.68 (m, H-2b), 1.81
(m, H-3), 3.41 (m, H-5), 2.50 (dd, J = 18.2, 6.9 Hz, H-6a), 2.26 (dd, J = 18.2, 12.6 Hz, H-6b), 4.29 (s,
H-9), 1.84 (s, H-13), 0.49 (s, H-14), 5.01 (s, H-15a), 4.71 (s, H-15b), 1.69 (m, H-1′), 0.70 (m, H-2′a),
0.56 (m, H-2′b), 1.99 (m, H-3′), 2.31 (dd, J = 12.5, 6.8 Hz, H-5′), 2.44 (dd, J = 13.3, 6.8 Hz, H-6′a),
1
3
1
1.99 (t, J = 13.2 Hz, H-6′b), 5.14 (s, H-9′), 1.95 (s, H-13′), 1.13 (s, H-14′), 1.61 (s, H-15′). C{ H}
NMR (125 MHz, CDCl ) δ 23.7 (C-1), 15.7 (C-2), 23.6 (C-3), 151.2 (C-4), 50.4 (C-5), 22.2 (C-6),
54.3 (C-7), 109.8 (C-8), 85.3 (C-9), 42.8 (C-10), 127.9 (C-11), 171.1 (C-12), 8.8 (C-13), 17.2 (C-14),
3
1
1
1
06.8 (C-15), 27.6 (C-1′), 7.8 (C-2′), 29.4 (C-3′), 92.7 (C-4′), 48.3 (C-5′), 27.3 (C-6′), 128.4 (C-7′),
67.3 (C-8′), 109.8 (C-9′), 49.0 (C-10′), 138.0 (C-11′), 170.2 (C-12′), 16.3 (C-13′), 16.5 (C-14′), 31.2
+
(C-15′) , 52.7 (C-16′). HRMS (ESI-TOF) m/z: [M + Na] Calcd for C31
H
36
O
8
Na 559.2302; Found
5
59.2301.
1
9
3
′R-III: H NMR (600 MHz, CDCl ) δ 1.97 (m, H-1), 0.89 (m, H-2a), 0.76 (q, J = 4.0 Hz, H-2b),
2
4
.02 (m, H-3), 3.15 (m, H-5), 2.61 (ddd, J = 18.3, 6.5, 2.4 Hz, H-6a), 2.27 (dd, J = 18.2, 12.4 Hz, H-6b),
.37 (s, H-9), 1.84 (s, H-13), 0.50 (s, H-14), 5.07 (s, H-15a), 4.77 (s, H-15b), 1.58 (m, H-1′), 0.71 (td, J
= 8.5, 6.0 Hz, H-2′a), 0.57 (td, J = 6.0, 4.2 Hz, H-2′b), 1.99 (m, H-3′), 2.44 (dd, J = 12.5, 6.9 Hz, H-5′),
2.49 (dd, J = 12.5, 6.9 Hz, H-6′a), 1.95 (m, H-6′b), 5.55 (s, H-9′), 2.05 (s, H-13′), 1.08 (s, H-14′), 1.68
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(
(
(
s, H-15′); 13C{ H} NMR (150 MHz, CDCl
3
) δ 22.8 (C-1), 16.2 (C-2), 23.9 (C-3), 150.8 (C-4), 53.4
C-5), 22.2 (C-6), 154.6 (C-7), 110.3 (C-8), 88.0 (C-9), 51.6 (C-10), 126.7 (C-11), 171.0 (C-12), 8.8
C-13), 18.4 (C-14), 107.1 (C-15), 28.4 (C-1′), 7.8 (C-2′), 29.6 (C-3′), 93.2 (C-4′), 46.8 (C-5′), 27.8 (C-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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7
8
9
0
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0
6′), 129.2 (C-7′), 168.1 (C-8′), 115.4 (C-9′), 43.6 (C-10′), 137.7 (C-11′), 170.1 (C-12′), 16.2 (C-13′),
+
17.6 (C-14′), 30.8 (C-15′), 52.7 (C-16′). HRMS (ESI-TOF) m/z: [M + Na] Calcd for C31
H
36
O
8
Na
559.2301; Found 559.2301.
Acetalization of Compounds II and IVa. Following the above procedure, the reactants were II (2.50
mg, 0.010 mmol), IVa (5.00 mg, 0.019 mmol), and 0.001 mmol p-TsOH, HCOOH, or HAc in 2 ml of
toluene. The mixtures were dissolved in MeOH and detected by using HPLC (Figure S1-5). The
product from the reaction with 10 mol% p-TsOH was detected by using LC/MS (Figure S1-6), and the
molecular formula of compound IIIa was assigned as C30
m/z 522.2481 [M+NH , 522.2486).
]+ (calcd for C30
32 7
H O based on the pseudomolecular ion at
4
H33NO
7
Plant Material. The leaves of Sarcandra glabra (Thunb.) Nakai were collected from Guangxi
Province in China in September 2016 and were identified by Professor Mian Zhang of China
Pharmaceutical University. A voucher specimen (No. SG201609) was deposited in the Department of
Natural Medicinal Chemistry, China Pharmaceutical University.
Extraction and Isolation. The petroleum ether (150.1 g) fraction was separated using silica gel CC
and eluted with a gradient of DCM-MeOH (1:0, 100:1, 50:1 and 25:1, v/v) to afford seven fractions (Fr.
A~G) based on a TLC analysis. Fr. D (35.1 g) and Fr. E (50.4 g) were successively subjected to
separation with an HW-40C gel eluted with DCM-MeOH (1:1) to afford Fr. D-1-~7 and Fr. E-1~2. Fr.
D-6 (2.8 g) was applied to a LH-20 gel (DCM-MeOH, 1:1) and further purified using preparative
HPLC with 85% MeOH-H
2
O to obtain 4 (128.2 mg) and 5 (6.5 mg). Fr. E-2 was subjected to MCI gel,
eluted with MeOH-H O (30%, 50%, 70%, and 90%) to afford nine subfractions: Fr. E-2-1~9. Then, Fr.
2
E-2-7 (3.5 g) and Fr. E-2-8 (5.6 g) were separately submitted to separation on an LH-20 gel with
MeOH and then purified by using preparative HPLC to yield 1 (7.2 mg), 2 (21.2 mg), 3 (13.5 mg), 6
(
105.3 mg), 7 (5.1 mg), 8 (4.0 mg) and 10 (2.0 mg). The dichloromethane (80.5 g) fraction was
separated on silica gel CC and eluted with a gradient of DCM-MeOH (1:0, 50:1, 25:1 and 0:1 v/v) to
afford five fractions (Fr. a~e) based on a TLC analysis. Fr. b (5.0 g) was subjected to separation on an
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HW-40C gel, LH-20 gel and preparative HPLC to yield 9 (33.2 mg), 11 (8.2 mg), 12 (1.9 mg) and 13
(2.6 mg).
Spectroscopic Data of Isolated Compounds.
Sarglalactone A (1): white amorphous powder; [α]
2
D
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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4
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7
8
9
0
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3
4
5
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7
8
9
0
-35.6 (c 0.12, MeOH); IR (KBr) νmax 3435,
-1
2924, 1776, 1602, 1094, 975 cm ; UV (MeOH) log (ε) λmax 200 (4.54) nm; CD (MeOH) λ (Δε) 228 (-
1
13
1
0.71), 253 (+0.44); for H (600 MHz) and C{ H} (150 MHz) NMR data, see Table 1; HRMS (ESI-
+
TOF) m/z: [M + Na] Calcd for C45
H
50
O
11Na 789.3245; Found 789.3240.
2
6
Sarglalactone B (2): white amorphous powder; [α]
D
-13.2 (c 0.49, MeOH); IR (KBr) νmax 3432,
-
1
2
2
925, 1770, 1096, 975 cm ; UV (MeOH) (log ɛ) λmax 209 (4.34) nm; CD (MeOH) λ (Δε) 230 (-0.20),
1
13
1
51 (+0.42); for H (600 MHz) and C{ H} (150 MHz) NMR data, see Table S1-2; HRMS (ESI-TOF)
+
m/z: [M + Na] Calcd for C45
H
50
O
11Na 789.3245; Found 789.3242.
2
6
Sarglalactone C (3): white amorphous powder; [α]
D
17.6 (c 0.20, MeOH); IR (KBr) νmax 3368,
-1
2
925, 1773, 1662, 1254, 1176, 1100, 973 cm ; UV (MeOH) log (ε) λmax 210 (4.48) nm; CD (MeOH) λ
1
13
1
(
Δε) 221 (-0.94), 253 (+0.47); for H (600 MHz) and C{ H} (150 MHz) NMR data, see Table S1-2;
+
HRMS (ESI-TOF) m/z: [M + Na] Calcd for C47
H
54
O
11Na, 817.3558; Found 817.3558.
26
Sarglalactone D (4): colorless needle crystals; mp 204-207 ºC; [α]
D
-56.8 (c 0.27, CHCl
3
); IR (KBr)
-1
ν
max 2968, 1765, 1277, 1103, 968, 926, 867 cm ; UV (CHCl ) (log ɛ) λmax 203 (4.29) nm; CD (MeOH)
3
1
13
1
λ (Δε) 213 (-5.59); for H (600 MHz) and C{ H} (150 MHz) NMR data, see Table 1; HRMS (ESI-
+
TOF) m/z: [M + Na] Calcd for C30
H
32
O
7
Na 527. 2040; Found 527.2041.
2
6
Sarglalactone E (5): white amorphous powder; [α] -20.8 (c 0.05, MeOH); IR (KBr) νmax 3393,
D
-1
2
2
922, 1770, 1384, 1279, 1102, 978 cm ; UV (MeOH) log (ε) λmax 203 (4.27) nm; CD (MeOH) λ (Δε)
1
13
1
16 (-0.84), 231 (-0.31), 246 (-0.44); for H (500 MHz) and C{ H} (125 MHz) NMR data, see Table
+
S1-3; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C30
Sarglalactone F (6): colorless needle crystals; mp 112-115 ºC; [α]
32 7
H O Na 527.2040; Found 527.2043.
2
6
D
-66.8 (c 0.20, MeOH); IR (KBr)
-1
ν
max 2981, 1778, 1730, 1307, 1269, 1183, 1091, 976 cm ; UV (MeOH) (log ɛ) λmax 210 (4.31) nm; CD
1
13
1
(MeOH) λ (Δε) 218 (-1.03), 252 (+0.62); for H (500 MHz) and C{ H} (125 MHz) NMR data, see
+
Table S1-3; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C32
H
38
O
8
Na 573.2459; Found 573.2462.
2
6
Sarglalactone G (7): white amorphous powder; [α]
D
-88.6 (c 0.25, MeOH); IR (KBr) νmax 2971,
-1
2
929, 1774, 1728, 1306, 1264, 1178, 1143, 1101, 1084, 976 cm ; UV (MeOH) log (ε) λmax 210 (4.28)
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1
13
1
nm; CD (MeOH) λ(Δε) 230 (+6.4), 253 (-7.3); for H (500 MHz) and C{ H} (125 MHz) NMR data,
+
see Table S1-3; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C32
H
38
O
8
Na 573.2459; Found 577.2461.
22.1 (c 0.16, MeOH); IR (KBr) νmax 2925,
853, 1733, 1593, 1461, 1384, 1310, 1172, 1103 cm ; UV (MeOH) (log ɛ) λmax 207 (4.20) nm; CD
2
6
Sarglalactone H (8): white amorphous powder; [α]
D
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1
2
1
13
1
(MeOH) λ (Δε) 242 (+5.26); for H (500 MHz) and C{ H} (125 MHz) NMR data, see Table S1-3;
+
HRMS (ESI-TOF) m/z: [M + Na] Calcd for C32
H
38
O
9
Na 589.2408; Found 589. 2407.
-19.1 (c 0.1, MeOH); IR (KBr) νmax 3449, 2977, 1729, 1640,
1386, 1273,1085 cm ; UV (MeOH) log (ε) λmax 208 (3.86) nm; CD (MeOH) λ (Δε) 211 (-5.57), 236
2
7
Sarglalactone I (9): colorless oil; [α]
D
-1
1
13
1
(
+15.8); for H (600 MHz) and C{ H} (150 MHz) NMR data, see Table S1-4; HRMS (ESI-TOF)
+
m/z: [M + Na] Calcd for C17
H
22
O
5
Na 329.1359; Found 329.1361.
2
7
Sarglalactone J (10): colorless oil; [α]
D
22.6 (c 0.20, MeOH); IR (KBr) νmax 3451, 1722, 1650, 1384,
-1
1
13
1
1
314, 1087, 1038 cm ; UV (MeOH) (log ɛ) λmax 203 (3.88) nm; for H (600 MHz) and C{ H} (150
+
MHz) NMR data, see Table S1-4; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C16
H
20
O
6
Na 331.1152;
Found 311.1150.
27
Sarglalactone K (11): colorless oil; [α]
D
8.4 (c 0.11, MeOH); IR (KBr) νmax 3446, 2924, 1733, 1637,
-1
1
384, 1266, 1100 cm ; UV (MeOH) log (ε) λmax 208 (3.86) nm; CD (MeOH) λ (Δε) 211 (-8.47), 252
1
13
1
(
+2.8); for H (500 MHz) and C{ H} (125 MHz) NMR data, see Table S1-4; HRMS (ESI-TOF) m/z:
+
[
M + H] Calcd for C16
H
21
O
4
277.1434; Found 277.1432.
2
7
Sarglalactone L (12): colorless oil; [α]
D
-16.6 (c 0.01, MeOH); IR (KBr) νmax 3449, 2923, 1637,
-1
1
13
1
1
384, 1080 cm ; UV (MeOH) (log ɛ) λmax 203 (3.88) nm; for H (500 MHz) and C{ H} (125 MHz)
+
NMR data, see Table S1-4; HRMS (ESI-TOF) m/z: [M + Na] Calcd for C18
26 6
H O Na 361.1622; Found
361.1622.
2
7
Sarglalactone M (13): colorless oil; [α]
D
-16.7 (c 0.16, MeOH); IR (KBr) νmax 3454, 1725, 1639,
-1
1
1384, 1307, 1271, 1181,1082 cm ; UV (MeOH) log (ε) λmax: 208 (3.86) nm; for H (600 MHz) and
1
3
1
+
C{ H} (150 MHz) NMR data, see Table S1-4; HRMS (ESI-TOF) m/z: [M + Na] Calcd for
Na 375.1778; Found 375.1778.
X-ray Crystallographic Analysis.
The signal crystals of 4 (C30
19 28 6
C H O
32 7 38 8 2
H O ) and 6 (C32H O ) were obtained from MeOH/H O (10:1) and
acetone solutions, respectively. Suitable crystals were selected and then measured using a Bruker
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APEX-II CCD diffractometer with Cu Kα radiation. Crystal data for compound 4 (M = 504.56 g/mol):
1 1 1
orthorhombic, space group P2 2 2 (no. 19), a = 7.5297(6) Å, b = 18.5627(15) Å, c = 18.7260(15) Å, V
3
-1
3
=
2617.4(4) Å , Z = 4, T = 150 K, μ(CuKα) = 0.741 mm , Dcalc = 1.280 g/cm , 42067 reflections
°
°
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measured (6.7 ≤ 2Θ ≤ 131.98 ), 4499 unique (Rint = 0.0431, Rsigma = 0.0169) which were used in all
calculations. The final R
1
was 0.0278 (>2 sigma (I)) and wR
2
was 0.0711 (all data). Crystal data for
compound 6 (M = 550.62 g/mol): monoclinic, space group P2
1
(no. 4), a = 7.510(5) Å, b = 35.163(11)
3
-1
Å, c = 10.914(3) Å, β = 91.743(14)°, V = 2881(2) Å , Z = 4, T = 220.0 K, μ(CuKα) = 0.741 mm ,
3
°
°
Dcalc = 1.270 g/cm , 37278 reflections measured (5.026 ≤ 2Θ ≤ 142.172 ), 9954 unique (Rint = 0.0685,
sigma = 0.0636) which were used in all calculations. The final R was 0.0473 (I > 2σ(I)) and wR was
.1154 (all data). The crystallographic data for compounds 4 and 6 have been deposited in the
R
1
2
0
Cambridge Crystallographic Data Centre (Nos. 1893947 and 1893946, respectively) and are included
in the Supporting Information.
Cytotoxicity Assays. MCF-7/DOX and U2OS cells were cultured in RPMI-1640 medium
supplemented with 10% fetal bovine serum, harvested with trypsin, and resuspended at a final density
of 4.5 × 104 cells/ml. Aliquots (0.1 ml) of cell suspensions were seeded evenly into 96-well culture
2
plates and incubated in a 37 °C incubator with a 5% CO atmosphere for 24 h. A series of
concentrations of the compounds in DMSO were added to designated wells. After 48 h, an MTT assay
was performed as described previously.18
MDR Reversal Assays. MCF-7/DOX cells were distributed into 96-well culture plates at a density
of 4.5 × 103 cells per well. A full range of concentrations of doxorubicin with or without 30 or 10 μM
compounds or 10 μM verapamil (positive control) were added to the cells. After 48 h, the MTT assay
was performed as described above. IC50 values for doxorubicin were calculated from plotted results by
setting untreated cells to 100%. The reversal fold, in terms of potency of reversal, was calculated using
the following formula: reversal fold (RF) = IC50 (MCF-7/DOX cells)/IC50 (MCF-7/DOX cells in
combination with compounds). All assays were performed in triplicate. 18
Synergistic Cytotoxicity Assays.
MTT Growth Assay to Evaluate the Synergistic Effect. U2 OS cells were cultured in RPMI-1640
medium supplemented with 10% fetal bovine serum, harvested with trypsin, and resuspended at a final
density of 4.5 × 104 cells/ml. A full range of concentrations of doxorubicin with or without 50 μM
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compounds were added to the cells. After 48 h, the MTT assay was performed as described above.
Synergistic effects of the compounds and doxorubicin were quantified by calculating the
combined index (CI) using the following equation: CI = D
were the doses of the test compound and doxorubicin that produced 50% inhibition alone, respectively;
and DDOX were the doses of the test compound and doxorubicin that produced 50% inhibition in the
A B
/IC50, A + D /IC50, DOX. IC50, A and IC50, DOX
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D
A
combination system, respectively.19
High Content Imaging to Detect the Levels of HMGB1. Immunofluorescence staining was performed
using a previously described method.20 U2 OS cells were cultured on a 96-well glass-bottom culture
plate. Then, 10 μM doxorubicin with or without 50 μM compounds were added to the cells and
incubated for 48 h. Cells were fixed with warm 4% paraformaldehyde for 15 min at room temperature.
Next, the fixative was removed, and cells were washed with PBS 3 times for 5 min each. Cells were
permeabilized with 0.5% Triton X-100 for 10 min and blocked with 5% BSA for 60 min. Cells were
incubated with primary antibodies against HMGB1 (diluted with 5% BSA at 1:400) overnight at 4 °C.
After the incubation, cells washed with PBS 3 times for 5 min each. Cells were then incubated with an
Alexa Fluor-conjugated secondary antibody for 1.5 h at room temperature in the dark. Afterwards, cells
were washed with PBS 3 times for 5 min each. The nuclei were stained with DAPI (Beyotime, Haimen,
China) for 10 min before imaging. Images were captured using an ImageXpressei Micro High Content
(Molecular Devices, USA).
Western blot analysis. Western blot analyses were performed as previously described.22 U2 OS cells
and their corresponding parental cells were incubated with various concentrations of compound 3 and
1
0 μM DOX, or 0.1% DMSO for 48 h. Cells were harvested by trypsinization and treated with 1×
RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40, 1
mM EDTA and protease inhibitors) (Amresco, Solon, USA) to extract the total proteins. An aliquot of
proteins from the total cell lysates (30 to 40 μg/lane) was separated by using sodium dodecyl sulfate
(8%, 12% or 15%) polyacrylamide gel electrophoresis (SDS–PAGE, Bio-Rad Laboratories, Hercules,
CA), wet-transferred to NC membranes (Bio-Rad Laboratories, Hercules, CA), blotted with primary
antibodies against cleaved HMGB1, and probed with secondary isotype-specific antibodies conjugated
to horseradish peroxidase (Cell Signaling Technology). Bound immuno-complexes were detected using
a ChemiDOC™ XRS+system (Bio-Rad Laboratories, Hercules, CA).
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Spectroscopic data including 1D and 2D NMR and HRMS spectra of synthetic and isolated
compounds; NMR, HPLC and LC-MS analyses of biomimetic conversion; Cytotoxicity of isolated
compounds (PDF)
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Crystallographic data of compounds 4 and 6 (CIF)
AUTHOR INFORMATION
Corresponding Author
*
*
E-mail: cpu_lykong@126.com
E-mail: luojun@cpu.edu.cn
ORCID
Lingyi Kong: 0000-0001-9712-2618
Jun Luo: 0000-0002-7892-011X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
This study was supported in part by grants from the National Natural Science Foundation of China
(81430092 and 81673554), the Outstanding Youth Fund of the Basic Research Program of Jiangsu
Province (BK20160077), the “Double First-Class” University Project (CPU2018GY08), the 111
Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of
China (B18056), and the Drug Innovation Major Project (2018ZX09711-001-007).
REFERENCES
(1) (a) Nozoe, S.; Uchida, M.; Kusano, G.; Kondo, Y.; Takemoto, T. Two New Sesquiterpenoids
from Chloranthus glaber Makino. Heterocycles 1978, 9, 139-144. (b) Tsui, W. Y.; Brown, G. D.
Cycloeudesmanolides from Sarcandra glabra. Phytochemistry 1996, 43, 819-821.
(2) (a) Takeda, Y.; Yamashita, H.; Matsumoto, T.; Terao. H. Chloranthalactone F, a Sesquiterpenoid
from the Leaves of Chloranthus glaber. Phytochemistry 1993, 33, 713-715. (b) Kawabata, J.; Fukushi,
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Page 21 of 23
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Y.; Tahara, S.; Mizutani, J. Shizukaol A, a Sesquiterpene Dimer from Chloranthus japonicus.
Phytochemistry 1990, 29, 2332-2334. (c) Kawabata, J.; Fukushi, E.; Mizutani, J. Symmetric
Sesquiterpene Dimer from Chloranthus serratus. Phytochemistry 1993, 32, 1347-1349. (d) Lucia, A.;
Sauvain, M.; Beck, S.; Moulis, C.; Gimenez A.; Jullian, V. Bolivianine, a New Sesquiterpene with an
Unusual Skeleton from Hedyosmum angustifolium, and Its Isomer, Isobolivianine. Org. Lett. 2007, 9,
4693-4696.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(3) (a) Ni, G.; Zhang, H.; Liu, H. C.; Yang, S. P.; Geng, M. Y.; Yue, J. M. Cytotoxic
Sesquiterpenoids from Sarcandra glabra. Tetrahedron 2013, 69, 564-569. (b) Fang, P. L.; Cao, Y. L.;
Yan, H.; Pan, L. L.; Liu, S. C.; Gong, N. B.; Lü, Y.; Chen, C. X.; Zhong, H. M.; Guo, Y. Lindenane
Disesquiterpenoids with Anti-HIV-1 Activity from Chloranthus japonicus. J. Nat. Prod. 2011, 74,
1
408-1413. (c) Wang, P.; Luo, J.; Zhang, Y. M.; Kong, L. Y. Sesquiterpene Dimers Esterified with
Diverse Small Organic Acids from the Seeds of Sarcandra glabra. Tetrahedron 2015, 46, 5362-5370.
d) Zhou, B.; Yan, W.; Dalal, S.; Merino, E. F.; Liu, Q. F.; Xu, C. H.; Tao, Y.; Jian, D.; Kingston, D. G.
(
I.; Cassera, M. B.; Yue, J. M. Nanomolar Antimalarial Agents against Chloroquine-Resistant
Plasmodium falciparum from Medicinal Plants and Their Structure–Activity Relationships. J. Nat.
Prod. 2017, 80, 96-107.
(4) (a) Yuan, C.; Du, B.; Deng, H.; Yi, M.; Liu, B. Total Syntheses of SarcandrolideꢀJ and Shizukaolꢀ
D: Lindenane Sesquiterpenoid [4+2] Dimers. Angew. Chem. Int. Ed. 2017, 56, 637-640. (b) Wu, J. L.;
Lu, Y. S.; Tang, B.; Peng, X. S. Total Syntheses of Shizukaols A and E. 2018, 9, 4040-4046. (c). Yang
G. X., Ma G. L., Li H., Huang T., Xiong J., Hu J. F. Advanced Natural Products Chemistry Research in
China between 2015 and 2017. Chin J. Nat. Med. 2018, 16, 0881-0906.
(5) (a) Chi, J.; Xu, W. J.; Wei, S. S.; Wang, X. B.; Li, J. X.; Gao, H. L.; Kong, L. Y.; Luo, J.
Chlotrichenes A and B, Two Lindenane Sesquiterpene Dimers with Highly Fused Carbon Skeletons
from Chloranthus holostegius. Org. Lett. 2019, 21, 789-792. (b) Wang, P.; Li, R. J.; Liu, R. H.; Jian, K.
L.; Yang, M. H.; Yang, L.; Kong, L. Y.; Luo, J. Sarglaperoxides A and B, Sesquiterpene-
Normonoterpene Conjugates with a Peroxide Bridge from the Seeds of Sarcandra glabra. Org. Lett.
2
016, 18, 832-835.
(6) (a) Sumioka, H.; Harinantenaina, L.; Matsunami, K.; Otsuka, H.; Kawahata, M.; Yamaguchi, K.
Linderolides A–F, Eudesmane-type Sesquiterpene Lactones and Linderoline, a Germacrane-type
ACS Paragon Plus Environment

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Page 22 of 23
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Sesquiterpene from the Roots of Lindera strychnifolia and Their Inhibitory Activity on NO Production
in RAW 264.7 Cells in Vitro. Phytochemistry 2011, 72, 2165-2171. (b) Huong, T. T.; Thong, N. V.;
Minh, T. T.; Tram, L. H.; Anh, N. T.; Cuong, H. D.; Cuong, P. V.; Ca, D. V. Chloranerectuslactone V
(I), a New Sesquiterpene from Chloranthus erectus Verdc. Lett. Org. Chem. 2014, 11, 639-642.
(7) Guo, Y. Q.; Tang, G. H.; Li, Z. Z.; Lin, S. L.; Yin, S. Chlojapolactone A, an Unprecedented 1,3-
Dioxolane Linked-lindenane Sesquiterpenoid Dimer from Chloranthus japonicus. RSC Adv. 2015, 5,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
03047-103051.
8) (a) Xia, N. H.; Joël, J. Flora of China; Science Press: Beijing, 1999, 4, 132. (b) Chinese
(
Pharmacopoeia Committee. Pharmacopoeia of People’s Republic of China, Part 1. The Medicine
Science and Technology Press of China, 2015, 223-224. (c) Tsai, Y. C.; Chen, S. H.; Lin, L. C.; Fu, S.
L. Anti-inflammatory Principles from Sarcandra glabra. J Agric Food Chem. 2017, 65, 6497-6505.
(
9) Monnier, J. R.; Peters, K. T.; Hartley, G. W. The selective epoxidation of conjugated olefins
containing allylic substituents and epoxidation of propylene in the presence of butadiene. J. Catal.
004, 225, 374-380.
10) Bryliakov, K. P. Catalytic Asymmetric Oxygenations with the Environmentally Benign
Oxidants H and O . Chem. Rev. 2017, 117, 11406 −11459.
11) (a) Margaros, I.; Montagnon, T.; Tofi, M.; Pavlakos, E.; Vassilikogiannakis, G. The power of
2
(
2
O
2
2
(
singlet oxygen chemistry in biomimetic syntheses. Tetrahedron 2006, 62, 5308-5317. (b)
Vassilikoginanakis, G.; Stratakis, M. Biomimetic Total Synthesis of Litseaverticillols A, C, D, F, and
G: Singlet-Oxygen-Initiated Cascades & dagger. Angew. Chem. 2003, 115, 5623-5626.
(12) Wang, K.; Meng, L. G.; Zhang, Q.; Wang, L. Direct Construction of 4-aryl Tetralones via
Visible-light-induced Cyclization of Styrenes with Molecular Oxygen. Green Chem. 2016, 18, 2864–
2
870.
13) Masaaki, U.; Yutaka, K.; Genjiro, K.; Yoshikazu, K.; Shigeo, N.; Chizuko, K.; Tsunematsu, T.
(
Studies on the Constituents of Chloranthus spp.Ⅲ. Six Sesquiterpenes from Chloranthus japonicus.
Chem. Pharm. Bull. 1980, 28, 92-102.
(14) (a) Zhang, C. X.; Wang, C. C.; Wang, Z. H.; Tanabe, G.; Muraoka, O.; Lin, A. J.; Xu, J. Y.; Wu
X. M.; Wu, L.; Xie, W. J. Total Synthesis, Structural Elucidation and Anti-inflammatory Activity
Evaluation of 2-deoxy-3,6-anhydro Hexofuranoside Derivatives Isolated from Sauropus rostratus. Org.
ACS Paragon Plus Environment

Page 23 of 23
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Biomol. Chem. 2016, 14, 10906-10913. (b) Camougis, G.; Takman, B. H.; Rene, J.; Tasse, P. Potency
difference between the zwitterion form and the cation forms of tetrodotoxin. Science 1967, 156, 1625-
1
627.
(15) Zhou, B.; Liu, Q. F.; Dalal, S.; Cassera, M. B.; Yue, J. M. Fortunoids A-C, Three
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Sesquiterpenoid Dimers with Different Carbon Skeletons from Chloranthus fortuna. Org. Lett. 2017,
19, 734-737.
(
16) Huang, J.; Ni, J.; Liu, K.; Yu, Y.; Xie, M.; Kang, R.; Vernon, P.; Cao, L.; Tang, D. HMGB1
Promotes Drug Resistance in Osteosarcoma. Cancer Res. 2012, 72, 230-238.
17) Soriano, A. F.; Helfrich, B.; Chan, D. C.; Heasley, L. E.; Jr, B. P.; Chou, T. C. Synergistic
(
Effects of New Chemopreventive Agents and Conventional Cytotoxic Agents against Human Lung
Cancer Cell Lines. Cancer Res. 1999, 59, 6178-6184.
(
18) Yang, L.; Wei, D. D.; Chen, Z.; Wang, J. S.; Kong, L. Y. Reversal of Multidrug Resistance in
Human Breast Cancer Cells by Curcuma wenyujin and Chrysanthemum indicum. Phytomedicine. 2011,
8, 710-718.
19) Yang, J.; Liu, R. H. Synergistic Effect of Apple Extracts and Quercetin 3-β-D-glucoside
1
(
Combination on Antiproliferative Activity in MCF-7 Human Breast Cancer Cells in vitro. J. Agric.
Food Chem. 2009, 57, 8581 -8586.
(20) Fujimoto, D.; Ueda, Y.; Hirono, Y.; Goi, T.; Yamaguchi, A. PAR1 Participates in the Ability of
Multidrug Resistance and Tumorigenesis by Controlling Hippo-YAP Pathway. Oncotarget. 2015, 6,
3
4788-34799.
21) Xia, Y. Z.; Yang, L.; Wang, Z. D.; Guo, C.; Zhang, C.; Geng, Y. D.; Kong, L. Y. Schisandrin A
(
Enhances the Cytotoxicity of Doxorubicin by the Inhibition of Nuclear Factor-kappa B Signaling in a
Doxorubicin-resistant Human Osteosarcoma Cell Line. Rsc Adv. 2015, 5, 13.
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