Triligustilides A and B: Two Pairs of Phthalide Trimers from Angelica sinensis with a Complex Polycyclic Skeleton and Their Activities

Article abstract of DOI:10.1021/acs.orglett.8b00017

Two pairs of enantiomeric phthalide trimers [(-)/(+) triligustilides A (1a/1b) and (-)/(+) triligustilides B (2a/2b)] with complex polycyclic skeletons simultaneously possessing bridged, fused, and spiro ring systems were isolated from Angelica sinensis, together with two pairs of new phthalide dimers. The biogenetic pathways of new phthalides were proposed, and their bioactivities were also evaluated. This is the first time optically pure polymeric phthalides have been obtained from racemates, and their absolute configurations are reported.

Full text of DOI:10.1021/acs.orglett.8b00017

Letter
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Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Triligustilides A and B: Two Pairs of Phthalide Trimers from Angelica
sinensis with a Complex Polycyclic Skeleton and Their Activities
Jian Zou,†,§ Guo-Dong Chen,†,§ Huan Zhao,†,‡ Ying Huang,† Xiang Luo,† Wei Xu,† Rong-Rong He,†
Dan Hu,† Xin-Sheng Yao,† and Hao Gao*,†
†Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy/Guangdong Province Key Laboratory of
Pharmacodynamic Constituents of TCM and New Drugs Research, and ‡College of Traditional Chinese Medicine, Jinan University,
Guangzhou 510632, People’s Republic of China
S
* Supporting Information
ABSTRACT: Two pairs of enantiomeric phthalide trimers [(−)/(+) triligustilides
A (1a/1b) and (−)/(+) triligustilides B (2a/2b)] with complex polycyclic
skeletons simultaneously possessing bridged, fused, and spiro ring systems were
isolated from Angelica sinensis, together with two pairs of new phthalide dimers.
The biogenetic pathways of new phthalides were proposed, and their bioactivities
were also evaluated. This is the first time optically pure polymeric phthalides have
been obtained from racemates, and their absolute configurations are reported.
Angelica sinensis (Apiaceae) is a perennial herb mainly cultivated
in Gansu, Yunnan, and Sichuan Provinces in China.1 Its dried
root was documented in the ancient Chinese materia medica
book “Shen Nong’s Herbal Classic” in the Qin Han dynasty (300
A.D.)1 and has been used as a famous traditional Chinese
medicine (TCM) as Dang-Gui (Angelicae Sinensis Radix).
According to classical works, it was commonly used for the
treatments of gynecological disorders (e.g., menstrual disorders,
amenorrhea, dysmenorrhea, anemia, premenstrual syndrome,
and menopause).2 Modern pharmacology reports have demon-
strated that it possesses a series of bioactivities, including
antiplatelet aggregation, anti-inflammatory, anticancer, and
neuroprotective effects.3 It is not only one of the most famous
medicinal plants but also one of the most popular edible plants,
which was widely marketed as a health food for women’s care in
Asia and as a dietary supplement in Europe and America.4
A large number of phytochemical investigations have been
carried out on A. sinensis, and over 150 compounds have been
found from it, including phthalides, phenylpropanoids, terpe-
noids, alkynes, alkaloids, and other constituents.5 Among them,
phthalides (such as Z-ligustilide, Z-butylidenephthalide, and n-
butylphthalide) are the major and characteristic constituents in
Dang-Gui.6 Modern pharmacology reports have shown that
phthalides exhibit diverse bioactivities, including antiasthma,
anticonvulsion, inhibition of platelet aggregation, enhancement
of blood flow volume, etc.7 In particular, n-butylphthalide (NBP)
has been approved as a modern drug for the treatment of
ischemic stroke by the State Food and Drug Administration of
China in 2005.8 Moreover, phthalides exhibit abundant structural
diversity, which not only originates from the derivatization of
monomeric phthalides such as hydroxylation, glycosidation, and
esterification6 but also depends on the dimerization ([2 + 2] or
[4 + 2] cycloaddition) with diverse linkage styles forming a
number of complex polycyclic skeletons with multichiral
centers.9 Phthalides have attracted much attention due to their
various bioactivities and complicated chemical structures. To
date, the reported polymeric phthalides deriving from achiral
monomers exist in a racemic manner. However, chiral separation
of these racemic polymeric phthalides has not been carried out
yet. Thus, their corresponding optically pure polymeric
phthalides have not been obtained. Their absolute configurations
remain unsolved.
To search for interesting phthalides from nature, establish the
absolute configurations of polymeric phthalides, and explore
their bioactivities, a chemical investigation on Dang-Gui was
carried out. The dried root of A. sinensis were extracted with
EtOH−H2O (95:5 v/v). The extract was separated by
continuously different column chromatography leading to the
isolation of two pairs of novel enantiomeric trimeric phthalides
[(−) triligustilide A (1a)/(+) triligustilide A (1b), and (−)
triligustilide B (2a)/(+) triligustilide B (2b)], two pairs of new
related enantiomeric dimeric phthalides [(+) tokinolide A (3a)/
(−) tokinolide A (3b), and (−) riligustilide (4a)/(+) riligustilide
(4b)], and the precursor [Z-ligustilide (5)] (Figure 1). Among
them, the trimeric phthalides (1a, 1b, 2a, and 2b) possess new
and complex polycyclic skeleton, which was composed of three
Z-ligustilide (5) units in an unique linkage style (2−4′/7−3′,
3″−8′/4′′−9′). Herein, we describe the isolation, structure
elucidation, and bioassays (including anti-inflammatory, anti-
platelet aggregation, cytotoxic and antimicrobial activities) of all
phthalides (1−5). Moreover, the plausible biogenetic pathways
of 1−4 are proposed.
Triligustilide A (1) was isolated as colorless crystals. The
molecular formula of 1 was established as C36H42O6 (16 degrees
of unsaturation) from its HR-ESI-MS (m/z 593.2892 [M + Na]+,
calcd for C36H42O6Na 593.2879). The 13C NMR spectrum
Received: January 3, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b00017
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Letter
structure was established as shown in Figure 2. The geometries of
the double bonds of Δ8 and Δ8″ in 1 were assigned as Z
configurations by the NOESY correlations between H-9 and Ha-
6, between H-9″ and Ha-6′′, and between H-9′′ and Hb-6′′. The
assignments of all proton and carbon resonances are provided in
Table S1. In the bicyclo[4.2.0]octane system, the relative
configurations of H-3″ and H-4″ should be fixed as 3″R*,4″S*.
Furthermore, the NOESY correlations between H-3 and H-4′,
between Ha-6 and H-3′, between Hb-6 and H-3′, between Ha-10
and Hb-11′, between Hb-10 and Hb-11′, between Hb-6′ and H-
9′, and between Hb-10′ and H-4″ indicated that the relative
configuration of 1 was 2S*,7R*,3′R*,4′S*,8′S*,9′S*,3″R*,4″S*
(Figure 3). The single-crystal X-ray diffraction experiment
(Figure 4) confirmed the above deduction.
Figure 3. Key NOESY correlations of 1 and 2.
Figure 1. Chemical structures of 1−4.
showed 36 carbon signals. Combined with the DEPT-135
experiment, these carbons can be categorized as three carbonyl
carbons (δC 175.7, 171.6, 167.0), 10 aromatic or olefinic carbons,
three sp3 quaternary carbons [including one oxygenated (δC
92.2)], five sp3 methine carbons, 12 sp3 methylene carbons, and
1
three methyl carbons. In the H NMR spectrum of 1, the
characteristic protons for three methyl groups [δH 0.83 (t, J = 7.3
Hz), 0.88 (t, J = 7.2 Hz), and 0.95 (t, J = 7.4 Hz)] and four olefinic
protons [δH 4.66 (dd, J = 8.4, 6.5 Hz), 5.33 (t, J = 7.9 Hz), 5.92
(br d, J = 9.7 Hz), and 6.06 (ddd, J = 9.7, 4.9, 3.6 Hz)] were
observed. All of the proton resonances were assigned to relevant
carbon atoms through the HSQC experiment. The H−1H
1
Figure 4. X-ray crystallographic analyses of 1 and 2.
COSY data of 1 revealed the presence of five subunits as shown in
bold red lines in Figure 2. The key HMBC correlations (Figure
2) revealed the partial structure of 1. On the basis of the
molecular formula information, the degree of unsaturation, and
the above analyses of 1H−1H COSY and HMBC data, the planar
Triligustilide B (2) was isolated as colorless crystals. The
molecular formula of 2 was established as C36H42O6 (16 degrees
of unsaturation) from its HR-ESI-MS (m/z 593.2875 [M + Na]+,
calcd for C36H42O6Na 593.2879). The NMR data of 2 are very
similar to those of 1. Further detailed NMR analyses involving
1H−1H COSY and HMBC established the same planar structure
as that of 1. The geometries of the double bonds of Δ8 and Δ8″ in
2 were assigned as Z configurations using the NOESY
correlations between H-9 and Ha-6, between H-9″ and H-6′′a,
and between H-9′′ and H-6′′b. The assignments of all proton
and carbon resonances are provided in Table S2. In the
bicyclo[4.2.0]octane system, the relative configurations of H-3″
and H-4″ should be 3″S*,4″R*. Furthermore, the NOESY
correlations between H-3 and H-4′, between Ha-6 and H-3′,
between Hb-6 and H-3′, between Ha-10 and H2-11′, between
Figure 2. Key 1H−1H COSY and HMBC correlations of 1.
B
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Letter
Hb-10 and H2-11′, between H2-6′ and H-9′, between H2-6′ and
H-3″, between H-9′ and H-3′′, and between Hb-10′ and Hb-5′′
revealed the relative configuration of 2 to be 2S*,7R*,3′R*,-
4′S*,8′S*,9′S*,3″S*,4″R* (Figure 3). The above result was
confirmed by the single-crystal X-ray diffraction experiment
(Figure 4).
Compound 3 was isolated as a yellow oil. The molecular
formula of 3 was established as C24H28O4 (11 degrees of
unsaturation) from its HR-ESI-MS (m/z 381.2060 [M + H]+,
calcd for C24H29O4 381.2066). Compound 3 was identified as
tokinolide A by comparison of the NMR data with literature
values.10 Detailed analyses of the 1D and 2D NMR data of 3
confirmed the above deduction, and the relative configuration of
3 was first determined as 2S*,7R*,3′R*,4′S* (Table S3).
Compound 4 was isolated as colorless crystals. The molecular
formula of 4 was established as C24H28O4 (11 degrees of
unsaturation) from its HR-ESI-MS (m/z 381.2067 [M + H]+,
calcd for C24H29O4 381.2066). Compound 4 was identified as
riligustilide by comparison of the NMR data with literature
values.9b The single-crystal X-ray diffraction experiment
confirmed the above deduction, and unambiguously determined
the relative configuration of 4 as 3S*,4R*,8′S*,9′S* (Table S4).
The specific optical rotations of 1−4 were close to zero,
indicating that 1−4 might be mixtures of enantiomers. This
deduction was confirmed by the observations of 1−4 in the chiral
HPLC analyses. These four pairs of enantiomers [(−)-1a/
(+)-1b, (−)-2a/(+)-2b, (+)-3a/(−)-3b, and (−)-4a/(+)-4b]
were obtained using a Phenomenex Amylose-2 column (5 μm,
250 × 4.6 mm) (Figure S1). Moreover, the quantum chemical
calculations of the electronic circular dichroic (ECD) spectra
were used to determine the absolute configurations of 1a and 1b.
Since the flexible side chains that are far away from the chiral
carbons in 1 had an insignificant effect on the ECD spectrum,11
the simplified structures of (2S,7R,3′R,4′S,8′S,9′S,3″R,4″S)-1′
and (2R,7S,3′S,4′R,8′R,9′R,3″S,4″R)-1′ (Figure S6) were used
for the ECD calculations. The predicted ECD curves of
(2S,7R,3′R,4′S,8′S,9′S,3″R,4″S)-1′ and (2R,7S,3′S,4′R,8′R,9′R,-
3″S,4″R)-1′ were almost identical to the experimental ones of 1a
and 1b, respectively. Therefore, the absolute configurations of 1a
and 1b were established as (2S,7R,3′R,4′S,8′S,9′S,3″R,4″S)-1a
and (2R,7S,3′S,4′R,8′R,9′R,3″S,4″R)-1b, respectively (Figure 1).
By the same procedure of ECD calculations as that of 1 (Figure
S8), the absolute configurations of 2a and 2b were determined as
(2S,7R,3′R,4′S,8′S,9′S,3″S,4″R)-2a and (2R,7S,3′S,4′R,8′R,9′R,-
3″R,4″S)-2b, respectively (Figure 1). The absolute configu-
rations of 3a and 3b, 4a and 4b were established as
(2S,7R,3′R,4′S)-3a and (2R,7S,3′S,4′R)-3b, (3S,4R,8′S,9′S,)-4a
and (3R,4S,8′R,9′R)-4b by the same procedure as that of 1,
respectively (Figures S10 and S12).
Figure 5. Effects of compounds 1, 4, and 5 and on the inhibition of
TNF-α (a) and IL-6 (b). The data represent the mean SD of three
experiments. *p < 0.1, **p < 0.01, ***p < 0.001 compared with the LPS
group; #p < 0.001 compared with the control group.
indicated that polymerization would weaken the activity of
inhibition of platelet aggregation (Table 1).
Table 1. Inhibition of Platelet Aggregation in Vitro Induced
by AA of compounds 1−5
compd
inhibit rate (100 μM)
compd
inhibit rate (100 μM)
1
2
3
31.4 5.0
27.1 5.2
41.7 5.9
4
45.1 4.6
89.9 0.2
95.4 0.2
5
aspirin
Furthermore, compounds 1−5 were also evaluated for
cytotoxicities against four human tumor cell line (SW480,
PANC-1, MCF-7, and HepG2) and antimicrobial assays against
two bacteria (Staphylococcus aureus 209P, Escherichia coli
ATCC0111) and two fungi (Canidia albicans FIM709,
Aspergillus niger R330). None of the compounds possessed
cytotoxic and antimicrobial activities (Tables S9 and S11).
As the major and characteristic constituents in Dang-Gui,
phthalides not only show a variety of bioactivities but also possess
abundant structural diversity.6c In particular, a large number of
novel dimeric phthalides are isolated from Dang-Gui, such as
E232, gelispiroloide, and sinaspirolide.9 In this present research,
two pairs of novel enantiomeric trimeric phthalides 1−2 were
isolated. These were composed of three Z-ligustilide (5) units in
an unprecedented linkage style (2−4′/7−3′, 3″−8′/4′′−9′)
forming complex polycyclic skeleton simultaneously possessing
bridged-ring, fused-ring, and spiro-ring systems. In addition, two
pairs of new biosynthetically related enantiomeric dimeric
phthalides 3−4 were also obtained. Furthermore, bioactivities
screening showed that major monomeric phthalide Z-ligustilide
(5) had significant inhibition of platelet aggregation, and all
compounds displayed different levels of anti-inflammatory
effects, which indicatied that monomeric and polymeric
phthalides are the beneficial constituents for the classical effects
of Dang-Gui.
The plausible biogenetic pathways of 1−4 are proposed as
shown in Scheme 1. Dimeric phthalides (tokinolide A (3) and
riligustilide (4)) with two different linkages (type A, 2−4′/7−3′
for 3; type B, 3−8′/4−9′ for 4) derive from two molecules of Z-
ligustilide (5) through [2 + 2] cycloaddition,12 whereas
triligustilides A (1) and B (2) with a new linkage style (type
(A + B): 2−4′/7−3′, 3″−8′/4′′−9′) originate from three
molecules of Z-ligustilide (5) through [2 + 2] /[2 + 2]
cycloaddition. The structural shapes of polymeric phthalides are
intriguingly similar to two or three flying Apsaras getting
together. For the aesthetic representation of 1−2, see Figure S14.
According to classical effects of Dang-Gui and the established
bioassay models in our group, bioassays (including anti-
inflammatory, antiplatelet aggregation, cytotoxic, and antimicro-
bial activities) of all phthalides have been carried out.
In the anti-inflammatory assay in LPS-stimulated RAW264.7
macrophages, compounds 1−5 exhibited different levels of anti-
inflammatory effects against the production of the pro-
inflammatory cytokines (TNF-α, IL-6). Among them, com-
pounds 1, 4, and 5 showed inhibition in a dose-dependent
manner (Figure 5). All compounds were also evaluated for their
inhibition in vitro against platelet aggregation in rat plasma
induced with arachidonic acid (AA). Compound 5 exhibited
significant antiplatelet aggregation effects, and the results
C
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Scheme 1. Plausible Biosynthetic Pathways of Compounds 1−
4
ACKNOWLEDGMENTS
■
This work was financially supported by grants from the National
Natural Science Foundation of China (81422054 and
3171101305), Chang Jiang Scholars Program (Hao Gao, 2017)
from the Ministry of Education of China, the Guangdong Natural
Science Funds for Distinguished Young Scholar
(S2013050014287), Guangdong Special Support Program
(2016TX03R280), Guangdong Province Universities and
Colleges Pearl River Scholar Funded Scheme (Hao Gao,
2014), K. C. Wong Education Foundation (Hao Gao, 2016),
and the high-performance computing platform of Jinan
University. We are grateful to Prof. M. Banwell at the University
of Australian National for the biogenetic pathway advices.
Assistance with the proper usage of scientific English was
provided by Dr. L. J. Sparvero at the University of Pittsburgh.
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* Supporting Information
■
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tghao@jnu.edu.cn.
ORCID
Xin-Sheng Yao: 0000-0003-1603-4873
Hao Gao: 0000-0003-1178-0121
Author Contributions
§J.Z. and G.-D.C. contributed equally.
Notes
The authors declare no competing financial interest.
D
DOI: 10.1021/acs.orglett.8b00017
Org. Lett. XXXX, XXX, XXX−XXX