Nitrogen-containing dihydro-β-agarofuran derivatives from Tripterygium wilfordii

Article abstract of DOI:10.1021/np500280x

Thunder god vine, the dried roots of Tripterygium wilfordii, is a widely used traditional Chinese medicine. More than 200 bioactive complex natural products have been isolated from this herb. Inspired by the diversity of chemical structures and bioactivities of the components of this herb, the investigation to mine new chemical entities as potential drug leads led to the identification of 36 nitrogen-containing compounds. Among them, 18 new dihydro-β-agarofuran alkaloids (tripterygiumines A-L (1-12), M-Q (22-26), and R (33)) were identified from the spectroscopic data and chemical degradation studies. Tripterygiumine Q (26) exhibited immunosuppressive activity against human peripheral mononuclear cells with an IC₅₀ value of 8.67 μM and showed no cytotoxicity, even at 100 μM, indicating that 26 may represent a novel scaffold for the development of new immunosuppressants.

Full text of DOI:10.1021/np500280x

Article
pubs.acs.org/jnp
Nitrogen-Containing Dihydro-β-agarofuran Derivatives from
Tripterygium wilfordii
Yinggang Luo,*,† Xiang Pu,†,‡ Guoyong Luo,†,‡ Min Zhou,† Qi Ye,† Yan Liu,† Jian Gu,† Huayi Qi,†
Guoyou Li,† and Guolin Zhang*,†
†Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, 9 Section 4, Renmin Road
South, Chengdu 610041, People’s Republic of China
‡University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, People’s Republic of China
S
* Supporting Information
ABSTRACT: Thunder god vine, the dried roots of Tripterygium wilfordii,
is a widely used traditional Chinese medicine. More than 200 bioactive
complex natural products have been isolated from this herb. Inspired by
the diversity of chemical structures and bioactivities of the components of
this herb, the investigation to mine new chemical entities as potential drug
leads led to the identification of 36 nitrogen-containing compounds.
Among them, 18 new dihydro-β-agarofuran alkaloids (tripterygiumines
A−L (1−12), M−Q (22−26), and R (33)) were identified from the
spectroscopic data and chemical degradation studies. Tripterygiumine Q
(26) exhibited immunosuppressive activity against human peripheral
mononuclear cells with an IC50 value of 8.67 μM and showed no
cytotoxicity, even at 100 μM, indicating that 26 may represent a novel
scaffold for the development of new immunosuppressants.
he air-dried root of Tripterygium wilfordii (Celastraceae),
Tknown as thunder god vine, is a traditional Chinese
medicine that has been widely used for more than 400 years to
treat inflammation, cancers, and autoimmune diseases,
including rheumatoid arthritis, multiple sclerosis, and lupus.1
A number of sesqui-, di-, and triterpenoids, steroids, and
sesquiterpenoid pyridine alkaloids exhibiting beneficial and/or
adverse effects have been isolated from this plant.2 The
sesquiterpenoid pyridine alkaloids possess a characteristic
macrocyclic dilactone skeleton consisting of a dicarboxylic
acid moiety, 2-(carboxyalkyl)nicotinic acid, and a polyoxy-
genated dihydro-β-agarofuran sesquiterpenoid. The hydroxy
groups of the latter are usually esterified by various organic
acids including acetic, benzoic, furanoic, nicotinic, and cinnamic
acids. The 2-(carboxyalkyl)nicotinic acid moiety originates
from evoninic acid, wilfordic acid, hydroxywilfordic acid, or
their congeners.2
Our previous phytochemical investigation of aqueous EtOH
extracts of the dried roots of T. wilfordii yielded five new and 13
known alkaloids.3 Some of these alkaloids exhibited moderate
inhibition against herpes simplex virus type II.3 As a
continuation of the chemical studies of Tripterygium alkaloids
to mine new chemical entities with drug potential, 36
sesquiterpenoid alkaloids, of which 18 are new nitrogen-
containing derivatives, were identified. Herein, the isolation and
structure elucidation of the 18 new alkaloids are reported. The
immunosuppressive activities of these alkaloids were evaluated
for their therapeutic potential.
RESULTS AND DISCUSSION
■
The CHCl3-soluble extract of T. wilfordii was separated by
repeated column chromatography on silica gel. Semipreparative
HPLC of the subfractions afforded 36 compounds. The
detailed extraction and isolation procedures of each compound
are presented in the Supporting Information.
Compound 1 was isolated as a white powder. The molecular
formula of C50H57NO21 was determined by 13C NMR (Table
1) and HRESIMS data [m/z 1030.3320 ([M + Na]+)] and
revealed 23 indices of hydrogen deficiency. The IR spectrum
indicated the presence of hydroxy (3437 cm−1), carbonyl (1740
cm−1), and pyridine (1631 cm−1) groups, and the UV spectrum
suggested the presence of aromatic groups (244 and 262 nm).
The NMR spectroscopic data (Table 1) indicated the presence
of three acetyls, two oxygenated methylenes, three aliphatic
methylenes, five aliphatic methines, six oxygenated methines,
five methyls, one methoxy, one 2,3-disubstituted pyridine, and
one benzoyl group. In addition to the resonances of the
aforementioned groups, resonances for a ketocarbonyl, five
ester carbonyls, three oxygenated quaternary carbons, and one
aliphatic quaternary carbon were observed in the 13C NMR
spectrum of 1. A polyoxygenated dihydro-β-agarofuran skeleton
was established from the 1H−1H COSY cross signals for the H-
1/H-2/H-3 and H-5/H-6/H-7/H-8 coupling systems and the
following HMBC correlations: H-1/C-9; H-2/C-9; H-3/C-4,
Received: March 28, 2014
Published: June 25, 2014
© 2014 American Chemical Society and
American Society of Pharmacognosy
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C-10; H-5/C-7, C-10, C-13; H-6/C-7; H-7/C-9; H-8/C-1, C-9,
C-11; H-11/C-8, C-9; H-12/C-3, C-4, C-10; H-14/C-6, C-13,
C-15; and H-15/C-13, C-14 (Figure 1A). A 2-(3-carboxybutan-
2-yl)nicotinic acid moiety was established based on the 2,3-
disubstituted pyridine and the −CH(CH3)−CH(CH3)−
moieties corresponding to the HMBC correlations of H-4′/
C-2′, C-6′, C-12′; H-5′/C-3′; H-6′/C-4′; H-7′/C-2′, C-8′, C-
9′, C-11′; H-8′/C-2′, C-11′; H-9′/C-2′, C-7′, C-8′; and H-10′/
C-7′, C-8′, C-11′ (Figure 1A). A monoterpene tricarboxylic
H-2/C-72‑OBz (Figure 1A). A free hydroxy group was assigned
to C-4 by comparing the 13C NMR chemical shift of C-4 (δC
70.50) with the reported value3,4 and was confirmed by the
HMBC cross signals of 4-OH/C-12 and H-12/C-3, C-4, C-10
(Figure 1A).
The relative configuration of the polyoxygenated dihydroa-
garofuran moiety of 1 was established by the NOESY
correlations of H-8/H-1, H-14; H-5/H-11, H-12; and H-11/
H-12 (Figure 1B). Owing to the flexibility of the macrocyclic
rings, the relative configurations of the substituted pyridine and
the monoterpene tricarboxylic acid moieties could not be
determined by NOESY experiments. However, J-based
configuration analysis is a powerful tool for the stereochemical
determination of macrocyclic structures.5 Coupling constants
were calculated using HETLOC6 and HSQC-HECADE7
techniques. The C-7′−C-10′ and C-2″−C-4″ moieties are
characteristic of the 2,3-dimethyl- and 2-methylbutane systems,
1
acid moiety (C-1″−C-10″) was established from the H−1H
COSY cross signals for the H-10″/H-2″/H-3″/H-4″ coupling
systems and the following HMBC correlations: H-10″/C-1″, C-
3″; H-4″/C-2″, C-5″; H-6″/C-5″, C-7″, C-8″, C-9″; and H-7″/
C-5″, C-6″, C-8″, C-9″ (Figure 1A). These data suggested that
compound 1 was a dimacrocyclic sesquiterpenoid pyridine
alkaloid. The HMBC cross signals of H-3/C-11′ and H-15/C-
12′ confirmed the C-3−O−C-11′ and C-15−O−C-12′ link-
ages, respectively, for the first macrocyclic system between the
polyoxygenated dihydro-β-agarofuran and the substituted
nicotinic acid moieties (Figure 1A). The linkages of the second
macrocyle were between the dihydro-β-agarofuran and the
monoterpene tricarboxylic acid moiety at C-7−O−C-1″ and C-
11−O−C-9″ and were established from the HMBC cross
signals of H-7/C-1″ and H-11/C-9″, respectively (Figure 1A).
The third carboxy group of the tricarboxylic acid moiety was a
methyl ester, as confirmed by the HMBC correlation of OCH3/
C-8″ (Figure 1A). The 1-OAc, 5-OAc, 8-OAc, and 2-OBz
substituents were determined from the corresponding HMBC
correlations of H-1/C-21‑OAc, H-5/C-25‑OAc, H-8/C-28‑OAc, and
3
2,3
respectively. The JH,H and
J
C,H
coupling constants of the C-
7′−C-10′ moiety are summarized in Figure 1C. Comparison of
3
2,3
the reported JH,H and
J
C,H
values for the 2,3-disubstituted
and 2-substituted butane systems with methyl/hydroxy,
alkyloxy, or acyloxy groups permitted the deduction of the
relative configurations of the C-7′−C-10′ and C-2″−C-4″
moieties, which were consistent with those of triptonine A. The
relative configuration of triptonine A had been previously
established by X-ray crystallography analysis.8a Thus, com-
pound 1 was identified as 2-de-O-acetyl-2-O-benzoyltriptonine
A and was named tripterygiumine A. Besides triptonines A and
B,8 1 is the third reported dimacrocyclic structure in which the
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a
Table 1. NMR Spectroscopic Data for Tripterygiumine A (1)
position
δH (J in Hz)
δC, type
position
10′
11′
12′
1″
δH (J in Hz)
δC, type
9.69, CH3
173.87, C
1
2
3
4
5
6
5.68, d (4.6)
5.53, m
73.82, CH
69.83, CH
75.78, CH
70.50, C
1.20, d (7.2)
4.85, d (2.4)
168.53, C
175.67, C
37.32, CH
28.06, CH2
7.14, s
73.41, CH
50.22, CH
2″
3″
2.62, m
1.98, m
1.83, m
3.47, m
2.97, m
2.51, d (3.6)
7
5.52, m
69.18, CH
4″
42.12, CH2
8
5.37, d (6.1)
70.86, CH
52.01, C
5″
6″
7″
8″
204.19, C
51.92, CH
32.64, CH2
171.82, C
9
4.82, t (7.3)
3.07, d (7.4)
10
11
94.07, C
5.67, d (13.6)
4.36, d (13.6)
1.63, s
62.13, CH2
12
13
14
15
22.71, CH3
84.50, C
9″
10″
4-OH
167.84, C
1.19, d (7.0)
4.61, s
18.00, CH3
1.72, s
18.54, CH3
69.65, CH2
6.04, d (11.7)
3.74, d (11.6)
8″-OMe
3.70, s
52.0, CH3
2′
3′
4′
5′
6′
7′
8′
9′
165.43, C
1-OAc
1.79, s
2.26, s
1.92, s
20.34, CH3; 168.94, C
21.67, CH3; 170.15, C
20.36, CH3; 168.66, C
124.97, C
124.97, C
5-OAc
8.09, dd (7.7, 1.4)
7.28, dd (7.7, 4.8)
8.70, dd (4.8, 1.5)
4.68, q (6.8)
137.76,CH
121.09, CH
151.53, CH
36.41, CH
45.03, CH
11.74, CH3
8-OAc
2-OBz-1
2-OBz-2,6
2-OBz-3,5
2-OBz-4
2-OBz-7
8.12, d (7.5)
7.50, t (7.7)
7.62, t (7.5)
130.10, CH
128.70, CH
2.58, q (7.4)
133.72, CH
1.40, d (7.0)
164.73, C
a
Recorded in CDCl3 at 600 MHz for 1H and 150 MHz for 13C NMR spectra. Chemical shifts and coupling constants (in parentheses) are given in
ppm and Hz, respectively.
polyoxygenated sesquiterpene is esterified with a monoterpene
carboxylic acid.
indices of hydrogen deficiency. The IR and UV data of 3 were
similar to those of 2. Comparison of the NMR spectroscopic
data of 3 (Tables S1 and S2) with those of 2 suggested that two
acetyl groups and one benzoyl group in 2 were replaced by two
benzoyl groups and one acetyl group, respectively, in 3. 2D
NMR experiments established the planar structure of 3 (Figure
S1A). The relative configuration of the polyoxygenated
dihydroagarofuran moiety of 3 was established by the
NOESY correlations of H-8/H-1, H-14; H-5/H-11, H-12;
and H-11/H-12 (Figure S1B). The relative configuration of the
2-(3-carboxybutan-2-yl)nicotinic acid was determined by
comparison of the NMR data of 3 with those of 1 and 2
based on their similar biosynthetic origins.
Compound 4 (tripterygiumine D) was shown to have the
molecular formula C33H39NO13 by 13C NMR (Table S2) and
HRESIMS data [m/z 680.2293 ([M + Na]+)]. The IR and UV
data of 4 were similar to those of 2. Comparison of the NMR
spectroscopic data of 4 (Tables S1 and S2) with those of 2 and
3 revealed that only the 5-OH group was benzoylated in 4. The
planar structure and relative configuration of 4 were established
by the methods similar to those used for 3 (Figure S1).
Alkaloid 5 (tripterygiumine E) was shown to have the
molecular formula C39H45NO18 by 13C NMR (Table S2) and
HRESIMS data [m/z 838.2553 ([M + Na]+)]. The IR and UV
data of 5 were similar to those of 2. Comparison of the NMR
spectroscopic data of 5 (Tables S1 and S2) with those of 2
suggested that the 5-OH group was free. The 2-OAc and 8-OBz
groups in 2 were replaced with 2-OFu and 8-OAc groups,
respectively, in 5. The planar structure and relative config-
Tripterygiumine B (2) was shown to have the molecular
formula C43H49NO18 by 13C NMR (Table 2) and HRESIMS
data [m/z 890.2819 ([M + Na]+)], revealing 20 indices of
hydrogen deficiency. The IR, UV, and NMR spectroscopic data
(Table 2) of 2 were similar to those of 1 and the alkaloids
reported previously.3 The same polyoxygenated dihydro-β-
agarofuran and 2-(3-carboxybutan-2-yl)nicotinic acid moieties
as those in 1 were established on the basis of spectroscopic data
analysis (Figure 1D). The HMBC cross signals of H-3/C-11′
and H-15/C-12′ confirmed the C-3−O−C-11′ and C-15−O−
C-12′ linkages between the dihydro-β-agarofuran and the
substituted nicotinic acid moieties, respectively. A free hydroxy
group was assigned to C-4 by comparison of the 13C NMR
chemical shift of C-4 (δC 70.68) with the reported value3,4 and
was confirmed by the HMBC cross signals of 4-OH/C-12 and
H-12/C-3, C-4, C-10 (Figure 1D). The presence of 1-OAc, 2-
OAc, 5-OAc, 8-OAc, and 7-OBz substituents was determined
on the basis of the corresponding HMBC correlations of H-1/
C-21‑OAc, H-2/C-22‑OAc, H-5/C-25‑OAc, H-8/C-28‑OAc, and H-7/
C-77‑OBz (Figure 1D). The relative configuration of the
polyoxygenated dihydroagarofuran moiety of 2 was established
by the NOESY correlations of H-8/H-1, H-14; H-5/H-11, H-
12; and H-11/H-12 (Figure 1E). The relative configuration of
the C-7′−C-10′ moiety was determined by J-based config-
uration analysis (Figure 1F).
Compound 3, tripterygiumine C, was shown to have the
molecular formula C48H51NO18 by 13C NMR (Table S2) and
HRESIMS data [m/z 952.2998 ([M + Na]+)], revealing 24
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1
Figure 1. Key HMBC (curved arrows), H−1H COSY (bold bonds), and NOESY (curved double arrows) correlations and J-based configuration
analysis (Newman projection) of alkaloids 1, 2, and 22.
uration of 5 were established by the methods described above
(Figure S1).
Alkaloid 8 was shown to have the molecular formula
C28H37NO13 by 13C NMR (Table S2) and HRESIMS data [m/
z 618.2165 ([M + Na]+)]. Comparison of the NMR
spectroscopic data of 8 (Tables S1 and S2) with those of 4
suggested that the benzoyl group in 4 was replaced by an acetyl
group in 8. The planar structure and relative configuration of 8
were established by the methods described above (Figure S1).
The structure of 8 was identical to the methanolysis product of
cathedulin K2.9a However, only the 1H NMR data in methanol-
d4 were reported for the methanolysis product of cathedulin
K2. Herein, 8 is reported as a naturally occurring
sesquiterpenoid pyridine alkaloid and was named tripterygiu-
mine H.
Alkaloid 6 (tripterygiumine F) was shown to have the
molecular formula C46H49NO19 by 13C NMR (Table S2) and
HRESIMS data [m/z 942.2797 ([M + Na]+)]. The IR and UV
data of 6 were similar to those of 3. Comparison of the NMR
spectroscopic data of 6 (Tables S1 and S2) with those of 3
suggested that the C-5 benzoyl group in 3 was replaced by a
furanoyl group in 6. The planar structure and relative
configuration of 6 were established by the method described
above (Figure S1).
Alkaloid 7 (tripterygiumine G) was shown to have the
molecular formula C46H49NO19 by 13C NMR (Table S2) and
HRESIMS data [m/z 942.2783 ([M + Na]+)]. The
spectroscopic data (Tables S1 and S2) suggested that 7 was
isomeric with alkaloid 6. The spectroscopic data revealed that
alkaloid 7 possessed 2-OFu and 5-OBz substituents instead of
the 2-OBz and 5-OFu groups in 6 (Figure S1).
Alkaloid 9 (tripterygiumine I) was shown to have the
molecular formula C41H47NO17 by 13C NMR (Table S2) and
HRESIMS data [m/z 848.2772 ([M + Na]+)]. The IR and UV
data of 9 were similar to those of 5. Comparison of the NMR
spectroscopic data of 9 (Tables S1 and S2) with those of 5
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a
Table 2. NMR Spectroscopic Data for Tripterygiumine B (2)
position
δH (J in Hz)
δC, type
position
6′
7′
8′
9′
10′
11′
12′
4-OH
1-OAc
2-OAc
5-OAc
δH (J in Hz)
δC, type
151.50, CH
1
5.69, d (4.0)
5.52, m
73.22, CH
69.28, CH
75.73, CH
70.68, C
8.70, dd (4.8, 1.7)
4.66, q (6.9)
2.60, q (7.2)
1.39, d (7.0)
1.22, d (7.1)
2
36.48, CH
44.92, CH
11.90, CH3
9.70, CH3
173.88, C
168.47, C
3
4.86, d (2.3)
4
5
7.03, s
73.74, CH
50.58, CH
68.95, CH
70.46, CH
52.13, C
6
2.36, d (3.9)
5.54, m
7
8
5.40, d (6.0)
4.53, s
1.81, s
2.16, s
2.22, s
9
20.41, CH3; 169.10, C
20.98, CH3; 169.86, C
21.60, CH3; 170.01, C
10
11
94.04, C
5.45, d (13.4)
4.39, d (13.4)
1.66, s
60.47, CH2
12
13
14
15
23.26, CH3
84.17, C
8-OAc
1.94, s
2.28, s
20.41, CH3; 168.92, C
21.18, CH3; 170.33, C
128.90, C
11-OAc
7-OBz-1
7-OBz-2,6
1.70, s
18.52, CH3
69.87, CH2
5.97, d (11.6)
3.71, d (11.6)
8.10, d (7.3)
129.98, CH
2′
3′
4′
5′
165.36, C
125.07, C
137.71, CH
121.06, CH
7-OBz-3,5
7-OBz-4
7-OBz-7
7.51, t (7.7)
7.62, t (7.4)
128.72, CH
133.65, CH
164.81, C
8.07, dd (7.8, 1.7)
7.27, dd (7.8, 4.8)
a
Recorded in CDCl3 at 600 MHz for 1H and 150 MHz for 13C NMR spectra. Chemical shifts and coupling constants (in parentheses) are given in
ppm and Hz, respectively.
a
Table 3. NMR Spectroscopic Data for Tripterygiumine M (22)
position
δH (J in Hz)
δC, type
position
5′
6′
7′
δH (J in Hz)
δC, type
121.26, CH
1
2
3
5.89, d (3.4)
5.42, t (3.1)
5.21, d (3.0)
71.77, CH
69.85, CH
74.31, CH
7.29, dd (7.9, 4.7)
8.77, dd (4.7, 1.7)
4.12, m
153.68, CH
33.00, CH2
2.89, m
4
71.96, C
8′
2.39, m
33.44, CH2
1.90, m
5
5.37, brs
3.18, brs
75.90, CH
64.52, CH
197.61, C
78.70, CH
52.5, C
9′
10′
11′
12′
4-OH
5-OH
1-OAc
2.38, m
38.17, CH
18.92, CH3
174.99, C
167.14, C
6
1.22, d (6.4)
7
8
5.63, s
9
6.59, s
6.22, s
1.90, s
10
11
94.10, C
4.91, d (12.8)
4.69, d (12.8)
1.98, s
60.69, CH2
20.11, CH3; 169.30, C
12
13
14
15
23.94, CH3
86.37, C
8-OAc
1.99, s
1.99, s
20.48, CH3; 169.64, C
20.54, CH3; 169.33, C
128.56, C
11-OAc
2-OBz-1
2-OBz-2,6
1.63, s
19.33, CH3
71.33, CH2
5.92, d (12.4)
3.78, d (12.4)
8.02, d (7.3)
129.73, CH
2′
3′
4′
164.97, C
123.63, C
138.65, CH
2-OBz-3,5
2-OBz-4
2-OBz-7
7.64, t (7.4)
7.52, t (7.8)
128.94, CH
133.94, CH
165.32, C
8.37, dd (7.9, 1.7)
a
Recorded in CDCl3 at 600 MHz for 1H and 150 MHz for 13C NMR spectra. Chemical shifts and coupling constants (in parentheses) are given in
ppm and Hz, respectively.
suggested that the furanoyl group in 5 was replaced by a
benzoyl group in 9. The planar structure and relative
configuration of 9 were established using the methods
described above (Figure S1).
Alkaloid 10 (tripterygiumine J) was shown to have the
molecular formula C34H43NO16 by 13C NMR (Table S2) and
HRESIMS data [m/z 744.2480 ([M + Na]+)]. Comparison of
the NMR spectroscopic data of 10 (Tables S1 and S2) with
those of 9 suggested that the benzoyl group in 9 was replaced
by an acetyl group in 10, with free 5-OH and 7-OH groups.
The planar structure and relative configuration of 10 were
established by the methods described above (Figure S1).
Alkaloid 11 (tripterygiumine K) was shown to have the
molecular formula C39H45NO16 by 13C NMR (Table S2) and
HRESIMS data [m/z 806.2638 ([M + Na]+)]. Comparison of
the NMR spectroscopic data of 11 (Tables S1 and S2) with
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those of 9 suggested that the 11-OAc group was replaced by a
benzoyl group in 11, as well as free hydroxy groups at C-2 and
C-5. The planar structure and relative configuration of 11 were
established by the methods described above (Figure S1).
Alkaloid 12 (tripterygiumine L) was shown to have the
molecular formula C40H46N2O17 by 13C NMR (Table S2) and
HRESIMS data [m/z 849.2725 ([M + Na]+)]. Comparison of
the NMR spectroscopic data of 12 (Tables S1 and S2) with
those of 9 revealed that the 1-OAc and 2-OBz groups of 9 were
replaced by nicotinoyl and acetyl groups, respectively, in 12.
The planar structure and relative configuration of 12 were
established using the methods described above (Figure S1).
Tripterygiumines A−L (1−12) contain the same 2-(3-
carboxybutan-2-yl)nicotinic acid moiety; the relative config-
uration of this moiety was determined from the NOESY data, J-
based configuration analysis, and analysis of the biosynthetic
pathway. Owing to the limited amounts of these new alkaloids,
we performed only chemical degradation of alkaloid 4
(Supporting Information), using a reported procedure.9 The
2-(3-carboxybutan-2-yl)nicotinic acid moiety of 4 was defined
as (1′S,2′S)-evoninic acid. These results were consistent with
the NMR spectroscopic data.
11, H-12; and H-11/H-12 (Figure S1B). The relative
configuration of the 2-(3-carboxybutan-1-yl)nicotinic acid
moiety was established by comparing the NMR spectroscopic
data of 23 with those of 22 and 7-epi-mekongensine, the
configuration of which had been previously confirmed by X-ray
crystallography analysis,4c and based on their similar bio-
synthetic origins.
Alkaloid 24 (tripterygiumine O) was shown to have the
molecular formula C46H49NO20 by 13C NMR (Table S4) and
HRESIMS data [m/z 958.2772 ([M + Na]+)]. Comparison of
the NMR spectroscopic data of 24 (Tables S3 and S4) with
those of 23 suggested that the C-7 carbonyl group in 23 had
been reduced and acetylated to a 7-OAc group. In addition, the
2-OBz and 9′-OFu substituents in 23 were replaced with 2-
OFu and 9′-OBz groups in 24. The planar structure and relative
configuration of 24 were established using the methods
described above (Figure S1).
Tripterygiumine P (25) was shown to have the molecular
formula C35H41NO15 by 13C NMR (Table S4) and HRESIMS
data [m/z 738.2348 ([M + Na]+)]. Comparison of the NMR
spectroscopic data of 25 (Tables S3 and S4) with those of 24
revealed that only the 5-OH and 9′-OH groups were esterified
by acetyl and benzoyl groups, respectively, in 25. The planar
structure and relative configuration of 25 were established using
the methods described above (Figure S1).
Compound 26, tripterygiumine Q, was shown to have the
molecular formula C33H39NO16 by 13C NMR (Table S4) and
HRESIMS data [m/z 728.2185 ([M + Na]+)]. Comparison of
the NMR spectroscopic data of 26 (Tables S3 and S4) with
those of 25 suggested that the benzoyl group in 25 was
replaced by a furanoyl group in 26. The planar structure and
relative configuration of 26 were established using the methods
described above (Figure S1).
Compound 22 was shown to have the molecular formula
C39H43NO16 by 13C NMR (Table 3) and HRESIMS data [m/z
804.2511 ([M + Na]+)], revealing 19 indices of hydrogen
deficiency. The IR, UV, and NMR spectroscopic data (Table 3)
of 22 were similar to those of the alkaloids reported previously.3
A polyoxygenated dihydroagarofuran-7-one skeleton and a 2-
(3-carboxybutan-1-yl)nicotinic acid moiety were evident from
1
the H−1H COSY, HSQC, and HMBC correlations (Figure
1G). The HMBC cross signals of H-3/C-11′ and H-15/C-12′
confirmed the C-3−O−C-11′ and C-15−O−C-12′ linkages
between the dihydroagarofuran and the substituted nicotinic
acid moieties, respectively (Figure 1G). The 1-OAc, 8-OAc, 11-
OAc, and 2-OBz substituents were established from the
corresponding HMBC correlations of H-1/C-21‑OAc, H-8/C-
28‑OAc, H-11/C-211‑OAc, and H-2/C-72‑OBz (Figure 1G). The
presence of a free C-4 hydroxy group was determined by
comparison of the 13C NMR chemical shift of C-4 (δC 71.96)
with reported chemical shifts3,4 and confirmed by the HMBC
cross signals of 4-OH/C-12 and H-12/C-3, C-4, C-10 (Figure
1G). A free C-5 hydroxy group was evident by comparison of
the 13C NMR chemical shift of C-5 (δC 75.90) with the
reported values and the molecular composition of 22.
Compound 33 was isolated as a white powder. A molecular
formula of C28H33NO7 was determined by 13C NMR and
HRESIMS data [m/z 496.2324 ([M + H]+)] and revealed 13
indices of hydrogen deficiency. The IR spectrum showed
absorption bands at 3437, 1723, and 1630 cm−1, corresponding
to hydroxy, carbonyl, and aromatic groups, respectively. The
NMR spectroscopic data were similar to those for wilforcidine
(35),11 suggesting that 33 was a nitrogen-containing
sesquiterpenoid ester derivative. The planar structure of 33
1
was determined from the H−1H COSY, HSQC, and HMBC
correlations (Figure S1A). The relative configuration of 33 was
established by the NOESY correlations of H-1/H-3 and H-5/
H-7, H-11, H-12 (Figure S1B). Compound 33 was named
tripterygiumine R.
The relative configuration of the polyoxygenated dihydroa-
garofuran moiety of 22 was established by the NOESY
correlations of H-8/H-1, H-14; H-5/H-11, H-12; and H-12/
H-11, H-3 (Figure 1H). The relative configuration of the C-7′−
C-10′ moiety was determined by comparing the chemical shifts
and coupling constants of the H-7′/H-8′/H-9′/H-10′ spin
system with those of ebenifolin W-I. The configuration of
ebenifolin W-I had been previously determined by NMR
spectroscopic analysis and confirmed by X-ray crystallography
analysis.10 Thus, compound 22 was named tripterygiumine M.
Alkaloid 23 (tripterygiumine N) was shown to have the
molecular formula C44H45NO19 by 13C NMR (Table S4) and
HRESIMS data [m/z 914.2503 ([M + Na]+)]. Comparison of
the NMR spectroscopic data of 23 (Tables S3 and S4) with
those of 22 suggested the presence of an additional furanoyl
group in 23. The 2D NMR experiments established the planar
structure of 23 (Figure S1A). The relative configuration of the
polyoxygenated dihydroagarofuran moiety of 23 was estab-
lished by the NOESY correlations of H-8/H-1, H-14; H-5/H-
By comparing the spectroscopic and physicochemical data
with the reported data, the 18 known nitrogen-containing
dihydroagarofuran derivatives were identified as hyponine D
(13),12 2-O-benzoyl-2-deacetylmayteine (14),4c hexadesacety-
leuomynine (15),13 euojaponine A (16),14 neoeuonymine
(17),13 hyponine C (18),15 7-(acetyloxy)-O11-benzoyl-O2,11
-
deacetyl-7-deoxoevonine (19),16 4-hydroxy-7-epi-chuchuhua-
nine E-V (20),17 wilfornine F (21),18 triptonine B (9′-O-(3-
furoyl)wilfortrine, 27),19 1-desacetylwilforgine (28),4b alat-
amine (29),20 alatusinine (30),21 wilforzine (31),22 wilforjine
(32),23 1β,5α,11-triacetoxy-7β-benzoyl-4α-hydroxy-8β-
nicotinoyldihydroagarofuran (34),24 wilforcidine (35),11 and
5α-benzoyl-4α-hydroxy-1β,8α-dinicotinoyldihydroagarofuran
(36).24
The immunosuppressive activities of compounds 1−36 were
evaluated against human peripheral mononuclear cells, as
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Journal of Natural Products
Article
previously described.25 Tripterygiumine I (9), alatusinine (30),
and wilforcidine (35) exhibited weak immunosuppressive
activity and cytotoxicity (Table S5). Triptonine B (9′-O-(3-
furoyl)wilfortrine, 27) exhibited immunosuppressive activity
with an IC50 value of 4.95 μM but also showed cytotoxicity with
an IC50 value of 26.41 μM (Table S5). Tripterygiumine Q (26)
exhibited immunosuppressive activity with an IC50 value of 8.67
μM, and no cytotoxicity was observed even at a dose of 100
μM, indicating that 26 is an ideal lead for the development of
novel immunosuppressive drugs (Table S5). This work thus
validates chemical mining as an efficient strategy for the
discovery of new chemical entities with therapeutic potential.
3437, 1748, 1631, 1371, 1270, 1251, 1228, 1168, 1114, 1061, 713, 618,
and 510 cm−1; 1H and 13C NMR data, see Tables S1 and S2,
respectively; ESIMS m/z 848 [M + Na]+; HRESIMS m/z 848.2772
(calcd for C41H47NO17+Na+, 848.2736), error −4.3 ppm.
Tripterygiumine J (10): white powder; [α]2D0 −20 (c 0.2, CHCl3);
UV (MeOH) λmax (log ε) 220 (3.64) and 245 (3.51) nm; IR (KBr)
νmax 3422, 1748, 1434, 1371, 1235, 1171, 1116, 911, and 871 cm−1; 1H
and 13C NMR data, see Tables S1 and S2, respectively; ESIMS m/z
744 [M
+
Na]+; HRESIMS m/z 744.2480 (calcd for
C34H43NO16+Na+, 744.2474), error −0.8 ppm.
Tripterygiumine K (11): white powder; [α]2D0 +16 (c 0.3, CHCl3);
UV (MeOH) λmax (log ε) 205 (4.21), 243 (4.03), and 262 (3.03) nm;
IR (KBr) νmax 3436, 1744, 1632, 1373, 1274, 1251, 1200, 1117, 1072,
and 715 cm−1; 1H and 13C NMR data, see Tables S1 and S2,
respectively; ESIMS m/z 806 [M + Na]+; HRESIMS m/z 806.2638
(calcd for C39H45NO16+Na+, 806.2631), error −0.9 ppm.
EXPERIMENTAL SECTION
■
General Experimental Procedures and Plant Material. See ref
3.
Tripterygiumine L (12): white powder; [α]2D0 +9 (c 0.1, CHCl3); UV
(MeOH) λmax (log ε) 201 (4.12) and 242 (3.88) nm; IR (KBr) νmax
3437, 1745, 1631, 1456, 1433, 1372, 1276, 1252, 1229, 1170, 1114,
Extraction and Isolation. See the Supporting Information.
Tripterygiumine A (1): white powder; [α]2D0 −20 (c 0.2, CHCl3);
UV (MeOH) λmax (log ε) 204 (4.23), 244 (3.59), and 262 (3.25) nm;
IR (KBr) νmax 3437, 1740, 1631, 1384, 1319, 1268, 1232, 1164, and
1094 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 1030 [M
+ Na]+; HRESIMS m/z 1030.3320 (calcd for C50H57NO21+Na+,
1030.3315), error −0.5 ppm.
1060, 738, and 617 cm−1; H and 13C NMR data, see Tables S1 and
1
S2, respectively; ESIMS m/z 849 [M + Na]+; HRESIMS m/z
849.2725 (calcd for C40H46N2O17+Na+, 849.2689), error −4.2 ppm.
Tripterygiumine M (22): white powder; [α]2D0 +35 (c 0.2, CHCl3);
UV (MeOH) λmax (log ε) 204 (4.17) and 250 (3.50) nm; IR (KBr)
νmax 3436, 1739, 1632, 1451, 1384, 1264, 1241, 1153, 1131, 1092, 862,
Tripterygiumine B (2): white powder; [α]2D0 +6 (c 0.3, CHCl3); UV
(MeOH) λmax (log ε) 204 (4.27), 243 (3.59), and 262 (3.17) nm; IR
(KBr) νmax 3453, 1746, 1633, 1432, 1371, 1314, 1231, 1171, 1095,
1070, and 901 cm−1; 1H and 13C NMR data, see Table 2; ESIMS m/z
712, and 624 cm−1; H and 13C NMR data, see Table 3; ESIMS m/z
1
804 [M
+
Na]+; HRESIMS m/z 804.2511 (calcd for
C39H43NO16+Na+, 804.2474), error −4.6 ppm.
Tripterygiumine N (23): yellowish powder; [α]2D0 −29 (c 0.4,
CHCl3); UV (MeOH) λmax (log ε) 205 (4.37) and 252 (3.62) nm; IR
(KBr) νmax 3437, 1741, 1632, 1384, 1306, 1227, 1129, 713, and 618
890 [M
+
Na]+; HRESIMS m/z 890.2819 (calcd for
C43H49NO18+Na+, 890.2842), error −2.6 ppm.
Tripterygiumine C (3): yellow powder; [α]2D0 +2 (c 0.2, CHCl3); UV
(MeOH) λmax (log ε) 205 (4.38), 244 (3.56), and 262 (3.05) nm; IR
(KBr) νmax 3438, 1745, 1632, 1452, 1433, 1372, 1314, 1253, 1170,
1116, 1071, 895, and 713 cm−1; 1H and 13C NMR data, see Tables S1
and S2, respectively; ESIMS m/z 952 [M + Na]+; HRESIMS m/z
952.2998 (calcd for C48H51NO18+Na+, 952.2998), error 0.0 ppm.
Tripterygiumine D (4): yellowish powder; [α]2D0 −36 (c 0.3,
CHCl3); UV (MeOH) λmax (log ε) 202 (4.25), 243 (4.19), and 262
(3.12) nm; IR (KBr) νmax 3436, 1718, 1632, 1358, 1278, 1118, 1063,
895, 714, and 619 cm−1; 1H and 13C NMR data, see Tables S1 and S2,
respectively; ESIMS m/z 680 [M + Na]+; HRESIMS m/z 680.2293
(calcd for C33H39NO13+Na+, 680.2314), error 3.0 ppm.
1
cm−1; H and 13C NMR data, see Tables S3 and S4, respectively;
ESIMS m/z 914 [M + Na]+; HRESIMS m/z 914.2503 (calcd for
C44H45NO19+Na+, 914.2478), error −2.7 ppm.
Tripterygiumine O (24): yellowish powder; [α]2D0 −41 (c 0.2,
CHCl3); UV (MeOH) λmax (log ε) 206 (4.18) and 252 (3.49) nm; IR
(KBr) νmax 3436, 1742, 1632, 1440, 1373, 1289, 1256, 1230, 1130,
1055, 1003, 715, and 604 cm−1; 1H and 13C NMR data, see Tables S3
and S4, respectively; ESIMS m/z 958 [M + Na]+; HRESIMS m/z
958.2772 (calcd for C46H49NO20+Na+, 958.2740), error −3.4 ppm.
Tripterygiumine P (25): white powder; [α]2D0 −42 (c 0.1, CHCl3);
UV (MeOH) λmax (log ε) 200 (4.28) and 244 (3.66) nm; IR (KBr)
νmax 3437, 1717, 1637, 1384, 1293, 1250, 1172, 1136, 1110, 1061, 987,
Tripterygiumine E (5): white powder; [α]2D0 +3 (c 0.2, CHCl3); UV
(MeOH) λmax (log ε) 202 (4.25), 243 (4.19), and 262 (3.12) nm; IR
(KBr) νmax 3436, 1748, 1633, 1497, 1456, 1433, 1373, 1307, 1253,
884, 761, 719, and 618 cm−1; H and 13C NMR data, see Tables S3
1
and S4, respectively; ESIMS m/z 738 [M + Na]+; HRESIMS m/z
738.2348 (calcd for C35H41NO15+Na+, 738.2368), error 2.7 ppm.
Tripterygiumine Q (26): yellowish powder; [α]2D0 −15 (c 0.2,
CHCl3); UV (MeOH) λmax (log ε) 250 (3.60) nm; IR (KBr) νmax
1
1230, 1160, 1116, 1079, 1060, 785, 751, and 605 cm−1; H and 13C
NMR data, see Tables S1 and S2, respectively; ESIMS m/z 838 [M +
Na]+; HRESIMS m/z 838.2553 (calcd for C39H45NO18+Na+,
838.2529), error −2.8 ppm.
3437, 1731, 1631, 1374, 1243, 1115, 720, and 618 cm−1; H and 13C
1
NMR data, see Tables S3 and S4, respectively; ESIMS m/z 728 [M +
Na]+; HRESIMS m/z 728.2185 (calcd for C33H39NO16+Na+,
728.2161), error −3.3 ppm.
Tripterygiumine F (6): white powder; [α]2D0 −4 (c 0.4, CHCl3); UV
(MeOH) λmax (log ε) 205 (4.31), 242 (4.12), and 262 (3.08) nm; IR
(KBr) νmax 3436, 1744, 1632, 1384, 1313, 1268, 1248, 1227, 1160,
Tripterygiumine R (33): white powder; [α]2D0 +48 (c 0.1, CHCl3);
UV (MeOH) λmax (log ε) 202 (4.04) and 241 (3.57) nm; IR (KBr)
1
1118, and 627 cm−1; H and 13C NMR data, see Tables S1 and S2,
respectively; ESIMS m/z 942 [M + Na]+; HRESIMS m/z 942.2797
(calcd for C46H49NO19+Na+, 942.2791), error −0.7 ppm.
1
νmax 3437, 1723, 1630, 1384, 1290, 1119, and 618 cm−1; H NMR
Tripterygiumine G (7): white powder; [α]2D0 +10 (c 0.2, CHCl3);
UV (MeOH) λmax (log ε) 205 (4.35), 244 (4.13), and 262 (3.02) nm;
IR (KBr) νmax 3437, 1744, 1631, 1384, 1305, 1254, 1228, 1117, 912,
(CDCl3) δ 5.63 (dd, J = 12.0, 4.2 Hz, H-1), 2.16 (m, H-2a), 1.61 (m,
H-2b), 2.02 (m, H-3a), 1.76 (dt, J = 13.3, 3.2 Hz, H-3b), 4.50 (d, J =
5.5 Hz, H-5), 2.23 (brs, H-6), 2.33 (ddd, J = 11.1, 7.5, 3.6 Hz, H-7a),
2.16 (dd, J = 11.1, 3.0 Hz, H-7b), 5.09 (d, J = 7.3 Hz, H-8), 1.55 (s, H-
14), 1.60 (s, H-15), 1.65 (s, H-12), 1.51 (s, H-11), 3.27 (s, OH-4),
5.12 (d, J = 5.5 Hz, OH-5), 8.64 (brs, H-1Nic), 7.75 (d, J = 5.0 Hz, H-
4Nic), 7.19 (dd, J = 7.5, 5.0 Hz, H-5Nic), 8.68 (brs, H-6Nic), 7.76 (d, J =
7.4 Hz, H-2Bz, 6Bz), 7.32 (t, J = 7.7 Hz, H-3Nic, 5Nic), and 7.50 (t, J =
7.5 Hz, H-4Nic); 13C NMR (CDCl3) δ 73.96 (C-1), 23.47 (C-2), 37.22
(C-3), 73.03 (C-4), 91.55 (C-10), 79.71 (C-5), 50.17 (C-6), 32.05 (C-
7), 73.40 (C-8), 50.63 (C-9), 84.68 (C-13), 26.62 (C-14), 30.13 (C-
15), 23.80 (C-12), 20.24 (C-11), 153.10 (C-2Nic), 125.59 (C-3Nic),
136.32 (C-4Nic), 122.87 (C-5Nic), 150.41 (C-6Nic), 164.23 (C-7Nic),
129.20 (C-1Bz), 129.74 (C-2Bz, 6Bz), 128.10 (C-3Bz, 5Bz), 133.13 (C-
1
714, and 618 cm−1; H and 13C NMR data, see Tables S1 and S2,
respectively; ESIMS m/z 942 [M + Na]+; HRESIMS m/z 942.2783
(calcd for C46H49NO19+Na+, 942.2791), error 0.9 ppm.
Tripterygiumine H (8): white powder; [α]2D0 −24 (c 0.2, CHCl3);
UV (MeOH) λmax (log ε) 242 (3.46), and 261 (2.96) nm; IR (KBr)
νmax 3437, 1722, 1632, 1384, 1319, 1279, 1200, 1120, 1060, 881, 786,
and 577 cm−1; 1H and 13C NMR data, see Tables S1 and S2,
respectively; ESIMS m/z 618 [M + Na]+; HRESIMS m/z 618.2165
(calcd for C28H37NO13+Na+, 618.2157), error −1.2 ppm.
Tripterygiumine I (9): white powder; [α]2D0 +20 (c 0.2, CHCl3); UV
(MeOH) λmax (log ε) 220 (3.91) and 246 (3.50) nm; IR (KBr) νmax
1656
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Journal of Natural Products
Article
4Bz), and 165.17 (C-7Bz); ESIMS m/z 496 [M + H]+; HRESIMS m/z
496.2324 (calcd for C28H33NO7+H+, 496.2330), error 1.1 ppm.
Immunosuppressive Activity Assay. The immunosuppressive
activity assays were performed according to a published procedure25
with minor modifications. Briefly, human peripheral mononuclear cells
(PBMCs) were isolated from healthy donors by density-gradient
centrifugation with Lymphoprep and resuspended in complete
medium. To evaluate the proliferative activity of the isolated
compounds, PBMCs (106 cells/mL) were stimulated by plate-bound
anti-CD3 (2 μg/mL) and soluble anti-CD28 (1 μg/mL) monoclonal
antibodies and incubated for 96 h in the presence of varying
concentrations of the isolated compound dissolved in DMSO (less
than 0.025%) or vehicle alone. PBMC proliferation was analyzed by
flow cytometry on an Accuri C6 system (Becton Dickinson). To
evaluate the cytotoxicity of the isolated compounds, PBMCs (106
cells/mL) were incubated in the presence of different concentrations
of compound or vehicle alone for 96 h. Cell survival was evaluated by
flow cytometry on an Accuri C6 system.
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ASSOCIATED CONTENT
* Supporting Information
Extraction and isolation, methanolysis/hydrolysis of tripter-
■
S
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1
S1−S5, and spectroscopic data, including H NMR, 13C NMR,
1H−1H COSY, HSQC, HMBC, NOESY, and HRESIMS data
for all new compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*(Y. Luo) Tel: +86-28-82890813. Fax: +86-28-82890288. E-
mail: yinggluo@cib.ac.cn.
■
(16) Nakagawa, H.; Takaishi, Y.; Fujimoto, Y.; Duque, C.; Garzon,
C.; Sato, M.; Okamoto, M.; Oshikawa, T.; Ahmed, S. U. J. Nat. Prod.
2004, 67, 1919−1924.
*(G. Zhang) Tel: +86-28-82890996. Fax: +86-28-82890288. E-
mail: zhanggl@cib.ac.cn.
Notes
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̃
da Silva, M. F. G. F. Phytochemistry 2001, 58, 1205−1207.
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Jia, Y. F.; Li, D. J. Nat. Prod. 2001, 64, 582−587.
(19) Morota, T.; Yang, C. X.; Ikeya, Y.; Qin, W. Z.; Nishimura, H.;
Xu, L. H.; Ando, M.; Miao, K. L.; Maruno, M.; Yang, B. H.
Phytochemistry 1995, 39, 1219−1222.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for financial support from the National Basic
Research Program of China (973 Program, 2009CB522804),
the National New Drug Innovation Major Project of China
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