Homosecoiridoids from the flower buds of Lonicera japonica

Article abstract of DOI:10.1021/np2004566

Homosecoiridoids (1-15) were isolated from the flower buds of Lonicera japonica. Compounds 1-4, designated as loniphenyruviridosides -D, possess unprecedented skeletons featuring phenylpyruvic acid derived moieties coupled with an iridoid or a secoiridoid nucleus. Compounds 5-15 (lonijaposides D-N) are additional examples of the unusual pyridinium alkaloid-coupled secoiridoids (lonijaposides -C). The validity of the CD data to determine the configuration of the secoiridoid derivatives is discussed on the basis of detailed CD data analysis and semisynthesis of 2 and 3 with the co-occurring secologanic acid. The configuration of secologanic acid was determined by a single-crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation. Biosynthetic pathways of the homosecoiridoids were postulated. Compounds 1-4 inhibited STAT-3 activity of HELF cells, and lonijaposides F (7), H (9), I (10), and K (12) showed activity against the release of glucuronidase in rat polymorphonuclear leukocytes induced by platelet-activating factor.

Full text of DOI:10.1021/np2004566

ARTICLE
pubs.acs.org/jnp
Homosecoiridoids from the Flower Buds of Lonicera japonica
Yang Yu, Weixia Song, Chenggen Zhu,* Sheng Lin, Feng Zhao, Xiuli Wu, Zhenggang Yue, Bo Liu,
Sujuan Wang, Shaopeng Yuan, Qi Hou, and Jiangong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, and Key Laboratory of Bioactive Substances and
Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica, Chinese Academy of Medical
Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S Supporting Information
b
ABSTRACT:
Homosecoiridoids (1ꢀ15) were isolated from the flower buds of Lonicera japonica. Compounds 1ꢀ4, designated as loniphenyr-
uviridosides AꢀD, possess unprecedented skeletons featuring phenylpyruvic acid derived moieties coupled with an iridoid or a
secoiridoid nucleus. Compounds 5ꢀ15 (lonijaposides DꢀN) are additional examples of the unusual pyridinium alkaloid-coupled
secoiridoids (lonijaposides AꢀC). The validity of the CD data to determine the configuration of the secoiridoid derivatives is
discussed on the basis of detailed CD data analysis and semisynthesis of 2 and 3 with the co-occurring secologanic acid. The
configuration of secologanic acid was determined by a single-crystal X-ray crystallographic analysis using anomalous scattering of Cu
Kα radiation. Biosynthetic pathways of the homosecoiridoids were postulated. Compounds 1ꢀ4 inhibited STAT-3 activity of
HELF cells, and lonijaposides F (7), H (9), I (10), and K (12) showed activity against the release of glucuronidase in rat
polymorphonuclear leukocytes induced by platelet-activating factor.
onicera japonica Thunb. (Caprifoliaceae) is widely cultivated
in Shandong and Henan Provinces of China. Its flower buds
lonijaposides AꢀC.6 We report herein the isolation, structure
determination, and biological activity of the new isolates. In
addition, the validity of the CD data to determine the configura-
tion of the secoiridoid derivatives is discussed on the basis of
systematic comparison of the CD data combined with a single-
crystal X-ray crystallographic analysis of the co-occurring secolo-
ganic acid using anomalous scattering of Cu Kα radiation.
L
and stems are used in traditional Chinese medicine for the
treatment of various diseases and are among the most common
ingredients of formulations used for treating influenza, cold,
fever, and infections.1,2 Chemical and pharmacological studies
resulted in characterization of constituents with different struc-
tural features and biological activities from the flower buds of L.
japonica, including caffeoyl quinic acids, secoiridoids, flavonoids,
saponins, cerebrosides, and polyphenols.3,4 As part of a program
to assess the chemical and biological diversity of traditional
Chinese medicines,5 three pyridinium alkaloid-coupled secoir-
idoids (lonijaposides AꢀC) with an unusual skeleton of an
N-substituted nicotinic acid nucleus coupled with a secoiridoid,
together with seven known iridoid derivatives, were reported
from a water extract of the flower buds of L. japonica.6 Focusing
our continuing investigation on the minor components of the
extract, 15 new homosecoiridoids (1ꢀ15) have been isolated.
Compounds 1ꢀ4, named loniphenyruviridosides AꢀD, possess
unprecedented skeletons featuring phenylpyruvic acid derived
moieties coupled with an iridoid or a secoiridoid nucleus, and
compounds 5ꢀ15 (lonijaposides DꢀN) are analogues of
’ RESULTS AND DISCUSSION
Loniphenyruviridoside A (1) showed IR absorptions for
hydroxy, carboxylic, conjugated double bond, and aromatic ring
functional groups. Its molecular formula, C24H28O10, was in-
dicated by HR-ESIMS combined with NMR data (Table 1 and
Experimental Section). The NMR spectra of 1 showed reso-
nances (Table 1) attributable to two trisubstituted double bonds,
two methylenes, and five methines (two oxygen-bearing), in
addition to diagnostic resonances for phenyl and β-glucopyr-
anosyl moieties. These data indicated that 1 was an unusual
Received: June 2, 2011
Published: September 26, 2011
r
Copyright
2011 American Chemical Society and
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Scheme 1. Plausible Biosynthetic Pathways of 1ꢀ4
analogue of the co-occurring secoiridoids6 having an additional
trisubstituted double bond and phenyl units. The presence of a β-
glucopyranosyl unit was confirmed by enzymatic hydrolysis of 1
with β-glucosidase, which produced glucose, identified by TLC
comparison with an authentic sugar sample. The glucose isolated
from the hydrolysate gave a positive optical rotation, [α]20
D
+41.1 (c 0.06, H2O), indicating that it was β-D-glucose.7 The
structure of the aglycone moiety of 1 was finalized by 2D NMR
data analysis (Supporting Information, Figures S8ꢀS10). The
proton and protonated carbon resonances in the NMR spectra
Figure 1. ORTEP diagram of secologanic acid.
NOE enhancements, combined with coupling constants of the
vicinal protons, indicated that these protons were cofacial on the
cyclohexane ring. The coupling constant for J1,9 and J8,9 (≈0.0
Hz) in the 1H NMR spectrum indicated that the torsion angles
between H-1 and H-9 and between H-8 and H-9 were around
90°. This suggested that H-1 was oriented on the other side of the
cis-fused ring system based on molecular modeling. The CD
spectrum of 1 displayed positive Cotton effects at 272 nm and
1
were assigned by the gHSQC data. 1Hꢀ H gCOSY cross-peaks
of H-9/H-5/H2-6/H-7/H2-10/H-8/H-9 indicated the presence
of a 1,2,3,5-tetrasubstituted cyclohexane ring in 1. HMBC
correlations of H-1/C-3, C-5, and C-10; H-3/C-1, C-4, C-5,
and C-11; H-5/C-3, C-4, and C-9; H-9/C-1, C-4, and C-5; and
H-10/C-1, in combination with their shifts, revealed a 3-carboxy-
6-β-glucopyranosyl-4,5-dihydro-[6H]-pyrano moiety sharing
C-5 and C-9 with the cyclohexane ring. This was supported by
gCOSY cross-peaks of H-1/H-9 and H-3/H-5 (a long-range W-
type coupling) and gHMBC correlations of H-1/C-8, H-5/C-6,
C-7, and C-8, H-8/C-5, and H2-10/C-6, C-7, and C-9. In
addition, gHMBC correlations of H-200 and H-600/C-400 and
C-700; H-300 and H-500/C-100; and H-800/C-100 and C-700, together
with the shifts of H-800 and C-800 and the quaternary nature of
C-700, indicated the occurrence of a 3-phenyl-4,5-dihydro-[6H]-
pyrano moiety in 1. Meanwhile, gHMBC correlations of H-8/
C-800, H-800/C-7 and C-8, and H2-10/C-700, combined with the
molecular composition, demonstrated a connection of C-7 and
C-700 and an oxygen bridge between C-8 and C-800. Accordingly,
the planar structure of 1 was elucidated as shown. In the NOE
difference spectrum of 1, irradiation of H-6b gave enhancements
of H-5, H-7, H-9, and H-10a (endo); in turn, when H-9 was
irradiated H-5, H-6b, H-8, and H-10a were enhanced. These
1
negative at 227 and 244 nm, arising from the Lb band of the
styrene chromophore8 of the 3-phenyl-4,5-dihydro-[6H]-pyrano
moiety and an overlap of the πfπ* and nfπ* transitions of the
3-carboxy-4,5-dihydro-[6H]-pyrano chromophore,9 respec-
tively. On the basis of the empirical helicity rule for the styrenes,8
in combination with the relative configuration and lowest energy
conformation analysis (MM2), the longer wavelength Cotton
effect predicts the 7R,8R configuration for 1. When applying the
octant rule to the conjugated carbonyl chromophore10 and the
helicity rule to the 3-carboxy-4,5-dihydro-[6H]-pyrano chromo-
phore,9 the shorter wavelength Cotton effects predict the 1S,5S,
9S configuration. The predicted configuration is consistent with
that based on a biogenetic postulation of 1 (Scheme 1) combined
with the above NOE enhancements. The configuration of the
proposed biosynthetic precursor, the co-occurring secologanic
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Table 3. 13C NMR Spectroscopic Data (δ) for Lonijaposides
D (5), E (6), and HꢀN (9ꢀ15)a
addition, H-1 and H-6b were enhanced upon irradiation of H-8.
These enhancements indicated that H-5, H-6a, H-7, and H-9,
opposite of H-8 and H-1, were cofacial. In the NOE difference
spectrum of methyl ester 2a, irradiation of H-200 and/or H-600 did
not enhance the COOCH3 (Supporting Information, Figure S29),
indicating an E geometry of the C-700ꢀC-800 double bond that was
supported by absorptions at 1678 and 1635 cmꢀ1 in the IR
spectrum of 2.11 The CD spectrum of 2 displayed positive Cotton
effects at 281 nm and negative at 232 nm, resulting from the 1Lb
band of the phenylpropenoic chromophore12 and the πfπ*
transition of the 4,5-dihydro-[6H]-pyrano chromophore.9 The
shorter wavelength Cotton effect predicts the 1S,5S,9S configura-
tion by application of the helicity rule to the 4,5-dihydro-[6H]-
pyrano chromophore.9 With this information and the knowledge
of relative configuration, the absolute configuration at C-7 could
be determined as R. The absolute configuration was confirmed by
semisynthesis of 2 using the co-occurring secologanic acid and the
commercially available phenylpyruvic acid (Experimental Section).
Thus, the structure of compound 2 was determined and desig-
nated as loniphenyruviridoside B.
The spectroscopic data of compound 3 (Table 1 and Experi-
mental Section) indicated that it was an isomer of 2. Comparison of
the NMR data of 2 and 3 indicated that H-6a and H-7 and C-6 and
C-7 in 3 were shielded, whereas H-1, H-5, H-8, H-9, and H2-10
were deshielded. This suggested that 3 was a C-7 epimer of 2 and
was proved by 2D NMR, NOE difference, and CD data
(Supporting Information, Figures S40ꢀS44), as well as by semi-
synthesis of 3 (Experimental Section). Particularly, the CD spec-
trum of 3 displayed a negative Cotton effect at 282 nm, opposite
that of 2, suggesting the 7S configuration (Supporting Information,
Figure S44). Therefore, the structure of compound 3 was assigned
for loniphenyruviridoside C, as confirmed by semisynthesis with
the co-occurring secologanic acid (Experimental Section).
no.
5
6
9
10
11
12
13
14
15
1
99.8 100.2 100.3 99.6 100.1 99.7 99.7 99.7 100.2
N.D. 157.3 154.3 151.5 157.2 152.5b 151.6 151.7 157.3
115.8b 109.9 111.4 116.0 109.9 115.4 116.1 116.1 110.0
3
4
5
41.2
40.9 128.7 40.8 40.7 41.0 41.0 41.0 40.9
6
141.6 140.3 127.8 141.4 140.2 141.1 141.3 141.3 140.1
128.0 128.4 32.3 127.8 128.4 128.0 128.1 128.0 128.6
136.7 136.1 135.4 136.6 136.0 136.6 136.7 136.7 136.1
7
8
9
47.8
47.4 44.4 47.7 47.4 47.7 47.8 47.8 47.5
10
11
10
122.5 122.8 121.0 122.3 122.8 122.4 122.4 122.4 122.8
N.D. 172.2 171.3 177.8 172.2 177.7b 177.6b 177.4b 172.3
101.9 102.1 101.6 101.7 102.0 101.8 101.8 101.8 102.1
20
30
40
50
75.5
78.5
72.5
79.2
63.5
75.4 75.2 75.4 75.4 75.4 75.5 75.5 75.5
78.5 79.0 78.4 78.5 78.4 78.5 78.5 78.6
72.4 72.2 72.4 72.3 72.4 72.5 72.5 72.4
79.2 78.3 79.1 79.2 79.1 79.2 79.2 79.3
63.5 63.5 63.5 63.5 63.5 63.5 63.5 63.5
60
200
300
400
500
600
700
1000
2000
3000
4000
OMe
145.5 145.3 143.0 144.9 144.9 145.7 144.9 144.8 144.7
139.8 139.9 137.2 141.0 140.7 140.7 141.0 141.2 140.9
144.0 143.8 145.3 144.6 144.7 144.5 144.5 144.5 144.5
141.0 140.7 142.1 130.4 130.6 130.3 130.5 130.6 130.8
145.6 145.2 144.6 145.0 145.3 145.7 144.8 144.8 145.1
N.D. 170.8 170.6
N.D. 60.5 180.0b 63.1 63.1 173.7b 179.7b 180.2b 183.2
36.4
30.3
64.4
18.5 36.3 66.3 66.4 66.2 26.2 36.5 36.1
30.2
64.1
61.7 30.3 30.1
63.5 64.3
54.9
54.8 54.8
54.8
Compound 4 was another isomer of 2, as indicated by
spectroscopic data (Table 1 and Experimental Section). Com-
parison of the NMR data of 2 and 4 indicated that H-3 and C-3 of
4 were shielded by ΔδH ꢀ0.50 and ΔδC ꢀ6.9 ppm, respectively,
whereas C-4, C-11, and C-900 were deshielded by ΔδC +6.4, +4.7,
and +1.2 ppm. This suggested that 4 resulted from intramole-
cular trans-esterification of 2 with the lactone formed between
C-900 and C-7. This was confirmed by 2D NMR data of 4
(Supporting Information, Figures S59ꢀS61), particularly, by
gHMBC correlations of H-7/C-700, C-800, and C-900. In the
NOE difference spectrum of 4, irradiation of H-7 enhanced
H-5 and H-200/H-600, and irradiation of H-200/H-600 gave en-
hancements of H-5, H-6a, and H-7. In addition, H-1 and H-6b
were enhanced upon irradiation of H-8. These enhancements
demonstrated that free rotation about the C-5ꢀC-6ꢀC-7 bonds
in 4 was limited in the solution state and that the configuration at
C-1, C-5, and C-9 of 4 was identical to that of 2. The CD
spectrum of 4 showed a positive Cotton effect at 282 nm
a Data were measured in D2O at 125 MHz for 6, 9, 10, 11, 13, and 14 and
at 150 for 5, 12, and 15, respectively. The assignments were based on
1
DEPT, 1Hꢀ H COSY, HSQC, HMQC, and HMBC experiments, while
the data are presented as calculated using C-60 (δ 63.5 ppm) as the
reference. “N.D.” means that the carbon resonance was not observed in
the spectrum. b The resonance data were obtained from the HMBC
spectrum.
acid, was determined by a single-crystal X-ray crystallographic
analysis using anomalous scattering of Cu Kα radiation. An
ORTEP drawing with the atom-numbering indicated is shown in
Figure 1. Therefore, the structure of compound 1 was deter-
mined and designated as loniphenyruviridoside A.
By HR-ESIMS and NMR data (Table 1 and Experimental
Section) compound 2 had the molecular formula C25H28O12
with an additional CO2 unit compared to 1. The NMR data of 2
(Table 1) indicated that it consisted of 7-substituted secologanic
acid6 and 700,800-disubstituted phenylpropenoic acid moieties, as
verified by 2D NMR data analysis (Supporting Information,
Figures S20ꢀS22). In the gHMBC spectrum, long-range corre-
lations of H-6b/C-700; H-7/C-100, C-700, and C-800; and H-200 and
H-600/C-700, together with their shifts, revealed that the two
moieties were connected via a carbon bond between C-7 and
C-700, while correlations of H-7/C-11 and their shifts confirmed
the presence of the oxygen bridge between C-7 and C-11 forming
a lactone ring in the 7-substituted secologanic acid moiety. In the
NOE difference spectrum of 2, irradiation of H-7 enhanced H-5
and H-6a, and irradiation of H-9 gave enhancement of H-5. In
1
attributed to the Lb band of the phenylpropenoic chromo-
phore12 (Supporting Information, Figure S64), which was similar
to that of 2 but opposite that of 3. This suggested the 7R
configuration for 4. Therefore, the structure of compound 4 was
defined for loniphenyruviridoside D.
Compound 5, molecular formula C26H31NO13 by HR-ESIMS
and NMR data, showed IR absorptions for hydroxy and car-
boxylic groups (Tables 2 and 3 and Experimental Section). The
1H NMR data of 5 (Table 2) were similar to those of lonijapo-
side A6 except for the absence of the methyl moiety of the ester
group and a shielded shift of H-3 (ΔδH ꢀ0.37 ppm) in 5.
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This suggested that 5 was demethyl lonijaposide A, which was
verified by the HMBC data (Supporting Information, Figure
S70), as well as by alkaline hydrolysis of lonijaposide A that
produced 5. Thus, the structure of compound 5 was determined
and designated as lonijaposide D.
Compound 6, molecular formula C25H31NO11 by HR-ESIMS
and NMR data, exhibited spectroscopic data (Tables 2 and 3 and
Experimental Section) similar to those of lonijaposide B.6
Comparison of the NMR data of 6 with those of lonijaposide
B indicated that the N-(2-hydroxyethyl) unit in lonijaposide B
was replaced by an N-ethyl group in 6, as verified by 2D NMR
data (Supporting Information, Figures S78ꢀS80). In the HMBC
spectrum of 6, correlations of H-3 and CH3OCO/C-11; H-200
and H-600/C-1000; and H2-1000/C-200, C-600, and C-2000, together
with their shifts, confirmed the presence of the methoxycarbonyl
and N-ethyl units in 6. Therefore, the structure of compound 6
was assigned for lonijaposide E.
pyrano chromophores (Supporting Information, Figure S104),
respectively. When applying the helicity rule to the 4-methylene-
4,5-dihydro-[6H]-pyrano chromophore,9 the recorded negative
Cotton effect (232 nm) predicts the 1S,9R configuration for 9,
which is consistent with that of the reported natural glycosidic
secoiridoids.9,13,14 Therefore, the structure of compound 9 was
assigned for lonijaposide H.
Compound 10 had the molecular formula C23H29NO10, indi-
cated by HR-ESIMS and NMR data. Comparison of the NMR data
of 10 (Tables 2 and 3) and lonijaposide B6 showed the presence of a
3-substituted pyridine ring in 10 replacing the 3,5-disubstituted
nicotinic acid unit in lonijaposide B. This was verified by gCOSY
cross-peaks of H-400/H-500/H-600 (Supporting Information, Figure
S111) and gHMBC correlations of H-200/C-400, C-600, and C-7;
H-400/C-200, C-600, and C-7; H-500/C-300 and C-600; and H-600/C-200,
C-400, andC-500 (Supporting Information, Figure S113). Meanwhile,
a COSY cross-peak of H2-1000/H2-2000 and HMBC correlations of
H-200 and H-600/C-1000 and H-1000/C-200 and C-600 confirmed the
presence of the N-(2-hydroxyethyl) in 10. Hence, the structure of
compound 10 was assigned for lonijaposide H.
The spectroscopic data of compound 11 (Tables 2 and 3 and
Experimental Section) indicated that it was the methyl ester of
10. This was supported by a deshielded shift of H-3 (ΔδH +0.48
ppm) in 11 when compared with that of 10 and confirmed by
correlations from both H-3 and COOCH3 to C-11 in the HMBC
spectrum of 11 (Supporting Information, Figure S121). Com-
pound 11 was proposed to be a chloride salt based on the isotope
cluster of quasi-molecular ions at m/z 528/530/532 (3:2:1)
[M ꢀ H]ꢀ by negative ESIMS and a peak at m/z 494.2029
[M ꢀ Cl]+ (494.2021 calcd for C24H32NO10) by positive HR-
ESIMS. In addition, a quasi-molecular ion peak for the acetate
salt of 11, at m/z 552 [M ꢀ H]ꢀ, was also indicated by the
negative ESIMS (Supporting Information, Figure S118); how-
ever, it was an artifact formed in the isolation procedure by acetic
acid used in the HPLC separation. Therefore, the structure of
compound 11 was assigned for lonijaposide J.
Compound 12 was the analogue of 10 with an N-(carboxy-
methyl) replacing the N-(2-hydroxyethyl), which was demon-
strated by comparison of the spectroscopic data of 12 and 10
(Tables 2 and 3 and Experimental Section). In the HMBC
spectrum of 12 (Supporting Information, Figure S128), correla-
tions of H-2000/C-1000, C-200, and C-600 and H-200 and H-600/C-2000
combined with shifts of these proton and carbon resonances
verified the presence of the N-(carboxymethyl) moiety. In
addition, the shift of C-11 (δC 177.7) in 12 was in agreement
with that of 10, supporting that the inner salt was formed via the
11-carboxylate. Accordingly, the structure of compound 12 was
assigned for lonijaposide K.
Compound 7 had the molecular formula C24H29NO11 with
one less CH2 unit than 6 (HR-ESIMS and NMR data). The 1H
NMR spectrum of 7 (Table 2) was similar to that of 6 except for
the absence of the methyl group and a shielded shift of H-3 (ΔδH
ꢀ0.44 ppm) in 7. This revealed that 7 was the 11-carboxylic acid
analogue of 6, which was proved by alkaline hydrolysis of 6 that
gave 7. Thus, the structure of compound 7 was assigned for
lonijaposide F.
1
Comparison of the H NMR data of compounds 8 and 6
(Table 2) indicated replacement of the N-ethyl in 6 by an N-
methyl (δH 4.39) group in 8. This was supported by HR-ESIMS
of 8 (Experimental Section). Although the 13C NMR resonances
were not obtained because of the limited sample amount, the
gHMBC spectrum of 8 (Supporting Information, Figure S92)
showed correlations from COOCH3 to a carbon resonance at δC
172 (C-11) and from both N-CH3 and H-400 to a carbon
resonance at δC 145 (C-200/C-600). This combined with the
chemical shift of H-3 confirmed the presence of the methox-
ycarbonyl and N-methyl groups in 8. Accordingly, the structure
of compound 8 was assigned for lonijaposide G.
The spectroscopic data of compound 9 (Tables 2 and 3 and
Experimental Section) showed that it was an isomer of lonijapo-
side A.6 Comparison of the NMR data with that of lonijaposide A
indicated that the disubstituted C-6ꢀC-7-trans double bond in
lonijaposide A was replaced by a trisubstituted double bond in 9.
In addition, the methine (CH-5) in lonijaposide A was sub-
stituted by a methylene (CH2-7) group in 9. This suggested the
presence of the C-5ꢀC-6 double bond in 9, which was proved by
the 2D NMR data. A gCOSY cross-peak of H-6/H2-7
(Supporting Information, Figure S100) and gHMBC correla-
tions of H-6/C-4, C-7, C-9, and C-500; H2-7/C-5, C-6, and C-400;
and H-400 and H-600/C-7 (Supporting Information, Figure S102),
in combination with their shifts, confirmed the location of the
trisubstituted double bond in 9. In addition, gHMBC correla-
tions of H-200 and H-600/C-4000; H-4000/C-200 and C-600; and
COOCH3/C-11 demonstrated the presence of the N-(3-
carboxypropyl) and methoxycarbonyl units. In the NOE differ-
ence spectrum of 9 (Supporting Information, Figure S103),
irradiation of H-6 gave enhancements of H-400, H-600, and
COOCH3, demonstrating an E configuration for the trisubsti-
tuted double bond. The CD spectrum of 9 displayed positive
Cotton effects at 277 nm and negative at 247 and 232 nm,
probably resulting from the 1Lb band of the N-substituted
nicotinic chromophore and the nfπ* and πfπ* transitions
of the conjugated carbonyl and 4-methylene-4,5-dihydro-[6H]-
The HR-ESIMS of compound 13 indicated that it was a
homologue of 12 having the molecular formula C24H29NO11.
Comparison of the NMR data of 13 and 12 (Tables 2 and 3)
revealed the presence of an N-(2-carboxyethyl) in 13 replacing
the N-(carboxymethyl) in 12. Although resonances for the
carboxylic carbons (C-11 and C-1000) were not observed in the
13C NMR spectrum of 13, their shifts were assigned by correla-
tions from H-3 to a carbon resonance at δC 177.6 (C-11) and
from H2-2000 to a carbon resonance at δC 179.7 (C-1000), respec-
tively, in the HMBC spectrum (Supporting Information, Figure
S134). In addition, HMBC correlations of H2-3000/C-1000, C-2000,
C-200, and C-600 and H-200 and H-600/C-3000 confirmed the
presence of the N-(2-carboxyethyl) moiety in 13. Thus, the
structure of compound 13 was assigned for lonijaposide L.
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The spectroscopic data of compound 14 (Tables 2 and 3 and
Experimental Section) demonstrated that it was a homologue of
13 with an N-(3-carboxypropyl) unit substituting the N-(2-
carboxyethyl) unit. This was confirmed by correlations of H-200
and H-600/C-4000 and H2-4000/C-200 and C-600 in the HMBC
spectrum of 14 (Supporting Information, Figure S142). Hence,
the structure of compound 14 was defined for lonijaposide M.
Compound 15 was the methyl ester of 14, as indicated by
comparison of the spectroscopic data of 15 and 14 (Tables 2 and
3 and Experimental Section) and confirmed by the HMBC data
of 15 (Supporting Information, Figure S148) displaying a
correlation from COOCH3 to C-11. The carboxylate inner salt
was supported by the deshielded shift of C-1000(δC 183.2) in 15.
Therefore, the structure of compound 15 was assigned for
lonijaposide N and further confirmed by alkaline hydrolysis of
15 to produce 14.
68.6 ( 2.8%, 72.1 ( 2.6%, and 54.6 ( 2.3% inhibition rates,
respectively, while other isolates exhibited inhibition rates lower
than 30%. The positive control (ginkgolide B) gave an inhibition
rate of 84.3 ( 3.3% at the same concentration.6,17 In addition,
these compounds were also assessed for their activities against
several human cancer cell lines,18 neuroprotective activity against
glutamate-induced neurotoxicity in cultures of PC12 cells,19 and
antioxidant activity in Fe2+-cystine-induced rat liver microsomal
lipid peroxidation,20 as well as the inhibitory activity against
HIV-1 replication21 and protein tyrosine phosphatase 1B (PTP1B),20
but all were inactive at a concentration of 10 μM.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a PE model 343. UV and CD spectra were measured on
a JASCO J-810 spectropolarimeter. IR spectra were recorded on a
Nicolet 5700 FT-IR microscope spectrometer (FT-IR microscope
transmission). 1D- and 2D-NMR spectra were obtained at 500 or 600
MHz for 1H and 125 or 150 MHz for 13C, respectively, on INOVA 500
MHz or SYS 600 MHz spectrometers with solvent peaks as references
(unless otherwise noted). ESIMS data were measured with a Q-Trap
LC/MS/MS (Turbo ionspray source) spectrometer. HR-ESIMS data
were, in turn, measured on an AccuToFCS JMS-T100CS spectrometer.
Column chromatography was performed with silica gel (200ꢀ300 mesh,
Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China)
and Pharmadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden).
HPLC separation was performed on an instrument with a Waters 600
controller, a Waters 600 pump, and a Waters 2487 dual λ absorbance
detector on an Prevail (250 ꢁ 10 mm i.d.) semipreparative column
packed with C18 (5 μm). Glass precoated silica gel GF254 plates were
used for TLC. Spots were visualized under UV light or by spraying with
7% H2SO4 in 95% EtOH followed by heating.
The acids (lonijaposide C6 and compounds 1ꢀ5, 7, 10, and
12ꢀ14) and methyl esters (lonijaposides A and B6 and com-
pounds 6, 8, 9, 11, and 15) were considered to be natural
products since hydrolysis of the esters and methylation of the
acids were unlikely to occur in the isolation procedure and under
simulated conditions, heating H2O solutions of the esters or
MeOH solutions of the acids, either with or without SP-700
resin, silica gel, Pharmadex LH-20, or reversed C18 silica gel at
45 °C for 48 h.
The biosynthetic precursors of 1ꢀ4 are proposed to be the co-
occurring secologanic acid and/or secologanin. A plausible path-
way to compounds 1ꢀ4 is postulated in Scheme 1. Condensa-
tion of secologanic acid with phenylpyruvic acid by an aldol-like
reaction would yield a key conjugated enone intermediate.15 The
intermediate may be cyclized through a sequentially or simulta-
neously intramolecular hetero-DielsꢀAlder addition and decar-
boxylation to yield 1, while it undergoes re- and si-1,4-additions
of the conjugated enone with the carboxylic alcohols to generate
2ꢀ4, respectively. In addition, an enzyme-catalyzed intramole-
cular ester exchange of 2ꢀ4 would be an alternative for their
formation. The biosynthetic pathway of 5ꢀ15 would be similar
to that postulated for lonijaposides AꢀC.6 Compounds 5ꢀ9
may be derived from an enzyme-catalyzed coupling between
nicotinic acid with γ-hydroxy/γ-aminobutyric acid (5 and 9),
ethanol/ethylamine (6 and 7), or methanol/methylamine (8)
and the co-occurring secologanic acid (5 and 7) or secologanin
(6, 8 and 9), and compounds 10ꢀ15 from coupling between
pyridine with 2-aminoethanol/1,2-ethandiol (10 and 11), 2-hy-
droxy/2-amino acetic acid (12), 3-hydroxy/3-amino propionic
acid (13), or γ-hydroxy/γ-amino butyric acid (14 and 15) and
the co-occurring secologanic acid (10 and 12ꢀ14) or secologa-
nin (11 and 15). An enzyme-catalyzed methylation or demethy-
lation would be an alternative for the mutual conversion between
the 11-carboxylic acids and the methyl 11-carboxylates.
In the preliminary in vitro assays, compounds 1ꢀ4, at 10 μM,
inhibited STAT-3 (signal transducers and activators of transcrip-
tion 3, which has been reported to play critical roles of regulating
tyrosine kinase correlative cytokine signaling pathways in the
pathogenesis of autoimmune and inflammation diseases and
cancer16) activity in human embryonic lung fibroblast (HELF)
cells. As compared with the blank, control compounds 1ꢀ4
showed 45.7 ( 12.5%, 25.8 ( 2.0%, 30.9 ( 1.8%, and 48.8 (
8.7% inhibition, respectively. Lonijaposides F (7), H (9), I (10),
and K (12), at 10 μM, showed inhibitory activity against
the release of glucuronidase in rat polymorphonuclear leuko-
cytes induced by platelet-activating factor with 87.6 ( 1.6%,
Plant Material. See ref 6.
Extraction and Isolation. For extraction and preliminary fractio-
nation of the extract, see ref 6. Fraction B3-10 (22.1 g) was separated by
MPLC over RP silica gel, eluting with a gradient of EtOH (0ꢀ50%) in
H2O to give subfractions B3-10-1ꢀB3-10-13. B3-10-6 (1.9 g) was further
separated by flash chromatography over RP silica gel, eluting with a
gradient of CH3CN (0ꢀ30%) in H2O to obtain fractions B3-10-6-
1ꢀB3-10-6-6. B3-10-6-3 (28 mg) was subjected to RP-HPLC, using
CH3CNꢀH2O (5:95) containing 0.5% HOAc as the mobile phase, to
afford 6 (6 mg, 0.00005%), 7 (1 mg, 0.0000083%), and 8 (0.8 mg,
0.0000067%). Fraction B3-11 (4.6 g) was separated by flash chroma-
tography over RP silica gel, eluting with a gradient of EtOH (0ꢀ50%) in
H2O to give subfractions B3-11-1ꢀB3-11-4. B3-11-1 (62 mg) and B3-11-
4 (42 mg) were subjected separately to RP-HPLC, using CH3CNꢀH2O
(7:93) containing 0.5% HOAc as the mobile phase, from B3-11-1 to yield
5 (1.2 mg, 0.00001%), 9 (5.4 mg, 0.000045%), and 15 (8.8 mg,
0.000073%) and from B3-11-4 to obtain 10 (5.6 mg, 0.000047%) and
11 (2.1 mg, 0.000018%). Fraction B3-13 (7.6 g) was separated by flash
chromatography over RP silica gel, eluting with a gradient of EtOH
(0ꢀ50%) in H2O, to give subfractions B3-13-1ꢀB3-13-15. B3-13-6 (16
mg) and B3-13-8 (2860 mg) were subjected separately to RP-HPLC, for
B3-13-6 using CH3CNꢀH2O (3:97) containing 0.5% HOAc as the
mobile phase to afford 12 (3.9 mg, 0.000033%) and 13 (5 mg,
0.000042%) and for B3-13-8 using CH3CNꢀH2O (7:93) containing
0.5% HOAc as the mobile phase to afford 14 (370.2 mg, 0.0031%).
Subfraction B4 (86 g) was chromatographed over a RP silica gel column,
eluting with a gradient of EtOH (0ꢀ100%) in H2O, to yield subfractions
B4-1ꢀB4-7, of which subfraction B4-7 (1.4 g) was further separated by
flash chromatography over RP silica gel, eluting with a gradient of
MeOH (0ꢀ50%) in H2O to give subfractions (B4-7-1ꢀB4-7-4). B4-7-3
(76 mg) and B4-7-4 (108 mg) were separately subjected to RP-HPLC,
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Journal of Natural Products
ARTICLE
for B4-7-3 using CH3OHꢀH2O (1:1) containing 0.5% HOAc as the
mobile phase, to afford 1 (5 mg, 0.000042%) and 2 (22 mg, 0.00018%),
and for B4-7-4 using CH3OHꢀH2O (6:4) with 0.1% HOAc as the mobile
phase, to afford 3 (16 mg, 0.00013%) and 4 (4.5 mg, 0.000038%).
Lonijaposide G (8): beige, amorphous solid; [α]20 ꢀ103.4 (c 0.04,
D
H2O); UV (H2O) λmax (log ε) 205 (4.12, sh), 230 (3.97, sh), 261 (3.81,
sh), 3.06 (3.41) nm; CD (H2O) 196 (Δε ꢀ0.75), 213 (Δε + 0.04), 227
(Δε ꢀ0.80), 238 (Δε ꢀ0.57), 247 (Δε ꢀ0.65), 266 (Δε ꢀ1.71); IR
(KBr) νmax 3391, 2959, 2922, 2852, 1730, 1668, 1577, 1543, 1465, 1420,
1381, 1264, 1121, 1073, 1038, 958, 863, 801, 743, 704 cmꢀ1; 1H NMR
(D2O, 600 MHz) data, see Table 2; positive ESIMS m/z 530 [M + Na]+;
positive HR-ESIMS m/z 508.1812 [M + H]+ (calcd for C24H30NO11,
508.1813).
Loniphenyruviridoside A (1): white, amorphous powder; [α]20
D
ꢀ72.1 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 202 (4.42), 220
(4.13, sh), 274 (3.71, sh) nm; CD (MeOH) 244 (Δε ꢀ8.59), 272 (Δε
+6.91); IR (KBr) νmax 3330, 2921, 2851, 1732, 1641, 1588, 1499, 1402,
1277, 1195, 1156, 1063, 985, 930, 761, 698 cmꢀ1; 1H NMR (methanol-
d4, 600 MHz), see Table 1; 13C NMR (methanol-d4, 125 MHz), see
Table 1; negative ESIMS m/z 475 [M ꢀ H]ꢀ; negative HR-ESIMS m/z
475.1603 [M ꢀ H]ꢀ (calcd for C24H27O10, 475.1610).
Lonijaposide H (9): beige, amorphous solid; [α]20 ꢀ140 (c 0.1,
D
H2O); UV (H2O) λmax (log ε) 193 (4.57), 210 (4.27, sh), 265
(4.03) nm; CD (H2O) 200 (Δε ꢀ2.74), 206 (Δε ꢀ2.58), 213 (Δε
ꢀ3.05), 227 (Δε ꢀ0.92), 239 (Δε ꢀ0.89), 247 (Δε ꢀ1.16), 277 (Δε +
1.21), 308 (Δε + 0.21); IR (KBr) νmax 3420, 2128, 1644, 1564, 1414,
1299, 1077, 1020, 932, 657 cmꢀ1; 1H NMR (D2O, 600 MHz) data, see
Table 2; 13C NMR (D2O, 125 MHz) data, see Table 3; negative ESIMS
m/z 578 [M ꢀ H]ꢀ; positive HR-ESIMS m/z 580.2039 [M + H]+
(calcd for C27H34NO13, 580.2025).
Loniphenyruviridoside B (2): white, amorphous powder; [α]20
D
ꢀ50.2 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (4.42), 220
(4.09, sh), 284 (3.86) nm; CD (MeOH) 232 (Δε ꢀ 8.26), 281 (Δε +
8.93); IR (KBr) νmax 3361, 2920, 2850, 1740, 1678, 1635, 1541, 1500,
1395, 1307, 1279, 1163, 1074, 1020, 927, 882, 768, 696 cmꢀ1; 1H NMR
(methanol-d4, 600 MHz), see Table 1; 13C NMR (methanol-d4, 125
MHz), see Table 1; negative ESIMS m/z 519 [M ꢀ H]ꢀ; negative HR-
ESIMS m/z 519.1490 [M ꢀ H]ꢀ (calcd for C25H27O12, 519.1508).
Loniphenyruviridoside C (3): white, amorphous powder; [α]20D ꢀ52
(c 0.3, MeOH); UV (MeOH) λmax (log ε) 202 (4.48), 220 (4.22, sh),
284 (4.12) nm; CD (MeOH) 214 (Δε ꢀ10.45), 244 (Δε +2.06), 282
(Δε ꢀ17.15); IR (KBr) νmax 3329, 2920, 2852, 1741, 1676, 1628, 1541,
1499, 1395, 1311, 1281, 1158, 1073, 940, 873, 766, 695 cmꢀ1; 1H NMR
(methanol-d4, 600 MHz), see Table 1; 13C NMR (methanol-d4, 125
MHz), see Table 1; negative ESIMS m/z 519 [M ꢀ H]ꢀ; positive HR-
ESIMS m/z 521.1664 [M + H]+ (calcd for C25H29O12, 521.1654).
Lonijaposide I (10): beige, amorphous solid; [α]20D ꢀ237.2 (c 0.05,
H2O); UV (H2O) λmax (log ε) 227 (4.18), 252 (3.95, sh), 302
(3.54) nm; CD (H2O) 197 (Δε ꢀ2.15), 215 (Δε ꢀ0.80), 228 (Δε
ꢀ1.70), 243 (Δε ꢀ0.77), 265 (Δε ꢀ2.72); IR (KBr) νmax 3349, 2928,
1643, 1538, 1412, 1346, 1189, 1160, 1075, 1045, 1022, 951, 796, 659,
1
617 cmꢀ1; H NMR (D2O, 500 MHz) data, see Table 2; 13C NMR
(D2O, 125 MHz) data, see Table 3; positive ESIMS m/z 480 [M + H]+,
502 [M + Na]+; negative ESIMS m/z 478 [M ꢀ H]ꢀ; positive HR-
ESIMS m/z 480.1873 [M + H]+ (calcd for C23H30NO10, 480.1864).
Lonijaposide J (11): beige, amorphous solid; [α]20D ꢀ219.2 (c 0.05,
H2O); UV (H2O) λmax (log ε) 229 (4.08), 252 (3.93, sh), 299
(3.46) nm; CD (H2O) 206 (Δε + 0.01), 221 (Δε ꢀ1.91), 235 (Δε
ꢀ5.64), 244 (Δε ꢀ4.36), 260 (Δε ꢀ11.63); IR (KBr) νmax 3352, 2920,
2851, 1691, 1631, 1567, 1414, 1306, 1253, 1200, 1164, 1076, 1045, 947,
771, 720, 656, 621 cmꢀ1; 1H NMR (D2O, 500 MHz) data, see Table 2;
13C NMR (D2O, 125 MHz) data, see Table 3; positive ESIMS m/z 494
[M ꢀ Cl]+; negative ESIMS m/z 528/530/532 (3:2:1) [M ꢀ H]ꢀ;
positive HR-ESIMS m/z 494.2029 [M ꢀ Cl]+ (calcd for C24H32NO10,
494.2021).
Loniphenyruviridoside D (4): white, amorphous powder; [α]20
D
ꢀ209.5 (c 0.4, MeOH); UV (MeOH) λmax (log ε) 203 (4.52), 220
(4.19, sh), 283 (3.89) nm; CD (MeOH) 211 (Δε ꢀ9.85), 244 (Δε +
1.16), 282 (Δε +11.81); IR (KBr) νmax 3337, 2955, 2919, 2851, 1737,
1642, 1598, 1548, 1499, 1396, 1073, 1019, 927, 883, 771, 721, 699 cmꢀ1
;
1H NMR (methanol-d4, 600 MHz), see Table 1; 13C NMR (methanol-d4,
125 MHz), see Table 1; negative ESIMS m/z 519 [M ꢀ H]ꢀ; positive
HR-ESIMS m/z 521.1652 [M + H]+ (calcd for C25H29O12, 521.1654).
Lonijaposide D (5): beige, amorphous solid; [α]20 ꢀ117.2 (c 0.05,
D
Lonijaposide K (12): beige, amorphous solid; [α]20 ꢀ81 (c 0.32,
H2O); UV (H2O) λmax (log ε) 205 (4.23, sh) nm, 230 (3.97, sh) nm, 264
(3.74, sh) nm, 307(3.42, sh) nm; CD (H2O) 201 (Δε ꢀ4.59), 212 (Δε
ꢀ1.73), 226 (Δε ꢀ3.91), 244 (Δε ꢀ1.95), 265 (Δε ꢀ3.02); IR (KBr)
νmax 3383, 2958, 2918, 2851, 1728, 1598, 1543, 1360, 1288, 1121, 1074,
D
H2O); UV (H2O) λmax (log ε) 230 (4.15), 257 (3.93), 299 (3.39) nm;
IR (KBr) νmax 3360, 2925, 1636, 1546, 1507, 1381, 1190, 1162, 1074,
1042, 942, 795, 716, 670, 613 cmꢀ1; 1H NMR (D2O, 600 MHz) data,
see Table 2; 13C NMR (D2O, 150 MHz) data, see Table 3; positive
ESIMS m/z 494 [M + H]+; positive HR-ESIMS m/z 494.1675 [M + H]+
(calcd for C23H28NO11, 494.1662).
1
1041, 833, 796, 742, 611 cmꢀ1; H NMR (D2O, 600 MHz) data, see
Table 2; 13C NMR (D2O, 150 MHz) data, see Table 3; positive ESIMS
m/z 566 [M + H]+; negative ESIMS m/z 564 [M ꢀ H]ꢀ; positive HR-
ESIMS m/z 566.1862 [M + H]+ (calcd for C26H32NO13, 566.1868).
Lonijaposide L (13): beige, amorphous solid; [α]20 ꢀ68 (c 0.47,
D
Lonijaposide E (6): beige, amorphous solid; [α]20 ꢀ169.2 (c 0.2,
H2O); UV (H2O) λmax (log ε) 226 (3.85), 257 (3.61), 299 (3.09) nm;
IR (KBr) νmax 3366, 2955, 2199, 2164, 2094, 1641, 1562, 1411, 1248,
D
H2O); UV (H2O) λmax (log ε) 207 (4.41, sh) nm, 231 (4.34, sh) nm, 261
(4.19, sh) nm, 306 (3.67) nm; CD (H2O) 197 (Δε ꢀ2.17), 205 (Δε
ꢀ0.54), 215 (Δε ꢀ0.38), 229 (Δε ꢀ2.39), 240 (Δε ꢀ2.11), 267 (Δε
ꢀ6.13); IR (KBr) νmax 3363, 2922, 1700, 1638, 1612, 1592, 1437, 1385,
1303, 1261, 1165, 1076, 1046, 938, 894, 788, 770, 681 cmꢀ1; 1H NMR
(D2O, 600 MHz) data, see Table 2; 13C NMR (D2O, 125 MHz) data, see
Table 3; positive ESIMS m/z 522 [M + H]+, 544 [M + Na]+, and 560 [M +
K]+; negative ESIMS m/z 520 [M ꢀ H]ꢀ, 556 [M + Cl]ꢀ; positive HR-
ESIMS m/z 522.1978 [M + H]+ (calcd for C25H32NO11, 522.1970).
Lonijaposide F (7): beige, amorphous solid; [α]20D ꢀ105.4 (c 0.04,
H2O); UV (H2O) λmax (log ε) 229 (4.03, sh), 263 (3.82), 305
(3.54) nm; CD (H2O) 197 (Δε ꢀ1.63), 212 (Δε ꢀ0.47), 225 (Δε
ꢀ1.04), 246 (Δε ꢀ0.21), 269 (Δε ꢀ1.07); IR (KBr) νmax 3397, 2958,
2917, 2851, 1731, 1578, 1542, 1512, 1469, 1423, 1381, 1288, 1262,
1
1162, 1103, 1073, 952, 929, 840, 802, 687, 652, 619 cmꢀ1; H NMR
(D2O, 500 MHz) data, see Table 2; 13C NMR (D2O, 125 MHz) data,
see Table 3; positive ESIMS m/z 508 [M + H]+; negative ESIMS m/z
506 [M ꢀ H]ꢀ, 542 [M + Cl]ꢀ; positive HR-ESIMS m/z 508.1819
(calcd for C24H30NO11, 508.1819).
Lonijaposide M (14): beige, amorphous solid; [α]20D ꢀ169 (c 0.24,
H2O); UV (H2O) λmax (log ε) 227 (4.26), 257 (4.04), 296 (3.53) nm;
IR (KBr) νmax 3408, 2936, 1643, 1560, 1401, 1188, 1159, 1075, 1042,
941, 800, 683 cmꢀ1; 1H NMR (D2O, 500 MHz) data, see Table 2; 13
C
NMR (D2O, 125 MHz) data, see Table 3; positive ESIMS m/z 522 [M +
H]+, 544 [M + Na]+; negative ESIMS m/z 520 [M ꢀ H]ꢀ; positive HR-
ESIMS m/z 522.1958 [M + H]+ (calcd for C25H32NO11, 522.1975).
Lonijaposide N (15): beige, amorphous solid; [α]20D ꢀ218.4 (c 0.25,
H2O); UV (H2O) λmax (log ε) 229 (4.50), 250 (4.35, sh), 300 (3.78,
sh) nm; CD (H2O) 207 (Δε ꢀ0.40), 219 (Δε ꢀ1.21), 235 (Δε ꢀ3.73),
244 (Δε ꢀ2.75), 263 (Δε ꢀ8.81); IR (KBr) νmax 3386, 2920, 1701,
1632, 1574, 1507, 1439, 1400, 1304, 1249, 1199, 1162, 1077, 1047, 938,
1
1199, 1183, 1118, 1074, 1028, 802, 742, 718 cmꢀ1; H NMR (D2O,
600 MHz) data, see Table 2; negative ESIMS m/z 506 [M ꢀ H]ꢀ;
positive HR-ESIMS m/z 508.1816 [M + H]+ (calcd for C24H30NO11,
508.1813).
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ARTICLE
891, 822, 795, 771, 686, 637 cmꢀ1; 1H NMR (D2O, 600 MHz) data, see
Table 2; 13C NMR (D2O, 125 MHz) data, see Table 3; positive ESIMS
m/z 536 [M + H]+; negative ESIMS m/z 534 [M ꢀ H]ꢀ; positive HR-
ESIMS m/z 536.2137 [M + H]+ (calcd for C26H34NO11, 536.2126).
Enzymatic Hydrolysis of Compound 1. A solution of compound 1
(3 mg) in H2O (1 mL) was treated with β-glucosidase from almonds
(Fluka) (5 mg) at 37 °C for 24 h. The reaction mixture was extracted
with EtOAc (2 ꢁ 3 mL). The aqueous phase of the hydrolysate was dried
using a stream of N2 and then subjected to column chromatography over
silica gel, eluting with CH3CNꢀH2O (8:1) to yield glucose (0.6 mg). It
gave a positive specific rotation, [α]20D +41.1 (c 0.06, H2O). The solvent
system CH3CNꢀH2O (6:1) was used for TLC identification of glucose
(Rf = 0.34).
X-ray Crystallography of Secologanic Acid. C16H22O10, M = 374.34,
orthorhombic, P212121, a = 8.460(2) Å, b = 11.033(1) Å, c = 18.202(1)
Å, α = β = γ = 90o, V = 1699.0(1) Å3, Z = 4, Dcalcd = 1.463 g cmꢀ3, 2966
reflections independent, R = 0.0682, wR2 = 0.1612, S = 1.043.
The data were collected on a MACDIP-2030K diffractometer with
Cu Kα radiation by using the ω and k scan technique to a maximum 2θ
value of 141.74°. The crystal structures were solved by direct methods by
using SHELXS-97, and all non-hydrogen atoms were refined anisotro-
pically using the least-squares method. All hydrogen atoms were
positioned by geometric calculation and difference Fourier overlapping
calculation. The absolute configuration was determined on the basis of
the Flack parameter, 0.2(4). Crystallographic data for the structure of
secologanic acid have been deposited with the Cambridge Crystal-
lographic Data Centre as supplementary publication CCDC 786003.
Copies of these data can be obtained free of charge via www.ccdc.cam.ac.
uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)
1223-336-033; or deposit@ccdc.cam.ac.uk).
STAT-3 Reporter Activity Assay (ref 21). Human embryonic lung
fibroblast (HELF) cells were grown in DMEM supplemented with 10%
FCS and maintained in a 5% CO2 humidified atmosphere. The cells
were transfected with pSTAT-3-TA-luc luciferase reporter plasmid by
lipofectamine 2000 reagent according to previous reports. After 24 h, the
cells were treated in 5% FBS with chemicals at varying concentrations or
DMSO for 24 h. The STAT-3 luciferase activity was measured in cell
lysates by a microplate luminometer using the firefly luciferase assay kit
(Promega, Madison, WI, USA). The STAT-3 inhibition activity of the
treated cells is reported relative to pSTAT-3-TA-luc transfected cells
treated with DMSO arbitrarily set at 100%.
Anti-inflammatory Activity Assay. See refs 6 and 17.
Cells, Culture Conditions, and Cell Proliferation Assay. See ref 18.
Neuroprotective Activity Assay. See ref 19.
Antioxidative and PTP1B Inhibition Assays. See ref 20.
HIV-1 Replication Inhibition Assay. See ref 21.
’ ASSOCIATED CONTENT
S
Supporting Information. Copies of UV, IR, MS, 1D and
b
2D NMR, and CD spectra for compounds 1ꢀ15; copies of
spectra of 2a, 3a, S2, and S3; X-ray crystallographic data of
secologanic acid. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 86-10-83154789. Fax: 86-10-63017757. E-mail: zhuchenggen@
imm.ac.cn and shijg@imm.ac.cn.
Methylation of Compounds 2 and 3. To a solution of 2 (3 mg) or 3
(4 mg) in anhydrous MeOH (5 mL) were added EDCI (5 mg) and
DMAP (2.5 mg). After the solution was stirred for 20 min at rt, H2O
(5 mL) was added and evaporated under reduced pressure to give a
residue. The residue was subjected to RP-HPLC, using CH3CNꢀH2O
(3:10) containing 1% HOAc as the mobile phase, to afford 2a (1.9 mg)
from 2 and 3a (2.3 mg) from 3, respectively. 2a: white, amorphous
powder; 1H NMR (methanol-d4, 600 MHz) and 13C NMR (methanol-
’ ACKNOWLEDGMENT
Financial support from the National Natural Sciences Foun-
dation of China (NNSFC; grant nos. 20772156, 30825044, and
20932007) and the National Science and Technology Project of
China (no. 2009ZX09311-004) is acknowledged.
1
d4, 150 MHz), see Table 1. 3a: white, amorphous powder; H NMR
(methanol-d4, 600 MHz) and 13C NMR (methanol-d4, 150 MHz) data,
see Table 1.
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