Anti-hepatitis B virus activities and absolute configurations of sesquiterpenoid glycosides from Phyllanthus emblica

Article abstract of DOI:10.1039/c4ob01196a

During the process exploring anti-viral compounds from Phyllanthus species, eight new highly oxygenated bisabolane sesquiterpenoid glycoside phyllaemblicins G1-G8 (1-8) were isolated from Phyllanthus emblica, along with three known compounds, phyllaemblicin F (9), phyllaemblic acid (10) and glochicoccin D (11). Phyllaemblicin G2 (2), bearing a tricyclo [3.1.1.1] oxygen bridge ring system, is an unusual sesquiterpenoid glycoside, while phyllaemblicins G6-G8 (6-8) are dimeric sesquiterpenoid glycosides with two norbisabolane units connecting through a disaccharide. All the structures were elucidated by the extensive analysis of HRMS and NMR data. The relative configuration of phyllaemblicin G2 was constructed based on heteronuclear coupling constants measurement, and the absolute configurations for all new compounds were established by calculated electronic circular dichroism (ECD) using time dependent density functional theory. The sesquiterpenoid glycoside dimers 6-9 displayed potential antihepatitis B virus (HBV) activities, especially for the new compound 6 with IC₅₀of 8.53 ± 0.97 and 5.68 ± 1.75 μM towards the HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg) secretion, respectively.

Full text of DOI:10.1039/c4ob01196a

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Anti-hepatitis B virus activities and absolute
configurations of sesquiterpenoid glycosides
from Phyllanthus emblica†
Cite this: Org. Biomol. Chem., 2014,
12, 8764
Jun-Jiang Lv,‡a,b Ya-Feng Wang,‡c Jing-Min Zhang,c,d Shan Yu,a,b Dong Wang,a
Hong-Tao Zhu,a Rong-Rong Cheng,a Chong-Ren Yang,a Min Xu*a and
Ying-Jun Zhang*a
During the process exploring anti-viral compounds from Phyllanthus species, eight new highly oxyge-
nated bisabolane sesquiterpenoid glycoside phyllaemblicins G1–G8 (1–8) were isolated from Phyllanthus
emblica, along with three known compounds, phyllaemblicin F (9), phyllaemblic acid (10) and glochicoc-
cin D (11). Phyllaemblicin G2 (2), bearing a tricyclo [3.1.1.1] oxygen bridge ring system, is an unusual ses-
quiterpenoid glycoside, while phyllaemblicins G6–G8 (6–8) are dimeric sesquiterpenoid glycosides
with two norbisabolane units connecting through a disaccharide. All the structures were elucidated by
the extensive analysis of HRMS and NMR data. The relative configuration of phyllaemblicin G2 was
constructed based on heteronuclear coupling constants measurement, and the absolute configurations
for all new compounds were established by calculated electronic circular dichroism (ECD) using time
dependent density functional theory. The sesquiterpenoid glycoside dimers 6–9 displayed potential anti-
hepatitis B virus (HBV) activities, especially for the new compound 6 with IC50 of 8.53 0.97 and 5.68
1.75 μM towards the HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg) secretion,
respectively.
Received 10th June 2014,
Accepted 9th September 2014
DOI: 10.1039/c4ob01196a
www.rsc.org/obc
resistance to these drugs stimulated chemists to explore new
anti-HBV agents with novel mechanisms. High levels of HBV
Introduction
Despite the effective vaccine programs available, hepatitis B surface antigen (HBsAg)-bearing subviral particles in the
virus (HBV) infection is still a major world health problem. serum of chronically infected individuals play a role in sup-
About 400 million people in the world suffer from HBV infec- pressing the HBV immune response and current therapeutics
tion.1 The present approved HBV chemotherapies are all are not directed at reducing this antigenemia.3–5
nucleoside or nucleoside-based polymerase inhibitors,2 and
Phyllanthus emblica Linn, a Euphorbiaceae plant, is an
important traditional medicinal plant in China. In our pre-
vious studies, nine bisabolane and norbisabolane sesquiterpe-
noid glycosides were isolated and reported: phyllaemblic acid,6
phyllaemblic acids B and C,7 and phyllaemblicins A–F.8,9
Among them, phyllaemblicin B exhibited significant anti-cox-
sackievirus B3 activity.9 In order to explore novel antiviral com-
pounds from P. emblica, a further chemical study was carried
out, leading to the isolation of eight new highly oxygenated
bisabolane sesquiterpenoid glycosides (1–8) from the roots of
the title plant, along with three known ones (9–11). Their
structures were elucidated by HRMS and NMR experiments,
and the absolute configurations were established by calculated
ECD using time-dependent density functional theory (TDDFT).
The isolated compounds were evaluated for their anti-HBV
aState Key Laboratory of Phytochemistry and Plant Resources in West China,
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
People’s Republic of China. E-mail: zhangyj@mail.kib.ac.cn, minxu@mail.kib.ac.cn;
Fax: +86-871-6522-3235; Tel: +86-871-6522-3235
bUniversity of Chinese Academy of Sciences, Beijing 100049, People’s Republic of
China
cSchool of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001,
People’s Republic of China
dHenan Key Laboratory for Pharmacology of Liver Diseases, Academy of Medical and
Pharmaceutical Sciences, Zhengzhou University, 40 Daxue Road, Zhengzhou 450052,
People’s Republic of China
†Electronic supplementary information (ESI) available: MS, 1D and 2D NMR
spectra of compounds 1–8 and 2A, together with the ECD calculation data are
provided. See DOI: 10.1039/c4ob01196a
‡These authors contributed equally to this work.
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activities against HBsAg and HBeAg secretion and the results spectrum of 1 (Table 1) displayed the existence of one benzoyl
obtained are discussed herein.
group [δH 8.12 and 7.53 (each 2H, J = 8.0 Hz), δH 7.68 (1H, t, J =
7.4 Hz)] and two anomeric protons [δH 5.55 and 4.26 (each 1H,
d, J = 8.0 Hz)]. In addition to the signals of one benzoyl and
two hexosyl groups the 13C NMR and DEPT spectra (Table 1)
showed 15 carbon signals arising from one methyl (δC 13.0),
Results and discussion
The 70% ethanol extract of the air-dried roots of P. emblica was five methylenes including two oxygen-bearing ones (δC 63.2
subjected to various column chromatography, followed with and 63.4), five methines including three oxymethines, one
preparative HPLC (p-HPLC) to afford 11 sesquiterpenoids ketal carbon (δC 107.7), one carboxyl (δC 175.9) group and two
(1–11). Compounds 9–11 were identified as the known phyl- oxygen-bearing quaternary carbons (δC 81.1 and 87.8). These
laemblicin F (9),9 phyllaemblic acid (10),6 and glochicoccin D NMR features were comparable to those of phyllaemblicin B, a
(11),10 respectively, by directly comparing with authentic known norbisabolane glycoside from the titled plant,8 indicat-
samples and their spectroscopic and physical data with those ing that 1 was a highly oxygenated bisabolane sesquiterpenoid
previously reported in the literatures.
glycoside. The main difference was the appearance of an oxy-
Phyllaemblicin G1 (1) was obtained as a white amorphous genated quaternary carbon (δC 87.8) and an additional oxy-
powder, with a molecular formula C34H48O20, as established methylene (δC 63.4) in 1, instead of the C-7 ketone (δC 213.7) in
from the HRESIMS m/z 775.2657 [M − H]−. The 1H NMR phyllaemblicin B. The signals δC 63.4 and δC 87.8 were
Table 1 13C and 1H NMR spectroscopic data for compounds 1–3 in CD3OD (δ in ppm)
1
2
3
a
a
a
c
b
b
No.
δC
δH
δC
δH
δC
δH
1
73.0, CH
31.7, CH2
3.90 brs
2.02d
75.4, CH
27.6, CH2
4.32 brs
72.1, CH
29.1, CH2
3.81 dd (4.9, 10.2)
1.60 ddd (9.2, 10.2, 13.9)
1.96d
2.58 dddd (5.6, 5.6, 9.2, 11.2)
1.87 ddd (3.3, 11.2, 14.3)
1.96d
2a
2b
3
4a
4b
5
1.63d
2.24 brd (14.4)
3.03 tt (4.0, 13.0)
1.49 ddd (1.4, 13.0 14.1)
1.63d
34.6, CH
29.3, CH2
2.85 m
33.5, CH
28.4, CH2
34.3, CH
27.7, CH2
1.85 ddd (4.0, 11.0, 14.3)
2.07d
80.5, CH
81.1, C
4.09 t (3.8)
74.9, CH
77.1, C
3.84 brs
82.0, CH
76.5, C
4.07 brs
6
7
8
87.8, C
107.7, C
34.2, CH2
82.1, C
107.9, C
32.0, CH2
76.0, CH
102.4, C
36.0, CH2
3.82 s
2.17d
9a
9b
10
11
12a
12b
13
14
15a
15b
1′
1.95 dd (3.1, 15.1)
2.28 dd (2.8, 15.1)
5.28 brs
2.21 dd (10.0, 15.5)
2.16 dd (2.3, 15.5)
5.67 ddd (2.3, 5.8, 10.0)
2.03 m
71.8, CH
34.1, CH
63.2, CH2
72.7, CH
41.7, CH
64.8, CH2
72.0, CH
34.2, CH
63.1, CH2
5.29 brs
2.08d
2.15d
3.56 dd (4.8, 11.2)
4.05 dd (11.2, 11.5)
3.45d
3.64 dd (4.7, 11.4)
4.07 dd (11.4, 11.4)
3.67 dd (5.8, 11.0)
175.9, C
13.0, CH3
63.4, CH2
176.4, C
13.6, CH3
61.0, CH2
177.4, C
13.0, CH3
0.86 d (7.2)
3.69d
1.03 d (6.9)
3.81 d (11.8)
3.88 d (11.8)
0.93 d (6.8)
3.92d
132.3, C
132.6, C
132.1, C
2′,6′
3′,5′
4′
130.8, CH
129.8, CH
134.3, CH
168.0, C
8.12 d (8.0)
7.53 t (8.0)
7.64 t (7.4)
130.9, CH
129.6, CH
134.0, CH
168.4, C
8.06 d (8.0)
7.47 t (8.0)
7.57 t (7.4)
130.7, CH
129.5, CH
134.1, C
8.12 d (8.0)
7.50 t (8.0)
7.63 t (7.4)
7′
168.1, C
1″
2″
3″
4″
93.7, CH
82.6, CH
77.8, CH
70.7, CH
78.8, CH
62.3, CH2
5.55 d (8.0)
3.35d
3.61 dd (9.0, 9.0)
3.43 dd (9.0, 9.0)
3.37d
94.3, CH
82.0, CH
77.9, CH
71.1, CH
79.0, CH
62.7, CH2
5.59 d (7.5)
52.3, CH3
3.37 s
3.66d
3.64 dd (9.0, 9.0)
3.42d
5″
3.44 ddd (3.0, 5.0, 9.0)
3.76 dd (5.0, 12.2)
3.86 dd (3.0, 12.2)
4.71 d (8.0)
3.21 dd (8.0, 9.5)
3.46 dd (9.5, 9.5)
3.38 dd (9.5, 9.5)
3.46d
6″a
6″b
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′a
6′′′b
3.72 dd (5.0, 12.1)
3.87 dd (2.1, 12.1)
4.26 d (8.0)
3.13 dd (8.0, 9.2)
3.28d
105.8, CH
75.8, CH
77.8, CH
70.8, CH
77.7, CH
61.9, CH2
105.4, CH
76.5, CH
78.0, CH
71.6, CH
77.8, CH
62.7, CH2
3.28d
2.88 ddd (2.9, 2.9, 9.2)
3.62 m
3.62 m
3.77 dd (4.8, 12.2)
3.85 dd (3.0, 12.2)
a Data were recorded for 13C NMR at 125 MHz, or 1H NMR at 500 MHz. b Data were recorded for 13C NMR at 150 MHz, or 1H NMR at 600 MHz.
c Data were recorded at 800 MHz. d Overlapped signals.
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Fig. 2 Experimental (1) and calculated (the aglycone of 1) ECD curves.
The absolute configuration of the aglycone of 1 was deter-
mined by comparing the calculated ECD curve with its experi-
mental ECD spectrum, as there was a benzoate chromophore
in the rigid C ring connecting to the C*-10 hydroxyl group. A
negative and a positive Cotton effect appeared at 230 and
250 nm arising from the π–π* excitation of the benzene ring.
The calculated ECD curve agreed well with the experimental
result as shown in Fig. 2. Thus, the absolute configurations of
Fig. 1 Key 1H–1H COSY, HMBC, ROESY correlations (A–B), and ring C
conformations (C–D) of 1.
assigned to C-15 and C-7, respectively, based on the HMBC
correlations from H-15 to C-7 and C-8, and H-1 to C-7 (Fig. 1).
Other HMBC correlations confirmed the planar structure of 1
as shown in Fig. 1.
The relative configuration of 1 was established by proton C-10 and C-11 were determined as 10S,11R. The rigid tetra-
coupling constants and ROESY experiment (Fig. 1). The coup- hydropyran ring C in 1 was of chair conformation with
ling constants of J3,4a (11.0 Hz), and J4,5 (3.8 Hz) indicated the 8R,10S,11R (Fig. 1C) and 8S,10S,11R (Fig. 1D) configurations,
axially α-orientated H-3 and β-orientated H-5. The broad which were confirmed by the Monte Carlo search with MMFF
singlet of H-1 suggested its β-orientation. This was further con- method and DFT/B3LYP 6-311G(d,p) optimization. The 10-H
firmed by the ROESY correlations of H-3 with H-4b (δH 2.07), and 11-H in 8S,10S,11R isomer were equatorially and axially
H-1 and H-5 with H-4a. The ROESY correlation of H-15 with orientated, respectively, which were consistent with that of
H-5 indicated that C-15 methylene was β-orientated. The coup- compound 1. However, they were axially and equatorially orien-
ling constants of H-12b (δH 4.05, 1H, dd, J = 11.2, 11.5 Hz) tated in the 8R,10S,11R isomer, which were opposite to those
suggested the axially orientated H-11, and the small coupling of 1. On the basis of the above evidence, the absolute configur-
constants of H-10 (δH 5.28, 1H, brs) implied its equatorial ation of C-8 was assigned as S.
orientation and location on the same face as H-11. The relative
Phyllaemblicin G2 (2) has the same molecular formula
configuration of 1 (except C-8) was therefore established as C34H48O20 as 1, as deduced from the HRESIMS (m/z 775.2653
1
shown in Fig. 1.
[M − H]−). The H and 13C NMR spectroscopic data (Table 1)
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Fig. 3 Key 1H–1H COSY, HMBC and ROESY correlations of 2.
were similar to those of 1, suggesting a close relationship
between these two compounds. In the 13C NMR spectrum, sig-
nificant up-field shifts of C-5, C-6 and C-7 (ΔδC −5.6, −4.0,
−5.7) and down-field shifts of C-1 and C-11 (ΔδC 2.4 and 7.6)
in 2, related to 1, were observed. In the HMBC experiment, the
strong correlation from H-12 to C-8 in 1 disappeared in 2,
instead of weak correlations from H-1 and H-5 to C-8 (δC 107.9)
(Fig. 3). This suggested that two oxygen bridges were formed
between C-1 and C-5 with C-8 in 2, and ring C was opened to
form an aliphatic chain. In order to confirm the rearranged
ring system in 2, the aglycone (2A) was obtained after hydro-
lysis using 0.3 M NaOH aqueous solution. The 1H–1H COSY
spectrum of 2A in DMSO-d6 (see ESI Fig. S23–27†) indicated
that both H-12 and H-15 were mutually coupled with their
corresponding hydroxyl protons at δH 4.29 (1H, t, J = 6.4 Hz,
12-OH) and δH 4.82 (1H, brs, 15-OH), respectively, indicating
that both C-12 and C-15 connected with a free hydroxy group.
Together with other 1H–1H COSY and HMBC correlations
(Fig. 3) of 2, the planar structure of 2 was constructed as
shown in Fig. 3.
The large coupling constant 13.0 Hz of H-3 suggested its
axial orientation, while the small coupling constants of H-1
and H-5 revealed their equatorial orientations. H-3 in A ring
with chair conformation was on the opposite face to H-1 and
H-5. The stereochemistry of C-7 could be deduced as being the
same as compound 1, considering the biosynthetic reason.
This was confirmed by the ROESY correlations of H-15b (δH
3.88) and H-9b. Together with the ROESY correlations of H-3
with H-10 and H-2′,6′ of the benzoyl group, it allowed the con-
struction of the relative configuration of 2 (Fig. 3). The value of
J9a,10 = 10.0 Hz indicated an anti-orientation of H-10 and H-9a
(δH 2.21). The relative configurations of C-10 and C-11 in the
aliphatic chain were determined using Murata’s method,11 as
Fig. 4 Newman projection of all possible staggered rotamers of threo
and erythro configurations viewed down bonds C11–C10. The JH,H and
3JC,H values allowed the assignment of rotamers of B2/B3, and the
3
ROESY correlations were drawn in double-sided arrows.
blished as 10S* and 11R*, respectively. In this case, the third
rotamer B1 existed in a comparable population, which meant
3
2
the JC-14,H-10 and JC-10,H-11 do not accurately correspond to
the rotamers B2/B3. This situation was mentioned by Murata
et al.,11 and the predominant B2/B3 rotamers deduced from
3
3JH-11,C-9 and JH-10,C-12 could lead to the correct answer. Using
Monte Carlo search with MMFF method and DFT/B3LYP
6-311G(d,p) optimization, the populations of the three rotamers
B1, B2 and B3 could be estimated as 44.1%, 34.2%, and 21.5%,
respectively (see ESI Fig. S74†).
Although the aliphatic chain on which the benzoate
chromophore was located was not a rigid system, all the lower
energy conformers displayed similar Cotton effects as that of
the Boltzmann averaged curve (Fig. 5 and ESI Fig. S74†).
The configurations of the aglycone part of phyllaemblicin
G2 (2) was determined on the basis of ECD calculation.
The absolute configurations of C-10 and C-11 were consistent
with those of 1 and the other bisabolane sesquiterpenoid
glycosides.6,8
this is a methyl and oxygen substituted 1,2-methine system in
2,3
an aliphatic chain. All
J
C,H
values were accurately measured
by 2D NMR of HETLOC (see ESI Fig. S15–18†). As shown in
Fig. 4, the medium 3JH-11,H-10 (5.8 Hz), medium 3JH-10,C-12 (4.2 Hz),
3
and small JH-11,C-9 (3.0 Hz) indicated that rotamers B2/B3
were the correct rotamer pair based on Murata’s rules. This
was confirmed by the ROESY correlations of H-10 with H-11
and H-14 in B2, and H-9 with H-14 in B3. Thus, the relative
configurations of C-10 and C-11 in compound 2 were esta-
Fig. 5 Experimental and calculated ECD curves of 2.
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Phyllaemblicin G3 (3) had a molecular formula C22H28O9, HRESIMS. Detailed analysis of NMR data (Table 2) revealed
as determined from the HRESIMS. Its NMR data were that both the compounds 4 and 5 had the same aglycone as
similar to those of phyllaemblic acid (10). However, instead compound 3. The only difference was the different saccharide
of a ketone signal (δC 213.9, C-7) in 10, the 13C NMR spec- moieties of 4 and 5. The saccharide moiety of 4 was deter-
trum displayed an oxymethine signal (δC 76.0) in 3. In the mined to be S1, which is the same as that of 1. In the case
HMBC spectrum, its corresponding proton at δH 3.82 (1H, s) of compound 5, the 1H and 13C NMR spectra displayed the
showed correlations with C-6, C-8 and C-9, and the signal δC presence of a glucosyl [anomeric proton at δH 4.10 (1H, d, J =
76.0 was assigned to be C-7. Moreover, the methoxy group 8.0 Hz)] unit and a set of signals arising from a hexaoxycyclo-
(δC 52.3, δH 3.37) was assigned to connect to C-13 as a hexane residue, which was linked to C-13, based on the
methyl ester by the HMBC correlation from δH 3.37 to the HMBC correlation from H-1″ (δH 4.80) to C-13 (δC 177.1). The
carboxyl carbon C-13. Ring C in compound 3 had the same hexaoxycyclohexane moiety was determined to be myo-inositol
relative configurations as 1. The large coupling constants of on the basis of 1H–1H COSY correlations of H-1″/H-2″/H-3″/
JH-1,H-2a (10.2 Hz), JH-2a,H-3 (9.2 Hz), and JH-4a,H-3 (11.2 Hz) H-4″/H-5″/H-6″ and their corresponding coupling con-
indicated the axial orientations of H-1 and H-3, while H-5 stants (Table 2). Moreover, the HMBC correlation from δH
with a small coupling constant was assigned to be equatorial 4.10 (H-1′′′) to C-2″ (δC 83.2) confirmed the saccharide moiety
orientation. The NOE effects of H-1 and H-5 with H-4a (δH in 5 as myo-inositol-2-O-β-glucopyranosyl group. The ECD
1.87), H-3 with H-2a (δH 1.60) and H-4b allowed the assign- spectra of 4 and 5 displayed the same Cotton effects as that
ment of H-1 and H-5 on the opposite face to H-3, indicating of 3, indicating the same absolute configuration as that of 3
that ring A was of boat conformation (Fig. 6). Taking into (Fig. 6).
account of the ROESY correlations of H-7 with H-9 and H-2a,
Phyllaemblicins G6–G8 (6–8) were dimeric sesquiterpenoid
the configuration of 3 was determined. The calculated ECD glycosides with two norbisabolane units connecting through a
curve agreed well with the experimental ECD spectrum. disaccharide moiety. Their molecular formulas were assigned
Thus, the configuration of
1S,3S,5R,6S,7R,8S,10S,11R.
3
was determined as as C54H68O27 (6), C54H66O28 (7), and C54H66O28 (8) based on
HRESIMS. The 1D NMR spectra (Tables 3 and 4) of 6 displayed
The molecular formulas of phyllaemblicins G4–G5 (4–5) the presence of two hexosyl units [anomeric centers at δH 5.64
were determined as C33H46O19 and C33H46O19, respectively, by and 4.14 (each 1H, d, J = 8.0 Hz, H-1″,1′′′), δC 93.8 (C-1″) and
106.2 (C-1′′′)]. The 13C NMR signals arising from the aglycone
part were similar to those of phyllaemblic acid (10). However,
they appeared in pairs or overlapped, suggesting that there
were two norbisabolane sesquiterpenoid units in 6, taking
1
account of the molecular formula. In the H–1H COSY experi-
ment, two proton spin systems, H-1″/H-2″ and H-4″/H-5″/H-6″
arising from a hexosyl unit, could be constructed. Together
with the HMBC correlations from H-3″ to C-2″ and C-5″, this
hexosyl moiety was determined to be a β-glucopyranosyl group
based on the coupling constants (Table 3). In HMBC spectrum,
H-1″ and H-6″ were correlated with two aglycone carboxyl
carbons at δC 176.0 (C-13, part A) and δC 177.4 (C-13, part B)
indicating that the sesquiterpenoid parts A and B were
connected to this glucosyl C-1″ and C-6″ through ester
linkage, respectively. Another hexosyl group in 6 was also
assigned as β-glucopyranosyl unit, and the connectivity of
the saccharide was confirmed by the HMBC correlation from
H-1′′′ to C-2″. The extensive analysis of the 2D NMR data
(Fig. 7) allowed the assignment of the signals arising from
aglycone moieties (parts A and B), which were determined to
be phyllaemblic acid (10, part A) and 7-hydroxyphyllaemblic
acid (part B). Therefore, the structure of 6 was determined as
shown.
Phyllaemblicin G7 (7) had similar NMR spectra to 6. The
obvious difference was the appearance of a p-hydroxybenzoyl
[δH 7.90 and 7.07 (each 2H, d, J = 8.5 Hz, H-2′,6′, H-3′,5′)] and a
ketone [δC 214.0] group in 7, instead of the benzoyl and C-7
oxymethine in 6. The ketone group at δC 214.0 was assigned to
be C-7 of part B based on its HMBC correlations with H2-9 (δH
1.91 and 2.10). The p-hydroxybenzoyl group was assigned to
Fig. 6 Key ROESY correlations of 3, and calculated (3) and experimental
ECD spectra of 3–5.
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Table 2 13C and 1H NMR spectroscopic data for compounds 4–5 in CD3OD (δ in ppm)
4 a
5 b
No.
δC
δH
δC
δH
1
72.1, CH
28.6, CH2
3.85 dd (4.6, 10.2)
1.60 ddd (9.4, 10.2, 14.0)
1.91 m
72.3, CH
28.4, CH2
3.84 dd (3.7, 9.4)
1.60 ddd (9.7, 9.7, 14.1)
2.20 m
2.71 m
1.93 m
2.20 m
4.13 dd (2.5, 2.5)
2a
2b
3
4a
4b
5
34.7, CH
28.1, CH2
2.67 dddd (5.6, 5.6, 9.4, 11.2)
34.5, CH
28.6, CH2
1.96c
2.16c
82.3, CH
76.7, C
4.11 brs
82.3, CH
76.6, C
6
7
8
9
10
11
12a
12b
13
14
1′
72.0, CH
102.5, C
36.1, CH2
72.0, CH
34.1, CH
63.0, CH2
3.83 s
75.8, CH
102.8, C
36.1, CH2
71.9, CH
34.2, CH
63.1, CH2
3.85 s
2.12c
2.15c
5.30 brs
2.11 m
5.33 m
2.15c
3.60c
3.64c
4.04 dd (11.6, 11.6)
4.09 dd (12.0, 12.0)
175.9, C
13.1, CH3
132.1, C
177.1, C
13.1, CH3
132.2, C
0.88 d (6.8)
0.90 d (6.9)
2′,6′
3′,5′
4′
131.0, CH
129.7, CH
134.4, CH
168.1, C
8.13 d (7.8)
7.52 t (7.8)
7.64 t (7.4)
130.9, CH
129.8, CH
134.6, CH
168.0, C
8.17 d (8.0)
7.56 t (8.0)
7.76 t (7.4)
7′
1″
2″
3″
4″
94.0, CH
82.9, CH
77.6, CH
70.7, CH
78.8, CH
62.2, CH2
5.55 d (7.9)
3.41 dd (7.9, 9.5)
3.61c
75.4, CH
83.2, CH
73.6, CH
73.5, CH
73.3, CH2
72.7, CH
4.80 t (9.6)
3.78 dd (9.6, 9.6)
3.60 dd (2.8, 9.6)
4.02 dd (2.8, 2.8)
3.43 dd (2.8, 9.6)
3.79 dd (9.6, 9.6)
3.38 dd (9.5, 9.5)
3.35 m
5″
6″a
6″b
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′a
6′′′b
3.78c
3.86c
105.8, CH
76.0, CH
77.8, CH
70.7, CH
77.8, CH
62.2, CH2
4.24 d (7.7)
3.14 dd (7.7, 8.8)
3.28c
106.1, CH
76.0, CH
78.1, CH
70.7, CH
77.6, CH
62.1, CH2
4.10 d (8.0)
3.11 dd (8.0, 9.0)
3.24 dd (9.0, 9.0)
3.31 dd (9.0, 9.0)
2.66 dt (3.0, 9.5)
3.44c
3.30c
2.86 ddd (3.2, 6.3, 9.1)
3.62c
3.70 dd (5.2, 12.8)
3.87c
a Data were recorded at 400 MHz for 1H NMR and 100 MHz for 13C NMR. b Data were recorded at 500 MHz for 1H NMR and 125 MHz for
13C NMR. c Signals were overlapped.
connect to the C-10 hydroxyl group of part B, based on the Antiviral activities of sesquiterpenoid glycosides
HMBC correlations from H-10 and H-2′,6′ to C-7′ of part
B. Detailed analysis of the 2D NMR data of 7 allowed the The isolated compounds 1, 2, 6–10 were evaluated for their
assignments of parts A and B as phyllaemblic acid (10) and antiviral activities against four types of virus, e.g. herpes
glochicoccin D (11), respectively.
simplex virus type 1 (HSV-1), enterovirus 71 (EV71), hepatitis B
Extensive analysis of 2D NMR spectra of phyllaemblicin G8 (HBV) and influenza A virus (H3N2). The sesquiterpenoid
(8) revealed that 8 had S1 as saccharide moiety, and phyl- dimers 6–9 can selectively affect HBsAg and HBeAg secretions,
laemblic acid (10, part A) and glochicoccin D (11, part B) as phyllaemblicin G6 (6) in particular showed strong inhibition
aglycone parts, which were the same as those of 7. In the towards HBsAg and HBeAg secretion with IC50 values of 8.53
HMBC spectrum of 8, correlations from H-1″ to C-13 of part A 0.97 and 5.68 1.75 μM compared with the positive control,
(δC 175.7), and the H-6′′′ to C-13 of part B (δC 176.6) indicated lamivudine with inhibition 44.66 2.09% and 25.96 9.59%
that part B of 8 located on the second glucosyl C-6′′′, which is towards HBsAg and HBeAg, respectively (Table 5). Even at a
different from that of 7.
concentration of 100 μM, and the inhibitory ratio of lamivu-
Since the sesquiterpenoid parts in compounds 6–8 dine cannot reach 50%. However, the aglycone 10 only dis-
were also obtained in the study, their relative and absolute played weak inhibition effect. The sesquiterpenoid 1 and 2
configurations can be proposed to be the same as those of were both ineffective to HBsAg and HBeAg secretion above the
their corresponding monomers. This was supported by the cytotoxic concentrations, implying that the structure character-
similar ECD spectra of 6–8 to those of phyllaemblic acid (10) istics of the sesquiterpenoid glycoside dimers (6–9) were
(Fig. 8).
related to the HBsAg and HBeAg secretion activity. It is
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Table 3 1H NMR Spectroscopic data for compounds 6–8 in CD3OD (δ in ppm)
6 a
7 a
8 a
No.
Part A
Part B
Part A
Part B
Part A
Part B
1
3.91 brs
3.83 m
3.90 brs
3.92 brs
1.78b
3.90 brs
3.90 brs
1.77b
2a
2b
3
1.76 dt (1.2, 13.1)
1.60 ddd (9.1, 10.1, 14.0)
1.82b
1.77b
2.04b
2.03b
1.91 dd (3.3, 12.5)
2.89 tt (3.5, 13.5)
1.72 ddd
2.02b
1.94b
2.85 tt (3.0, 13.0)
1.84 ddd
1.94b
2.90 tt (3.0, 12.5)
1.86 ddd
2.65 m
2.98 tt (3.5, 13.0)
2.99 tt (3.0, 13.0)
2.01 m
4a
1.98b
2.00b
(3.9, 12.4, 14.3)
2.29 brd (14.3)
4.22 brs
(4.0, 13.1, 14.6)
(3.6, 13.0, 15.5)
2.29 brd (15.5)
4.17 brs
4b
5
7
9a
9b
10
2.23 dd (3.2, 14.5)
4.04 brs
2.26b
2.91 m
4.29 brs
2.59 brd (15.5)
4.25 brs
4.07 brs
3.80 s
1.98b
2.01b
1.92b
1.91b
1.94b
2.15 dd (3.3, 15.2)
5.31 brs
1.90 dd (3.3, 14.8)
2.11 dd (3.3, 14.8)
5.24 brs
2.23b
2.12b
2.00b
2.10b
5.33 brs
5.27 brs
5.28 brs
5.29 brs
11
2.13b
2.04b
2.06b
2.00b
2.11 m
2.02 m
12a
12b
14
2′,6′
3′,5′
4′
1″
2″
3″
4″
3.51b
3.46b
3.40 dd (4.5, 11.5)
3.60 t (11.5)
0.73 d (7.0)
7.90 d (8.5)
7.07 d (8.5)
3.15b
3.45b
3.26b
3.86b
3.77b
3.38b
3.78 t (11.5)
0.79 d (7.0)
8.09 d (8.0)
7.63 t (8.0)
7.70 t (7.5)
3.45b
0.84 d (7.0)
8.09 d (8.0)
7.52 t (8.0)
7.56b
5.64 d (8.0)
3.44 dd (8.0, 9.0)
3.64 dd (9.0, 9.0)
3.50b
3.62 dt (2.4, 9.5)
4.28 brd (11.7)
4.50 dd (5.1,11.7)
4.14 d (8.0)
3.11 dd (8.0, 8.0)
3.22 dd (9.0, 9.0)
3.23 dd (9.0, 90)
2.65 ddd (3.2, 5.5, 9.0)
3.49 m
0.78 d (7.0)
8.12 d (8.0)
7.56 t (8.0)
7.65 t (7.1)
5.80 d (8.1)
3.78 dd (8.0, 9.2)
3.70 dd (9.2, 9.2)
3.83 dd (9.2, 9.2)
3.70 m
4.23 dd (1.5, 12.5)
4.76 dd (3.0,12.5)
4.05 d (7.7)
3.11 dd (7.7, 9.2)
3.16 dd (9.2, 9.2)
3.24 dd (9.2, 9.2)
2.27 m
0.60 d (7.0)
8.25 d (8.0)
7.70 t (8.0)
7.65 t (7.5)
0.68 d (7.0)
8.05 d (8.5)
6.97 d (8.5)
5.72 d (8.1)
3.43 dd (8.1, 9.3)
3.67 dd (9.3, 9.3)
3.21 dd (9.3, 9.3)
2.49 dt (2.5, 9.3)
3.39 dd (2.3, 12.1)
3.46 m
5″
6″a
6″b
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′a
6′′′b
4.02 d (8.0)
3.09 dd (8.0, 9.0)
3.18 dd (9.0, 9.0)
3.59 dd (9.0, 9.0)
3.70 m
4.36 dd (1.5, 12.0)
4.59 dd (4.5, 12.0)
3.21 m
3.36 m
3.49 m
a Data were recorded at 500 MHz. b Signals were overlapped.
believed that high levels of HBsAg-bearing particles in the necting through a disaccharide. All the structures were eluci-
serum of chronically infected individuals play a role in sup- dated by the detailed analysis of HRESIMS and NMR data. The
pressing the HBV immune system.3 Discovery of anti-HBV absolute configurations were determined by TDDFT calculated
compounds aimed at potentiating the immune response by ECD. The relative configuration of compound 2 was resolved
suppressing antigenemia became an alternative of anti-HBV by J-based analysis and quantum chemical calculations. The
therapeutic research. However, there are rare examples except sesquiterpenoid dimers 6–9 displayed potential inhibitory
that anti-HBV lead compound PBHBV-2-15 was discovered activity towards HBsAg and HBeAg secretion.
based on the target of HBsAg secretion.4 The sesquiterpenoid
glycosides dimers 6–9, with unusual structures, displayed sig-
nificant HBsAg and HBeAg reduction activity, which were
Experimental section
promising for the development as antigenemia agents for anti-
General procedures
HBV therapeutics.
Optical rotations were performed on a P-1020 polarimeter
(JASCO, Tokyo, Japan). IR and UV spectra were measured on a
Bruker Tensor 27 spectrometer with KBr pellets and Shimadzu
Conclusions
UV 2401PC, respectively. 1D and 2D NMR spectra were run on
During the process of exploring novel antiviral compounds Bruker DRX-400, 500, AVANCE III-600 and AVANCE-800 NMR
from Phyllanthus spp., eight new (1–8) and three known (9–11) spectrometers operating at 400, 500 and 600 and 800 MHz for
highly oxygenated bisabolane sesquiterpenoids were isolated 1H, and 100, 125 and 150 and 200 MHz for 13C, respectively.
from P. emblica. Phyllaemblicin G2 (2), bearing a tricyclo Coupling constants are expressed in Hertz and chemical shifts
[3.1.1.1] oxygen bridge ring system, is an unusual sesquiterpe- are given on ppm scale with solvents as internal standard.
noid glycoside, while phyllaemblicins G6–G8 (6–8) are dimeric ESI-MS and HRESIMS were measured at Bruker HCT/Esquire
sesquiterpenoid glycosides with two norbisabolane units con- and Agilent G6230. ECD was detected at Applied Photophysics.
8770 | Org. Biomol. Chem., 2014, 12, 8764–8774
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Paper
Table 4 13C NMR spectroscopic data for compounds 6–8 in CD3OD (δ in ppm)
6 a
7 b
8 b
No.
Part A
Part B
Part A
Part B
Part A
Part B
1
2
3
4
5
6
71.7, CH
32.9, CH2
32.4, CH
29.6, CH2
76.3, CH
75.6, C
72.3, CH
29.2, CH2
34.6, CH
28.0, CH2
81.8, CH
76.5, C
71.5, CH
31.3, CH2
32.2, CH
30.3, CH2
75.9, CH
75.4, C
71.8, CH
32.6, CH2
32.3, CH
29.6, CH2
76.5, CH
75.9, C
71.4, CH
32.6, CH2
32.1, CH
29.5, CH2
76.1, CH
75.4, C
71.4, CH
31.7, CH2
32.2, CH
29.2, CH2
76.2, CH
75.6, C
7
8
9
213.6, C
100.7, C
32.9, CH2
71.1, CH
34.4, CH
63.5, CH2
176.0, C
13.8, CH3
132.4, C
131.1, CH
129.7, CH
134.4, CH
168.0, C
93.8, CH
83.3, CH
77.9, CH
71.0, CH
76.6, CH
64.3, CH2
106.2, CH
76.1, CH
78.0, CH
70.9, CH
77.9, CH
62.1, CH2
76.3, CH
102.5, C
36.3, CH2
72.0, CH
34.2, CH
63.2, CH2
177.4, C
13.8, CH3
132.3, C
131.0, CH
130.0, CH
134.6, CH
168.2, C
213.6, C
100.5, C
32.9, CH2
70.1, CH
34.3, CH
63.3, CH2
175.9, C
13.3, CH3
123.2, C
133.6, CH
117.1, CH
163.5, C
168.0, C
94.0, CH
84.6, CH
77.4, CH
70.0, CH
76.9, CH
63.4, CH2
107.2, CH
75.9, CH
78.1, CH
70.0, CH
77.5, CH
61.1, CH2
214.0, CH
100.6, C
33.1, CH2
71.0, CH
34.0, CH
63.1, CH2
176.9, C
13.6, CH3
132.2, C
131.6, CH
129.9, CH
134.8, CH
167.2, C
213.6, C
100.4, C
32.7, CH2
70.9, CH
34.2, CH
63.3, CH2
175.7, C
13.2, CH3
132.5, C
131.1, CH
130.1, CH
134.2, CH
167.8, C
93.5, CH
83.8, CH
77.4, CH
70.3, CH
77.4, CH
61.4, CH2
106.4, CH
75.8, CH
77.8, CH
70.6, CH
76.3, CH
63.7, CH2
213.9, CH
100.5, C
32.7, CH2
70.3, CH
34.0, CH
63.2, CH2
176.6, C
13.3, CH3
123.1, C
133.4, CH
116.2, CH
163.4, C
168.1, C
10
11
12
13
14
1′
2′,6′
3′,5′
4′
7′
1″
2″
3″
4″
5″
6″
1′′′
2′′′
3′′′
4′′′
5′′′
6′′′
a Data were recorded at 125 MHz. b Data were recorded at 100 MHz.
Table 5 Anti-HBV activities of compounds 6–10 (μM)
HBVa
HBsAg
HBeAg
IC50/SI
IC50/SIc
CC50
b
No.
6
7
8
9
8.53 0.97/4.46
26.30 3.72/2.97
25.80 3.18/4.65
11.54 1.35/3.73
149.87 8.28/6.18
5.68 1.75/6.70
21.38 2.56/3.65
20.98 3.23/5.72
14.08 2.02/3.06
568.80 15.91/1.63
38.04 2.38
78.15 7.03
119.97 5.93
43.03 3.32
926.24 17.58
10
Fig. 7 Key 1H–1H COSY and HMBC correlations of 6.
a Lamivudine was tested as the positive control, the inhibition ratios
against HBsAg and HBeAg were 44.66 2.09 and 25.96 9.59 (100 μM);
31.56
9.21 and 18.67
3.16 (10 μM). b Compound concentration
reducing the viability of HepG2.2.2.15 cells culture by 50%. c SI =
CC50/IC50
.
The apparatus of p-HPLC was an Agilent 1260 with DAD detector.
Column chromatography (CC) was performed with Sepha-
dex LH-20 (Pharmacia Fine Chemical Co., Ltd Uppsala,
Sweden), Diaion HP20SS (Mitsubishi Chemical Co., Tokyo,
Japan), Rp-8 gel (40–60 µm, Merck, Darmstadt, Germany),
Toyopearl HW-40C (TOSOH, Japan), silica gel (200–300 mesh,
Qingdao Hailang Group Co., Ltd Qingdao, People’s Republic
of China) and a 250 × 9.4 mm, i.d., 5 µm Sunfire C18 column
(Waters). TLC was carried out on precoated silica gel GF254
Fig. 8 Experimental ECD curves of 6–8 and 10.
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plates, which were visualized by ultraviolet light and spraying 2931, 1715, 1630, 1283, 1074 cm−1
;
1H NMR (CD3OD,
with 10% sulphuric-ethanol solution. The quantum chemical 500 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1;
calculations were carried out at HPC Center, Kunming Insti- MS (ESI): m/z: 775 [M − H]−, HRMS (ESI): m/z 775.2657
tute of Botany (KIB), Chinese Academy of Sciences (CAS), [M − H]− (calcd for C34H47O20, 775.2661).
China.
Phyllaemblicin G2 (2): white amorphous powder; [α]D25
=
−24.9 (c = 1.1 in methanol); UV (MeOH) λmax (log ε) 199.8
(1.07), 228.4 (1.08), 271.8 (0.08) nm; ECD (in MeOH, λmax [nm],
Plant materials
The roots of P. emblica were collected in Baoshan City, Yunnan Φ [mdeg]) 227 (−3.7), 249 (−0.2), 274 (−1.0); IR (KBr) νmax
Province, People’s Republic of China, in 2010, and identified 3427, 2923, 1710, 1630, 1285, 1077 cm−1 1H NMR (CD3OD,
;
by Prof. C. R. Yang (KIB, CAS).
A
voucher specimen 800 MHz) and 13C NMR (CD3OD, 125 MHz) data, see Table 1;
(KIB-ZL-0100020) has been deposited in State key laboratory of MS (ESI): m/z: 799 [M + Na]+; HRMS (ESI): m/z 775.2653
phytochemistry and plant resource in West China at Kunming [M − H]− (calcd for C34H47O20, 775.2661).
Institute of Botany, Chinese Academy of Sciences.
Phyllaemblicin G3 (3): white amorphous powder; [α]D25
=
+31.3 (c = 0.8 in methanol); UV (MeOH) λmax (log ε) 201.0
Extraction and isolation
(1.07), 227.4 (1.09), 270.8 (0.02) nm; ECD (in MeOH, λmax [nm],
1
The air-dried roots of P. emblica (109 kg) were extracted three Φ [mdeg]) 225 (−5.6), 245 (3.4), 280 (−0.8); H NMR (CD3OD,
times with 70% reflux ethanol solution to give the extract 600 MHz) and 13C NMR (CD3OD, 150 MHz) data, see Table 1;
(7.8 kg), which was suspended into H2O (22.5 L) and partici- MS (ESI): m/z: 459 [M + Na]+; HRMS (ESI): m/z 459.1637
pated with n-BuOH. The organic layer was concentrated in [M + Na]+ (calcd for C22H28O9Na, 459.1631).
vacuum and subjected to a Diaion HP20SS column chromato-
Phyllaemblicin G4 (4): white amorphous powder; [α]D25
=
graphy (CC) eluting with CH3OH–H2O (0%–100%) to afford +21.1 (c = 0.8 in methanol); UV (MeOH) λmax (log ε) 199.8
four major fractions (Fr.1–4). Fr.3 (400 g) was applied to a (1.07), 228.6 (1.09), 272.4 (0.02) nm; ECD (in MeOH, λmax [nm],
silica gel column eluting with CHCl3–CH3OH–H2O Φ [mdeg]) 225 (−7.7), 246 (3.3), 281 (−1.1); IR (KBr) νmax 3429,
(50 : 1 : 0–7 : 3 : 0.5) to give seven sub-fractions (Fr.A–Fr.G). Fr.A 2930, 1712, 1632, 1281, 1077 cm−1
;
1H NMR (CD3OD,
was subjected to Sephadex LH-20 (CH3OH–H2O, 0–70%) to 400 MHz) and 13C NMR (CD3OD, 100 MHz) data, see Table 2;
afford two fractions: Fr.A1 and Fr.A2. Fr.A1 (3.2 g) was chro- MS (ESI): m/z: 781 [M + Cl]−; HRMS (ESI): m/z 791.2602
matographed over MCI CHP-20P (CH3OH–H2O, 30%–90%) to [M + HCOO]− (calcd for C34H47O21, 789.2610).
give four fractions (Fr.A1.1–Fr.A1.4). Fr.A1.2 was separated by
Phyllaemblicin G5 (5): white amorphous powder; [α]D25
=
Toyopearl HW-40C (CH3OH–H2O, 0%–30%), followed by +5.3 (c = 1.3 in methanol); UV (MeOH) λmax (log ε) 200.4 (1.03),
p-HPLC (CH3CN–H2O, 15%–30%) to yield 2 (32 mg). Fr.A1.4 was 228.0 (0.92) nm; ECD (in MeOH, λmax [nm], Φ [mdeg]) 225
purified by Toyopearl HW-40C (CH3OH–H2O, 0%–30%) to give (−6.7), 247 (3.0), 281 (−1.1); IR (KBr) νmax 3424, 2921, 1716,
1
5 (5 mg). Fr.B passaged over Sephadex LH-20 (CH3OH–H2O, 1628, 1281, 1116, 1075 cm−1; H NMR (CD3OD, 500 MHz) and
30%–70%) to afford Fr.B1–Fr.B2. Fr.B1 was fractioned on MCI 13C NMR (CD3OD, 125 MHz) data, see Table 2; MS (ESI): m/z:
CHP-20P column (CH3OH–H2O 30%–80%) to afford Fr.B1.1– 769 [M + Na]+; HRMS (ESI): m/z 745.2547 [M − H]− (calcd for
Fr.B1.6. Fr.B1.5 was purified by Toyopearl HW-40C (CH3OH– C33H45O19, 745.2633).
H2O 0%–30%) and p-HPLC (CH3CN–H2O, 15%–30%) to afford
Phyllaemblicin G6 (6): white amorphous powder; [α]D25
=
3 (1 mg) and 4 (115 mg). Fr.C was fractioned on Sephadex +19.1 (c = 1.2 in methanol); UV (MeOH) λmax (log ε) 200.4 (1.4),
LH-20 (CH3OH–H2O, 30%–70%) to afford Fr.C1–Fr.C4. Fr.C1 227.8 (1.5), 269.6 (0.6) nm; ECD (in MeOH, λmax [nm], Φ
was separated over MCI CHP-20P (CH3OH–H2O, 30%–70%) to [mdeg]) 226 (−7.4), 247 (5.7), 322 (−3.6); IR (KBr) νmax 3433,
1
give Fr.C1.1–1.6, and Fr.C1.5 was chromatographed on Rp-8 2931, 1718, 1278, 1078 cm−1; H NMR (CD3OD, 500 MHz) and
(CH3OH–H2O, 30%–80%) to afford Fr.C1.5.1–1.5.4. Fr.C1.5.4 13C NMR (CD3OD, 125 MHz) data, see Tables 4 and 3; MS
was purified by Toyopearl HW-40C (CH3OH–H2O, 0%–30%) (ESI): m/z: 1183 [M + Cl]−; HRMS (ESI): m/z 1193.3915 [M +
and p-HPLC (CH3CN–H2O, 15%–30%) to afford 1 (8 mg). Fr.E HCOO]− (calcd for C55H69O29, 1193.3925).
(24.4 g) passaged over Sephadex LH-20 (CH3OH–H2O, 30%–
Phyllaemblicin G7 (7): white amorphous powder; [α]D25
=
100%) to afford Fr.E1–Fr.E3. Fr.E1 was fractioned on Rp-8 +5.7 (c = 1.0 in methanol); UV (MeOH) λmax (log ε) 201.0 (1.5),
(CH3OH–H2O, 30%–80%) to afford Fr.E1.1–Fr.E1.8. Fr.E1.2 231.2 (1.2), 258.4 (1.2) nm; ECD (in MeOH, λmax [nm], Φ
and Fr.E1.3 were separately chromatographed on Toyopearl [mdeg]) 212 (19.1), 231 (−6.2), 253 (3.0), 322 (−9.2); IR (KBr)
HW-40C (CH3OH–H2O, 0%–30%), followed by p-HPLC νmax 3435, 2929, 1714, 1610, 1280, 1079 cm−1 1H NMR
;
(CH3CN–H2O, 15%–30%) to afford 7 (30 mg), 8 (24 mg), 6 (CD3OD, 500 MHz) and 13C NMR (CD3OD, 100 MHz) data, see
(8 mg) and 9 (500 mg). Fr.G was chromatographed on Toyo- Tables 4 and 3; MS (ESI): m/z: 1161 [M − H]−; HRMS (ESI): m/z
pearl HW-40C (CH3OH–H2O, 0%–30%) and p-HPLC (CH3CN– 1161.3659 [M − H]− (calcd for C54H65O28, 1161.3662).
H2O 15%–30%) to afford 10 (51 mg) and 11 (15 mg).
Phyllaemblicin G8 (8): white amorphous powder; [α]D25
+6.0 (c = 1.5 in methanol); UV (MeOH) λmax (log ε) 201.0 (1.5),
=
Phyllaemblicin G1 (1): white amorphous powder; [α]D25
=
+29.8 (c = 1.2 in methanol); UV (MeOH) λmax (log ε) 199.8 230.2 (1.2), 258.0 (1.2) nm; ECD (in MeOH, λmax [nm], Φ
(1.06), 228.6 (1.08), 272.6 (0.02) nm; ECD (in MeOH, λmax [nm], [mdeg]) 209 (9.8), 228 (−3.5), 253 (1.2), 321 (−6.2); IR (KBr)
Φ [mdeg]) 224 (−7.3), 245 (3.6), 280 (−1.1); IR (KBr) νmax 3426, νmax 3433, 2931, 1713, 1609, 1281, 1079 cm−1 1H NMR
;
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(CD3OD, 500 MHz) and 13C NMR (CD3OD, 100 MHz) data, see each conformer by introducing the Gaussian Function. The
Tables 4 and 3; MS (ESI): m/z: 1161 [M − H]−; HRMS (ESI): m/z final ECD spectrum of each compound was obtained by aver-
1161.3660 [M − H]− (calcd for C54H65O28, 1161.3662).
aging all the simulated ECD spectra of all the conformers
according to their excited energies and Boltzmann distri-
bution.14 The band shapes of the calculated ECD curves were
Alkaline hydrolysis of 2
Compound 2 (5 mg) was added to 2 mL NaOH solution (0.2 M), all 0.3 eV.
and incubated in a 60 °C water bath for 30 min. Then, the
solution was cooled to room temperature, and neutralized by
Amberlite IR 120. The elution was evaporated to dryness and
subjected to chromatography over silica gel (CHCl3–MeOH–
H2O 8 : 2 : 0.2) to afford 2A (1.5 mg): white amorphous powder;
Anti-virus assay as previously reported.15
Acknowledgements
[α]2D5 = −4.9 (c = 1.0 in methanol); IR (KBr) νmax 3431, 2926, The authors are grateful to the members of the analytical
1620, 1423, 1384, 1077, 1027 cm−1 1H NMR (DMSO-d6, group at State Key Laboratory of Phytochemistry and Plant
;
800 MHz): δH 3.92 (1H, brs, H-1), 1.48 (1H, m, H-2a), 1.87 (1H, Resources in West China, Kunming Institute of Botany,
brd, H-2b), 2.00 (1H, m, H-3), 1.52 (1H, m, H-4a), 1.96 (1H, m, Chinese Academy of Sciences, for measuring the spectroscopic
H-4b), 4.22 (1H, brs, H-5), 1.68 (1H, dd, J = 9.6, 15.2 Hz, H-9a), data. We also thank Fudan University for part of the antiviral
1.83 (1H, brd, J = 15.2 Hz, H-9b), 3.88 (1H, m, H-10), 1.56 (1H, assay. This work was supported by the NSFC 21002105, the 973
m, H-11), 3.24 (1H, dt, J = 10.4, 6.4 Hz, H-12a), 3.45 (1H, dt, J = Program of Ministry of Science and Technology of P. R. China
10.4, 4.8 Hz, H-12b), 0.80 (3H, d, J = 7.2 Hz, H-14), 3.64 (1H, d, (2011CB915503), the National Science and Technology Support
J = 12 Hz, H-15a), 3.75 (1H, d, J = 12.0 Hz, H-15b), 4.82 (1H, Program of China (2013BAI11B02), the Fourteenth Candidates
brs, 15-OH), 4.29 (1H, t, J = 5.1 Hz, 12-OH); 13C NMR (CD3OD, of the Young Academic Leaders of Yunnan Province (Min Xu,
200 MHz): 75.9 (C-1), 29.4 (C-2), 35.4 (C-3), 28.9 (C-4), 76.0 2011CI044), and the West Light Foundation of the Chinese
(C-5), 77.2 (C-6), 81.9 (C-7), 108.8 (C-8), 34.9 (C-9), 71.0 (C-10), Academy of Sciences. The calculation sections were supported
42.5 (C-11), 65.8 (C-12), 175.1 (C-13), 13.6 (C-14), 61.0 (C-15); by the High Performance Computing Center of Kunming Insti-
MS(ESI) m/z 347 [M − H]−; HRMS (ESI): m/z 347.1347 [M − H]− tute of Botany, Chinese Academy of Sciences.
(calcd for C15H23O9, 347.1348).
Acid hydrolysis of compound 7
Notes and references
Hydrochloride solution (2 M, 400 μL) was added to compound
7 (4 mg) in a vial and the vial was sealed. After incubation at
80 °C for 6 h, the reaction mixture was cooled to room temp-
erature and extracted with CHCl3. The aqueous layer was neu-
tralized by passage through a small column with Amberlite
IRA-401. TLC was used to identify the type of monosaccharide
by comparing with the authentic sugars with elution system
chloroform–n-butanol–methanol–acetic acid–water 17 : 10 : 6 : 2 : 3,
and Rfs of 0.35 for glucose, [α]2D5 = +94 (c = 1.5 in H2O);
1H NMR (pyridine-d5, 800 MHz) spectrum, see ESI Fig. S72.†
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