Prenylated benzoylphloroglucinols and xanthones from the leaves of garcinia oblongifolia with antienteroviral activity

Article abstract of DOI:10.1021/np500124e

An acetone extract of the leaves of Garcinia oblongifolia showed antiviral activity against enterovirus 71 (EV71) using a cytopathic effect inhibition assay. Bioassay-guided fractionation yielded 12 new prenylated benzoylphloroglucinols, oblongifolins J-U (1-12), and five known compounds. The structures of 1-12 were elucidated by spectroscopic analysis including 1D- and 2D-NMR and mass spectrometry methods. The absolute configurations were determined by a combination of a Mosher ester procedure carried out in NMR tubes and ECD calculations. Compared to ribavirin (IC₅₀ 253.1 μM), compounds 1, 4, and 13 exhibited significant anti-EV71 activity in vitro, with IC₅₀ values of 31.1, 16.1, and 12.2 μM, respectively. In addition, the selectivity indices of these compounds were 1.5, 2.4, and 3.0 in African green monkey kidney (Vero) cells, respectively.

Full text of DOI:10.1021/np500124e

Article
pubs.acs.org/jnp
Prenylated Benzoylphloroglucinols and Xanthones from the Leaves
of Garcinia oblongifolia with Antienteroviral Activity
Hong Zhang,†,∥ Ling Tao,†,‡,∥ Wen-Wei Fu,† Shuang Liang,† Yi-Fu Yang,† Qing-Hong Yuan,§
Da-Jian Yang,‡ Ai-Ping Lu,*,⊥ and Hong-Xi Xu*,†
†School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Cai Lun Lu 1200, Shanghai 201203, People’s Republic of
China
‡Chongqing Academy of Chinese Materia Medica, Nanshan Road 34, Chongqing 400065, People’s Republic of China
§Department of Physics, East China Normal University, Dongchuan Road 500, Shanghai 200241, People’s Republic of China
⊥School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR, People’s Republic of China
S
* Supporting Information
ABSTRACT: An acetone extract of the leaves of Garcinia oblongifolia
showed antiviral activity against enterovirus 71 (EV71) using a
cytopathic effect inhibition assay. Bioassay-guided fractionation yielded
12 new prenylated benzoylphloroglucinols, oblongifolins J−U (1−12),
and five known compounds. The structures of 1−12 were elucidated by
spectroscopic analysis including 1D- and 2D-NMR and mass
spectrometry methods. The absolute configurations were determined
by a combination of a Mosher ester procedure carried out in NMR
tubes and ECD calculations. Compared to ribavirin (IC50 253.1 μM),
compounds 1, 4, and 13 exhibited significant anti-EV71 activity in vitro,
with IC50 values of 31.1, 16.1, and 12.2 μM, respectively. In addition,
the selectivity indices of these compounds were 1.5, 2.4, and 3.0 in
African green monkey kidney (Vero) cells, respectively.
All compounds were evaluated for their antiviral activities
against EV71 using the CPE inhibition assay by an MTT
method. Herein, the isolation, structure elucidation, and
antiviral evaluation of the isolates obtained are described.
nterovirus 71 (EV71) is a small, positive-sense, single-
Estranded RNA virus in the genus Enterovirus, family
Picornaviridae.1 Infection with EV71 is of particular concern
because this causes severe neurological complications and may
result in death in infants and young children.2 Currently, no
vaccines or antiviral drugs against EV71 are clinically available.1
Thus, it is imperative to develop new and effective antiviral
drugs to combat human EV71 infections. In a program for
discovering new compounds with anti-EV71 activities from
Chinese herbs, an acetone extract of the leaves of Garcinia
oblongifolia Champ. ex Benth (Clusiaceae) demonstrated anti-
EV71 activity using an EV71 infection-induced cytopathic effect
(CPE) assay (Figure S1, Supporting Information). Thus, it was
speculated that the leaves of G. oblongifolia might contain
secondary metabolites with anti-EV71 activity.
RESULTS AND DISCUSSION
■
The petroleum ether-soluble part of the acetone extract of G.
oblongifolia was purified by column chromatography over MCI
gel, silica gel, reversed-phase C18 silica gel, Sephadex LH-20,
and preparative HPLC to afford 17 compounds, including 12
new (1−12) and five known compounds. The structures of the
known compounds were identified as euxanthone (13),6
oblongifolin C,4a garcihombronone C,7 oblongixanthone B,4b
and dulxanthone-B8 by comparison of their spectroscopic data
with published values.
G. oblongifolia, a tree or medium-sized shrub, is widely
distributed in southern mainland China and northern Vietnam.3
Previous phytochemical investigations on this species resulted
in the isolation of compounds including polycyclic prenylated
acylphloroglucinols,3,4 prenylated xanthones,3,4 and biphenyls.5
In this study, the chemical constituents of the acetone extract of
the leaves of G. oblongifolia were investigated to find potential
anti-EV71 lead compounds. As a result, a series of prenylated
benzoylphloroglucinols, including 12 new compounds (1−12)
and one known analogue, along with four known xanthones,
were identified from the active petroleum ether-soluble fraction.
Oblongifolin J (1) was isolated as a light brown gum and
showed a pseudomolecular ion peak at m/z 503.2772 [M + H]+
(calcd 503.2797) in the HRESIMS, corresponding to the
molecular formula C32H38O5. The 1H NMR spectrum revealed
the presence of a benzene ring [δH 7.51 (1H, m), 7.35 (2H, t, J
= 7.6 Hz), and 7.18 (2H, d, J = 7.6 Hz)], a vinyl proton [δH
5.16 (1H, m)], two methine carbons [δH 3.95 (1H, m) and
1.88 (1H, m)], three methylene carbons [δH 2.48 and 2.43
Received: February 8, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Chart 1
Figure 1. Calculated ECD spectra of 1 and 3 and their experimental curves.
(each 1H, m), 2.49 (2H, m), and 2.96 and 2.46 (each 1H, m)],
and seven methyl groups [δH 2.22 (3H, s), 1.70 (6H, s), 1.68
(3H, s), 1.62 (3H, s), 1.23 (3H, s), and 1.19 (3H, s)]. The 13C
NMR and DEPT spectra indicated the presence of four
carbonyl carbons (δC 208.4, 204.4, 203.7, and 203.0), a benzoyl
group (δC 195.8, 135.9, 134.1, 130.5 × 2, and 129.3 × 2), two
double bonds [δC 135.8, 135.5, 120.8, and 120.2], four
quaternary carbons [δC 80.1, 78.4, 70.5, and 55.2], two
methines, four methylenes, and seven methyls. On the basis
of these data, compound 1 was proposed as a trioxygenated
adamantyl ketone bearing two prenyl units.9 When the NMR
data of 1 were compared with those of (+)-hyperibone K
(Figure S3, Supporting Information),9b it was found that they
exhibited very similar structures. The only structural difference
between the two compounds was the presence of an acetonyl
group (CH3−CO−CH2−) in 1 [as shown by the presence of
the methyl resonance (δC 30.5; δH 2.22) and a methylene (δC
43.1; δH 2.96, 2.46), which showed HMBC correlations to the
low-field ketone carbonyl (δ 208.4)] instead of a 2-
methylpropenyl group in (+)-hyperibone K. The location of
the acetonyl group was assigned at C-24 by the HMBC
correlations from H-25 (δH 2.96, 2.46) to C-3 (δC 80.1), C-7
(δC 47.2), and C-24 (δC 49.3). Other key HMBC correlations
are shown in Figure S4 (Supporting Information).
The relative configurations at the chiral centers C-1, C-3, C-
5, and C-7 were evident for the adamantyl core.9c,10 The
relative configuration of the remaining chiral carbon at C-24
was deduced from the NOE correlations of H-8a/CH3-23 and
H-25b/CH3-22 (Figure S4, Supporting Information). Thus,
there were two possible isomers: (1S,3R,5R,7S,24S)-1 and
B
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(1R,3S,5S,7R,24R)-1. The absolute configuration of 1 was
determined by comparison of their experimentally measured
electronic circular dichroism (ECD) curves with the TDDFT-
predicted curves. The results showed that the calculated ECD
curve of (1S,3R,5R,7S,24S)-1 was consistent with the
experimental ECD spectrum of 1 (Figure 1). Thus, the
structure of 1 was assigned as shown.
experiments. Key HMBC correlations are shown in Figure 2.
The relative configuration was assigned in a similar manner to
Oblongifolin K (2) was obtained as a light brown gum. The
HRESIMS data of 2 exhibited an ion at m/z 533.2562 [M −
H]− (calcd for C32H37O7 533.2539), suggesting a molecular
formula of C32H38O7, 32 mass units greater than that of 1. The
1
similarities of the H and 13C NMR spectroscopic data of 2
(Table 1) when compared to 1 indicated that they possess a
similar structure, and the only difference observed was that the
benzoyl group (δC 195.8, 135.9, 134.1, 130.5 × 2, and 129.3 ×
2) in 1 was replaced by a 3,4-dihydroxybenzoyl group (δC
151.8, 146.3, 128.2, 124.5, 117.5, and 115.0) in 2. The planar
structure of 2 was confirmed by DEPT, HSQC, and HMBC
Figure 2. Key correlations observed in the HMBC and NOESY NMR
spectra of 2.
1, and key NOESY correlations are shown in Figure 2. In order
to determine the absolute configuration of 2, ECD calculations
were conducted. The calculated ECD spectrum of
(1S,3R,5R,7S,24R)-2 matched well with the experimental
ECD spectrum of 2 (Figure S5, Supporting Information),
thus establishing the structure and absolute configuration of 2
as depicted.
1
Table 1. H and 13C NMR Data (400 and 100 MHz,
a
CD3OD) for Compounds 1 and 2
1
2
Compounds 1 and 2 both contain an acetonyl functionality
in their molecules, consistent with these two compounds being
isolated from an acetone-soluble fraction, so they could be
considered as extraction artifacts. To determine if 1 and 2 are
artifacts, another sample of the leaves of G. oblongifolia was
extracted using petroleum ether, and subsequent isolation
procedures and analytical processes were carried out in the
absence of acetone (Experimental Section, Supporting
Information). As shown in Figure S2 (Supporting Informa-
tion), 1 and 2 could also be detected in this new plant extract
by UPLC-ESIMS/MS, thus indicating that they are natural
products rather than extraction artifacts.
position
δH (J in Hz)
δC
δH (J in Hz)
δC
1
2
3
4
5
6
7
8
70.5
203.7
80.1
70.3
203.9
79.8
203.0
78.4
203.0
78.3
55.2
54.9
1.88, m
2.62, m
2.37, m
47.2
1.79, m
47.3
43.8
2.58, m
2.30, m
43.7
9
204.4
195.8
135.9
130.5
129.3
134.1
129.3
130.5
24.3
204.7
194.4
128.2
117.5
146.3
151.3
115.0
124.5
24.3
10
11
12
13
14
15
16
17
Oblongifolin L (3) was obtained as a yellow gum. Its
negative-ion HRESIMS revealed a pseudomolecular ion [M −
7.18, d (7.6)
7.35, t (7.6)
7.51, m
6.89, d (1.9)
1
H]− consistent with the molecular formula, C33H42O4. The H
NMR data (Table 3) indicated that 3 possesses a
monosubstituted benzene ring [δH 7.58 (2H, m), 7.55 (1H,
m), and 7.41 (2H, m)]. There were also resonances for two
angular methyl groups [δH 1.23 and 0.98 (each 3H, s)], three
olefinic protons [δH 5.17 (1H, m), 5.04 (1H, m), and 5.02 (1H,
m)], five vinylic methyl groups [δH 1.72, 1.65, 1.62 (3H each,
s), and 1.57 (6H, s)], and four allylic methylene carbons, which
suggested the presence of one prenyl group and one geranyl
group. The 13C NMR spectrum showed resonances for six
aromatic carbons and a conjugated carbonyl group (δC 198.1),
revealing the occurrence of a benzoyl group. Resonances for a
bicyclo[3.3.1]nonane ring system including a nonconjugated
ketone (δC 207.4), an enolized 1,3-diketone (δC 196.9, 185.7,
and 118.9), two quaternary carbons (δC 65.3 and 45.5), two
methines (δC 69.8 and 42.4), and a methylene (δC 43.2) were
observed. In addition, a characteristic proton single peak was
present at δH 3.06, demonstrating HSQC correlation with a
carbon signal at δC 69.8. This suggested that a methine group
occurs at C-5 in 3,11 which was confirmed by the HMBC
correlations from the proton signal at δH 3.06 to C-1 (δC 65.3),
C-3 (δC 118.9), C-4 (δC 185.7), C-6 (δC 45.5), C-9 (δC 207.4),
C-17 (δC 27.6), and C-18 (δC 21.2). These data suggested that
3 is an isoprenyl benzophenone derivative bearing both a
prenyl group and a geranyl group. Comparison of the NMR
data with those of oblongifolin B (Figure S3, Supporting
7.35, t (7.6)
7.18, d (7.6)
2.48, m
6.57, d (8.4)
6.25, dd (1.9, 8.)
2.46, m
2.43, m
2.36, m
b
18
19
20
21
22
23
24
25a
25b
26
27
28
4.92, m
120.8
135.5
26.4
18.4
22.9
23.8
49.3
43.1
4.85, m
121.1
135.2
26.4
18.4
22.9
23.8
49.8
43.3
1.68, s
1.62, s
1.23, s
1.19, s
3.95, m
2.96, m
2.46, m
1.65, s
1.59, s
1.17, s
1.13, s
3.87, m
2.93, dd (18.1, 3.2)
2.34, m
208.4
30.5
28.9
208.4
30.5
28.9
2.22, s
2.17, s
2.49, m
2.50, m
2.46, m
29
30
31
32
5.16, m
120.2
135.8
26.3
5.14, t (6.8)
120.4
135.8
26.3
1.70, s
1.70, s
1.67, s
1.67, s
18.3
18.3
a
Assignments based on HSQC, HMBC, and NOESY experiments.
Chemical shifts in ppm. Overlapped with the solvent peak.
b
C
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a
1
Table 2. H NMR Data (400 MHz, CD3OD/0.1% TFA) for Compounds 3−6 and 8−10
position
3
4
5
6
8
9
10
5
7
8
3.06, s
2.93, s
3.06, s
3.12, s
3.08, s
2.88, s
2.89, s
1.73, m
2.02, m
1.99, m
7.58, m
7.41, m
7.55, m
7.41, m
7.58, m
1.23, s
2.27, m
1.95, m
1.43, m
8.15, m
7.53, m
7.68, m
7.53, m
8.15, m
1.21, s
1.72, m
1.96, m
1.48, m
7.59, m
7.42, m
7.55, m
7.42, m
7.59, m
1.22, s
2.58, m
1.91, m
1.56, m
7.72, m
7.48, m
7.59, m
7.48, m
7.72, m
1.19, s
1.76, m
2.02, m
1.51, m
7.60, m
7.44, m
7.58, m
7.44, m
7.60, m
1.25, s
2.06, m
2.38, m
1.85, m
7.82, m
7.49, m
7.64, m
7.49, m
7.82, m
0.97, s
1.57, m
3.02, dd (14.4, 4.4)
1.55, m
7.84, m
7.52, m
7.65, m
7.52, m
7.84, m
0.94, s
12
13
14
15
16
17
18
19
0.98, s
0.94, s
0.97, s
1.02, s
1.01, s
1.20, s
1.15, s
2.16, m
1.74, m
5.02, m
1.57, s
2.20, m
1.78, m
5.12, m
1.63, s
2.16, m
1.76, m
5.05, m
1.61, s
2.81, m
2.57, m
2.20, m
1.76, m
5.10, m
1.62, s
2.34, m
1.88, m
2.12, m
2.07, m
5.24, m
1.68, s
20
22
5.26, t (7.1)
1.67, s
6.12, s
5.83, s
23
24
1.97, m
2.01, m
2.25, t (7.4)
2.30, t (7.3)
2.05, m
1.98, m
2.45, m
2.39, m
5.18, m
1.74, s
2.08, m
2.06, m
2.46, m
2.38, m
5.17, m
1.72, s
2.44, m
2.35, m
5.07, m
1.65, s
2.46, m
2.36, m
5.16, m
1.72, s
2.41, m
2.75, dd (13.0, 10.9)
1.95, dd (13.0, 5.6)
4.74, dd (10.9, 5.6)
1.09, s
2.82, dd (15.1, 3.8)
1.72, m
25
27
28
29
5.15, m
1.71, s
1.63, s
2.30, m
2.08, m
5.09, m
1.67, s
3.73, m
1.25, s
1.62, s
1.65, s
1.62, s
1.64, s
1.09, s
1.00, s
2.06, m
1.98, m
5.04, m
1.65, s
2.10, m
2.80, t (7.4)
1.60, m
2.14, m
2.26, m
2.09, m
1.85, m
30
32
5.10, m
1.69, s
3.95, t (6.1)
4.89, s
5.11, m
5.15, m
6.03, s
5.80, s
1.81, s
1.61, s
1.65, s
4.80, s
33
1.57, s
1.61, s
1.58, s
1.70, s
1.58, s
1.60, s
a
Assignments based on DEPT, HSQC, and HMBC experiments. Chemical shifts in ppm, J in Hz.
Information) revealed that the structure of 3 was closely
comparable to that of oblongifolin B,4a except for the
unsubstituted benzoyl group and the absence of a prenyl
group at C-5. All these assignments were confirmed by DEPT,
HSQC, and HMBC NMR experiments. Key HMBC
correlations are shown in Figure 3. The relative configurations
of H-5, H-7, and CH3-18 were assigned using NOE
correlations, as shown in Figure 3. Furthermore, the calculated
ECD curve (Figure 1) for 3 led to the absolute structure being
determined as shown.
Oblongifolin M (4) was obtained as a pale yellow gum. The
1H and 13C NMR data (Tables 2 and 3) were similar to those
of 3, except for the differences of C-3, C-4, and C-10 (4: δC
130.8, 169.9, and 165.8, respectively; 3: δC 118.9, 185.7, and
198.1, respectively). In addition, the HRESIMS showed an ion
peak at m/z 517.2950 [M − H]−, consistent with a molecular
formula of C33H42O5. The molecular weight was found to differ
from that of 3 by 16 Da, corresponding to the presence of an
additional oxygen. These findings indicated that 4 is a derivative
of 3 with an additional oxygen located between C-3 and C-10
or between C-10 and C-11.12 The HMBC correlations showed
long-range correlations from the proton signal at δH 8.15 (2H,
m, H-12 and H-16) to C-10 (δC 165.8), as well as from δH 2.93
(H-5) to C-3 (δC 130.8) and C-4 (δC 169.9) (Figure S7,
Supporting Information), so the additional oxygen atom was
assigned between C-3 and C-10. In addition, an alkaline
hydrolysis reaction was conducted for 4, and its hydrolysate
analyzed by UPLC-ESI-QTOF-MS (Experimental Section,
Supporting Information). A deprotonated molecular ion peak
at m/z 121.0268 for [M − H]− was detected, which was
assigned unambiguously as benzoic acid by comparing the
retention times and m/z value with a reference compound
(Figure S6, Supporting Information).
Oblongifolin N (5) showed a deprotonated molecular ion
peak at m/z 515.2789 [M − H]− in the HRESIMS,
corresponding to the elemental formula, C33H40O5 (calcd
515.2797). Comparison of its NMR data (Tables 2 and 3) with
those of 3 revealed these compounds to be closely related,
except for differences between C-29 (δC 37.2), C-30 (δC
203.7), C-31 (δC 145.9), and C-32 (δC 126.0) of 5 and C-29
(δC 27.6), C-30 (δC 125.3), C-31 (δC 132.5), and C-32 (δC
26.1) of 3. This indicated the presence of a 2-carbonyl-3-
methylbutenyl group at C-23 of 5. This conclusion was
supported by HMBC correlations between H-23/C-30, H-23/
C-29, H-23/C-20, H-23/C-22, H-29/C-30, H-33/C-31, H-33/
C-32, H-32/C-30, and H-32/C-31 (Figure S8, Supporting
Information). The planar structure of oblongifolin N was
further confirmed by DEPT, HSQC, and HMBC experiments.
Oblongifolin O (6) gave the same molecular formula
(C33H40O5) as 5 (m/z 515.2806 [M − H]− (calcd
515.2797)) by HRESIMS. The 1H and 13C NMR spectra
were very similar to those of 5, suggesting that 6 is also a
prenylated benzoylphloroglucinol with a terminal double bond
and one additional carbonyl group in the side chain. Key
HMBC correlations (Figure 5) indicated that the carbonyl
group and the terminal double bond were located at C-20 and
C-21 in 6, respectively. This differed from 5, since these
functionalities occurred at C-30 and C-31.
D
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Table 3. 13C NMR Data (100 MHz, CD3OD/0.1% TFA) for
a
Compounds 3−6 and 8−10
position
3
4
5
6
8
9
10
1
65.3
196.9
118.9
185.7
69.8
63.4
186.8
130.8
169.9
69.5
65.3
196.9
119.0
185.8
70.1
65.3
196.1
119.3
185.1
70.1
65.6
197.1
119.1
185.7
70.1
62.7
180.4
117.6
193.8
75.0
51.4
174.9
127.2
194.1
75.4
2
3
4
5
6
45.5
42.9
45.4
44.8
45.4
44.5
44.1
7
42.4
40.7
42.4
37.5
42.5
42.9
42.3
8
43.2
41.1
43.1
43.0
43.1
39.4
42.9
9
207.4
198.1
138.9
129.9
129.3
133.9
129.3
129.9
27.6
207.4
165.8
130.6
131.3
129.5
134.6
129.5
131.3
27.2
207.7
198.2
139.1
129.9
129.3
133.9
129.3
129.9
27.5
206.9
198.1
139.2
130.1
129.3
133.9
129.3
130.1
27.4
207.6
198.2
139.1
129.9
129.3
133.9
129.3
129.9
27.5
203.7
194.3
138.7
130.4
129.9
134.9
129.9
130.4
20.9
206.5
195.9
138.8
130.2
129.9
135.0
129.9
130.2
20.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
Figure 4. Experimental ECD spectra of 3−7.
21.2
20.1
27.2
21.4
21.2
27.5
27.3
28.8
28.8
28.9
38.7
29.0
28.5
28.4
123.8
138.2
16.4
124.1
137.7
18.2
124.3
137.4
16.7
202.2
150.2
126.0
32.4
123.9
138.0
16.5
123.7
138.5
16.5
123.9
138.4
16.6
40.9
40.9
35.7
36.8
40.9
41.3
Figure 5. Key correlations observed in the HMBC NMR spectra of 6
and 7.
31.2
30.6
31.2
30.9
31.1
29.8
30.8
121.1
135.5
26.4
121.1
134.6
26.2
121.1
135.5
26.4
121.0
135.5
26.3
121.1
135.6
26.3
94.8
69.6
71.1
86.9
26.1
27.3
4), two quaternary carbons at δC 65.6 (C-1) and 45.6 (C-6), a
methylene carbon (δC 45.7), two methine carbons (δC 69.7 and
35.9), three methyl groups at δC 27.6 (C-17), 20.4 (C-18), and
14.9 (C-19), and five other signals that were attributed to an
isoprenyl group. On comparing the NMR data of 7 with those
of 3, it was found that the two compounds have the same
benzoylphloroglucinol skeleton, but a difference was observed
only in the structure of the side chain at C-7, since the geranyl
group of 3 was replaced at this position by a methyl group in 7.
The HMBC correlations from this methyl to C-7, C-6, and C-8
confirmed its location at C-7. Other key HMBC correlations
are shown in Figure 5.
According to the observed NOESY correlations (Figures
S7−S10, Supporting Information), the relative configurations
of compounds 4−7 were deduced to be the same as those of 3,
with H-5, H-7, and CH3-18 all on the same side of the 2,2-
dimethylbicyclo[3.3.1]nonane ring. In addition, the experimen-
tal ECD spectra of 4−7 were in near agreement with that of 3
(Figure 4), thus suggesting that the absolute configurations of
4−7 are the same as that of 3.
18.4
18.2
18.3
18.3
18.2
25.9
23.9
27.6
27.6
37.2
28.4
34.6
27.7
28.2
125.3
132.5
25.1
125.3
132.3
25.9
203.9
145.9
126.0
17.9
124.8
133.3
26.0
76.3
125.3
132.4
26.0
125.5
132.4
26.0
148.9
177.0
17.8
18.4
17.7
18.0
17.9
17.8
a
Assignments based on DEPT, HSQC, and HMBC experiments.
Chemical shifts in ppm.
Oblongifolin P (7) was obtained as a yellow gum, and its
molecular formula, C24H28O5, was determined on the basis of
the HRESIMS peak at m/z 379.1913 [M − H]−. The 1H NMR
spectrum indicated that 7 possesses one olefinic proton,
attributed to a prenyl group, three methyl groups on sp3
carbons, and two vinyl methyl groups. There were also
resonances evident for a monosubstituted benzene ring. The
13C NMR spectrum of 7 exhibited the presence of a
nonconjugated carbonyl group at δC 207.4 (C-9), an enolized
1,3-diketone group (δC 197.3, C-2; δC 119.1, C-3; δC 185.8, C-
Figure 3. Key correlations observed in the HMBC and NOESY NMR spectra of 3.
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Oblongifolin Q (8) was isolated as a light yellow gum. The
HRESIMS of 8 exhibited a deprotonated molecular ion [M −
H]− at m/z 517.2993 (calcd 517.2954), consistent with a
(1R,5R,7R,30S)-8 and (1S,5S,7S,30S)-8, were considered, and
the ECD spectra were calculated. Visual inspection suggested
that the (1R,5R,7R,30S)-8 curve was similar to the experimental
curve (Figure 7). Thus the structure of 8 was designated as
shown.
1
molecular formula of C33H42O5. The H NMR data of this
compound were similar to values obtained for 3, except for
differences between H-30 [δH 3.95 (1H, t, J = 6.1 Hz)] and H-
32 [δH 4.89 (1H, s); δH 4.80 (1H, s)] in 8 and H-30 [δH 5.04
(1H, m)] and H-32 [δH 1.65 (3H, s)] in 3. This information
indicated the occurrence of a 2-hydroxy-3-methylbutenyl group
at C-23 in 8.13 This finding was also supported by the 13C
NMR data of C-30 (δC 76.3, CH), C-31 (δC 148.9, C), and C-
32 (δC 117.0, CH2) and HMBC correlations between H-30/C-
33, H-32/C-30, H-32/C-31, H-32/C-32, H-33/C-30, H-33/C-
31, and H-33/C-32. The relative configuration of 8 was
determined by NOESY experiments. The NOE correlations of
H-5/CH3-18, CH3-18/H-7, and CH3-17/H-18a indicated that
H-5, CH3-18, and H-7 are all oriented on the same side of the
2,2-dimethylbicyclo[3.3.1]nonane ring, while CH3-17 and the
geranyl group are oriented on the opposite side. However, the
configuration of C-30 could not be assigned from the NOESY
spectrum. Thus, a convenient Mosher ester procedure carried
out in NMR tubes14 combined with calculated ECD data was
used to establish the absolute configuration of 8. Compound 8
were treated with (R)-(−)-α- and (S)-(+)-α-methoxy-α-
(trifluoromethyl)phenylacetyl chloride in deuterated pyridine
directly in separate NMR tubes at room temperature
(Experimental Section, Supporting Information), which
afforded the (S)- and (R)-MTPA ester derivatives (8S and
8R, respectively) of 8. The 1H NMR signals of the two MTPA
esters were assigned unambiguously based on their DEPT and
HSQC spectra. A consistent distribution of positive and
negative ΔδH values around C-30 allowed the assignment of
the S-configuration for C-30 (Figure 6). Two possible isomers,
Oblongifolin R (9) was isolated as a yellow gum. The
molecular formula, C33H42O5, was deduced by HRESIMS at m/
z 517.2949 [M − H]−, which was the same as that of the
known compound guttiferone R.11 The H and 13C NMR data
1
(Tables 2 and 3) were almost identical with those of guttiferone
R (Figure S3, Supporting Information),11 suggesting that 9 is
also a prenylated benzoylphloroglucinol derivative with a (2-
hydroxyisopropyl)dihydrofuran ring moiety. In the 13C NMR
spectrum, the signals of the carbons C-5 (δC 75.0) and C-21
(δC 138.5) in 9 differed significantly from C-5 (δC 69.7) and C-
21 (δC 133.8) in guttiferone R, suggesting these compounds to
differ structurally only in the structure of the side chain. HMBC
correlations as shown in Figure S11 (Supporting Information)
indicated gem-dimethyl substitution at C-5 and a geranyl
substituent at C-7 (C-23 was substituted by a prenyl to form
the geranyl) in 9, whereas one of these methyl groups was
substituted with a prenyl group and C-7 was substituted with a
prenyl group in guttiferone R. The relative configuration of 9
was determined by a NOESY experiment. Key NOE
correlations are shown in Figure S11 (Supporting Information).
Oblongifolin S (10) was obtained as a colorless gum. The
positive ion at m/z 519.3128 [M + H]+ indicated a molecular
1
formula of C33H42O5, which is similar to 9. The H and 13C
NMR data (Tables 2 and 3) showed close similarities to those
of 9, with the exception of differences at C-1 (10: δC 51.4, CH;
9: δC 62.7, CH), C-3 (10: δC 127.2, CH; 9: δC 117.6, CH), C-
25 (10: δC 69.6, CH; 9: δC 94.8, CH), and C-26 (10: δC 86.9,
C; 9: δC 71.1, C). This indicated that a 2,2-dimethyl-3-
hydroxypyran moiety at C-1 and C-2 of 10 replaced the (2-
hydroxyisopropyl)dihydrofuran ring moiety in 9.11,15 HMBC
correlations between H-24/C-1, H-24/C-2, H-24/C-25, H-24/
C-26, H-25/C-1, H-27/C-25, H-27/C-26, H-28/C-25, and H-
28/C-26 confirmed the presence of a 2,2-dimethyl-3-hydrox-
ypyran moiety fused with the phloroglucinol moiety at C-1 and
C-2. Key HMBC and NOE correlations are shown in Figure
S12 (Supporting Information). By comparing experimental and
calculated ECD spectra (Figure S13, Supporting Information),
the absolute configurations of 9 and 10 were both determined
to be 1R,5S,7S,25S.
Figure 6. Δδ (δS − δR) values (in ppm) for the MTPA ester of 8.
Figure 7. Calculated ECD spectra of 8 and 12 and their experimental curves.
F
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Oblongifolin T (11) was obtained as a yellow gum, and its
molecular formula of C38H50O7 was established by the negative
HRESIMS ion at m/z 617.3505 [M − H]− (calcd for
This conclusion was supported by HMBC correlations between
H-35/C-28, H-35/C-34, H-35/C-37, and H-35/C-38. In the
NOESY spectrum, the correlations of CH2-17/CH3-23, CH3-
23/CH2-24, and CH3-22/H-7 indicated that CH2-17, CH3-23,
and CH2-24 are all oriented on the same side of the 2,2-
dimethylbicyclo[3.3.1]nonane ring, while CH3-22 and H-7 are
oriented on the opposite side. The absolute configuration of 11
was assigned in a similar manner to 8. The configuration of C-
35 was determined to be R by a convenient Mosher ester
method (Figure S14, Supporting Information). Furthermore,
the ECD experiment and ECD calculation of 11 were
conducted. The experimental ECD spectrum of 11 was in
accordance with the calculated ECD spectrum for
(1R,5R,7S,35R)-11 (Figure S15, Supporting Information),
thus establishing the assignment of the absolute configuration
of 11 as depicted.
1
C38H49O7, 617.3478). The H and 13C NMR data (Table 4)
were identical with values reported for oblongifolin A (Figure
S3, Supporting Information),4a except for differences between
C-34 (δC 34.3), C-35 (δC 76.0), C-36 (δC 149.1), and C-37 (δC
111.7) of 11 and C-34 (δC 27.5), C-35 (δC 125.1), C-36 (δC
132.1), and C-37 (δC 26.0) of oblongifolin A, suggesting the
existence of a 2-hydroxy-3-methylbutenyl group at C-23 in 11.
1
Table 4. H and 13C NMR Data (400 and 100 MHz,
a
CD3OD) for Compounds 11 and 12
11
δH (J in Hz)
12
δH (J in Hz)
position
δC
δC
Oblongifolin U (12) gave the molecular formula C38H50O5,
as determined by HRESIMS (m/z 585.3585 [M − H]−, calcd
585.3580). A comparison of the spectroscopic data of 12 with
those reported for oblongifolin E (Figure S3, Supporting
Information)4b revealed these compounds to have very similar
structures and to represent a pair of epimers with a prenylated
benzoylphloroglucinol backbone. The only structural difference
between 12 and oblongifolin E found was the opposite
configuration of C-7. This was deduced by the different 13C
NMR chemical shifts of C-7 and CH3-22 [12: C-7 (δC 44.3),
CH3-22 (δC 16.4); oblongifolin E: C-7 (δC 47.7), CH3-22 (δC
27.3)].16 The structure of 12 was confirmed by DEPT, HSQC,
and HMBC experiments. To determine the relative config-
uration of 12, a NOESY experiment was performed. NOE
correlations between CH2-17/CH3-23 and CH2-24/CH3-23
suggested that CH2-17, CH3-23, and CH2-24 are on the same
side of the molecule. The absolute configuration of 12 was also
assigned by experimental and theoretically calculated ECD
methods. As shown in Figure 7, the absolute configuration of
12 was determined as shown.
1
2
3
4
5
6
7
8
62.2
195.4
117.9
194.9
68.2
63.8
194.7
119.4
193.0
69.8
49.4
48.7
1.55, m
47.7
1.64, m
2.07, m
2.04, m
44.3
2.18, m
2.09, m
40.8
40.9
9
210.1
195.9
129.5
117.5
146.4
152.8
115.2
125.4
27.2
208.9
198.8
140.7
116.3
158.7
120.7
129.9
121.4
27.7
10
11
12
13
14
15
16
17
7.17, d (1.9)
6.69, d (8.3)
7.02, brs
6.99, m
7.18, m
6.92, m
2.06, m
2.01, m
5.17, m
6.96, dd (8.3, 1.9)
2.74, m
2.58, m
18
19
20
21
22
23
24
5.17, m
120.8
135.7
26.5
18.4
23.4
27.5
30.1
121.0
135.5
26.1
18.4
16.4
23.2
29.3
All isolates were evaluated for their anti-EV71 activity using a
cytopathic effect inhibition assay determined using an MTT
method. When compared to ribavirin (IC50 253.1 μM),
compounds 1, 4, and 13 exhibited significant protection against
EV71-induced CPE in Vero cells, with IC50 values of 31.1, 16.1,
and 12.2 μM, respectively. Their selectivity indices (SI) were
1.5, 2.4, and 3.0 in Vero cells, respectively (Table 5).
1.72, s
1.68, s
1.25, s
1.03, s
2.14, m
2.08, m
4.92, m
1.73, s
1.70, s
0.82, s
1.19, s
2.18, m
2.15, m
5.02, m
25
26
27
28
29
125.9
137.2
16.6
124.1
138.1
16.5
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using an Autopol VI polarimeter. Ultraviolet absorption
spectra were recorded on a UV-2401 PC spectrophotometer. ECD
spectra were recorded on a JASCO J-810 spectrometer. IR spectra
1.48, s
1.58, s
1.99, m
2.52, m
2.42, m
4.95, m
37.1
1.52, m
2.50, m
43.4
32.4
31.4
30
31
32
33
34
120.8
135.9
26.5
4.92, m
121.1
135.6
26.3
Table 5. Anti-EV71 Activity, Cytotoxicity, and Selectivity
a
Index of Compounds 1, 4, and 13
1.66, s
1.67, s
1.55, m
1.67, s
1.67, s
2.71, m
2.65, m
5.07, m
b
b
c
18.4
18.3
compound
CC50 (μM)
IC50 (μM)
SI
d
d
34.3
26.2
1
47.0 7.7
37.8 0.9
36.5 5.3
31.1 2.5
16.1 0.5
12.2 3.3
253.1 13.0
1.5
2.4
4
35
36
37
3.97, dd (7.4, 5.7)
76.0
149.1
111.7
125.4
132.4
26.1
13
3.0
ribavirin
>1000
>4.0
4.91, s
4.79, s
1.70, s
1.67, s
1.60, s
a
Compounds 2, 3, 5−12, oblongifolin C, garcihombronone C,
b
38
17.8
17.9
oblongixanthone B, and dulxanthone-B are inactive. Cytotoxicity
(CC50) and antiviral activity (IC50) were determined using an MTT
a
c
Assignments based on HSQC, HMBC, and NOESY experiments.
Chemical shifts in ppm.
assay on Vero cells. Selectivity index (SI) is the ratio of CC50 to IC50.
d
Values represent the mean SD of three independent experiments.
G
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were obtained from a Perkin-Elmer 577 spectrometer. NMR spectra
were measured on a Bruker AV-400 spectrometer and calibrated based
on the solvent peak used. Mass spectrometry was performed on a
Waters Q-TOF Premier spectrometer (Micromass MS Technologies,
Manchester, UK), with an electrospray ion source (Waters, Milford,
MA, USA) connected to a lock-mass apparatus, which performed real-
time calibration correction. Column chromatography was performed
with CHP20P MCI gel (75−150 μm, Mitsubishi Chemical
Corporation, Tokyo, Japan), silica gel (100−200 or 200−300 mesh,
Qingdao Haiyang Chemical Co., Ltd., Qingdao, People’s Republic of
China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden),
and reversed-phase C18 silica gel (50 μm, YMC, Kyoto, Japan).
Precoated TLC sheets of silica gel 60 GF254 (Qingdao Haiyang
Chemical Co., Ltd., Qingdao, People’s Republic of China) were used.
A Waters 2535 series machine equipped with an Xbridge C18 column
(4.6 × 250 mm, 5 μm) was used for HPLC analysis, and a preparative
Xbridge Prep C18 OBD column (19 × 250 mm, 5 μm) was used for
the sample preparation. Ribavirin (Sigma-Aldrich, St. Louis, MO,
USA) was used as a positive compound with anti-EV71 activity.
Plant Material. The leaves of G. oblongifolia were collected at
Bobai, Guangxi Province, People’s Republic of China, in December
2005. The sample was identified by Dr. Chun-Feng Qiao. A voucher
specimen (herbarium no. 20120843) has been deposited at the
Innovative Research Laboratory of TCM, Shanghai University of
Traditional Chinese Medicine.
Extraction and Isolation. Air-dried and powdered leaves of the
plant (4.97 kg) were extracted with acetone (3 × 20 L, each two days)
at room temperature. The combined extracts were evaporated to
dryness under vacuum to afford the acetone-soluble portion (347.8 g).
The residue was suspended in H2O (3 L) and extracted in turn with
petroleum ether (5 × 3 L) and EtOAc (5 × 3 L) to obtain the dried
petroleum ether- (80 g), EtOAc- (200 g), and H2O-soluble (63 g)
extracts. The petroleum ether-soluble extract demonstrated anti-EV71
activity using cytopathic effect inhibition assays (Figure S16,
Supporting Information). This bioactive extract was subjected to
passage over a chromatography column (CC) on MCI and
successively eluted with H2O, 95% EtOH, and EtOAc. The 95%
EtOH-eluting fraction was shown to have anti-EV71 activity in the
CPE inhibition assay (Figure S2, Supporting Information). The
bioactive fraction was chromatographed by silica gel CC using a
gradient of petroleum ether−acetone (100:0 to 0:100, v/v) and
yielded 12 fractions, A−L, according to the analysis of their TLC
profiles.
Fraction E (3.7 g) was subjected to reversed-phase C18 silica gel CC
and eluted in a step-gradient manner with MeOH−H2O (60:40 to
100:0), to obtain 15 subfractions, Ea−Er, and compound 13 (25 mg).
Subfraction Eb was further separated by preparative HPLC (MeOH−
MeCN−H2O, 6.5:58.5:35, with 0.1% formic acid in H2O, 20 mL/min)
to give compound 7 (4 mg). Subfractions Eh and Ej were purified by
preparative HPLC (MeOH−MeCN−H2O, 7:63:30, with 0.1% formic
acid in H2O, 20 mL/min) to yield compounds 1 (5 mg) and 4 (68
mg), respectively. Subfraction Em was chromatographed on a
Sephadex LH-20 column (MeOH) to obtain compound 3 (108
mg). Compound 12 (7 mg) was obtained by preparative HPLC
(MeCN−H2O, 75:25, with 0.1% formic acid in H2O, 20 mL/min)
from subfraction En. Fraction F (7.9 g) was separated using a reversed-
phase C18 silica gel column eluted with MeOH−H2O (60:40 to 100:0)
as a gradient system to obtain 20 subfractions (Fa−Ft) and
oblongifolin C (45 mg). Garcihombronone C (5 mg), oblongix-
anthone B (7 mg), and dulxanthone-B (8 mg) were obtained from
subfractions Fe, Fm, and Fn by recrystallization in acetone,
respectively. Subfraction Fh was purified by preparative HPLC
(MeOH−MeCN−H2O, 6:54:40, with 0.1% formic acid in H2O, 20
mL/min) to yield compounds 10 (2.8 mg), 9 (25 mg), 5 (17 mg), and
6 (8 mg). Subfraction H (3.8 g) was subjected to reversed-phase C18
silica gel CC and eluted in a step-gradient manner with MeOH−H2O
(55:45 to 100:0), to obtain subfractions Ha−Ho. Subfractions Hf and
Hl were further purified by preparative HPLC (MeOH−H2O, 75:25,
and MeOH−H2O, 85:15, respectively, both with 0.1% formic acid in
H2O, 20 mL/min) to obtain compounds 2 (3.2 mg) and 11 (6 mg),
respectively. Subfraction G (3.9 g) was subjected to CC on reversed-
phase C18 silica gel, eluted with MeOH−H2O in a gradient (50:50 to
100:0), to obtain 20 subfractions (Ga−Gt). Compound 8 (8 mg) was
obtained from subfraction Gg by preparative HPLC (MeCN−H2O,
55:45, with 0.1% formic acid in H2O, 20 mL/min).
Oblongifolin J (1): light brown gum; [α]25 +8.6 (c 0.03, MeOH);
D
UV (MeOH) λmax (log ε) 246 (4.28) nm; ECD (c 5.18 × 10−4 M,
MeOH) λmax nm (Δε) 199 (+0.74), 240 (+0.95), 284 (−0.94); IR
(KBr) νmax 2956, 2925, 2852, 1743, 1700, 1596, 1448, 1376, 1253,
1
1172 cm−1; H NMR (CD3OD, 400 MHz) data, see Table 1; 13C
NMR (CD3OD, 100 MHz) data, see Table 1; HRESIMS m/z
503.2772 [M + H]+ (calcd for C32H39O5, 503.2797).
Oblongifolin K (2): light brown gum; [α]25D +55.4 (c 0.07, MeOH);
UV (MeOH) λmax (log ε) 236 (4.06), 284 (3.91), 315 (3.86) nm;
ECD (c 8.80 × 10−4 M, MeOH) λmax nm (Δε) 201 (+5.12), 247
(−0.38), 278 (+2.97), 334 (−5.12); IR (KBr) νmax 3403, 2964, 2919,
1
1745, 1700, 1600, 1521, 1438, 1376, 1288, 1189, 773 cm−1; H NMR
(CD3OD, 400 MHz) data, see Table 1; 13C NMR (CD3OD, 100
MHz) data, see Table 1; HRESIMS m/z 533.2562 [M − H]− (calcd
for C32H37O7, 533.2539).
Oblongifolin L (3): yellow gum; [α]25D −15.1 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 247 (4.04) nm; ECD (c 12.5 × 10−4 M, MeOH)
λmax nm (Δε) 214 (+20.44), 245 (−16.03), 314 (+5.79); IR (KBr)
νmax 3399, 2964, 2929, 2875, 1721, 1673, 1635, 1594, 1573, 1521,
1
1448, 1376, 1286, 1120, 1074 cm−1; H NMR (CD3OD/0.1% TFA,
400 MHz) data, see Table 2; 13C NMR (CD3OD/0.1% TFA, 100
MHz) data, see Table 3; HRESIMS m/z 501.2997 [M − H]− (calcd
for C33H41O4, 501.3005).
Oblongifolin M (4): pale yellow gum; [α]25D −4.2 (c 0.05, MeOH);
UV (MeOH) λmax (log ε) 232 (4.12), 269 (3.94) nm; ECD (c 9.36 ×
10−4 M, MeOH) λmax nm (Δε) 196 (+8.69), 212 (+4.84), 261
(−4.92), 312 (+1.24); IR (KBr) νmax 3423, 2967, 2917, 1745, 1671,
1602, 1452, 1376, 1263, 1209, 1122, 1022, 703 cm−1; 1H NMR
(CD3OD/0.1% TFA, 400 MHz) data, see Table 2; 13C NMR
(CD3OD/0.1% TFA, 100 MHz) data, see Table 3; HRESIMS m/z
517.2950 [M − H]− (calcd for C33H41O5, 517.2954).
Oblongifolin N (5): pale yellow gum; [α]25 −17.5 (c 0.04,
D
MeOH); UV (MeOH) λmax (logε) 259 (3.57) nm; ECD (c 10.85 ×
10−4 M, MeOH) λmax nm (Δε) 216 (+9.46), 247 (−6.53), 316
(+2.36); IR (KBr) νmax 3430, 2971, 2937, 1675, 1598, 1448, 1378,
1207, 1139, 844, 802 cm−1; 1H NMR (CD3OD/0.1% TFA, 400 MHz)
data, see Table 2; 13C NMR (CD3OD/0.1% TFA, 100 MHz) data, see
Table 3; HRESIMS m/z 515.2789 [M − H]− (calcd for C33H40O5,
515.2797).
Oblongifolin O (6): pale yellow gum; [α]25 −20.8 (c 0.05,
D
MeOH); UV (MeOH) λmax (log ε) 257 (3.93) nm; ECD (c 15.5 ×
10−4 M, MeOH) λmax nm (Δε) 215 (+13.55), 245 (−9.70), 319
(+3.79); IR (KBr) νmax 3409, 2965, 2917, 1731, 1673, 1598, 1569,
1
1448, 1376, 1261, 1203, 1139, 804 cm−1; H NMR (CD3OD/0.1%
TFA, 400 MHz) data, see Table 2; 13C NMR (CD3OD/0.1% TFA,
100 MHz) data, see Table 3; HRESIMS m/z 515.2806 [M − H]−
(calcd for C33H39O5, 515.2797).
Oblongifolin P (7): orange gum; [α]25D −56.3 (c 0.05, MeOH); UV
(MeOH) λmax (log ε) 254 (4.03) nm; ECD (c 13.16 × 10−4 M,
MeOH) λmax nm (Δε) 217 (+12.20), 244 (−7.16), 315 (+3.92); IR
(KBr) νmax 3442, 2967, 2921, 1733, 1677, 1635, 1554, 1450, 1382,
1209, 1143, 842, 804 cm−1; 1H NMR (CD3OD/0.1% TFA, 400 MHz)
δH 7.61 (2H, m, H-12 and H-16), 7.57 (1H, m, H-14), 7.44 (2H, m,
H-13 and H-15), 5.19 (1H, t, J = 6.7 Hz, H-21), 3.10 (1H, s, H-5),
2.49 (1H, m, H-20a), 2.41 (1H, m, H-20b), 1.91 (1H, m, H-7), 1.87
(1H, m, H-8a), 1.74 (3H, s, H-23), 1.65 (3H, s, H-24), 1.60 (1H, m,
H-8b), 1.19 (3H, s, H-17), 0.95 (3H, s, H-18), 0.90 (3H, s, H-19); 13C
NMR (CD3OD/0.1% TFA, 100 MHz) δC 207.4 (C-9), 198.2 (C-10),
197.3 (C-2), 185.8 (C-4), 138.9 (C-11), 135.5 (C-22), 133.9 (C-14),
129.9 (C-12 and C-16), 129.3 (C-13 and C-15), 121.0 (C-21), 119.1
(C-3), 69.7 (C-5), 65.6 (C-1), 45.7 (C-8), 45.1 (C-6), 35.9 (C-7), 31.1
(C-20), 27.6 (C-17), 26.4 (C-23), 20.4 (C-18), 18.2 (C-24), 14.9 (C-
19); HRESIMS m/z 379.1913 [M − H]− (calcd for C24H27O4,
379.1909).
H
dx.doi.org/10.1021/np500124e | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Oblongifolin Q (8): pale yellow gum; [α]25 −222.2 (c 0.03,
ASSOCIATED CONTENT
D
■
MeOH); UV (MeOH) λmax (log ε) 249 (3.90) nm; ECD (c 8.69 ×
10−4 M, MeOH) λmax nm (Δε) 215 (+15.36), 246 (−11.99), 317
(+4.22); IR (KBr) νmax 3428, 2969, 2937, 1733, 1673, 1598, 1556,
S
* Supporting Information
Detailed bioassay protocols used; analysis of compounds 1 and
2 in a new plant extract by UPLC-ESIMS/MS; computational
details for compounds 1−3 and 8−12; alkaline hydrolysis of
compound 4 and UPLC-ESI-QTOF-MS analysis; preparation
of the (S)- and (R)-MTPA ester derivatives of 8 and 11;
HRESIMS, ECD, NMR, and IR spectra of compounds 1−12.
These materials are available free of charge via the Internet at
http://pubs.acs.org.
1
1448, 1396, 1205, 1141, 804, 721 cm−1; H NMR (CD3OD/0.1%
TFA, 400 MHz) data, see Table 2; 13C NMR (CD3OD/0.1% TFA,
100 MHz) data, see Table 3; HRESIMS m/z 517.2993 [M − H]−
(calcd for C33H41O4, 517.2954).
Oblongifolin R (9): light brown gum; [α]25 −54.5 (c 0.03,
D
MeOH); UV (MeOH) λmax (log ε) 255 (4.26) nm; ECD (c 8.40 ×
10−4 M, MeOH) λmax nm (Δε) 211 (−14.19), 241 (+3.51), 269
(+8.15), 298 (−4.67); IR (KBr) νmax 3430, 2967, 2921, 1737, 1679,
1
1623, 1450, 1370, 1286, 1213, 1170, 997, 842, 690 cm−1; H NMR
AUTHOR INFORMATION
■
(CD3OD/0.1% TFA, 400 MHz) data, see Table 2; 13C NMR
(CD3OD/0.1% TFA, 100 MHz) data, see Table 3; HRESIMS m/z
517.2949 [M − H]− (calcd for C33H41O5, 517.2954).
Corresponding Authors
*(A.-P. Lu) Tel: +852-34112456. Fax: +852-34112461. E-mail:
aipinglu@hkbu.edu.hk.
*(H.-X. Xu) Tel: +86-21-51323089. Fax: +86-21-51323089. E-
mail: xuhongxi88@gmail.com.
Oblongifolin S (10): light brown gum; [α]25 −37.8 (c 0.04,
D
MeOH); UV (MeOH) λmax (log ε) 253 (3.80) nm; ECD (c 5.02 ×
10−4 M, MeOH) λmax nm (Δε) 210 (−6.16), 249 (+4.50), 270
(+4.16), 315 (+1.53); IR (KBr) νmax 3411, 2967, 2931, 1729, 1677,
Author Contributions
∥H. Zhang and L. Tao contributed equally to this work.
1
1594, 1448, 1363, 1249, 1122 cm−1; H NMR (CD3OD/0.1% TFA,
400 MHz) data, see Table 2; 13C NMR (CD3OD/0.1% TFA, 100
MHz) data, see Table 3; HRESIMS m/z 519.3128 [M + H]+ (calcd
for C33H43O5, 519.3110).
Notes
The authors declare no competing financial interest.
Oblongifolin T (11): yellow gum; [α]25 +10.8 (c 0.05, MeOH);
D
ACKNOWLEDGMENTS
■
UV (MeOH) λmax (log ε) 244 (3.80), 327 (3.56) nm; ECD (c 8.98 ×
10−4 M, MeOH) λmax nm (Δε) 193 (+5.73), 222 (+2.27), 257
(−2.50), 351 (+0.30); IR (KBr) νmax 3423, 2960, 2921, 1635, 1540,
This research was supported by the National Natural Science
Foundation of China (No. 81273403), National Specific
Program on TCM (No. 200907001), and the Innovation
Ability Construction Project for Chongqing Scientific Research
Institutes of Chongqing (Grant No. cstc2012 pt-kyys10001).
The authors thank Prof. H.-Z. Wang (Wuhan Institute of
Virology, Chinese Academy of Sciences) for providing the
EV71/BrCr virus. We are grateful to Dr. C.-F. Qiao (Institute
of Chinese Medical Sciences, University of Macau) for
collecting the plant material. The authors are grateful to B.
Xia, J. Yang, J.-W. Zhou, T. Li, S.-D. Zhang, and S.-D. Gong for
fruitful discussions about ECD calculations.
1
1454, 1378, 1292, 1168 cm−1; H NMR (CD3OD/0.1% TFA, 400
MHz) data, see Table 4; 13C NMR (CD3OD/0.1% TFA, 100 MHz)
data, see Table 4; HRESIMS m/z 617.3505 [M − H]− (calcd for
C38H49O7, 617.3478).
Oblongifolin U (12): orange gum; [α]20 +48.2 (c 0.08, MeOH);
D
UV (MeOH) λmax (log ε) 261 (3.90) nm; ECD (c 6.57 × 10−4 M,
MeOH) λmax nm (Δε) 213 (+4.54), 248 (−2.25), 318 (+0.82); IR
(KBr) νmax 3423, 2967, 2925, 1681, 1554, 1448, 1384, 1209, 1143, 844,
1
725 cm−1; H NMR (CD3OD/0.1% TFA, 400 MHz) data, see Table
4; 13C NMR (CD3OD/0.1% TFA, 100 MHz) data, see Table 4;
HRESIMS m/z 585.3585 [M − H]− (calcd for C38H49O5, 585.3580).
Antiviral Testing. Vero cells (ATCC CCL-81) were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum. After being dissolved in DMSO, the test
extracts (100 mg/mL) and compounds (100 mM) were stored at −20
°C. Ribavirin, used as a positive control, was dissolved in H2O at a
concentration of 400 mM and stored at −20 °C. After being treated or
untreated with compounds for four days, the viabilities of Vero cells
were assessed by the MTT method (Experimental Section, Supporting
Information).17 The concentration of 50% cellular cytotoxicity (CC50)
of each test compound was calculated using the Forecast function of
Microsoft Excel.18
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