Diterpenoids from aerial parts of Flickingeria fimbriata and their nuclear factor-kappaB inhibitory activities

Article abstract of DOI:10.1016/j.phytochem.2015.07.005

Abstract Chemical investigation of the aerial parts of Flickingeria fimbriata (Bl.) Hawkes resulted in isolation of sixteen ent-pimarane diterpenoids, including five rare 16-nor-ent-pimarane diterpenoids, two 15,16-dinor-ent-pimarane diterpenoids and one ent-pimarane diterpenoid. Structures were mainly elucidated by extensive spectroscopic analysis, and their absolute configurations were unequivocally determined by the exciton chirality method, the modified Mosher's method, the CD experiments (including Snatzke's method) and chemical transformations, respectively. All the isolated compounds were screened for inhibitory effects on the nuclear factor-kappaB (NF-κB) in lipopolysaccharide (LPS) induced murine macrophage RAW264.7 cells, using a NF-κB-dependent luciferase reporter gene assay. Several of these compounds displayed comparable or even better activities than the positive control pyrrolidinedithiocarbamate (PDTC) (IC₅₀ = 26.3 μM) with IC₅₀ values in the range of 14.7-29.2 μM and structure-activity relationships are briefly proposed.

Full text of DOI:10.1016/j.phytochem.2015.07.005

Phytochemistry 117 (2015) 400–409
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Diterpenoids from aerial parts of Flickingeria fimbriata and their nuclear
factor-kappaB inhibitory activities
Hang Li a, Jing-jun Zhao a, Jin-long Chen b, Long-ping Zhu a,c, Dong-mei Wang a,c, Lin Jiang a,c
,
De-po Yang a,c, , Zhi-min Zhao a,c,
⇑
⇑
a School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510006, China
b School of Environmental and Chemical Engineering, Nanchang University, Nanchang, Jiangxi 330031, China
c Guangdong Technology Research Center for Advanced Chinese Medicine, Guangzhou, Guangdong 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical investigation of the aerial parts of Flickingeria fimbriata (Bl.) Hawkes resulted in isolation of six-
teen ent-pimarane diterpenoids, including five rare 16-nor-ent-pimarane diterpenoids, two 15,16-dinor-
ent-pimarane diterpenoids and one ent-pimarane diterpenoid. Structures were mainly elucidated by
extensive spectroscopic analysis, and their absolute configurations were unequivocally determined by
the exciton chirality method, the modified Mosher’s method, the CD experiments (including Snatzke’s
method) and chemical transformations, respectively. All the isolated compounds were screened for inhi-
Received 10 January 2015
Received in revised form 1 July 2015
Accepted 9 July 2015
Keywords:
Flickingeria fimbriata
Orchidaceae
Norditerpenoids
Absolute configuration
NF-jB
bitory effects on the nuclear factor-kappaB (NF-
phage RAW264.7 cells, using a NF- B-dependent luciferase reporter gene assay. Several of these
compounds displayed comparable or even better activities than the positive control pyrrolidinedithiocar-
bamate (PDTC) (IC50 = 26.3 M) with IC50 values in the range of 14.7–29.2 M and structure–activity rela-
tionships are briefly proposed.
jB) in lipopolysaccharide (LPS) induced murine macro-
j
l
l
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
Nuclear transcription factor kappa-B (NF-jB) is a transcription
factor that is involved in activating large numbers of genes corre-
sponding to challenges by infections, inflammation, and other
stressful situations; this results in rapid reprogramming of gene
expression (Rothwarf and Karin, 1999; DiDonato et al., 2012). As
Flickingeria fimbriata (Bl.) Hawkes belongs to the medically
important genus Flickingeria (Orchidaceae), which includes
approximately seventy species. It is well known as an effective suc-
cedaneum for the precious and scarce congener Dendrobium can-
didum for treatment of inflammatory diseases, such as
pneumonia, tuberculosis, bronchitis, asthma and pleurisy (Song,
1999). In a continuing search for bioactive metabolites from F. fim-
briata (Chen et al., 2014a, 2014b), five new norditerpenoids (1–5)
representing rare examples of 16-nor-ent-pimaranes, two new
dinorditerpenoids (6–7) possessing a skeleton of 15,16-dinor-ent-
pimarane, and one new ent-pimarane diterpenoid glycoside (11),
along with eight known analogs (8–10 and 12–16) were isolated.
Their structures, including absolute configurations, were deter-
mined on the basis of detailed spectroscopic analysis and various
forms of chemical evidence. The absolute configuration of the
known compound 10 was first elucidated by a CD experiment.
a key regulator, NF-jB regulate many pro-inflammatory pathways
via the regulation of genes that encode pro-inflammatory cytoki-
nes, adhesion molecules, chemokines, growth factors and inducible
enzymes (Lawrence et al., 2001; Tornatore et al., 2012); therefore,
its inhibition results in anti-inflammatory effects.
Combined with the widely reported anti-inflammatory activi-
ties of ent-pimarane diterpenoids in vitro (Wu et al., 2014;
Costantino et al., 2009) as well as in animal models (Possebon
et al., 2014), compounds 1–16 were evaluated for their NF-
signaling pathway suppression effects on LPS-induced murine
macrophage RAW264.7 cells using the NF- B-dependent luciferase
jB
j
reporter gene assay. Compounds 11, 13 and 15–16 exhibited
comparable inhibitory activities with IC50 values between 26.9
and 29.2
positive control PDTC (IC50 = 26.3
14.7–19.2 M. Herein, details of the isolation, structural elucida-
tion, and NF- B inhibitory activity as well as preliminary struc-
ture–activity relationships of these compounds are described.
l
M; compounds 4 and 6–7 were more active than the
lM) with IC50 values in the range
l
⇑
Corresponding authors at: School of Pharmaceutical Sciences, Sun Yat-sen
j
University, Guangzhou, Guangdong 510006, China.
E-mail addresses: lssydp@mail.sysu.edu.cn (D.-p. Yang), zhaozhm2@mail.sysu.
edu.cn (Z.-m. Zhao).
http://dx.doi.org/10.1016/j.phytochem.2015.07.005
0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

H. Li et al. / Phytochemistry 117 (2015) 400–409
401
2. Results and discussion
2.1. Structure elucidation and identification
The methanol extract of the dried aerial parts of F. fimbriata was
suspended in H2O and successively partitioned with EtOAc and n-
BuOH. Various column chromatographic separations (MCI, silica
gel, Sephadex LH-20 and a reversed-phase ODS-A) of the EtOAc
and n-BuOH extracts afforded sixteen ent-pimarane diterpenoids,
including five new norditerpenoids (1–5), two new dinorditer-
penoids (6–7) and one new diterpenoid glycoside (11) (Fig. 1).
Compound 1 was obtained as a colorless oil. Its molecular for-
mula was established as C19H30O4 based on a quasimolecular ion
peak at m/z 345.2048 [M+Na]+ (calcd for C19H30O4Na, 345.2036)
in the positive HRESIMS measurement establishing five degrees
of unsaturation. The IR absorption bands at 3381 and 1708 cmꢀ1
indicated the presence of hydroxyl and carbonyl groups, respec-
tively. The 1H NMR spectrum of 1 showed four tertiary methyls
[dH 0.78, 0.83, 1.02 and 1.19, each (3H, s)], two oxygenated methi-
nes [dH 2.95 (1H, d, J = 9.6 Hz) and 3.56 (1H, ddd, J = 4.2, 9.6,
12.2 Hz)], one olefinic proton (dH 5.42, br s) and a series of aliphatic
methylene multiplets. The 13C NMR spectrum, in combination with
DEPT experiments, displayed 19 carbon resonances, including four
quaternary methyls, five methylenes, four methines (including two
oxygenated carbons), two olefinic carbon, one carboxyl carbon and
three quaternary carbons, respectively. As two of the five degrees
of unsaturation were accounted for by a double bond and a car-
boxyl group, the remaining three degrees of unsaturation required
a tricyclic nature of 1. The aforementioned spectrum showed high
similarity to that for the co-occurring known analog norflickin-
fimiod A (8) (Chen et al., 2014a) except for chemical shifts of A-ring
carbon atoms when combined with detailed 2D NMR spectroscopic
analyses (HSQC, 1H–1H COSY and HMBC) (Fig. 2), structure 1 was
confirmed as depicted. Its relative configuration was established
by the NOESY correlations (Fig. 2) and coupling constants. The
NOESY spectrum showed correlated signals of H-2/H3-19, H-
Fig. 2. Selected 2D NMR correlations of compound 1.
the large coupling constant between H-2 and H-3 (J3–2 = 9.6 Hz),
the OH-3 group was assigned as being . The b assignment of
a
protons H-5 and H-9 were deduced from the NOE correlations of
H-1b/H-5, H-5/H-7b and H-5/H-9. The absolute configuration at
C-13 was assigned as S by analysis of the CD spectrum of 1
(Supplementary material, S63) matched well with that of 8.
To determine the absolute configurations at C-2 and C-3, the
exciton chirality method (Harada and Nakanishi, 1972) was
applied. Benzoylation of 1 with benzoyl chloride afforded 2,3-O-
dibenzoate (1a). The CD spectrum of 1a (Fig. 3) in MeCN showed
a pair of exciton split Cotton effects with a positive one centering
at kmax 237 nm (De +12.0) and a negative one at kmax 222 nm
(
D
e ꢀ15.3), with a maximum absorption at kmax 227 nm in its UV
spectrum, which indicated that the transition dipole moments of
the two benzoyl groups should be oriented clockwise in space
and with a positive chirality (Fig. 3) on the basis of the dibenzoate
chirality rule. These analyses allowed us to definitely establish 2S
and 3S configurations. Considering the relative configuration
established by the NOESY spectrum, compound 1 was determined
as
(2S,3S,5S,9S,10S,13S)-2,3-dihydroxy-16-nor-ent-pimar-8(14)-
en-15-oic acid.
Compound 2, a colorless oil, had a molecular formula C19H30O3,
as determined by the quasimolecular ion peak at m/z 329.2098
[M+Na]+ (calcd for C19H30O3Na, 329.2087) in HRESIMS spectrum.
Comparision of the NMR spectroscopic data of 2 (Tables 1 and 2)
with those of 1 showed the differences, the main one being the
presence of the oxygenated methine carbon (C-2, dC 69.4) in 1
2/H3-20 and H-6
cofacial and axial oriented, assigned here as an
a
/H3-20, which indicated that these protons were
orientation.
a
Correspondingly, OH-2 and H3-18 were assigned to be in a b ori-
entation, suggesting a chair conformation for ring A. Based on
Fig. 1. Structures of isolated compounds 1–16.

402
H. Li et al. / Phytochemistry 117 (2015) 400–409
Fig. 3. CD and UV spectra of compound 1a in MeCN. Red arrow denotes the electric transition dipole of the chromophores. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Table 1
1H NMR spectroscopic data of compounds 1–7 and 11 (d in ppm, mult, J in Hz).
Position
1a
1.91 dd (4.2, 12.2) 1.69 m
1.13 d (3.2) 1.19 m
3.56 ddd (4.2, 9.6, 1.61 m
2a
3a
4a
5a
6b
7b
11a
1a
1.78 t (12.2)
1.58 m
5.14, dd (1.8,
12.6)
1.82 t (12.3)
1.61 m
5.24, ddd (2.5, 4.1, 1.64 m
1.72 m
1.18 m
2.28 m
1.81 m
2.26 m
1.26 br s
1.70 m
1.18 m
1b
2a
4.18 ddd (2.9, 4.4, 5.39 ddd (2.5, 4.4, 1.86 m
12.2)
2b
12.2)
1.59 m
12.6)
1.62 m
12.4)
1.62 m
3
2.95 d (9.6)
3.20 dd (6.2,
10.2)
3.50 br s
3.54 d (1.8)
4.50 dd (6.2,
10.2)
3.52 d (2.9)
3.59 d (2.5)
3.42 dd (4.3,
12.5)
5
1.15 m
1.08 dd (2.2,
12.2)
1.56 m
1.57 m
1.57 m
1.40 m
1.02 m
1.73 m
1.79 m
1.84 dd (2.4, 12.5) 1.17 dd (2.6,
11.4)
6a
1.42 dd (4.4, 12.7) 1.42 dd (4.2,
12.8)
1.63 m
2.33 ddd (1.8, 4.4, 2.34 dd
13.5) (2.4,14.1)
1.55 overlap
1.64 m
1.81 m
1.75 m
6b
1.64 m
1.45 m
2.38 d (13.7)
1.61 m
1.74 m
2.90 m
1.76 m
2.89 m
1.51 m
2.12 m
7a
2.35 dd (3.3, 14.0) 1.86 m
7b
2.06 td (5.4, 13.5) 2.05 td (5.1,
13.4)
2.12 m
2.10 m
1.68 m
2.85 m
2.85 m
2.42 m
9
11
11b
1.79 t (8.6)
1.48 td (3.3, 13.8) 1.48 m
1.65 m
1.71 m
1.94 t (8.4)
1.65 m
2.05 m
1.94 t (8.5)
1.66 m
1.42 m
1.58 m
1.42 m
1.37 m
1.78 m
1.19 m
1.61 dd (3.3,
10.4)
2.31 m
a
7.15 d (8.0)
6.94 dd (1.3, 8.0)
6.87 (1.3)
7.10 d (8.0)
6.93 dd (1.3, 8.0)
6.87 (1.3)
1.62 m
12
a
1.13 d (3.2)
1.08 dd (2.2,
12.2)
1.18 d (13.4)
1.16 m
1.43 m
12b
14
16a
16b
17
18
19
20
2-OOCH3
3-OOCH3
14-OOCCH3
Glc 10
20
2.16 dt (3.5, 12.9) 2.16 d (11.2)
5.42 br s
2.19 d (12.8)
5.46 br s
2.16 m
5.41 br s
1.80 m
4.85 br
1.12 m
5.51 br s
4.40 s
4.43 s
1.12 s
1.08 s
0.88 s
0.74 s
5.43 br s
1.19 s
1.02 s
0.83 s
0.78 s
1.17 s
1.00 s
0.81 s
0.75 s
1.23 s
1.03 s
0.98 s
0.86 s
2.08 s
1.18 s
1.02 s
0.99 s
0.83 s
1.07 s
1.02 s
0.95 s
0.92 s
2.26 s
1.09 s
0.97 s
1.21 s
2.27 s
1.10 s
1.03 s
1.27 s
2.13 s
2.03 s
2.18 s
4.35 d (7.7)
3.18 dd (7.7,
9.1)
3.39 dd (2.7,
4.0)
3.30 d (8.6)
3.27 dd (2.3,
5.6)
3.69 dd (5.7,
11.8)
30
40
50
60a
60b
3.88 dd (2.3,
11.8)
a
Measured in 400 MHz, in CH3OH.
Measured in 400 MHz, in CHCl3.
b
which was replaced the one sp3 methine carbon (C-2, dC 28.4) in 2.
The variation of the substituent at C-2 from compound 1 to 2 was
supported by the crucial 1H–1H COSY correlations of H-1/H2-2 and
H2-2/H-3. Detailed analysis of the NMR spectroscopic data of 2 also
suggested high structural similarity to the already known com-
pound 10, lonchophylloid B (Ma et al., 1998), with the major
differences due to the presence of one carbonyl (C-15) and one
oxygenated methylene (C-16) in 10 being replaced by one carboxyl
(C-15) in 2, as evidenced by the HMBC correlation from H3-17
(dH 1.17) to C-15 (dC 181.3) in 2. Structure 2 was thus deduced as
depicted. H-3 was assigned a b-equatorial orientation, based on
the coupling constants (J3–2 = 6.2, 10.2 Hz) and the NOESY

H. Li et al. / Phytochemistry 117 (2015) 400–409
403
Table 2
13C NMR data of compounds 1–7 and 11 (d in ppm, type).
Position
1a
2a
3a
4a
5a
6b
7b
11a
1
2
3
4
46.8, CH2
69.4, CH
84.3, CH
40.8, C
38.7, CH2
28.4, CH2
79.9, CH
40.2, C
37.7, CH2
72.3, CH
77.4, CH
40.1, C
37.8, CH2
72.3, CH
77.6, CH
40.2, C
38.5, CH2
24.5, CH2
82.2, CH
38.9, C
40.4, CH2
67.2, CH
79.0, CH
38.5, C
36.5, CH2
71.6, CH
76.8, CH
38.7, C
37.9, CH2
24.4, CH2
86.0, CH
39.5, C
5
6
7
8
55.5, CH
23.5, CH2
36.7, CH2
139.6, C
52.1, CH
40.2, C
21.7, CH2
34.4, CH2
44.2, C
55.7, CH
23.5, CH2
36.9, CH2
139.1, C
52.3, CH
39.5, C
21.7, CH2
34.9, CH2
44.8, C
48.9, CH
22.9, CH2
36.7, CH2
139.8, C
51.8, CH
40.4, C
21.5, CH2
34.4, CH2
44.1, C
48.9, CH
23.0, CH2
36.7, CH2
136.0, C
51.8, CH
40.6, C
21.5, CH2
34.5, CH2
44.4, C
54.9, CH
19.4, CH2
35.1, CH2
85.1, C
47.3, CH
37.6, C
19.5, CH2
28.6, CH2
47.0, C
78.7, CH
178.7, C
43.1, CH
18.6, CH2
30.2, CH2
134.8, C
146.4, C
38.7, C
124.2, CH
126.8, CH
135.1, C
129.8, CH
43.1, CH
18.5, CH2
30.1, CH2
134.9, C
146.2, C
38.8, C
124.1, CH
126.8, CH
135.2, C
129.9, CH
56.0, CH
23.4, CH2
36.9, CH2
143.1, C
52.2, CH
39.4, C
21.4, CH2
33.9, CH2
48.3, C
125.3, CH
215.7, C
66.8, CH2
27.9, CH3
29.2, CH3
17.4, CH3
15.1, CH3
9
10
11
12
13
14
15
16
17
18
19
20
127.8, CH
181.1, C
128.4, CH
181.3, C
127.8, CH
180.9, C
127.4, CH
179.3, C
28.3, CH3
29.7, CH3
17.7, CH3
15.9, CH3
28.7, CH3
29.2, CH3
16.6, CH3
15.3, CH3
28.3, CH3
29.4, CH3
22.8, CH3
15.9, CH3
21.4, CH3
28.1, CH3
29.4, CH3
22.8, CH3
15.8, CH3
19.0, CH3
29.0, CH3
17.6, CH3
15.6, CH3
21.0, CH3
28.5, CH3
21.8, CH3
25.8, CH3
21.0, CH3
28.4, CH3
21.9, CH3
25.7, CH3
21.6, CH3
2-OCCH3
2-OCCH3
3-OCCH3
3-OCCH3
14-OCCH3
172.7, C
170.5, C
21.2, CH3
172.9, C
20.9, CH3
171.6, C
14-OCCH3
Glc 10
20
102.1, CH
75.3, CH
78.4, CH
72.0, CH
77.8, CH
63.2, CH2
30
40
50
60
a
Measured in 100 MHz, in CH3OH.
Measured in 100 MHz, in CHCl3.
b
correlation of H-3/H-5. The relative configurations of other chiral
centers were the same as those of 1 based on the NOESY spectrum
(Supplementary material, S13). The absolute configuration at C-13
(13S) was the same as compound 1, this being confirmed by the
well matched CD curves (Supplementary material, S63). The abso-
lute configuration of the chiral centre at C-2 was confirmed by the
modified Mosher’s method (Ohtani et al., 1991; Wang et al.,
2013a,b). Esterification of 2 with R-(ꢀ)- and S-(+)-MTPA chlorides
directly in an NMR tube led to S-MTPA ester (2a) and R-MTPA ester
Compound 3,
a
colorless gum, had
a
molecular formula
C21H32O5 as determined by the m/z of 387.2146 [M+Na]+ (calcd
387.2142) from HRESIMS. Its NMR spectroscopic data (Tables 1
and 2) showed high similarity to those of compound 8 except for
the presence of an additional acetyl [dH 2.08 (s, 3H), dC 21.4 and
172.7] in 3, suggesting it was an acetylated derivative of 8. The ace-
toxy group was assigned at C-2 by the key HMBC correlation from
H-2 (dH 5.14) to the acetyl carbonyl at dC 172.7, which was further
supported by up-field shifts of C-1 and C-3 signals and severely
down field-shifted proton signal of H-2 (dH 5.14) in 3 with respect
to those in 8. The absolute configuration of 3 was determined to be
the same as that of 8 by a successful chemical conversion of 3 to 8
via alkaline hydrolysis. Therefore, structure 3 was characterized
as (2S,3R,5S,9S,10S,13S)-2-acetoxy-3-hydroxy-16-nor-ent-pimar-
8(14)-en-15-oic acid.
(2b), respectively. As shown in Fig. 4, the
D
dH
(D
dH = dS ꢀ dR) values
obtained from the proton signals assigned for these esters clearly
indicated that the absolute configuration at C-3 was R. Combined
with the relative configuration, compound 2 was established as
(3R,5S,9S,10S,13S)-2-hydroxy-16-nor-ent-pimar-8(14)-en-15-oic
acid.
Compound 4 was obtained as a colorless amorphous gum. Its
molecular formula was established as C28H36O5 based on the
positive-ion HRESIMS with a m/z of 475.2468 [M+Na]+. Its ESI mass
spectrum exhibited a peak (m/z 131, [C8H7CO]+) attributed to a
cinnamoyl group (Bai et al., 2012), this being assigned as a
E-cinnamoyl group by analysis of the 1H and 13C NMR data, which
included signals for five aromatic protons [dH 7.60 (2H, H-50 and
H-90) and 7.40–7.42 (3H, H-60, H-70 and H-80)], two olefinic protons
[dH 6.56 (1H, d, J = 16.0 Hz, H-20) and 7.74 (1H, d, J = 16.0 Hz, H-30)],
six aromatic carbons [dC 136.0 (C-40), 129.4 (C-50 and C-90), 130.2
(C-60 and C-80) and 131.6 (C-70)], two olefinic carbons [dC 119.5
(C-20) and 146.3 (C-30)] and a carbonyl carbon at dC 168.4 (C-10)
(Allouche et al., 2009; Wang et al., 2013a,b). The NMR spectro-
scopic data of 4 closely resembled those of 8, except for the
presence of an additional cinnamoyl group. The cinnamoyl group
was located at C-2 supported by the HMBC correlation from H-2
Fig. 4. The
D
dH values (
D
dH
=
D
dS
ꢀ
DdR) for S-/R-MTPA esters of compound 2.

404
H. Li et al. / Phytochemistry 117 (2015) 400–409
(dH 5.24) to the carbonyl carbon C-10 (dC 168.4). The relative config-
uration of 4 was the same as that of 8 according to NOESY correla-
tions (Supplementary material, S62). Likewise, the absolute
configuration at C-13 was assigned as S based on the negative
Cotton effect at 228 (
consistent with those of compounds 1–3.
D
e
ꢀ1.44) nm in its CD spectrum (Fig. 5) being
The absolute stereochemistry of 4 was also elucidated using the
CD exciton chirality method (Harada et al., 1981). Its UV spectrum
(Fig. 5) showed a strong absorption at kmax 274 (loge 4.39)
attributable to the trans-cinnamoyl group. Corresponding to this
UV maximum, the CD spectrum of 4 (Fig. 5) exhibited a negative
Fig. 6. Selected 2D NMR correlations of compound 6.
Cotton effect at kmax 280 nm (
vinyl benzene moiety, and a split Cotton effect between the two
different chromophores of the conjugated ,b-unsaturated ketone
p⁄ transition) and the D8,14 double bond
p⁄ transition) (Zhang et al., 2011; Liu et al.,
2012). This indicated that the transition dipole moments of the
two different chromophores were oriented clockwise in space
and with a positive chirality (Fig. 5). Thus, the absolute configura-
tions of the related chiral centers were concluded as being 2S, 3R,
5S, 9S and 10S. Ultimately, the stereostructure of 4 was further
supported by alkaline hydrolysis from 4 to afford 8. Compound 4
was named as (2S,3R,5S,9S,10S,13S)-2-O-E-cinnamoyl-3-hydroxy-
16-nor-ent-pimar-8(14)-en-15-oic acid.
D
e ꢀ19.14), this arising from the
Compound 6 was isolated as a colorless oil, whose molecular
formula was established as C18H26O2 based on a quasimolecular
ion peak at m/z 297.1836 [M+Na]+ (calcd for C18H26O2Na,
297.1825) in the positive HRESIMS measurements. Analysis of its
NMR spectroscopic data (Tables 1 and 2) indicated high structural
similarity with the known flickinflimilin B (Chen et al., 2014b),
except that the keto function at C-7 position of flickinflimilin B
was replaced by a sp3 methine carbon in 6. This variation was con-
firmed by the pivotal HMBC correlation from H-14 [dH 6.87 (1H, d,
J = 1.3 Hz)] to C-7 (dC 30.2) in 6. The relative configuration can also
be established by analyzing the NOESY correlations (Fig. 6) and
coupling constants. The NOESY spectrum showed correlated sig-
a
(260 nm,
(206 nm,
D
e +6.96,
p–
p–
D
e ꢀ9.41,
The HRESIMS analysis of compound 5 showed a molecular for-
mula of C23H34O6 as established by the m/z of 429.2264 [M+Na]+
(calcd for C23H34O6Na, 429.2248) with seven unsaturations.
Careful comparison of the 1H and 13C NMR spectroscopic data of
5 (Tables 1 and 2) with those of co-occurring compound 9, norflick-
inflimiod B (Chen et al., 2014a), established that the main differ-
ences were due to the oxygenated carbon at C-2 in 9 replaced by
one sp3 methine carbon (dC 24.5, C-2) and to the presence of two
additional acetyl groups [dH 2.03 (s, 3H), dC 21.2 and 172.9; dH
2.18 (s, 3H), dC 20.9 and 171.6] in 5. The two acetoxy groups were
connected to C-3 and C-14, respectively, as established by the core
HMBC correlations from H-3 (dH 4.50) to the acetyl carbonyl at dC
172.9 and from H-14 (dH 4.85) to the acetyl carbonyl at dC 171.6.
The determination of b-equatorial orientation of H-3 was also con-
cluded from the coupling constants (J3–2 = 6.2, 10.2 Hz) and the
NOESY correlations of H-3/H-5 and H-3/H-1b. The relative config-
uration of 14-OOCCH3 was assigned as b, this being deduced from
nals of H-2/H3-19, H-2/H3-20, H-6
a/H3-20, and therefore H-2
group was assigned as . Correspondingly, OH-2 and H3-18 were
a
b-oriented. The OH-3 group was assigned as being b-oriented on
the basis of the small coupling constants between H-2 and H-3
(J = 2.9 Hz). The b assignment of H-5 was deduced from the NOE
correlation of H-1b and H-5.
Since the diester derivative necessary for CD exciton chirality
method was difficult to synthesize probably due to the steric hin-
drance and since the Cotton effect was not observed in the CD
spectrum of 6, the exciton chirality method and ECD calculation
were not suitable for determination of the absolute configurations
of compound 6 at C-2 and C-3. Therefore, an in situ dimolybdenum
CD method developed by Snatzke and Frelek (Snatzke et al., 1981;
Frelek et al., 1999; Di Bari et al., 2001; Liu et al., 2014) was
employed. According to the empirical helicity rule proposed by
Snatzke, the conformation of a chiral metal complex required the
vicinal diol to be in a gauche arrangement and which would give
two diastereomorphous structures, where the favored conforma-
tion prefers the bulkier groups in a pseudo-equatorial position
away from the remaining portion of the metal complex.
Consequently, the positive Cotton effect at kmax 302 nm
the NOE correlations of 14-OOCCH3/H-9 and 14-OOCCH3/H-7b.
Other chiral centers were the same as for compound 9, as con-
firmed by the NOESY correlations (Supplementary material, S62).
The CD curve of 5 (Supplementary material, S63) was very similar
to that of 9, indicative of a 13S configuration. Thus, structure 5 was
(De +0.354) in the induced CD spectrum of the metal complex of
determined as
3a,14b-diacetoxy-16-nor-ent-pimar-15a,8-olide,
compound 6 in anhydrous DMSO (Fig. 7) unambiguously permitted
assignment of 2S and 3R configurations. Taking the relative config-
uration into account, the absolute configurations was assigned as
i.e. the corresponding diacetate derivative of norflickinflimiod C
(Chen et al., 2014a) previously reported from this plant.
Fig. 5. CD and UV spectra of compound 4 in MeCN. Red arrow denotes the electric transition dipole of the chromophores. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

H. Li et al. / Phytochemistry 117 (2015) 400–409
405
Fig. 7. The Mo2(OAc)4 induced CD spectrum of compound 6 in anhydrous DMSO and favored conformations of the Mo42+ complexes of compound 6 and its isomer.
2S, 3R, 5S and 10R and compound 6 was named as (2S,3R,5S,10R)-
2,3-dihydroxy-15,16-dinor-ent-pimar-8,11,13-triene.
conversion of the sugar to the thiocarbamoyl-thiazolidine deriva-
tive (Tanaka et al., 2007; Wang et al., 2013a,b). The CD spectrum
of 11 (Supplementary material, S64) displayed remarkably similar
Cotton effects with those of 10. Therefore, compound 11 was
identified and named as (3R,5S,9S,10S,13S)-3,16-dihydroxyl-15-
Compound 7 was isolated as a colourless oil and its molecular
formula was determined to be C20H28O3 by HRESIMS measure-
ments for the m/z 339.1935 (C20H28O3Na, calcd 339.1931) with
42 mass units higher than that of 6. Its 1H and 13C NMR spectro-
scopic data (Tables 1 and 2) closely resembled those of 6 except
for the presence of signals for an acetyl group [dH 2.13 (3H, s), dC
21.6 and 170.5], indicating that 7 was acetylated derivative of 6.
The acetoxy group was assigned at C-2 by the HMBC correlation
from H-2 (dH 5.39) to the acetyl carbonyl at dC 170.5, which was
further supported by the up-field shifts C-1 and C-3 signals and
the obvious down field-shifted proton signal of H-3 (dH 5.44) in 7
with respect to those in 6. The stereochemical structure of 7 was
also confirmed by alkaline hydrolysis of 7, which yielded the
hydrolysis product confirmed as compound 6 by 1H NMR, Rf values
and specific rotation. Then the structure of compound 7 was
(2S,3R,5S,10R)-2-acetoxy-3-hydroxy-15,16-dinor-ent-pimar-8,11,
13-triene.
one-ent-pimar-8(14)-ene-3-O-b-D-glucopyranoside.
The structures of known compounds were elucidated by spec-
trometric methods (1H NMR, 13C NMR, 2D NMR and ESIMS) and
by comparison with literature data as norflickinflimiod A (8), nor-
flickinflimiod B (9), flickinflimoside A (16) and flickinflimoside B
(15) previously reported from the dried stems of F. fimbriata
(Chen et al., 2014a), lonchophylloid B (10) (Ma et al., 1998) from
fresh stems of Ephemerantha lonchophylla, darutoside (13) from
aerial parts of Siegesbeckia pubescens (Wang et al., 2010),
2b,3b,15,16-tetrahydroxy-ent-pimar-8(14)-en (12) and 2b,3b,16-
trihydroxy-ent-pimar-8(14)-en-15-one (14) previously isolated
from aerial parts of Milleria quinqueflora (Jakupovic et al., 1987),
respectively (Fig. 1).
Known compound 10 was identified as lonchophylloid B by
comparison of its 1H, 13C NMR and optical rotation data with those
of the literature (Ma et al., 1998), while the relative configuration
of this compound has been determined by the NOE difference
spectrum and X-ray diffraction method, the determination of the
absolute configuration was achieved here by means of the CD
experiment. The observed CD curve of compound 10
(Supplementary material, S64) exhibited highly similar Cotton
effects with those of 2-acetyl-flickinflimiod (Chen et al., 2014a),
indicating that the absolute configuration at C-13 was S. Based
on this result, the absolute configuration can be determined as
3R, 5S, 9S, 10S and 13S.
2.2. Biological evaluation
Compounds 1–16 were tested for their abilities to inhibit the
NF-
NF-
j
j
B pathway in LPS stimulated RAW264.7 cells using the
B-dependent luciferase reporter gene assay (Moon et al.,
2001; Xu et al., 2013). Compounds 3, 5 and 8–9 were inactive
(<50% inhibition at 50 M), while compounds 4 and 6–7 exhibited
pronounced inhibition with IC50 values in the range of
14.7–19.2 M, i.e. being more active than the positive control
PDTC (IC50 = 26.3 M), a well-known NF- B inhibitor (Table 3).
Compounds 11, 13 and 15–16 showed potential inhibitory activi-
ties with IC50 values of 26.9–29.2 M, and compounds 1, 2, 10,
12 and 14 displayed moderate suppression with IC50 values rang-
ing from 30.4–43.7 M.
l
l
l
j
l
Compound 11 was obtained as an optically active colorless gum
with a molecular formula of C26H42O8 as determined by the m/z of
505.2760 [M+Na]+ (calcd 505.2772) from HRESIMS. Its 1H and 13C
NMR data (Tables 1 and 2) resembled those of 10, except for the
l
Preliminary structure–activity relationships could be concluded
from the inhibition results. For the 16-nor-ent-pimarane diter-
penoids, the orientation of 3-OH (a and b) had a slight impact on
presence of additional signals being attributed to
a typical
b-glucopyranose moiety [dH 4.35 (1H, d, J = 7.7 Hz, H-10), 3.69
(1H, dd, J = 5.7, 11.8 Hz, H-60a) and 3.88 (1H, dd, J = 2.3, 11.8 Hz,
H-60b); dC 102.1 (CH, C-10) and 63.8 (CH2, C-60)], indicating 11
was a glycosylated derivative of 10. The glucopyranose moiety
was connected to C-3 as evidenced by the mutual HMBC correla-
tions from H-3 (dH 3.42, dd, J = 4.3, 12.2 Hz) to C-10 (dC 102.1),
and from H-10 (dH 4.35, d, J = 7.7 Hz) to C-3 (dC 86.0).
Furthermore, this was supported by the down field-shifted carbon
signal of C-3 (dC 86.0) in 11, as compared to 10 (dC 79.2). Acid
hydrolysis of 11 with 2 M HCl gave a diterpene and glucose. The
diterpene was confirmed to be 10 by 1D NMR spectroscopic data
and optical rotation. The absolute configuration of the b-glucose
was identified to be of the D-configuration by HPLC analysis after
Table 3
IC50 values of the active compounds against NF-jB.
Compound
IC50
(lM)
Compound
IC50 (lM)
1
2
4
6
7
38.3 2.2
43.7 1.2
14.7 1.8
18.3 1.3
19.2 0.9
30.4 1.7
26.9 1.7
12
13
14
15
38.8 2.3
28.4 2.7
33.8 2.9
29.2 2.1
27.5 1.6
26.3 1.2
16
10
11
PDTC*
*
Positive control.

406
H. Li et al. / Phytochemistry 117 (2015) 400–409
inhibition (3 and 7, no activity; 1–2, moderate inhibition), and cin-
namoylation of the –OH group at C-2 resulted in a significant
has been deposited in the School of Pharmaceutical Sciences, Sun
Yat-sen University (voucher specimen number: GDLSJ201308).
increase of the inhibitory effect (4, IC50 = 14.9 lM); for the C-ring
aromatized-15,16-dinor-ent-pimarane diterpenoids, acetylation of
4.3. Extraction and isolation
the ꢀOH group at C-2 resulted in a tiny decrease of inhibition
(7, IC50 = 19.2 lM); for the ent-pimarane diterpenoids, glycosides
Air-dried powder of aerial parts of F. fimbriata (4.5 kg) was
extracted with MeOH (3 ꢁ 15 L) by maceration at room tempera-
ture to afford a crude extract (350 g), which was suspended in
H2O (2.5 L) and successively partitioned with EtOAc (3 ꢁ 2.5 L)
and n-BuOH (3 ꢁ 2.5 L). The EtOAc layer (83 g) was applied to
MCI column (L = 500 mm / = 100 mm) eluting with MeOH/H2O
(10:90–100:0) to afford five fractions (Fr. E1–E5) on the basis of
TLC analyses. Fr. E4 (12 g) was subjected to silica gel CC and eluted
with CH2Cl2/MeOH successively to afford five fractions (Fr.
E4a–E4e). Fr. E4c was separated by silica gel CC and Sephadex
LH-20 CC (CHCl3/MeOH, 1:1) to afford compound 7 (14 mg) and
another fraction, which was successively subjected to reversed-
phase ODS-A CC (MeOH/H2O, 60:40–100:0) and repeated silica
gel CC eluted with petroleum ether/EtOAc (6:1–1:2) to attain com-
pounds 6 (8 mg) and 5 (8 mg). Fr. E4e was purified by Sephadex
LH-20 CC (CHCl3/MeOH, 1:1) followed by reversed-phase ODS-A
CC (MeOH/H2O, 60:40–100:0) to afford compounds 10 (19 mg), 3
(37 mg) and 4 (7 mg). Fr. E3 (14 g) was separated on a reversed-
phase ODS-A column (MeOH/H2O, 50:50–100:0), followed by a
Sephadex LH-20 CC (MeOH) to afford compound 2 (12 mg). Fr. E2
was applied to silica gel CC and eluted with CH2Cl2/MeOH
(100:1–10:1) to give five fractions (Fr. E2a–E2e). Fr. E2a was
subjected to silica gel CC eluted with petroleum ether/EtOAc
(4:1–1:2) followed by reversed-phase ODS-A CC (MeOH/H2O,
50:50–100:0) to afford compounds 8 (20 mg) and 9 (23 mg). Fr.
E2b was further purified by silica gel CC (CH2Cl2/MeOH, 100:
1–10:1) to give compounds 14 (60 mg) and 1 (8 mg). Fr. E2c was
successively applied to a Sephadex LH-20 column (CHCl3/MeOH,
1:1), a silica gel column eluted with petroleum ether/EtOAc
(11, 13 and 15–16) displayed much better inhibitory activities than
the corresponding aglycones (10, 12 and 14), indicated that
D-glucose moiety is clearly necessary for inhibitory activity.
3. Conclusions
In this study, sixteen diterpenoids including five new rare
16-nor-ent-pimarane diterpenoids (1–5), two new 15,16-dinor-ent-
pimarane diterpenoids (6–7) and one new ent-pimarane
diterpenoid glycoside (11) were isolated and identified from the
aerial parts of F. fimbriata. In the luciferase assay, compounds 4
and 6–7 exhibited pronounced NF-
IC50 values in the range of 14.7–19.2
the positive control PDTC (IC50 = 26.3
j
l
l
B inhibitory activities with
M, being more active than
M). Up to now, only about
ten naturally occurring 16-nor-ent-pimarane diterpenoids have
been reported, thus this work extends the chemical and biological
diversity of this type of norditerpenoids. As the NF-jB-suppression
activity results in anti-inflammatory effects, the evaluation of
these active compounds may provide some scientific basis for the
anti-inflammatory efficacy of this medicinal plant.
In addition, to the best of our knowledge, ent-pimarane
norditerpenoids from the Flickingeria genus have only been
reported in F. fimbriata, suggesting these norditerpenoids could
be considered as chemotaxonomic markers.
4. Experimental
(4:1–1:2) and
a reversed-phase ODS-A column (MeOH/H2O,
4.1. General
50:50–100:0) to obtain compound 12 (8 mg). The n-BuOH layer
(170 g) was subjected to MCI CC eluting with MeOH/H2O
(0:100–100:0) to afford five fractions (Fr. B1–B5). Fr. B5 (17 g)
was separated by repeated reversed-phase ODS-A CC (MeOH/
H2O, 10:90–50:50), Sephadex LH-20 column (MeOH) and silica
gel CC eluted with CH2Cl2/MeOH (30:1–10:1) to obtain compounds
11 (30 mg), 13 (28 mg), 16 (37 mg) and 15 (40 mg), respectively.
Optical rotations were measured on a Rudolph Autopol I auto-
matic polarimeter, whereas UV spectra were obtained on
a
Shimadzu UV-2450 spectrophotometer. CD spectra were recorded
on an Applied Photophysics Chirascan spectrometer. IR spectra
were determined on a Perkin Elmer FT-IR Spectrum Two spectrom-
eter. NMR spectra were measured on a Bruker AM-400 spectrom-
eter at 25 °C. ESIMS was measured on a Finnigan LC QDECA
instrument, and HRESIMS was performed on a Waters-Micromass
Q-TOF. A Shimadzu LC-20 AT equipped with a SPD-M20A PDA
detector was used for HPLC. Silica gel (300–400 mesh, Qingdao
Haiyang Chemical Co., Ltd., Qingdao, China), C18 reversed-phase
silica gel (12 nm, S-50 mm, YMC Co., Ltd, Japan), Sephadex LH-20
gel (Amersham Biosciences) and MCI gel (CHP20P, 75–150 mm,
Mitsubishi Chemical Industries Ltd., Kyoto, Japan) were used for
column chromatography (CC). All solvents used were of analytical
grade (Guangzhou Chemical Reagents Company, Ltd., Guangzhou,
4.3.1. (2S,3S,5S,9S,10S,13S)-2,3-dihydroxy-16-nor-ent-pimar-8(14)-
en-15-oic acid (1)
Colorless oil; ½a D25
ꢀ21.0 (c 0.13, MeOH); UV (MeOH) kmax (loge)
ꢂ
205 (2.83) nm; CD (MeCN) (1.4 ꢁ 10ꢀ4 M) kmax
(D
e) 223 (ꢀ0.36)
nm; IR (microscope) mmax 3381, 2925, 2859, 1708, 1549, 1462,
1389, 1259, 1129, 1064, 951 and 757 cmꢀ1; for 1H and 13C NMR
spectroscopic data, see Tables 1 and 2; HRESIMS m/z 345.2048
[M+Na]+ (calcd for C19H30O4Na, 345.2036).
4.3.2. (3R,5S,9S,10S,13S)-2-hydroxy-16-nor-ent-pimar-8(14)-en-15-
China). For the NF-jB biological assay: RAW264.7 cells (The
oic acid (2)
Center of Cellular Resource, Chinese Academy of Science,
Shanghai, China), Dulbecco’s modified Eagle medium (DMEM)
(Invitrogen-Gibco, Grand Island, NY), FBS (Invitrogen-Gibco,
Grand Island, NY), Luciferase assay system reagent (Promega,
Madison, CA. USA), Automated microplate reader (Molecular
Devices, Sunnyvale, CA, USA).
Colorless oil; ½a D25
ꢀ14.6 (c 0.1, MeOH); UV (MeOH) kmax (loge)
ꢂ
204 (2.81) nm; CD (MeCN) (1.6 ꢁ 10ꢀ4 M) kmax
(D
e) 214 (ꢀ0.45)
nm; IR (microscope) mmax 3393, 2931, 2849, 2847, 1699, 1457,
1388, 1243, 1031, 932, 793 and 750 cmꢀ1; for 1H and 13C NMR
spectroscopic data, see Tables 1 and 2; HRESIMS m/z 329.2098
[M+Na]+ (calcd for C19H30O3Na, 329.2087).
4.2. Plant materials
4.3.3. (2S,3R,5S,9S,10S,13S)-2-acetoxy-3-hydroxy-16-nor-ent-pimar-
8(14)-en-15-oic acid (3)
Aerial parts of F. fimbriata were collected from Foshan,
Guangdong Province of China, in August 2013. The plant material
was authenticated by Professor De-po Yang and a plant specimen
Colorless gum; ½a D25
ꢀ54.1 (c 0.32, MeOH); UV (MeOH) kmax
ꢂ
(loge) 204 (2.89) nm; CD (MeCN) (1.2 ꢁ 10ꢀ4 M) kmax
(De) 222

H. Li et al. / Phytochemistry 117 (2015) 400–409
407
(ꢀ0.94) nm; IR (microscope) mmax 3381, 2931, 1701, 1454, 1367,
1243, 1026, 926 and 676 cmꢀ1; for 1H and 13C NMR spectroscopic
data, see Tables 1 and 2; HRESIMS m/z 387.2146 [M+Na]+ (calcd for
8 h at room temperature. After evaporating the solvent in vacuo,
the resulting crude oil was subjected to flash silica gel CC (petro-
leum ether/EtOAc, 4:1) to afford 1a (C33H38O6, 5.2 mg), which
was determined by NMR and MS spectra. CD (MeCN) (1.5 ꢁ 10ꢀ4
C21H32O5Na, 387.2142).
M) kmax
(D
e) 237 (+12.0), 222 (ꢀ15.3), 205 (ꢀ10.0), 196 (+13.4)
nm. 1H NMR (CD3OD, 400 MHz): dH 0.96 (3H, s, H-20), 1.03 (3H,
s, H-19), 1.15 (3H, s, H-18), 1.19 (3H, s, H-17), 5.24 (1H, d,
J = 9.4 Hz, H-3), 5.44 (1H, dd, J = 4.3, 9.4, 11.8 Hz, H-2), 5.46 (1H,
4.3.4. (2S,3R,5S,9S,10S,13S)-2-O-E-cinnamoyl-3-hydroxy-16-nor-ent-
pimar-8(14)-en-15-oic acid (4)
Colorless gum; ½a D25
ꢀ50.3 (c 0.16, MeOH); UV (MeOH) kmax
ꢂ
br s, H-14), 2.40 (1H, m, H-6
, H-12b and H-7b), 1.55 (1H, m, H-1b), 1.18 (1H, m, H-12
1.65 (1H, m, H-11 ), 1.46 (1H, m, H-11b), 1.73 (1H, m, H-6b),
1.52 (1H, m, H-6 ), 1.51 (1H, m, H-5), 1.97 (1H, t, J = 8.6 Hz, H-9),
a), 2.17 (3H, dd, J = 4.1, 12.3 Hz, H-
(loge) 275 (4.23), 217 (4.13), 206 (4.11) nm; CD (MeCN)
(5.3 ꢁ 10ꢀ5 M) kmax
(D
e) 288 (ꢀ19.14), 260 (+6.96), 228 (ꢀ1.44),
1a
a),
a
206 (ꢀ9.41) nm; IR (microscope) mmax 3415, 2943, 2872, 1711,
1636, 1450, 1310, 1280, 1201, 1165, 1027, 766, 710 and
684 cmꢀ1; for 1H and 13C NMR spectroscopic data, see Tables 1
and 2, the E-cinnamoyl group dH: 6.56 (1H, d, J = 16.0 Hz, H-20),
7.74 (1H, d, J = 16.0 Hz, H-30), 7.60 (2H, H-50 and H-90), 7.40–7.42
(3H, H-60, H-70 and H-80); dC: 168.4 (C-10), 119.5 (C-20), 146.3
(C-30), 136.0 (C-40), 129.4 (C-50 and C-90), 130.2 (C-60 and C-80),
131.6 (C-70); HRESIMS m/z 475.2468 [M+Na]+ (calcd for
a
[7.90 (2H, dd, J = 1.3, 8.3 Hz), 7.82 (2H, dd, J = 1.3, 8.3 Hz), 7.49
(2H, m), 7.35 (4H, m), 2,3-O-dibenzoyl groups]. 13C NMR (CD3OD,
100 MHz): dC 179.2 (C-15), 139.5 (C-8), 127.8 (C-14), 82.7 (C-3),
72.0 (C-2), 55.1 (C-5), 51.6 (C-9), 44.4 (C-13), 43.6 (C-1), 41.0
(C-4), 40.6 (C-10), 36.4 (C-7), 34.5 (C-12), 29.4 (C-18), 28.0
(C-17), 23.3 (C-6), 21.4 (C-11), 18.6 (C-19), 15.7 (C-20), [(168.1,
134.4, 131.4, 130.6, 130.6, 129.7, 129.7) and (167.6, 134.3, 131.3,
130.6, 130.6, 129.5, 129.5), 2,3-O-benzoyl groups]. ESIMS m/z
553.3 [M+Na]+, 529.3 [MꢀH]ꢀ.
C28H36O5Na, 475.2460).
4.3.5. 3a,14b-diacetoxy-16-nor-ent-pimar-15a,8-olide (5)
Colorless oil; ½a D25
ꢀ8.5 (c 0.16, MeOH); UV (MeOH) kmax (loge)
ꢂ
206 (3.21) nm; CD (MeCN) (9.6 ꢁ 10ꢀ5 M) kmax
(D
e) 224 (ꢀ0.13)
4.5. Preparation of (S)- and (R)-MTPA esters of 2
nm; IR (microscope) mmax 2922, 2850, 1779, 1732, 1646, 1368,
1254, 1224, 1214, 1141, 1062, 975, 953 and 755 cmꢀ1; for 1H
and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS
m/z 429.2264 [M+Na]+ (calcd for C23H34O6Na, 429.2248).
A solution of compound 2 (1.0 mg) in deuterated pyridine-d5
(0.5 mL) was treated with (R)-MTPA chloride (10 lL) under N2 in
an NMR tube and immediately shaken until uniformly mixed.
The mixture was stirred at room temperature for 8 h. The 1H
NMR spectrum, recorded directly from the reaction NMR tube,
showed the production of the corresponding (S)-MTPA ester (2a).
Preparation of (R)-MTPA ester (2b) was performed in the same
manner by treatment of 2 (1.0 mg) with (S)-MTPA chloride.
4.3.6. (2S,3R,5S,10R)-2,3-dihydroxy-15,16-dinor-ent-pimar-8,11,13-
triene (6)
Colorless oil; ½a D25
ꢂ
+14.6 (c 0.16, MeOH); UV (MeOH) kmax (loge)
216 (3.74), 207 (3.85) nm; Mo2(OAc)4-induced CD (DMSO) kmax
(De) 302 (+0.354) nm; IR (microscope) mmax 3384, 2944, 1618,
1499, 1457, 1377, 1280, 1131, 1043, 992, 940, 890, 810 and
678 cmꢀ1; for 1H and 13C NMR spectroscopic data, see Tables 1
and 2; HRESIMS m/z 297.1836 [M+Na]+ (calcd for C18H26O2Na,
297.1825).
4.5.1. (S)-MTPA ester (2a)
1H NMR (C5D5N, 400 MHz): dH 7.60–7.41 (5H, m, phenyl of
MTPA), 5.36 (1H, s, H-14), 4.92 (1H, J = 6.2, 10.0 Hz, H-3), 3.52
(3H, s, –OMe of MTPA), 1.28 (3H, s, H-17), 1.17 (3H, s, H-18),
0.92 (3H, s, H-19), 0.84 (3H, s, H-20), 2.42 (1H, m, H-7
(2H, m, H-7b and H-12b), 1.88 (1H, m, H-2 ), 1.82 (1H, m, H-1
1.73 (1H, m, H-9), 1.65 (2H, m, H-6b and H-11b), 1.54 (1H, m,
H-2b), 1.47 (2H, m, H-6 and H-11 ), 1.45 (1H, m, H-1b), 1.09
a
), 2.17
a
a),
4.3.7. (2S,3R,5S,10R)-2-acetoxy-3-hydroxy-15,16-dinor-ent-pimar-
8,11,13-triene (7)
Colorless oil; ½a D25
ꢂ
+25.5 (c 0.17, MeOH); UV (MeOH) kmax (loge)
a
a
(1H, m, H-12a), 1.06 (1H, m, H-5). HRESIMS m/z 545.2497
215 (3.73), 205 (3.84) nm; IR (microscope) mmax 3392, 2947, 1716,
1499, 1365, 1245, 1059, 1029, 860 and 814 cmꢀ1; for 1H and 13C
NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z
339.1935 [M+Na]+ (calcd for C20H28O3Na, 339.1931).
[M+Na]+ (calcd for C29H37F3O5Na, 545.2491).
4.5.2. (R)-MTPA ester (2b)
1H NMR (C5D5N, 400 MHz): dH 7.62–7.41 (5H, m, phenyl of
MTPA), 5.36 (1H, s, H-14), 4.92 (1H, J = 6.2, 10.0 Hz, H-3), 3.65
(3H, s, –OMe of MTPA), 1.28 (3H, s, H-17), 1.21 (3H, s, H-18),
4.3.8. (3R,5S,9S,10S,13S)-3,16-dihydroxyl-15-one-ent-pimar-8(14)-
ene-3-O-b- -glucopyranoside (11)
D
Colorless gum; ½a D25
ꢂ
ꢀ6.5 (c 0.23, MeOH); UV (MeOH) kmax
0.99 (3H, s, H-19), 0.83 (3H, s, H-20), 2.41 (1H, m, H-7
(2H, m, H-7b and H-12b), 1.81 (1H, m, H-2 ), 1.77 (1H, m, H-1
1.72 (1H, m, H-9), 1.66 (2H, m, H-6b and H-11b), 1.51 (1H, m,
H-2b), 1.47 (2H, m, H-6 and H-11 ), 1.43 (1H, m, H-1b), 1.08
a), 2.15
(loge) 204 (3.42) nm; CD (MeCN) (7.5 ꢁ 10ꢀ5 M) kmax
(De) 218
a
a),
(ꢀ1.00), 293 (+0.33); IR (microscope) mmax 3352, 2936, 1709,
1363, 1074, 1016 and 655 cmꢀ1; for 1H and 13C NMR spectroscopic
data, see Tables 1 and 2; HRESIMS m/z 505.2760 [M+Na]+ (calcd for
a
a
(1H, m, H-12a), 1.04 (1H, m, H-5). HRESIMS m/z 545.2501
[M+Na]+ (calcd for C29H37F3O5Na, 545.2491).
C26H42O8Na, 505.2772).
4.3.9. Lonchophylloid B (10)
4.6. Alkaline hydrolysis of 3, 4 and 7
Colorless needles (CHCl3/petroleum ether); ½a D25
ꢀ10.2 (c 0.13,
ꢂ
MeOH); CD (MeCN) (9.4 ꢁ 10ꢀ5 M) kmax
(+0.31) nm; ESIMS m/z 343.3 [M+Na]+.
(D
e) 216 (ꢀ1.19), 291
A solution of each diterpenoid (2 mg) in (MeOH/H2O, 1 mL, 1:1)
was stirred with aqueous NaOH (0.1 M, 0.5 ml) for 4 h at room
temperature. The mixture was then treated with HCl (0.1 M) until
pH 6–7 and subjected to Sephadex LH-20 using MeOH as eluent to
afford the pure corresponding hydrolysis product, which was iden-
tified on the basis of its 1H NMR spectrum, Rf values and specific
rotation.
4.4. Preparation of the 2,3-dibenzoate (1a)
Benzoyl chloride (100
lL) was added to a stirred solution of 1
(4.5 mg) in pyridine (1 mL) under N2 with the mixture stirred for

408
H. Li et al. / Phytochemistry 117 (2015) 400–409
4.7. Determination of absolute configuration of the diol unit in 6 by
Snatzke’s method
with LPS, LUMsample is the value of a tested sample and LUMblank is
the value of the cell without LPS stimulation. Nonlinear regression
(with sigmoidal dose response) was used to calculate the IC50 val-
ues using GraphPad Prism (GraphPad Software, Inc.). The values
were expressed as means standard deviation (SD) for triplicate
experiments.
According to the reported literature procedure, a solution of diol
(0.5 mg) in anhydrous DMSO (1 mL) was mixed with dimolybde-
num tetraacetate (1.0 mg). The first CD of the mixture (ca. molar
ratio 1:1.2 diol/dimolybdenum tetraacetate) was measured imme-
diately after mixing, and its time evolution was monitored until
stationary phase (about 10 min after mixing). The inherent CD
was subtracted. The observed sign of the diagnostic band corre-
sponding to the O–C–C–O dihedral angle at around 300 nm in
the induced CD spectrum was correlated to the absolute configura-
tion of the secondary alcohol.
Acknowledgements
This work was financially supported by Grants from the
National Natural Science Foundation of China (No. 81102782),
the Industry-University-Research Cooperation Program from
Science and Technology Department of Guangdong Province
(2013B090200059) and the Priming Scientific Research
Foundation for the Junior Teacher of Medicine in Sun Yat-sen
University.
4.8. Acid hydrolysis of 11 and determination of sugar configuration
Compound 11 (2 mg) was dissolved in 2 mL of 2 M HCl
(p-dioxane/H2O, 1:1) and heated until 100 °C and then reflux
began, this being maintained for 4 h. After removing the dioxane
in vacuo, the solution was then diluted with H2O and extracted
with CH2Cl2 (3 ꢁ 1 mL). The aqueous layer was evaporated in vacuo
to obtain a neutral residue, which was analyzed by silica gel TLC
(acetone/n-BuOH/H2O, 6:3:1) together with an authentic sugar
standard (glucose, Rf = 0.49). The remaining residue was dissolved
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2015.
07.005.
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