Stelleralides D-J and Anti-HIV Daphnane Diterpenes from Stellera chamaejasme

Article abstract of DOI:10.1021/acs.jnatprod.5b00660

Bioassay-guided fractionation of a petroleum ether extract of the roots of Stellera chamaejasme led to the isolation of seven new (stelleralides D-J, 1-7) and 12 known (8-19) daphnane diterpenoids. The structures and relative configurations of 1-7 were established on the basis of extensive spectroscopic analysis, including HRESIMS and comprehensive NMR techniques. All isolates were evaluated for anti-HIV activity in MT4 cells. All compounds tested, except 2, showed anti-HIV activity, and, especially, five 1α-alkyldaphnane diterpenoids (3, 4, 5, 10, and 11) exhibited extremely potent anti-HIV activity, with EC₅₀ values of 0.06-1.1 nM and selectivity index values of more than 10000.

Full text of DOI:10.1021/acs.jnatprod.5b00660

Article
pubs.acs.org/jnp
Stelleralides D−J and Anti-HIV Daphnane Diterpenes from Stellera
chamaejasme
Min Yan,† Yan Lu,† Chin-Ho Chen,‡ Yu Zhao,§ Kuo-Hsiung Lee,*,§,⊥ and Dao-Feng Chen*,†
†Department of Pharmacognosy, School of Pharmacy, Fudan University, Shanghai 201203, People’s Republic of China
‡Duke University Medical Center, Box 2926, SORF, Durham, North Carolina 27710, United States
§Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina 27599-7568, United States
⊥Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
S
* Supporting Information
ABSTRACT: Bioassay-guided fractionation of a petroleum ether extract of the roots of Stellera chamaejasme led to the isolation
of seven new (stelleralides D−J, 1−7) and 12 known (8−19) daphnane diterpenoids. The structures and relative configurations
of 1−7 were established on the basis of extensive spectroscopic analysis, including HRESIMS and comprehensive NMR
techniques. All isolates were evaluated for anti-HIV activity in MT4 cells. All compounds tested, except 2, showed anti-HIV
activity, and, especially, five 1α-alkyldaphnane diterpenoids (3, 4, 5, 10, and 11) exhibited extremely potent anti-HIV activity,
with EC50 values of 0.06−1.1 nM and selectivity index values of more than 10 000.
he rapid, worldwide spread of acquired immunodeficiency
Tsyndrome (AIDS) has prompted an intense research
effort to discover compounds that can inhibit effectively the
human immunodeficiency virus (HIV).1,2 Indeed, highly active
antiretroviral therapy (HAART), which combines three to four
antiretrovirals, can effectively control plasma viremia in many
patients, although the virus is suppressed rather than truly
eradicated.3−5 Thus, a major goal of current AIDS therapy
continues to be the development of new anti-HIV compounds
as well as drug regimens for eradication of the AIDS virus.
Stellera chamaejasme L. (Thymelaeaceae), a toxic perennial
herb, is distributed prevalently in northern and southwestern
mainland China and Nepal. Its dried roots, named “Rui-Xiang-
Lang-Du” in traditional Chinese medicine, have long been used
for the treatment of stubborn skin ulcers, tinea, scabies,
tuberculosis, and chronic tracheitis.6 Previous chemical
investigations on the roots of this plant have led to the
isolation of diverse secondary metabolites, including highly
functionalized daphnane diterpenoids,7−9 tigliane diterpe-
noids,10 lignans,11,12 biflavonoids,13−16 coumarins,17,18 and
sesquiterpenoids.11,19 In particular, certain daphnane and
tigliane diterpenoids have shown extremely potent anti-HIV
activity at low nanomolar concentrations with relatively low
cytotoxicity.7,10 However, these compounds are very difficult to
synthesize because of their complex structure. Accordingly,
further analysis of the plant material was conducted to acquire a
significant quantity of several known, as well as new, anti-HIV
diterpenoids from S. chamaejasme.
Consequently, the current research on anti-HIV diterpenoids
from S. chamaejasme led to the isolation of 19 daphnane
diterpenoids, including seven new [stelleralides D−J (1−7)]
and 12 known compounds [stelleralide C (8),7 pimelotide A
(9),20 gnidimacrin (10),7 pimelea factor P2 (11),7 wikstroelide
F (12),7 wikstroelide A (13),21 wikstroelide B (14),22
wikstrotoxin A (15),23 huratoxin (16),7 wikstroelide M
(17),22 wikstroelide J (18),22 and simplexin (19)7]. Herein,
are described the isolation and structural elucidation of the new
compounds together with the anti-HIV activity of all daphnane
diterpenoids isolated.
Received: July 28, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00660
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 1. H (600 MHz) and 13C (150 MHz) NMR
Spectroscopic Data for Compounds 1 and 2 in CDCl3
a
b
1
2
position
1
δH [J in (Hz)]
δC
δH [J in (Hz)]
δC
2.86 (1H, dd, 14.0,
4.0)
54.5 2.85 (1H, dd, 12.0,
4.0)
54.5
2
112.1
112.1
173.9
86.5
69.3
57.1
59.3
34.8
80.1
54.8
42.8
32.6
3
173.9
4
86.5
5
4.82 (1H, brs)
69.3 4.82 (1H, brs)
57.1
6
7
3.44 (1H, brs)
59.3 3.43 (1H, brs)
34.8 3.26 (1H, dd, 2.8, 1.6)
80.1
8
3.26 (1H, dd, 2.8, 1.6)
9
10
11
12
2.89 (1H, d, 4.0)
54.8 2.89 (1H, d, 4.2)
42.8 2.19 (1H, t, 6.8, 7.2)
2.19 (1H, t, 6.8, 6.8)
2.08 (1H, dd, 11.2,
6.8)
32.6 2.08 (1H, dd, 11.2,
6.8)
2.32 (1H, dd, 11.2,
6.8)
2.32 (1H, dd, 11.2,
6.8)
13
14
15
16
83.4
83.4
80.6
4.35 (1H, s)
80.6 4.35 (1H, s)
145.7
145.7
4.94 (1H, dd, 1.8, 1.8) 111.6 4.94 (1H, dd, 2.0, 2.0) 111.6
5.05 (1H, s)
1.79 (3H, s)
5.03 (1H, s)
RESULTS AND DISCUSSION
17
18
18.8 1.79 (3H, s)
18.7
69.5
■
4.54 (1H, dd, 12.4,
6.8)
69.5 4.54 (1H, dd, 12.4,
6.8)
In a bioassay-guided fractionation of an ethanolic extract (EC50
= 0.42 0.08 μg/mL, selectivity index (SI) ≥ 23) of the roots
of S. chamaejasme, three solvent fractions (petroleum ether,
EtOAc, n-BuOH) were obtained. The petroleum ether-soluble
fraction exhibited promising anti-HIV activity with an EC50
value of 0.06 0.007 μg/mL (SI ≥ 320), while the EtOAc-
soluble fraction had a higher EC50 value of 1.26 0.19 μg/mL
(SI ≥ 15), and the n-BuOH-soluble fraction showed no anti-
HIV activity. The active fraction was separated by repeated
column chromatography and preparative HPLC to afford 19
daphnane diterpenoids.
4.97 (1H, d, 12.5)
1.73 (3H, s)
4.97 (1H, d, 12.5)
19.6 1.74 (3H, s)
19
20
19.5
63.9
4.09, 4.68 (2H, d,
12.5)
63.9 4.09, 4.68 (2H, d,
12.5)
1′
2′
120.4
120.4
31.3
2.10 (1H, ddd, 12.0,
6.0, 3.0)
31.3 2.10 (1H, ddd, 12.0,
6.0, 3.0)
1.93 (1H, ddd, 12.0,
5.6, 3.0)
1.93 (1H, ddd, 12.0,
5.6, 3.0)
3′
4′
5′
6′
7′
8′
9′
10′
Bz-CO
Bz-1″
1.56, 1.76 (2H, m)
1.27, 1.62 (2H, m)
1.17, 1.48 (2H, m)
1.27, 1.52 (2H, m)
1.11, 1.52 (2H, m)
0.87, 2.27 (2H, m)
1.13 (1H, m)
22.2 1.56, 1.76 (2H, m)
25.1 1.27, 1.62 (2H, m)
24.2 1.17, 1.48 (2H, m)
26.6 1.27, 1.52 (2H, m)
26.9 1.11, 1.52 (2H, m)
32.6 0.87, 2.31 (2H, m)
37.8 1.13 (1H, m)
19.2 1.02 (3H,d, 6.4)
166.4
22.1
25.1
Compound 1 was obtained as a colorless oil. Its molecular
formula was determined as C53H76O12 based on an HRESIMS
ion at m/z 927.5235 [M + Na]+ (calcd for C53H76O12Na,
927.5229), indicating a hydrogen deficiency index (HDI) of 16.
The IR spectrum revealed the presence of hydroxy (3478
cm−1), carbonyl (1799 and 1718 cm−1), and aromatic groups
24.2
26.6
26.9
32.6
37.8
1
(1648 and 1452 cm−1). The H NMR data (Table 1) of 1
1.02 (3H, d, 6.4)
19.2
166.4
130.0
129.5
indicated two tertiary methyl (δH 1.73 and 1.79, each 3H, s),
one secondary methyl (δH 1.02, 3H, d, J = 6.4 Hz), one primary
methyl (δH 0.86, 3H, t, J = 8.0 Hz), two oxygenated methylenes
(δH 4.09, 4.54, 4.68, and 4.97), three oxygenated methines (δH
3.44, 4.35, and 4.82), a terminal double bond (δH 5.05 and
4.94), and five aromatic [δH 7.57 (1H, dd, J = 8.4, 1.2 Hz), 7.46
(2H, dd, J = 8.0, 8.0 Hz), 8.04 (2H, dd, J = 8.4, 1.2 Hz)]
protons. Analysis of the 13C NMR and DEPT spectra of 1
established the presence of daphnane ketal-lactone skeleton
resonances20 including a characteristic quaternary carbon
resonance at δC 120.4 (C-1′), an acetal carbon resonance at
δC 112.1 (C-2), a lactone carbonyl carbon resonance at δC
173.9 (C-3), and a methyl carbon resonance at δC 19.6 (C-19).
130.0
Bz-2″,
8.04 (2H, dd, 8.4, 1.2) 129.5 8.04 (2H, dd, 11.2,
1.2)
6″
Bz-3″,
5″
Bz-4″
7.46 (2H, dd, 8.0, 8.0) 128.5 7.45 (2H, dd, 7.6, 7.6) 128.5
7.57 (1H, dd, 7.2, 1.2) 133.2 7.57 (1H, dd, 7.6, 1.2) 133.2
a
Data for palmitic acid group: δH 2.37 (2H, t, 8.0, H-2‴), 1.62 (2H, m,
H-3‴), 1.23−1.30 (24H, m, H-4‴−H-15‴), 0.86 (3H, t, 8.0, H-16‴);
δC 173.8 (C-1‴), 34.1 (C-2‴), 24.7 (C-3‴), 29.0−29.8 (C-4‴−C-
b
13‴), 31.9 (C-14‴), 22.7 (C-15‴), 14.1 (C-16‴). Data for linoleic
acid group: δH 2.38 (2H, t, 8.0, H-2‴), 1.62 (2H, m, H-3‴), 1.23−1.30
(12H, m, H-4‴−H-7‴, H-16‴, and H-17‴), 2.04 (4H, m, H-8‴, H-
14‴), 5.32 (1H, m, H-9‴), 5.37 (1H, m, H-10‴), 2.76 (4H, t, 5.2, H-
11‴, H-15‴), 5.35 (1H, m, H-12‴), 5.33 (1H, m, H-13‴), and 0.88
(3H, t, 6.8, H-18‴); δC 174.0 (C-1‴), 33.9 (C-2‴), 24.7 (C-3‴),
29.0−29.8 (C-4‴−C-7‴), 27.1 (C-8‴, C-14‴), 127.9 (C-9‴), 130.2
(C-10‴), 25.6 (C-11‴, C-15‴), 130.1 (C-12‴), 128.1 (C-13‴), 31.7
(C-16‴), 22.5 (C-17‴), and 14.0 (C-18‴).
1
Comparison of the H and 13C NMR data (Table 1) of 1 with
those of stelleralide C (8)7 implied the similarity of essential
structural signals. Additionally, the presence of a fatty acid was
suggested [δC 173.8 (C-1‴), 34.1 (C-2‴), 24.7 (C-3‴), 29.0−
29.8 (C-4‴−C-13‴), 31.9 (C-14‴), 22.7 (C-15‴), 14.1 (C-
16‴)]. On the basis of the molecular formula, the fatty acid was
B
DOI: 10.1021/acs.jnatprod.5b00660
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proposed as palmitic acid. The linkage of palmitic acid at C-20
of the diterpenoid skeleton was confirmed by the HMBC
correlation between H2-20 (δH 4.09 and 4.68) and the carbonyl
carbon (δC 173.8) of palmitic acid (Figure 1) as well as the
downfield chemical shift of C-20 from δC 63.0 in 8 to δC 63.9 in
1. Other correlations in the HMBC spectrum confirmed the
connectivities in this compound.
(δH 4.09 and 4.68) to C-1‴ (δC 174.0). The relative
configurations of 2 were determined to be the same as those
of 1 by analysis of its NOESY spectrum and molecular
modeling, with β-orientations of H-1, H-7, H-8, H-14, H3-19,
and H-10′ and α-orientations of H-5, H-10, H2-18, and H-9′.
The structure of 2 (stelleralide E) was thus established as
shown.
Compound 3 was isolated as a white, amorphous powder. Its
HRESIMS exhibited a pseudomolecular peak at m/z 855.3557
[M + Na]+ (calcd for C46H56O14Na, 855.3562), corresponding
to the molecular formula C46H56O14. In accordance with the
molecular formula, 46 carbon signals were evident in the 13C
1
NMR spectrum. The H and 13C NMR spectra of 3 indicated
the presence of four methyl groups [δC 18.9, δH 1.80 (3H, s);
δC14.5, δH 1.18 (3H, d, J = 6.6 Hz); δC 18.5, δH 1.22 (3H, d, J =
7.2 Hz); δC 21.1, δH 2.10 (3H, s)], a terminal double bond [δC
145.4, 111.9; δH 5.15 (1H, brs), 4.92 (1H, dd, J = 1.2, 1.2 Hz)],
two hydroxymethyls [δC 66.8, δH 4.85 (1H, dd, J = 10.2, 3.0
Hz), 4.08 (1H, dd, J = 10.2, 10.2 Hz); δC 65.8, δH 3.83 (2H, d, J
= 7.2 Hz)], two benzoyl moieties (δC 166.1, 168.4, 129.3,
130.8, 130.2 × 2, 129.6 × 2, 128.5 × 2, 128.4 × 2, 132.8, 133.6),
an acetyl carbonyl resonance (δC 170.9), and, especially, a
typical quaternary carbon resonance at δC 118.4 (C-1′),
signifying that compound 3 is a 1α-alkyldaphnane deriva-
tive.25,26 The NMR spectra of 3 resembled closely those of
stelleralide A,7 suggesting that the two compounds have the
same molecular skeleton and substituent groups. However,
1
detailed comparison of their H and 13C NMR data (Table 2)
showed that an additional benzoyloxy group [δC 166.1 (Bz-
CO), 130.8 (Bz-1″), 129.6 (Bz-2″ and 6″), 128.4 (Bz-3″ and
5″), and 132.8 (Bz-4″) and δH 8.06 (H-2″ and 6″), 7.45 (H-3″
and 5″), and 7.56 (H-4″)] is present in 3. This conclusion was
also supported by the molecular formula of 3, which was 120
mass units greater than that of stelleralide A. The structure of 3
was fully determined by 2D NMR spectroscopic analysis. The
HMBC correlations (Figure 1) from H-10′ (δH 1.22) to C-1
(δC 49.2) and C-9′ (δC 27.9) and from H-7′ (δH 5.34) to C-5′
(δC 19.3), C-9′ (δC 27.9), and a carbonyl group (δC 166.1)
suggested that the benzoyloxy group is attached at C-7′. The
relative configuration of 3 was established by its NOESY
spectrum (Figure 1), comparison of its NMR data with those of
stelleralide A, and molecular modeling and determined to be
the same as that of stelleralide A with α-orientations of H-3, H-
5, H-10, H-2′, H-7′, and H-10′ and β-orientations for H-1, H-8,
H-7, H-14, and H-19. Consequently, compound 3 (stelleralide
F) was assigned as shown.
1
Figure 1. Key H−1H COSY, HMBC, and NOESY correlations of 1,
3, and 7.
The relative configuration of 1 was mainly established by
comparison of its spectroscopic data with those of stelleralide C
(8), as well as NOESY results (Figure 1) and molecular
modeling calculations. The NOESY correlations of H-1/H-19,
H-10′/H-1, H-7/H-8, and H-8/H-14 indicated that these
protons are cofacial and β-oriented, while the correlations of H-
10/H-9′, H-9′/H2-18, and H-5/H-10 suggested that these
protons are α-oriented. Thus, the structure of 1 (stelleralide D)
was established as shown.
The molecular formula of compound 2, obtained as a
colorless oil, was assigned as C55H76O12 according to its
HRESIMS (m/z 951.5184 [M + Na]+, calcd for C55H76O12Na,
951.5229), with an HDI of 18. The IR spectrum indicated the
presence of hydroxy (3459 cm−1), carbonyl (1797 and 1717
cm−1), and aromatic (1455 cm−1) groups. On the basis of the
1H and 13C NMR spectra (Table 1), it was evident that 2 has
the same daphnane ketal-lactone skeleton as 1. The key
difference between the two compounds was in the lipid motif.
The signals due to olefin protons (δH 5.32, 5.33, 5.35, and 5.37)
were consistent with the presence of an unsaturated fatty acid.
The lipid group was defined as linoleic acid by analysis of the
appropriate carbon signals [δC 174.0 (C-1‴), 33.9 (C-2‴), 24.7
(C-3‴), 29.0−29.8 (C-4‴−C-7‴), 27.1 (C-8‴, C-14‴), 127.9
(C-9‴), 130.2 (C-10‴), 25.6 (C-11‴, C-15‴), 130.1 (C-12‴),
128.1 (C-13‴), 31.7 (C-16‴), 22.5 (C-17‴), and 14.0 (C-
18‴)]. In order to confirm the structure of the linoleic acid
moiety, a GC-MS experiment24 was conducted (see Exper-
imental Section). The attachment of the linoleic acid group to
C-20 was assigned by the key HMBC correlations from H2-20
Compound 4 was assigned the molecular formula C51H48O14
based on a positive HRESIMS [M + Na]+ ion at m/z 917.3694.
The 1H and 13C NMR spectra of 4 and 3 were similar,
suggesting that 4 is also a 1α-alkyldaphnane derivative. Detailed
1
comparison of the H and 13C NMR data (Table 2) of 4 with
those of 3 indicated the only difference to be an acetyloxy
group at C-18 in 3 compared with a benzoyloxy group in 4.
This assignment was further confirmed by the HMBC
correlation between H2-18 (δH 4.41 and 5.11) and the
benzoyloxy carbonyl (δC 166.5). The relative configuration of
4 was assigned as being the same as that of 3 based on
comparison of their NMR data, NOESY experiments, and
molecular modeling. Therefore, the structure of 4 (stelleralide
G) was determined as shown.
Stelleralide H (5) was found to have the same molecular
formula, C44H54O12, as gnidimacrin (10), as determined by its
HRESIMS. Detailed comparison of the NMR data of 5 with
C
DOI: 10.1021/acs.jnatprod.5b00660
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1
Table 2. H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for Compounds 3−5 in CDCl3
a
b
c
3
4
5
position
δH [J in (Hz)]
δC
δH [J in (Hz)]
δC
δH [J in (Hz)]
δC
1
2.84 (1H, dd, 11.4, 11.4)
1.81 (1H, m)
49.2
37.4
82.4
79.4
73.5
60.4
63.5
36.6
81.3
48.1
40.9
29.2
3.00 (1H, dd, 11.2, 11.2)
1.87 (1H, m)
49.2
37.3
82.3
79.5
73.2
60.8
63.5
36.6
81.3
48.1
40.9
29.2
2.68 (1H, dd, 12.0, 12.0)
1.71 (1H, m)
48.1
37.4
79.5
78.5
70.9
60.3
63.5
36.6
80.9
47.9
41.1
29.5
2
3
4.90 (1H, d, 4.8)
4.96 (1H, d, 5.2)
3.81 (1H, d, 7.2)
4
5
4.06 (1H, d, 4.0)
4.12 (1H, d, 4.2)
3.79 (1H, s)
6
7
3.36 (1H, brs)
3.40 (1H, brs)
3.36 (1H, brs)
8
3.01 (1H, d, 1.8)
3.06 (1H, d, 1.8)
3.09 (1H, d, 1.8)
9
10
11
12
2.99 (1H, d, 12.0)
2.65 (1H, t, 7.8)
1.88 (1H, m)
3.01 (1H, d, 12.0)
2.85 (1H, t, 7.8)
1.98 (1H, m)
2.92 (1H, d, 14.4)
2.76 (1H, t, 7.8)
2.03 (1H, m)
2.21 (1H, d, 12.0)
2.35 (1H, d, 14.8)
2.32 (1H, d, 14.4)
13
14
15
16
84.4
81.3
84.5
81.3
84.5
81.4
4.37 (1H, d, 2.4)
4.43 (1H, d, 2.8)
4.40 (1H, d, 2.4)
145.4
111.9
145.4
111.7
145.6
111.9
4.93 (1H, dd, 1.2, 1.2)
5.15 (1H, s)
4.98 (1H, dd, 1.2, 1.2)
5.21 (1H, s)
4.97 (1H, 1.2, 1.2)
5.20 (1H, s)
17
18
1.80 (3H, s)
18.9
66.8
1.84 (3H, s)
18.8
67.2
1.84 (3H, s)
18.9
67.6
4.85 (1H, dd, 10.2, 3.0)
4.08 (1H, dd, 10.2, 10.2)
1.18 (3H, d, 6.6)
3.83 (2H, d, 7.2)
4.41 (1H, dd, 10.8, 3.8)
5.11 (1H, dd, 8.0, 8.0)
1.23 (3H, d, 6.8)
3.87 (2H, d, 7.2)
4.16 (1H, d, 12.0)
5.01 (1H, d, 12.0)
1.16 (3H, d, 6.0)
4.38 (1H, d, 10.2)
4.91 (1H, d, 10.2)
19
20
14.5
65.8
14.5
65.8
14.5
68.2
1′
2′
3′
4′
5′
6′
7′
8′
9′
10′
118.4
70.6
28.6
24.6
19.3
28.7
73.4
27.6
27.9
18.5
118.4
70.5
28.6
24.4
19.3
28.7
73.4
27.6
28.1
18.5
118.5
70.8
28.5
25.3
24.2
23.6
23.1
22.8
27.3
18.2
3.89 (1H, dd, 8.4, 3.0)
1.56, 1.66 (1H, m)
1.30, 1.66 (1H, m)
1.40, 1.53 (2H, m)
1.66, 1.87 (2H, m)
5.35 (1H, dd, 13.6, 6.8)
1.53, 1.96 (1H, m)
2.31 (1H, m)
3.95 (1H, dd, 7.6, 3.0)
1.59, 2.42 (1H, m)
1.36, 1.66 (1H, m)
1.48, 1.58 (2H, m)
1.71, 1.93 (2H, m)
5.39 (1H, dd, 13.6, 6.8,)
1.58, 2.01 (1H, m)
2.43 (1H, m)
3.90 (1H, dd, 10.8, 3.6)
1.71, 1.53 (1H, m)
1.27, 1.58 (1H, m)
1.20, 1.32 (2H, m)
1.36 (2H, m)
1.24, 1.34 (1H, m)
0.99, 1.59 (1H, m)
2.28 (1H, m)
1.22 (3H, d, 7.2)
1.30 (3H, d, 7.2)
0.99 (3H, d, 7.2)
a
Data for benzoyl and acetyl groups: δH 8.19 (2H, dd, 7.8, 1.2, H-2″, H-6″), 7.48 (2H, dd, 6.8, 7.8, H-3″, 5″), 7.62 (1H, dd, 6.8, 1.2, H-4″), 8.06 (2H,
dd, 10.4, 1.2, H-2‴, H-6‴), 7.45 (2H, dd, 10.4, 7.8, H-3‴, 5‴), 7.56 (1H, dd, 7.8, 1.0, H-4‴), 2.10 (3H, s, Ac-Me); δC 166.1, 168.4 (Bz-CO), 130.8
(C-1″), 130.2 (C-2″, 6″), 128.5 (C-3″, 5″), 133.6 (C-4″), 129.3 (C-1‴), 129.6 (C-2‴, 6‴), 128.4 (C-3‴, 5‴), 132.8 (C-4‴), 170.9 (Ac-CO), 21.1
b
(Ac-Me). Data for benzoyl group: δH 8.19 (2H, dd, 12, 1.8, H-2″, H-6″), 7.39 (2H, dd, 12.0, 10.8, H-3″, 5″), 7.62 (1H, dd, 10.8, 1.8, H-4″), 8.09
(2H, dd, 10.8, 2.4, H-2‴, H-6‴), 7.48 (2H, dd, 10.8, 11.4, H-3‴, 5‴), 7.58 (1H, dd, 11.4, 2.4, H-4‴), 8.16 (2H, dd, 10.8, 1.8, H-2⁗, H-6⁗), 7.51
(2H, dd, 10.8, 12, H-3⁗, 5⁗), 7.54 (1H, dd, 12.0, 1.8, H-4⁗); δC 166.1, 166.5, 168.4 (Bz-CO), 129.3 (C-1″), 129.9 (C-2″, 6″), 128.4 (C-3″, 5″),
133.5 (C-4″),130.8 (C-1‴), 129.4 (C-2‴, 6‴), 128.5 (C-3‴, 5‴), 132.8 (C-4‴), 130.5 (C-1⁗),129.4, (C-2⁗, 6⁗), 128.6 (C-3⁗, 5⁗), 133.0 (C-4⁗).
c
Data for benzoyl group: δH 8.08 (4H, dd, 7.8, 1.2, H-2″, 6″, 2‴, 6‴), 7.46 (4H, dd, 7.8, 7.2, H-3″, 5″, 3‴, 5‴), 7.58 (2H, dd, 7.2, 1.2, H-4″, 4‴); δC
166.5, 167.1 (Bz-CO), 129.6 (C-1″), 129.9 (C-2″, 6″), 128.4 (C-3″, 5″), 133.1 (C-4″), 130.1 (C-1‴), 129.7 (C-2‴, 6‴), 128.4, 128.4 (C-3‴, 5‴),
133.1 (C-4‴).
those of 10 suggested that these compounds are structural
analogues, differing only in the location of the benzoyl group.
The benzoyl group was located at C-20 in 5 rather than at C-3
in 10 based on the HMBC correlation between H2-20 (δH
4.38) and its corresponding benzoyl carbonyl (δC 167.5). The
configuration of 5 was elucidated by NOESY correlations to be
the same as that of 10. Thus, the structure of 5 (stelleralide H)
was established as shown.
rather than an acetyloxy group in 14. This assignment was
determined from the shifts in two carbon resonances: C-12 was
shifted upfield from δc 78.2 in 14 to 77.6 in 6, and C-13 was
shifted downfield from δc 83.4 in 14 to δc 85.0 in 6. The
location of the hydroxy group at C-12 was confirmed by the
HMBC correlations from H-12 (δH 3.94, 1H, brs) to C-9 (δC
78.5), C-11 (δC 44.9), C-13 (δC 85.0), C-14 (δC 80.6), and C-
15 (δC 144.7). In addition, in the NOESY spectrum, H-12
correlated with H-18 (δH 1.23, 3H, d, J = 7.6 Hz), indicating
that H-12 is α-oriented. Hence, the structure of compound 6
was fully established as shown.
Stelleralide I (6) was obtained as a white, amorphous
powder, and its molecular formula was deduced as C35H50O9
from an HRESIMS ion at m/z 637.3349 (calcd for
1
C35H50O9Na, 637.3347). The H and 13C NMR data (Table
Stelleralide J (7) was assigned a molecular formula of
3) of 6 were found to be closely similar to those of wikstroelide
B (14), a daphnanetoxin diterpene. The main difference
between them was the presence of a hydroxy group at C-12 in 6
C34H52O10, according to the HRESIMS (m/z 643.3455 [M +
1
Na]+, calcd for C34H52O10, 643.3453). On the basis of its H
and 13C NMR data (Table 3), 7 was considered to be a
D
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Journal of Natural Products
Article
1
Table 3. H (400 MHz) and 13C (100 MHz) NMR
between H3-18 (δH 1.06) and H-8 (δH 3.29), which indicated
that H-5, H-10, and H3-18 have α-orientations. Consequently,
compound 7 (Figure 1) was elucidated as shown.
The isolated compounds (1−19) were evaluated for anti-
HIV activity against NL4-3 virus replication in MT4
lymphocytes. Cytotoxicity [50% toxic concentration (CC50)]
was also assessed, and the results are summarized in Table 4.
Spectroscopic Data for Compounds 6 and 7 in CDCl3
6
δH [J in (Hz)]
7.59 (1H, brs)
7
position
δC
δH [J in (Hz)]
δC
1
160.9 7.73 (1H, s)
162.4
134.8
209.5
74.8
72.4
76.8
79.1
40.1
76.3
53.3
36.5
2
136.6
209.8
72.5
3
4
Table 4. Anti-HIV and Cytotoxicity Activities of Compounds
a
5
4.25 (1H, s)
72
3.83 (1H,s)
1−19
6
60.6
anti-HIV
cytotoxicity
selectivity index
CC50/EC50
7
3.55 (1H, d, 1.8)
3.78 (1H, brs)
64.3 4.31 (1H, s)
34.9 3.29 (1H, brs)
78.5
compound
EC50 (μM)
CC50 (μM)
8
9
1
0.59 0.18
6.71 2.46
≥11.1
≥10.8
≥12
≥11.2
≥12.9
≥16.3
≥4.4
≥13.8
≥18.3
≥1.29
≥15.7
≥15.3
≥15.6
≥15.3
≥16.7
≥14
≥16.6
≥15.2
≥18.8
≥3.74
≥18
≥1
≥12903
≥15342
≥13163
≥135
≥100
≥41
≥16
≥21500
≥14272
≥318
≥1300
≥392
≥1284
≥5384
≥281
10
11
3.85 (1H, t, 2.8)
47.6 3.63 (1H, d, 5.0)
44.9 2.24 (1H, m)
2
2.49 (1H, dd, 14.0,
6.8)
3
0.00093 0.00025
0.00073 0.00032
0.00098 0.00037
0.12 0.038
4
12
13
14
15
16
3.94 (1H, brs)
77.6 2.14 (2H, m)
85.0
37.5
74.4
5
6
4.74 (1H, d, 1.8)
5.14 (2H, d, 10.8)
80.6 5.65 (1H, s)
144.7
79.8
7
0.044 0.015
0.33 0.12
145.3
8
112.9 5.06 (1H, s), 5.17 (1H, 114.3
s)
9
1.12 0.30
10
11
12
13
14
15
16
17
18
19
AZT
0.00006 0.00002
0.0011 0.0004
0.048 0.0180
0.012 0.0041
0.039 0.0082
0.013 0.0041
0.0026 0.0009
0.059 0.0150
0.044 0.0118
0.047 0.0148
0.032 0.0082
17
18
19
20
1.89 (3H, s)
18.9 1.88 (3H, s)
18.9
17.6
9.8
1.23 (3H, d, 7.6)
1.82 (3H, s)
18.6 1.06 (3H, d, 8.5)
9.9 1.80 (3H, s)
3.78 (1H, d, 12.0)
3.94 (1H, d, 12.0)
65.0 3.77 (2H, d, 11.2)
67.0
1′
2′
3′
116.8
168.6
118.6
146.8
5.64 (1H, d, 15.2)
139.1 5.90 (1H, d, 16.0)
6.67 (1H, dd, 15.2,
10.4)
122.7 7.35 (1H, dd, 14.0,
7.6)
≥345
≥400
≥116
4′
5′
6′
5.86 (1H, dd, 14.4,
8.0)
128.6 7.35 (1H, dd, 14.0,
7.6)
128.2
146.3
31.9
5.86 (1H, dd, 14.4,
8.0)
134.9 6.21 (1H, dd, 14.0,
9.2)
a
The values are means SD (n = 3). AZT (zidovudine) was used as a
positive control.
2.11 (2H, dd, 14.4,
7.2)
32.7 1.29 (2H, m)
7′
8′
9′
10′
11′
12′
13′
14′
15′
1.27 (2H, m)
1.28 (2H, m)
1.28 (2H, m)
1.29 (2H, m)
1.30 (2H, m)
1.30 (2H, m)
1.31 (2H, m)
1.31 (2H, m)
0.90 (3H, t, 7.2)
29.2 1.30 (2H, m)
29.2 1.30 (2H, m)
29.3 1.30 (2H, m)
29.3 1.30 (2H, m)
29.5 1.30 (2H, m)
29.5 1.30 (2H, m)
31.9 1.30 (2H, m)
22.6 0.89 (3H, d, 6.4)
14.1
29.2
29.2
29.3
29.4
29.5
31.8
22.6
14.1
The 1α-alkyldaphnane diterpenoids (3, 4, 5, 10, and 11)
exhibited the most potent anti-HIV-1 activity, with EC50 values
of 0.06−1.1 nM (SI > 10 000). The daphnanetoxin diterpenes
(6, 7, 13−19) and compound 12 displayed lower, but still
potent anti-HIV-1 effects (EC50 2.6−120 nM, SI = 100−6000).
The least potent compounds were 1, 2, 8, and 9, which contain
a 2,4-epoxide moiety and lactone ring (SI < 50).
Structurally, the main difference between the most potent
(3−5, 10, 11) and the least potent (1, 2, 8, 9) compounds is in
ring A. These results indicated that the nature of ring A appears
to be responsible for the enhanced anti-HIV activity. In
addition, compounds 3−5, 10, and 11 with a cyclopentane ring
A were more potent than 6, 7, and 12−19 with a
cyclopentenone or cyclopentanone ring A, suggesting the
importance of a cyclopentane ring A for optimal anti-HIV
activity.
structural analogue containing 18 more mass units (H2O) than
wikstroelide M (16). A direct comparison of their 13C NMR
spectra showed that the carbon resonances at δC 61.9 (C-6)
and 63.6 (C-7) in 16 were shifted downfield to δC 76.8 and
79.1 in 7, suggesting that the 6,7-epoxide moiety in 16 is
replaced by two hydroxy groups in 7. This deduction was
confirmed from the HSQC and HMBC spectra, particularly
HMBC correlations (Figure 1) observed between H-7 (δH
4.31) and C-5 (δC 72.4), C-9 (δC 76.3), and C-20 (δC 67.0), as
well as H-8 (δH 3.29) and C-6 (δC 76.8) (Figure 1). In the
NOESY spectrum (Figure 1) of 7, H-8 (δH 3.29) showed
significant correlations with H-7 (δH 4.31) and H-14 (δH 5.65),
suggesting that H-7, H-8, and H-14 have β-orientations.
Correlation of H-7 (δH 4.31) and H2-20 (δH 3.77) indicated
that the hydroxymethyl at C-6 is also β-oriented, while the
hydroxy at C-6 was α-oriented (Figure 1). A correlation was
present between H-5 (δH 3.83) and H-10 (δH 3.63), but not
On the basis of their significant anti-HIV activity, compounds
3−5, 10, and 11 should be investigated in greater detail to
develop a deeper understanding of their anti-HIV character-
istics and potential. The new compounds likely share the same
mechanisms of action as previously investigated daphnane- and
tigliane-type diterpenes.7,10,27
EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on an Autopol V Plus instrument (Rudolph, Hackettstown,
NJ, USA). UV spectra were recorded in MeOH using a Lambda 25
■
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DOI: 10.1021/acs.jnatprod.5b00660
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
spectrophotometer (PerkinElmer, Wellesley, MA, USA). CD spectra
were obtained on a JASCO J-715 spectrometer. IR spectra were
measured on a PE Spectrum RXI spectrophotometer (PerkinElmer)
using KBr pellets. NMR spectroscopic data were recorded at room
temperature on Bruker AMX-400 MHz and AMX-600 MHz
instruments in CDCl3 with TMS as an internal standard. Standard
pulse sequences were employed for the measurement of 2D NMR
spectra (1H−1H COSY, HSQC, HMBC, and NOESY). ESIMS
analysis were carried out on a Dionex Ultimate 3000 UPLC
instrument with an LTQ Velos Pro MS spectrometer (Thermo Fisher
Scientific, USA). HRESIMS were acquired with a Bruker Daltonics
APEXIII 7.0 TESLA FTMS system (Bruker Daltonics, Billerica, MA,
USA). Analytical HPLC was carried out on an Agilent 1200 series LC
instrument with a DAD detector (Agilent Technologies, Palo Alto,
CA, USA) and a Symmetry C18 column (4.6 × 250 mm, 5 μm).
Preparative HPLC was performed on an Agilent 1100 (Agilent
Technologies) and a YMC-Pack Pro C18 RS column (20 × 250 mm, 5
μm). Silica gel (200−300 mesh, Qingdao Haiyang Chemical Co. Ltd.,
Qingdao, People’s Republic of China), Sephadex LH-20 (25−100 μm,
Pharmacia, Germany), and RP-C18 (30−50 μm, Fuji Silysia Chemical
Co. Ltd., Aichi, Japan) were used for column chromatography (CC).
The fractions were monitored by TLC (HSGF 254, Yantai, People’s
Republic of China), and detection was achieved by 10% H2SO4 in
EtOH. All solvents used for CC were of analytical grade (Shanghai
Chemical Reagents Co. Ltd., Shanghai, People’s Republic of China),
and solvents used for HPLC were of HPLC grade. Linoleic acid was
obtained from Sigma-Aldrich Company Ltd., Gillingham, United
Kingdom.
Stelleralide E (2): colorless oil; [α]2D0 −4.8 (c 0.10, CH2Cl2); UV
(CH2Cl2) λmax (log ε) 233 (4.16) nm; CD (MeOH, nm) λmax (Δε)
208 (4.67), 228 (7.78); IR (KBr) νmax 3459, 2923, 2854, 2359, 2341,
1797, 1717, 1455, 1395, 1173, 717 cm−1; 1H NMR (600 MHz,
CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 1;
HRESIMS m/z 951.5184 [M + Na]+ (calcd for C55H76O12Na,
951.5229).
Stelleralide F (3): white, amorphous powder; [α]2D0 −7.7 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 233 (4.02) nm; CD (MeOH, nm)
λmax (Δε) 215 (1.11), 236 (−1.14), 273 (0.87); IR (KBr) νmax 3463,
2925, 2860, 2360, 2340, 1731, 1712, 1457, 1384, 1279, 1025, 713
cm−1; 1H NMR (600 MHz, CDCl3) and 13C NMR (150 MHz,
CDCl3) data, see Table 2; HRESIMS m/z 855.3557 [M + Na]+ (calcd
for C46H56O14Na, 855.3562).
Stelleralide G (4): white, amorphous powder; [α]2D0 −16.7 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 233 (4.15) nm; CD (MeOH, nm)
λmax (Δε) 215 (1.07), 237 (−7.42); IR (KBr) νmax 3453, 2934, 2854,
1715, 1451, 1384, 1275, 1025, 712 cm−1; 1H NMR (600 MHz,
CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 2;
HRESIMS m/z 917.3694 [M + Na]+ (calcd for C51H48O14Na,
917.3719).
Stelleralide H (5): white, amorphous powder; [α]2D0 +4.6 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 233 (4.31) nm; CD (MeOH, nm)
λmax (Δε) 215 (4.07), 236 (−5.42); IR (KBr) νmax 3446, 2927, 2858,
1716, 1451, 1272, 1112, 1023, 711 cm−1; 1H NMR (600 MHz,
CDCl3) and 13C NMR (150 MHz, CDCl3) data, see Table 2;
HRESIMS m/z 775.3693 [M + H]+ (calcd for C44H54O12 775.3688).
Stelleralide I (6): white, amorphous powder; [α]2D0 +21.6 (c 0.10,
MeOH); UV (MeOH) λmax (log ε) 256 (3.98) nm; CD (MeOH, nm)
λmax (Δε) 226 (−12.1), 242 (5.82); IR (KBr) νmax 3435, 2920, 2843,
1709, 1616, 1463, 1375, 1019 cm−1; 1H NMR (400 MHz, CDCl3) and
13C NMR (100 MHz, CDCl3) data, see Table 3; HRESIMS m/z
637.3349 [M + Na]+ (calcd for C35H50O9Na, 637.3347).
Plant Material. The roots of S. chamaejasme (4 years old) were
purchased from Baotou, Inner Mongolia, People’s Republic of China,
in August 2011 and were authenticated by one of the authors (D.-
F.C.). A voucher specimen (DFC-YM-SC-2011-08) is deposited in the
Herbarium of Materia Medica, Department of Pharmacognosy, School
of Pharmacy, Fudan University, Shanghai, People’s Republic of China.
Extraction and Isolation. The dried roots of S. chamaejasme were
ground into a powder (40 kg), which was percolated with 95%
aqueous EtOH. After removal of the solvent under vacuum, the
residue was suspended in H2O and successively extracted with
petroleum ether, EtOAc, and n-BuOH. The petroleum ether-soluble
fraction (350 g) was subjected to VLC on silica gel using a stepwise
gradient elution of petroleum ether−Me2CO (30:1, 20:1, 10:1, 5:1,
2:1, and 1:1) to afford five subfractions (Fr.A−Fr.E). Fr.C (40 g) was
passed through a silica gel column eluted with petroleum ether−
EtOAc (30:1 to 10:1) to give seven fractions (Fr.C1−Fr.C7). Fr.C2 (7
g) was applied to CC on Sephadex LH-20 (n-hexane−CH2Cl2−
MeOH, 5:4:1) to obtain six fractions (Fr.C2a−Fr.C 2f). Fr.C2b (120
mg) was purified by preparative HPLC (10 mL/min, 50 min 85−95%
MeCN−H2O gradient elution) to yield 1 (10 mg) and 2 (8 mg).
Fr.C2f (400 mg) was separated by preparative HPLC (10 mL/min, 50
min 70−95% MeCN−H2O gradient elution) to acquire 12 (15 mg),
13 (35 mg), and 16 (40 mg). Fr.C3 (5 g) was chromatographed on an
RP-C18 silica gel column (MeOH−H2O, 70:30 to 100:0) to give five
fractions (Fr.C3a−Fr.C3e). Fr.C3d (1.2 g) was separated on a
preparative HPLC column (10 mL/min, 75% MeCN−H2O isocratic
elution) to obtain 3 (8 mg), 4 (5 mg), 9 (20 mg), 10 (15 mg), and 19
(25 mg). Separation of Fr.D (30 g) by MPLC (petroleum ether−
EtOAc, 15:1 to 0:1) gave six fractions (Fr.D1−Fr.D6). Fr.D3 was
purified by passage over Sephadex LH-20 (CHCl3−MeOH, 1:1) and
then by preparative HPLC (CH3CN−H2O, eluting from 65:35 to
90:10 for 40 min with a flow rate of 10 mL/min) to afford 6 (4 mg), 7
(7 mg), 8 (25 mg), 17 (12 mg), and 15 (9 mg). Using the same
purification procedures, Fr.D4 afforded 5 (9 mg), 11 (11 mg), 18 (7
mg), and 14 (20 mg).
Stelleralide J (7): white, amorphous powder; [α]2D0 +2.2 (c 0.12,
MeOH); UV (MeOH) λmax (log ε) 256 (4.01) nm; CD (MeOH, nm)
λmax (Δε) 243 (−1.69), 268 (3.08); IR (KBr) νmax 3405, 2925, 2854,
1
2361, 1701, 1639, 1006 cm−1; H NMR (400 MHz, CDCl3) and 13C
NMR (100 MHz, CDCl3) data, see Table 3; HRESIMS m/z 643.3455
[M + Na]+ (calcd for C34H50O10Na, 643.3453).
Base Hydrolysis of 2 and GC-MS Analysis. Compound 2 (2
mg) in CH2C12 (2 mL) was allowed to stand at room temperature for
3 h with 0.05 M NaOMe in MeOH (0.5 mL). The mixture was
neutralized, followed by addition of 10 mL of 14% BF3−MeOH, and
was heated at 80 °C for 5 min. Hexane (3 mL) was added to the above
mixture through the top of the condenser, and heating was continued
for 2 min. After dilution with a saturated NaCl solution, the organic
layer was collected and evaporated to dryness using N2. The residue
was redissolved in hexane and analyzed by GC-MS (Shimadzu,
GCMS-QP2010 Ultra) using an Inertcap column (0.25 mm × 30 m)
under the following conditions [injector temperature, 250 °C; initial
temperature, 80 °C (1 min), increased at 25 °C/min to 230 °C, held
for 10 min; carrier gas, He operated in the splitless mode; injection
size, 0.2 μL; MS conditions: EI voltage, 70 eV; scanned-mass range,
m/z 50−1000]. Identification of linoleic acid was carried out for 2,
giving a peak at 12.23 min. With authentic linoleic acid, a peak was
detected at 12.24 min.
Anti-HIV Assays. HIV-1 NL4-3 (multiplicity of infection = 0.001)
was used to infect MT4 cells in the presence of various concentrations
of compounds. Fresh medium, which contained appropriate
concentrations of the compounds, was added to the culture 48 h
after infection to maintain normal cell growth. Virus replication was
analyzed on day 4 postinfection using p24 ELISA kits from
PerkinElmer. The compound concentration that inhibited HIV-1
replication by 50% (EC50) was calculated by using the biostatistics
software CalcuSyn (Biosoft).
Stelleralide D (1): colorless oil; [α]2D0 +5.3 (c 0.10, CH2Cl2); UV
(CH2Cl2) λmax (log ε) 233 (4.38) nm; CD (MeOH, nm) λmax (Δε)
208 (4.06), 228 (7.70); IR (KBr) νmax 3478, 2924, 2854, 1799, 1718,
1648, 1452, 1396, 1270, 713 cm−1; 1H NMR (600 MHz, CDCl3) and
13C NMR (150 MHz, CDCl3) data, see Table 1; HRESIMS m/z
927.5235 [M + Na]+ (calcd for C53H76O12Na, 927.5229).
Cytotoxicity Assays. Cytotoxicity of the purified compounds
toward MT4 cells was determined by using a cell viability kit provided
by Promega. The CellTiter-Glo luminescent cell viability assay is a
simple method of determining the viability of the cells in culture based
on quantitation of ATP in metabolically active cells. The CellTiter-Glo
F
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reagent was added to MT4 cells that were cultured parallel to the
antiviral assays. The cytotoxic concentration that caused the reduction
of viable cells by 50% (CC50) was calculated from the dose−response
curve using CalcuSyn.
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ASSOCIATED CONTENT
* Supporting Information
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S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.5b00660.
1
1H NMR, 13C NMR, H−1H COSY, HSQC, HMBC,
NOESY, CD, and IR spectra for compounds 1−7 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*Tel (K.-H. Lee): +1-919-962-0066. Fax: +1-966-3893. E-mail:
khlee@unc.edu.
■
(26) Liao, S. G.; Chen, H. D.; Yue, J. M. Chem. Rev. 2009, 109,
1092−1140.
(27) Huang, L.; Ho, P.; Yu, J.; Zhu, L.; Lee, K. H.; Chen, C. H. PLoS
One 2011, 6, e26677.
*Tel (D.-F. Chen): +86-21-51980135. Fax: +86-21-51980017.
E-mail: dfchen@shmu.edu.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This investigation was supported by grants from the National
Natural Science Foundation of China (81273486), the
Research Fund for the Doctoral Program of Higher Education,
China (2012007113011), to D.F.C., and the National Institute
of Allergy and Infectious Diseases, NIH, Bethesda, MD, USA
(AI033066 to K.H.L.).
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DOI: 10.1021/acs.jnatprod.5b00660
J. Nat. Prod. XXXX, XXX, XXX−XXX