Carolignans from the Aerial Parts of Euphorbia sikkimensis and Their Anti-HIV Activity

Article abstract of DOI:10.1021/acs.jnatprod.5b01012

Seven new carolignans, including two pairs of enantiomers (±)-erythro-7′-methylcarolignan E (1a/1b) and (±)-threo-7′-methylcarolignan E (2a/2b), (+)-threo-carolignan E (3a), (+)-erythro-carolignan E (4a), and (-)-erythro-carolignan Z (5), together with four known lignans (3b, 4b, 6, and 7) and six polyphenols (8-13) were isolated from the aerial parts of Euphorbia sikkimensis. The structures of the new compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined by electronic circular dichroism calculations. Seven of the isolates were examined for anti-HIV effects, and compounds 1a and 1b showed moderate anti-HIV activity with EC₅₀ values of 6.3 and 5.3 μM.

Full text of DOI:10.1021/acs.jnatprod.5b01012

Article
pubs.acs.org/jnp
Carolignans from the Aerial Parts of Euphorbia sikkimensis and Their
Anti-HIV Activity
Cheng Jiang,† Pan Luo,† Yu Zhao,‡ Jialing Hong,† Susan L. Morris-Natschke,‡ Jun Xu,† Chin-Ho Chen,§
Kuo-Hsiung Lee,*,‡,⊥ and Qiong Gu*,†,‡
†Research Center for Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s
Republic of China
‡Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North
Carolina 27599, United States
§Duke University Medical Center, Box 2926, SORF, Durham, North Carolina 27710, United States
⊥Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 401, Taiwan
S
* Supporting Information
ABSTRACT: Seven new carolignans, including two pairs of
enantiomers ( )-erythro-7′-methylcarolignan E (1a/1b) and
( )-threo-7′-methylcarolignan E (2a/2b), (+)-threo-carolignan
E (3a), (+)-erythro-carolignan E (4a), and (−)-erythro-
carolignan Z (5), together with four known lignans (3b, 4b,
6, and 7) and six polyphenols (8−13) were isolated from the
aerial parts of Euphorbia sikkimensis. The structures of the new
compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined by electronic circular
dichroism calculations. Seven of the isolates were examined for anti-HIV effects, and compounds 1a and 1b showed moderate
anti-HIV activity with EC50 values of 6.3 and 5.3 μM.
isolation, structural elucidation, and anti-HIV activity of these
compounds.
he genus Euphorbia belongs to the family Euphorbiaceae,
Twhich is characterized by production of a white, milky
latex that is somewhat toxic. The family contains about 300
genera and 7000 species. Euphorbia is one of the largest genera
in the Euphorbiaceae family, with about 1600 species.
Numerous chemical studies on the Euphorbia genus have
been performed.1,2 Euphorbia sikkimensis Boiss. has been used
to treat poisoning, malaria, rheumatism, and jaundice.3 In 2013,
new and known diterpenes, triterpenoids, tocopherol deriva-
tives, and other compounds were isolated.4,5 Two diterpenoids
showed antiproliferative effects against the A549 cancer cell
line, while (−)-bornyl ferulate exhibited antiangiogenic activity
in a zebrafish model.5
In a continuing search for anti-HIV natural products from
herbal medicinal plants, anti-HIV screening showed that an
EtOAc-soluble fraction from an ethanol extract of the aerial
parts of E. sikkimensis had an EC50 value of 2.5 μg/mL and an SI
(selective index) value of more than 8.3. Prior literature
reported that diterpenoids from Euphorbia showed potent anti-
HIV activity at the nanomolar level,6,7 whereas one of these
diterpenoids, a phorbol ester, also showed tumor-promoting
activity.8 These results encouraged us to investigate the anti-
HIV-active constituents of the EtOAc-soluble fraction, which
led to the isolation and characterization of seven new lignans
(1−7) and six polyphenols (8−13). Compounds 1a and 1b
showed moderate anti-HIV activity with EC50 values of 6.3 and
5.3 μM, respectively. Herein are reported details of the
The known compounds were identified as (−)-(7′R,8′S)-
erythro-carolignan E (3b),9,10 (−)-(7′S,8′S)-threo-carolignan E
(4b),9,10 threo-guaiacylglycerol-β-O-4′-dihydroconiferyl alcohol
(6),11,12 erythro-guaiacylglycerol-β-O-4′-dihydroconiferyl alco-
hol (7),11,12 (E)-ferulic acid (8),13,14 ethyl ferulate (9),14
protocatechuic acid (10),15,16 vanillic acid (11),17,18 methyl
gallate (12),19 and ethyl gallate (13),19 based on spectroscopic
data analysis and comparison with reported literature values.
RESULTS AND DISCUSSION
■
Compound 1 (1a/1b) was obtained as a white, amorphous
solid. A molecular formula of C41H44O13 was deduced from a
quasimolecular ion peak at m/z 743.27039 (calcd 743.27091)
observed in the negative HRESIMS. The IR spectrum showed
absorption bands for OH (3407 cm−1), carbonyl (1704 cm−1),
and aromatic groups (1599, 1514, and 1460 cm−1). The H
1
NMR spectrum showed two pairs of doublets at δH 7.59 (1H, d,
J = 15.9 Hz, H-7‴) and 6.27 (1H, d, J = 15.9 Hz, H-8‴); 7.48
(1H, d, J = 15.9 Hz, H-7) and 6.23 (1H, d, J = 15.9 Hz, H-8),
together with six aromatic protons (ring A: δH 7.00 (1H, d, J =
Special Issue: Special Issue in Honor of John Blunt and Murray
Munro
Received: November 10, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b01012
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Table 1. 1H NMR Spectroscopic Data (400 MHz, in CDCl3)
for Compounds 1−5 (δH in ppm, J in Hz)
a
a
position
1a/1b
2a/2b
3a
4a
5
2
7.02 d (1.9) 7.00 d
(1.9)
7.01 d (1.9) 6.91 d
(1.8)
7.00 d
(2.0)
5
6.85 d (8.2) 6.89 d
(8.2)
6.88 d (8.3) 6.87 d
(8.3)
6.73 d
(8.3)
6
7.05 d (1.9, 7.06 d (1.9, 7.05 d (1.9, 7.00 m
7.05 d (2.0,
8.3)
8.2)
8.2)
8.3)
7
7.48 d
7.48 d
7.59 d
7.59 d
(15.9)
6.21 d
(15.9)
7.01 m
7.64 d
(15.9)
(15.9)
6.23 d
(15.9)
6.93 m
(15.9)
6.26 d
(15.9)
(15.9)
6.24 d
(15.9)
7.04 m
8
6.21 d
(15.9)
2′
5′
6′
7′
6.89 d
(1.8)
6.98 m
6.90 d (8.2) 6.87 d
(8.1)
6.87 d (8.1) 6.89 d
(8.1)
6.96 d
(8.2)
6.86 m
6.85 dd
(1.8, 8.1)
6.75 dd
(1.8, 8.1)
6.84 dd
(2.0, 8.0)
6.87 d (1.7,
8.2)
4.52 d (3.0) 4.25 t (6.3) 4.91 d (8.1) 4.90 d
(3.3)
4.88 d
(3.1)
8′
9′
4.48 m
4.43 m
4.13 m
4.20 m
4.44 m
4.26 m
4.55 m
4.11 dd
(4.7,
4.13 dd
(4.8,
4.83 d
(6.4)
11.6)
12.1)
4.31 dd
(2.8,
11.6)
4.44 m
4.34 dd
(3.4,
12.1)
4.48 m
2″
5″
6″
7″
8″
9″
6.68 d (1.8) 6.63 d
(1.8)
6.72 d (1.8) 6.75 d
(1.8)
6.60 d
(1.8)
1.8 Hz, H-2‴), 6.91 (1H, d, J = 8.1 Hz, H-5‴), 7.05 (1H, dd, J
= 1.8, 8.1 Hz, H-6‴); ring C: δH 7.02 (1H, d, J = 1.9 Hz, H-2),
6.85 (1H, d, J = 8.2 Hz, H-5), 7.05 (1H, dd, J = 1.9, 8.2 Hz, H-
6)). The 13C NMR spectrum exhibited two conjugated
carbonyl carbons at δC 167.1 and 167.5 ppm. The above data
indicated the presence of two feruloyl moieties. The 13C NMR
spectrum (Figure S6, Supporting Information) also displayed
two oxymethylene (CH2O, δC 63.9), two methylene (CH2, δC
32.1, 30.6), and two oxymethine (CHO, δC 83.5, 82.3) signals,
and the COSY correlations (Figure 1) suggested one CH2O−
6.90 m
6.84 d
(8.0)
6.91 d (8.0) 6.95 d
(8.0)
6.80 dd
(1.8, 8.1)
6.81 dd
(1.8, 8.0)
6.85 dd
(1.8, 8.0)
6.81 dd
(1.8, 8.0)
6.60 d
(1.8)
2.62 t (6.5) 2.58 m
2.68 t (7.9) 2.68 t (6.5) 6.63 d
(15.7)
6.30 d
(15.7)
1.96 m
1.91 m
2.00 m
1.99 m
4.15 t (6.5) 4.13 t (6.4) 4.20 t (6.0) 4.19 t (6.5) 4.25 d
(3.7)
4.44 m
2‴
5‴
6‴
7‴
8‴
7.00 d (1.8) 7.00 d
(1.8)
7.01 d (1.8) 7.01 m
6.93 d
(1.7)
6.91 d (8.1) 6.94 d
(8.1)
6.91 d (8.1) 6.95 d
(7.9)
6.75 d
(8.2)
7.05 dd
(1.8, 8.1)
7.59 d
(15.9)
6.27 d
(15.9)
7.02 dd
(1.8, 8.1)
7.59 d
(15.9)
6.28 d
(15.9)
7.01 dd
(1.8, 8.1)
7.51 d
(15.9)
6.28 d
(15.9)
7.06 dd
(1.8, 8.3)
7.49 d
(15.9)
6.28 d
(15.9)
7.08 dd
(1.7, 8.2)
7.69 d
(15.9)
6.23 d
(15.9)
OCH3-3
OCH3-3′
OCH3-3″
OCH3-3‴ 3.90 s
OCH3-5″
3.90 s
3.82 s
3.72 s
3.86 s
3.78 s
3.72 s
3.86 s
3.92 s
3.88 s
3.84 s
3.92 s
3.90 s
3.86 s
3.86 s
3.90 s
3.85 s
3.87 s
3.90 s
3.85 s
3.90 s
Figure 1. Key 1H−1H COSY () and HMBC (→) correlations of 1.
CH2−CH2 fragment and one CH2O−CHO−CHO group. The
1H and 13C NMR data (Tables 1 and 2) also indicated two
1,3,4-trisubstituted aromatic units (rings B and D) and five
methoxy groups. HMBC correlations (Figure 1) of H-9″ (δH
4.15, 2H, t, J = 6.5 Hz) with C-9‴ (δC 167.5) and H-9′ (δH
4.11, 1H, dd, J = 4.7, 11.6 Hz; 4.31, 1H, dd, J = 2.8, 11.6 Hz)
with C-9 (δC 167.1) showed that two feruloyl units were
connected at C-9 and C-9‴. All of the above data were closely
similar to those of the known compound erythro-carolignan
E,9,10 except for an additional methoxy group in the new
compound. The position of this methoxy group at C-7′ was
confirmed from the HMBC correlation of MeO (δH 3.27, s)
with C-7′ (δC 83.5, d). Its relative configuration was established
on the basis of NOESY correlations (Figure 2) and
interpretation of 1H−1H coupling constants. A coupling
constant of 15.9 Hz between H-8 (8‴) and H-7 (7‴) suggested
OCH3-7′
3.27 s
3.25 s
3.25 s
1
3.25 s
a
1a/1b and 2a/2b showed the same H NMR data.
an E-configuration for Δ7 and Δ7‴. The coupling constant
between H-7′ and H-8′ (3.0 Hz) implied an erythro-
configuration of these two protons.20−22 Thus, compound 1
(erythro-7′-methylcarolignan E) was fully identified.
The specific rotation of 1 approached zero, and no Cotton
effect was found in the electronic circular dichroism (ECD)
spectrum of 1, indicating a racemic mixture. Subsequent chiral
resolution of 1 afforded the anticipated enantiomers 1a and 1b,
which showed mirror image-like ECD curves (Figure 3a) and
specific rotations (1a: [α]20D −16.2; 1b: [α]20D +17.1). In order
to define the absolute configuration of the enantiomers 1a and
B
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Table 2. 13C NMR (100 MHz) Data (δ) of Compounds 1−5
(δC in ppm)
a
a
position
1a/1b
2a/2b
3a
4a
5
1
127.1
109.3
146.9
148.2
114.3
123.3
145.3
115.6
167.1
130.0
109.9
146.9
145.8
114.9
120.6
83.5
127.1
109.3
146.9
148.1
114.3
123.3
145.2
115.5
167.3
130.3
110.0
146.9
145.6
114.9
120.6
82.8
127.1
109.6
147.0
148.3
114.6
123.3
145.6
115.6
167.0
131.4
109.5
146.9
145.8
114.9
120.9
74.6
127.0
109.5
147.0
148.2
114.3
123.3
145.4
115.6
167.3
131.2
109.0
146.8
145.3
114.9
121.0
72.3
127.1
109.5
147.0
148.3
115.0
123.3
145.0
115.3
167.2
130.8
108.6
146.8
145.6
114.3
119.0
71.9
2
3
4
5
6
7
8
9
1′
2′
3′
4′
5′
6′
7′
Figure 2. Key NOE correlations (↔) of 1.
deduced from HRESIMS. The NMR data of 2 were almost
identical to those of 1, except for a slight discrepancy in the
positions of the oxygenated methines (C-7′ and C-8′). Detailed
2D NMR analysis (Figures S18−20, Supporting Information)
revealed that 2 shares the same planar structure as that of 1,
indicating 2 to be a stereoisomer of 1 with the configuration
changed at C-7′ or C-8′. A threo-configuration of 2 was further
determined by the coupling constants between H-7′ and H-8′
(6.3 Hz).20,23 Compound 2 also has negligible optical activity
and an ECD spectrum devoid of Cotton effects. Chiral isolation
of 2 afforded the enantiomers 2a and 2b. By comparison of the
calculated ECD spectra of 7′S,8′S-2 and 7′R,8′R-2 with the
experimental data of 2a and 2b (Figure 3b), the absolute
configurations of 2a and 2b were assigned as 7′S,8′S and
7′R,8′R. Compounds 2a and 2b were given the trivial names
(−)-(7′S,8′S)-threo-7′-methylcarolignan E and (+)-(7′R,8′R)-
threo-7′-methylcarolignan E, respectively.
8′
9′
1″
2″
3″
4″
5″
6″
7″
8″
9″
1‴
2‴
3‴
4‴
5‴
6‴
7‴
8‴
9‴
OCH3-3
OCH3-3′
OCH3-3″
OCH3-3‴
OCH3-5″
OCH3-7′
82.3
82.6
86.6
84.7
83.7
63.9
63.8
63.2
62.8
62.7
136.0
112.6
150.9
146.7
118.9
121.0
32.1
136.2
112.6
151.0
146.8
119.2
121.2
32.0
137.6
112.5
151.0
146.3
120.6
121.2
32.2
137.6
112.6
151.6
145.3
119.5
121.3
32.2
130.9
103.9
153.7
134.1
153.7
103.9
134.0
123.8
65.0
30.6
30.6
30.62
63.8
30.6
63.9
63.9
63.9
127.1
109.5
146.9
148.1
114.9
123.4
145.1
115.2
167.5
56.2
127.1
109.5
146.9
148.1
114.9
123.5
145.2
115.6
167.6
56.2
127.1
109. 6
147.0
148.2
114.9
123.4
145.2
114.9
167.5
56.2
127.1
109.6
147.0
148.2
115.0
123.4
145.2
115.2
167.5
56.2
127.2
109.4
147.0
148.1
114.9
123.3
145.0
115.6
167.2
56.2
Compound 3 (3a/3b), obtained as a white, amorphous solid,
gave a molecular formula C40H42O13 determined from the
HRESIMS ion at m/z 729.2542 [M − H]¯(calcd C40H41O13).
Its NMR data (Tables 1 and 2) were almost identical to those
of threo-carolignan E.9,10 Compound 3 showed a negligible
specific rotation and CD spectrum, indicating it to be a racemic
mixture. Subsequent chiral separation of 3 afforded a pair of
enantiomer 3a and 3b, which had opposite ECD curves (Figure
3c) and optical rotations (3a: [α]20D +30.4; 3b: [α]20D −29.8).
The absolute configurations of 3a and 3b were determined
using the same methods as described in 1a and 1b. Thus, 3a
was defined as (+)-(7′R,8′R)-threo-carolignan E, and 3b was
identified as the known compound (−)-(7′S,8′S)-threo-
carolignan E.10
Compound 4 (4a/4b), a white, amorphous solid, showed the
same molecular formula as that of 3. The two compounds had
similar NMR data, except for the position of the C-7′ and C-8′
oxygenated methines. Detailed 2D NMR analysis confirmed
that 4 and 3 shared the same planar structure, indicating that 4
is a stereoisomer of 3 with a different orientation of the
substituents at C-7′ or C-8′. The coupling constant J7′,8′ was 3.3
Hz, so therefore the relative configuration at C-7′ and C-8′ was
erythro. Compound 4 also showed a negligible optical activity
and an ECD spectrum devoid of Cotton effects. Chiral isolation
of 4 afforded the enantiomers 4a and 4b. By comparison of the
calculated ECD spectra of the 7′R,8′S and 7′S,8′R config-
urations of 4 with the experimental data of 4a and 4b (Figure
56.2
56.1
56.1
56.2
56.2
56.0
55.9
56.0
56.1
56.4
56.2
56.2
56.2
56.2
56.2
56.4
57.4
57.4
a
1a/1b and 2a/2b showed the same 13C NMR data.
1b, ECD calculations were performed for the two config-
urations (7′R,8′S)- and (7′S,8′R)-1 using the Gaussian 09
program at the TD-DFT-B3LYP/6-311++G(d,p) level in
MeCN. The calculation for the 7′R,8′S enantiomer agreed
with the experimental ECD data (Figure 3a) of 1a. Thus, 1a has
a 7′R,8′S-configuration. The calculated ECD spectrum for the
7′S,8′R-configuration was in good accordance with the
experimental spectrum of 1b (Figure 3a). Consequently, the
absolute configuration of 1b was unambiguously assigned with
the 7′S,8′R-configuration. Thus, compounds 1a and 1b were
given the trivial names (−)-(7′R,8′S)-erythro-7′-methylcaroli-
gnan E and (+)-(7′S,8′R)-erythro-7′-methylcarolignan E,
respectively.
Compound 2 (2a/2b) was isolated as a white, amorphous
solid, having the same molecular formula as that of 1 as
C
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Figure 3. (a) Experimental ECD spectra of (−)- and (+)-erythro-7′-methylcarolignan E (1a/1b) in MeCN and calculated ECD spectra of (S′,R′)-1
and (R′,S′)-1. (b) Experimental ECD spectra of (−)- and (+)-threo-7′-methylcarolignan E (2a/2b) in MeCN and calculated ECD spectra of (R′,R′)-
2 and (S′,S′)-2. (c) Experimental ECD spectra of (+)- and (−)-threo-carolignan E (3a/3b) in MeCN and calculated ECD spectra of (R′,R′)-3 and
(S′,S′)-3. (d) Experimental ECD spectra of (+)- and (−)-erythro-carolignan E (4a/4b) in MeCN and calculated ECD spectra of (S′,R′)-4 and
(R′,S′)-4. (e) Experimental ECD spectra of (−)-erythro-carolignan Z (5) in MeCN and calculated ECD spectra of (R′,S′)-5 and (S′,R′)-5. The
calculated ECD spectra were computed at the B3LYP/6-311++G(d,p) level.
3d), the absolute configurations of 4a and 4b were assigned as
7′S,8′R and 7′R,8′S, respectively. Thus, 4a was identified as
(+)-(7′S,8′R)-erythro-carolignan E, and 4b was elucidated as
the known compound (−)-(7′R,8′S)-erythro-carolignan E.10
Compound 5, a white, amorphous solid, gave the molecular
formula C41H42O14 as determined by the HRESIMS ion at m/z
757.24921 [M − H]¯(calcd 757.25018). The NMR data of 5
were similar to those of 1 with an additional methoxy group in
the B-ring and two olefinic carbons (δC 134.0, 123.8) rather
Figure 4. Key 1H−1H COSY () and HMBC (→) correlations of 5.
than two methylene signals. The B-ring proton signals at δH
6.60 (2H, d, J = 1.8 Hz) implied a symmetrical 1,3,4,5-
tetrasubstituted aromatic ring, consistent with the location of
the additional methoxy group in 5 at C-5″. This assignment was
confirmed by the HMBC correlations (Figure 4) of the
methoxy protons (δH 3.90) with C-6″ (δC 103.9). The location
of the double bond was determined by HMBC correlation from
the olefinic protons (δH 6.30, H-8″) with C-1″ (δC 130.9). The
overall structure was further established by analysis of its 2D
NMR spectra (Figures S46−49, Supporting Information). An
D
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submitted to separation over a Sephadex LH-20 column eluting with
CH2Cl2−MeOH (1:1) to give four subfractions (B1c1−B1c4).
Fraction B1c2 was further purified by semipreparative HPLC eluting
with MeOH−H2O (60:40) to produce compounds 3 (10 mg) and 4
(6.2 mg). Fractions B1d and B1e were combined and further subjected
to MPLC using CH2Cl2−EtOAc (50:1) and further purified with
semipreparative HPLC eluting with MeCN−H2O (55:45) to give
compounds 1 (8.2 mg) and 2 (15.8 mg). Compounds 5 (10.2 mg), 6
(1.0 mg), and 7 (0.8 mg) were isolated from fraction B1f by
semipreparative HPLC with MeCN−H2O (50:50). Fraction C was
chromatographed over a silica gel-containing column eluting with
CH2Cl2−MeOH (200:1, 20:1) to obtain compound 9 (4 mg).
Compounds 1−4 were further separated by semipreparative chiral
HPLC (CH3OH−H2O, 9:1, 3 mL/min) to give 1a (3.5 mg, tR 30.4
min), 1b (3.4 mg, tR 33.2 min), 2a (7.3 mg, tR 31.2 min), 2b (5.7 mg,
tR 34.6 min), 3a (2.9 mg, tR 28.2 min), 3b (5.5 mg, tR 25.7 min), 4a
(2.1 mg, tR, 29.4 min), and 4b (2.5 mg, tR, 26.8 min), respectively.
(−)-(7′R,8′S)-erythro-7′-Methylcarolignan E (1a): white, amor-
erythro-configuration of 5 was further determined by the
coupling constants between H-7′ and H-8′ (3.1 Hz).20−22 The
experimental ECD spectrum of 5 showed an ECD curve with
Cotton effects around 324 (−), 290 (−), and 242 (+) nm
(Figure 3e). The absolute configuration of 5 was assigned as
R,S through comparison with the calculated ECD spectra of
(7′R,8′S)-5 (Figure 3e). Thus, 5 was elucidated as (−)-7′R,8′S-
erythro-carolignan Z.
Seven compounds (1a, 1b, 2a, 2b, 5, 8, and 9) were tested
for in vitro inhibitory effects against HIV-1 replication in MT4
cell lines, with AZT used as the positive control. One pair of
enantiomers, 1a and 1b, showed more potent anti-HIV activity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 341 automatic polarimeter. UV spectra
were recorded using a Shimadzu UV-2450 spectrophotometer. CD
spectra were obtained on an Applied Photophysics Chirascan
spectrometer. IR spectra were determined on a Bruker Tensor 37
infrared spectrophotometer. The 1H (400 MHz), 13C (100 MHz), and
2D NMR spectra were obtained on a Bruker AM-400 with
tetramethylsilane as an internal reference at 25 °C. Chemical shifts
(δ) are expressed in ppm with reference to the solvent signals.
HRESIMS were acquired on a Shimadzu LCMS-IT-TOF instrument,
and the ESIMS data were measured on an Agilent 1200 series LC-MS/
MS system. Macroporous resin D101 (Sinopharm Chemical Reagent
Co. Ltd., Shanghai, People’s Republic of China), RP-C18 silica gel
(Fuji, 40−75 μm), MCI gel CHP20P (75−150 μm, Mitsubishi
Chemical Corporation, Tokyo, Japan), silica gel (200−300 mesh,
Marine Chemical Ltd., Qingdao, People’s Republic of China), and
Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Sweden) were used
for column chromatography. Analytical and semipreparative HPLC
separation were carried out on an LC-20AT Shimadzu liquid
chromatography system with a Zorbax SB-C18 column (250 × 9.4
mm, 5 μm) or an Agilent SB-C18 column connected with an SPD-
M20A diode array detector. Semipreparative chiral HPLC separation
was carried out on an LC-20AT Shimadzu liquid chromatography
system with a Phenomenex Lux cellulose-2 chiral-phase column (250
× 10 mm, 5 μm). Thin-layer chromatography (TLC) analysis was
carried out on silica gel plates (Marine Chemical Ltd.). Fractions were
monitored by TLC and visualized by heating plates sprayed with 5%
H2SO4 in EtOH. All solvents were of analytical grade (Guangzhou
Chemical Reagents Company Ltd., Guangzhou, People’s Republic of
China).
phous solid; [α]20 −16.2 (c 0.1, CHCl3); UV (MeOH) λmax (log ε)
D
231 (4.58), 288 (3.87), 327 (4.68) nm; ECD (MeCN) λmax (Δε) 232
(1.03), 211 (−3.72) nm; IR νmax 3407, 2962, 2930, 2853, 1704, 1633,
1599, 1514, 1460, 1428, 1262, 1096, 1027, 803 cm−1; 1H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Tables 1 and 2; HRESIMS m/z 743.27039 [M − H]¯ (calcd for
C41H43O13, 743.27091).
(+)-(7′S,8′R)-erythro-7′-Methylcarolignan E (1b): white, amor-
phous solid; [α]20 +17.1 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε)
D
232 (−1.03), 211 (3.80) nm; UV, IR, NMR, and HRESIMS were the
same as those of 1a.
(−)-(7′S,8′S)-threo-7′-Methylcarolignan E (2a): white, amorphous
solid; [α]20 −35.1 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 232
D
(4.36), 288 (2.92), 326 (4.64) nm; ECD (MeCN) λmax (Δε) 325
(−1.78), 295 (−1.80), 240 (−1.14) nm; IR νmax 3402, 2961, 2919,
2850, 1704, 1633, 1597, 1514, 1464, 1428, 1263, 1097, 1028, 803
cm−1; 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100
MHz) data, see Tables 1 and 2; HRESIMS m/z 743.26971 [M − H]¯
(calcd for C41H43O13, 743.27091).
(+)-(7′R,8′R)-threo-7′-Methylcarolignan E (2b): white, amorphous
solid; [α]20 +32.6 (c 0.2, CHCl3); ECD (MeCN) λmax (Δε) 325
D
(1.73), 294 (1.76), 240 (1.17) nm; UV, IR, NMR, and HRESIMS were
the same as those of 2a.
(+)-(7′R,8′R)-threo-Carolignan E (3a): white, amorphous solid;
[α]20 +30.4 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 333 (+1.08),
D
298 (+1.34), 240 (+1.58) nm; UV (MeOH) λmax (log ε) 230 (4.51),
284 (2.75), 320 (4.83) nm; IR νmax 3413, 2940, 2913, 2852, 1704,
1
1631, 1597, 1513, 1461, 1430, 1172, 1097, 1031, 801 cm−1; H NMR
(CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
Plant Material. The aerial parts of Euphorbia sikkimensis were
collected at Longli County of the Qiannan Buyi National Minority
Miao National Minority Autonomous Region, Guizhou Province,
People’s Republic of China, in July 2014. The sample was identified by
Dr. Qingwen Sun from Guiyang College of Traditional Chinese
Medicine, and a voucher specimen (GZQSY356) has been deposited
at the School of Pharmaceutical Science, Sun Yat-sen University.
Extraction and Isolation. The air-dried and powdered aerial parts
(20 kg) of E. sikkimensis were extracted with 95% EtOH (3 × 40 L) at
room temperature for 48 h. The solvent was concentrated under
reduced pressure to give a crude extract (2.562 kg). The 95% EtOH
extract was then suspended in H2O (2 L) and successively partitioned
with petroleum ether (3 × 8 L), EtOAc (3 × 8 L), and n-BuOH (3 × 8
L) to yield three corresponding portions. The EtOAc-soluble extract
(334 g) was chromatographed on D101 macroporous resin eluting
with a step gradient of EtOH−H2O (2:8, 5:5, 7:3, 10:0) to afford four
fractions (A−D). Fraction A was separated using MCI gel CHP20P
eluting with an increasing gradient of MeOH−H2O from 30% to 100%
and then further purified by silica gel columns to give compounds 8 (4
mg), 10 (6 mg), 11 (20 mg), 12 (2.0 g), and 13 (5.2 g). Fraction B
was subjected to an RP-18 column with MeOH−H2O (30−100%) as
eluent to afford four subfractions (B1−B4). Fraction B1 (16.4 g) was
chromatographed on a silica gel column using CH2Cl2−MeOH (1:0,
100:1, 15:1) to yield six subfractions (B1a−B1f). Fraction B1c was
¯
Tables 1 and 2; HRESIMS m/z 729.2542 [M − H] (calcd for
C40H41O13, 729.2553).
(−)-(7′S,8′S)-threo-Carolignan E (3b): white, amorphous solid;
[α]20 −29.8 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 334 (−1.08),
D
298 (−1.32), 240 (−1.54) nm; UV, IR, NMR, and HRESIMS were the
same as those of 3a.
(+)-(7′S,8′R)-erythro-Carolignan E (4a): white, amorphous solid;
[α]20 +16.6 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 234 (+3.18),
D
222 (−1.22) nm; UV (MeOH) λmax (log ε) 232 (4.36), 288 (2.92),
326 (4.64) nm; IR λmax 3512, 2941, 2917, 2850, 1700, 1631, 1592,
1513, 1464, 1430, 1260, 1090, 1030, 802 cm−1; 1H NMR (CDCl3, 400
MHz) and 13C NMR (CDCl3, 100 MHz) data, see Tables 1 and 2;
HRESIMS m/z 729.2542 [M − H]¯ (calcd for C40H41O13, 729.2553).
(−)-(7′R,8′S)-erythro-Carolignan E (4b): white, amorphous solid;
[α]20 −16.4 (c 0.1, CHCl3); ECD (MeCN) λmax (Δε) 234 (−3.20),
D
222 (+1.21) nm; UV, IR, NMR, and HRESIMS were the same as
those of 4a.
(−)-(7′R,8′S)-erythro-Carolignan Z (5): white, amorphous solid;
[α]23D −34.1 (c 0.24, CHCl3); ECD (MeCN) λmax (Δε) 324 (−2.54),
290 (−2.78), 242 (+1.28) nm; UV (MeOH) λmax (log ε) 219 (6.76),
286 (3.40), 326 (4.31) nm; IR λmax 3427, 2963, 2930, 2853, 1704,
1
1632, 1588, 1514, 1463, 1427, 1262, 1152, 1097, 1022, 801 cm−1; H
NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data, see
E
DOI: 10.1021/acs.jnatprod.5b01012
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Tables 1 and 2; HRESIMS m/z 757.24921 [M − H]¯ (calcd for
C41H41O14, 757.25018).
DEDICATION
■
Dedicated to Professors John Blunt and Murray Munro, of the
University of Canterbury, for their pioneering work on
bioactive marine natural products.
Multicycle Viral Replication in MT4 Cell Assay. The anti-HIV-1
assay was performed as previously described.24 HIV-1 NL4-3 Nanoluc-
sec at an infecting dose of 50 TCID50/well was used to infect MT4
cells (1 × 105 cells/mL) in the presence of compounds at various
concentrations in 96-well plates. On day 3 postinfection, supernatant
samples were harvested and assayed for luciferase activity using the
Promega Nano-Glo luciferase assay system. The antiviral potency is
defined as the drug concentration that reduces the luciferase activity by
50% (EC50).
Cytotoxicity Assay. A CytoTox-Glo cytotoxicity assay (Promega)
was used to determine the cytotoxicity of the isolates. MT4 cells were
cultured in the presence of various concentrations of the compounds
for 3 days. Cytotoxicity of the compounds was determined by
following the protocol provided by the manufacturer. The 50%
cytotoxic concentration (CC50) was defined as the concentration that
caused a 50% reduction of cell viability.
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Table 3. Anti-HIV Data of the Isolates from E. sikkimensis
number of
tests
SI
b
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EC50 (μM)
CC50 (μM) (CC50/EC50)
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ASSOCIATED CONTENT
* Supporting Information
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UV, IR, MS, and 1D and 2D NMR for compounds 1a,
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AUTHOR INFORMATION
Corresponding Authors
*Tel: 919-962-0066. Fax: 919-966-3893. E-mail: khlee@unc.
edu (K.-H. Lee).
■
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cn (Q. Gu).
Notes
(24) Dang, Z.; Zhu, L.; Lai, W.; Bogerd, H.; Lee, K. H.; Huang, L.;
Chen, C. H. ACS Med. Chem. Lett. 2016, in press.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported in part by the National High-Tech
R&D Program of China (863 Program) (2012AA020307), the
Guangdong Innovative Research Team Program (No.
2009010058), and the National Natural Science Foundation
of China (No. 81173470). Partial support was also received
from the National Institute of Allergy and Infectious Diseases
(NIAID) Grant AI33066 (K.H.L.).
F
DOI: 10.1021/acs.jnatprod.5b01012
J. Nat. Prod. XXXX, XXX, XXX−XXX