Polyacetylenes from Bupleurum longiradiatum

Article abstract of DOI:10.1021/np900534v

Eight new polyacetylenes (1-8) and six known polyacetylenes were isolated from the entire parts of Bupleurum longiradiatum, a poisonous plant. The structures of the new compounds were determined by spectroscopic data interpretation. The absolute configura

Full text of DOI:10.1021/np900534v

J. Nat. Prod. 2009, 72, 2153–2157
2153
Polyacetylenes from Bupleurum longiradiatum
Hai-Qiang Huang,† Xi Zhang,† Yun-Heng Shen,† Juan Su,† Xiao-Hua Liu,† Jun-Min Tian,† Sheng Lin,† Lei Shan,*,† and
Wei-Dong Zhang*,†,‡
Department of Natural Medicinal Chemistry, School of Pharmacy, Second Military Medical UniVersity, Shanghai 200433, People’s Republic of
China, and School of Pharmacy, Shanghai Jiao Tong UniVersity, Shanghai 200240, People’s Republic of China
ReceiVed September 2, 2009
Eight new polyacetylenes (1-8) and six known polyacetylenes were isolated from the entire parts of Bupleurum
longiradiatum, a poisonous plant. The structures of the new compounds were determined by spectroscopic data
interpretation. The absolute configuration of the known compound bupleurotoxin (9) was established by the modified
Mosher’s method. All isolates were also tested for their cytotoxicity against a human leukemia cell line (HL-60).
The genus Bupleurum (Apiaceae) includes about 200 species
widely distributed in Eurasia and North Africa.1 Certain species of
this genus, such as Bupleurum chinense, B. scorzonerifolium, and
B. falcatum, have been used as antiphlogistic, antipyretic, and
analgesic agents in traditional folk medicine preparations.2 However,
B. longiradiatum, found in northeastern mainland China, is a
poisonous species within the Bupleurum genus. This species bears
a general resemblance to other Bupleurum plants, but is not
permitted to be used as a herbal medicine due to its toxic
properties.3,4 Previous studies showed the ethyl ether extract of B.
longiradiatum had strong toxicity against mice, and this toxicity
was attributed to its high content of polyacetylenes.4,5 However,
the chemical profile of polyacetylenes in B. longiradiatum has not
been fully studied yet. Only four polyacetylenes were reported from
this plant so far.4 Moreover, the absolute configurations of the
known bupleurotoxin (9) and acetylbupleurotoxin (10) at the
stereogenic carbon at C-14 are not yet established. As part of our
interest on Bupleurum species,6,7 we undertook an investigation of
a dichloromethane extract of B. longiradiatum that led to the
isolation of eight new (1-8) and six known (9-14) polyacetylenes.
We report herein the isolation, structure elucidation, and cytotoxicity
of the isolated compounds, along with the determination of the
absolute configuration of bupleurotoxin (9). Compounds 1-5 are
the first polyacetylenes containing only a single acetylenic bond
isolated from the Bupleurum genus.
(H-8/H-9, J ) 15.6 Hz); δH 6.11, 5.80 (H-10/H-11, J ) 15.0 Hz)],
a singlet methyl, and a triplet methyl. The 13C NMR and DEPT
spectra of 1 (Table 1) gave 19 carbon resonances due to two
methyls, six methylenes, eight methines, and three quaternary
carbons, including a triple bond [δC 90.6 (C-6) and 93.0 (C-7)]
and an acetyl (δC 20.9, 170.7). The above data, along with the
HMBC correlations from H-4 (δH 6.53) to C-2 (δC 128.6) and C-6
(δC 90.6) and from H-9 (δH 6.58) to C-7 (δC 93.0) and C-11 (δC
138.7) indicated that 1 is a C17 ester containing a diene-yne-diene
moiety.12,13 In the H-1H COSY spectrum, the correlation from
1
an oxymethylene proton (δH 4.62, H-1) to an olefinic proton (δH
5.83, H-2) was observed, indicating that the oxymethylene was
linked to the double bond. In addition, the HMBC correlation from
H-1 at δH 4.62 to the ester carbonyl at δC 170.7 suggested that the
acetoxy group is connected to the C-1 position. Therefore,
compound 1 was determined as (2E,4E,8E,10E)-heptadecatetraen-
6-yn-1-yl acetate.
Compound 2 gave a molecular formula of C19H28O2, as evidenced
by the HREIMS at m/z 288.2089 [M]+, showing one less unsat-
uration degree than 1. The UV spectrum displayed absorption
maxima at 267 and 280 nm, indicating the presence of a diene-yne
system.12 The H and 13C NMR spectra (Table 2) revealed the
1
presence of a cis-disubstituted double bond [δH 5.43, 5.49 (H-9/
H-10, J ) 10.6 Hz)], two trans-disubstituted double bonds [δH 5.80,
6.29 (H-2/H-3, J ) 15.2 Hz); δH 6.50, 5.64 (H-4/H-5, J ) 15.4
Hz)], an acetylenic unit [δC79.1 (C-6) and 92.1 (C-7)], a deshielded
methylene [δH 3.09, H-8], an oxymethylene (δH 4.62, H-1), and an
acetyl (δC 20.9 and 170.7). The NMR data of 2 were quite similar
to those of 1, with the major difference being the presence of a
deshielded downfield methylene in 2. The 1H-1H COSY correlation
from this deshielded methylene proton (δH 3.09, H-8) to an olefinic
proton (δH 5.43, H-9), along with the HMBC correlations of H-8
(δH 3.09) with C-6 (δC 79.1) and C-10 (δC 132.2) and of H-9 (δH
5.43) with C-7 (δC 92.1), clearly established an yne-CH2-ene moiety
in 2. Therefore, the structure of 2 was established as (2E,4E,9Z)-
heptadecatrien-6-yn-1-yl acetate.
Results and Discussion
The dichloromethane extract of the whole plant of B. longira-
diatum was subjected to silica gel, RP-18, and Sephadex LH-20
column chromatographic purification, as well as repeated preparative
thin-layer chromatography (TLC) to yield eight new (1-8) and six
known polyacetylenes, namely, bupleurotoxin (9),4 acetylbupleu-
rotoxin (10),4 bupleuronol (11),4 bupleurynol (12),4,8 (2Z,9Z)-
heptadecadiene-4,6-diyn-1-ol (13),9,10 and (2Z,9Z)-pentadecadiene-
4,6-diyn-1-ol (14).11
Compound 1 exhibited a molecular ion peak [M]+ at m/z
286.1937 in the HREIMS, corresponding to the molecular formula
C19H26O2. The IR spectrum showed ester carbonyl (1741 cm-1),
Compound 3 was assigned the molecular formula C22H32O4, due
to the molecular ion peak at m/z 360.2309 in the HREIMS. Its UV,
IR, and 1H and 13C NMR spectroscopic data (Table 2) were similar
to those of 2. The main difference between the two compounds
was the absence of a triplet methyl and the presence of two
additional methylenes (including one oxymethylene), along with
an additional acetoxy group signal [δH 2.05 (3H, s); δC 171.2 (C),
21.0 (CH3)] in 3. These data and their mass spectra strongly
suggested that 3 is a C18 ester. The additional acetoxy was placed
at C-18 according to the HMBC correlation from H-18 (δH 4.05)
to the ester carbonyl (δC 171.2). Thus, compound 3 was defined
structurally as (2E,4E,9Z)-octadecatrien-6-yne-1,18-diyl diacetate.
triple-bond (2177 cm-1), and olefinic double-bond (1639 cm-1
)
absorptions. The UV spectrum showed absorption maxima at 316
and 338 nm, resembling that of a diene-yne-diene polyacetylene.12
1
The H NMR data of 1 (Table 1) indicated the presence of four
pairs of trans-disubstituted double bonds [δH 5.83, 6.33 (H-2/H-3,
J ) 15.2 Hz); δH 6.53, 5.79 (H-4/H-5, J ) 15.7 Hz); δH 5.63, 6.58
* To whom correspondence should be addressed. Tel: +86-21-81871244.
Fax: +86-21-81871244. E-mail: mailto:wdzhangY@hotmail.com.
† Second Military Medical University.
‡ Shanghai Jiao Tong University.
10.1021/np900534v  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/08/2009

2154 Journal of Natural Products, 2009, Vol. 72, No. 12
Huang et al.
Chart 1
Table 1. 1H (600 MHz) and 13C (150 MHz) NMR Data for Compounds 1, 6, 7, and 13 in CDCl3 (δ in ppm, J in Hz in parentheses)
1
6
7
13
position
δH
δC
δH
4.44 dd (6.4, 1.5)
δC
δH
4.41 dd (6.4, 1.5)
δC
δH
4.62 dd (6.4, 1.5)
δC
1
4.62 br d (6.5)
64.4 CH2
61.1 CH2
61.1 CH2
61.1 CH2
2
5.83 (overlapped)
128.6 CH 6.23 dt (11.0, 6.4)
144.9 CH 6.21 dt (11.0, 6.4) 145.0 CH 6.21 dt (11.0, 6.4) 145.0 CH
3
4
5
6
6.33 dd (15.2, 11.0) 133.3 CH 5.69 dd (11.0, 1.5)
109.6 CH 5.60 d (11.0)
77.6 C
79.9 C
75.1 C
109.5 CH 5.60 d (11.0)
109.5 CH
70.9 C
80.0 C
64.5 C
84.0 C
6.53 (overlapped)
5.79 dd (15.7, 2.1)
139.5 CH
113.0 CH
70.9 C
80.0 C
64.5 C
84.0 C
90.6 C
93.0 C
7
83.0 C
8
9
5.63 dd (15.6, 2.2)
6.58 (overlapped)
108.6 CH 5.56 d (15.4)
107.0 CH 3.09 br d (6.8)
18.0 CH2
3.09 br d (6.8)
18.0 CH2
142.4 CH 6.71 dd (15.4, 11.0) 145.7 CH 5.41 dt (10.5, 7.3) 122.0 CH 5.41 dt (10.5, 7.2) 122.0 CH
10
11
12
13
14
15
16
17
18
OAc-1
6.11 dd (15.0, 10.8) 129.8 CH 6.12 dd (15.2, 11.0) 129.4 CH 5.55 dt (10.5, 7.3) 133.1 CH 5.55 dt (10.5, 7.2) 133.1 CH
5.80 (overlapped)
2.11 q (7.2)
1.30 m
1.40 m
1.30 m
138.7 CH 5.90 dt (15.2, 7.2)
140.5 CH 2.05 q (7.3)
27.1 CH2
29.4 CH2
29.4 CH2
29.1 CH2
29.0 CH2
25.7 CH2
32.8 CH2
63.1 CH2
2.05 q (7.2)
1.30 m
1.30 m
1.30 m
1.30 m
27.2 CH2
29.2 CH2
29.2 CH2
29.1 CH2
31.8 CH2
22.6 CH2
14.1 CH3
32.8 CH2
29.0 CH2
28.8 CH2
31.7 CH2
22.6 CH2
14.0 CH3
2.14 q (7.2)
1.30 m
1.30 m
32.5 CH2
31.0 CH2
22.2 CH2
13.9 CH3
1.30 m
1.30 m
1.30 m
1.30 m
0.90 t (7.3)
1.30 m
0.89 t (7.0)
1.30 m
1.57 quint (7.0)
3.64 t (6.7)
1.30 m
0.89 t (7.2)
170.7 C
2.08 s
20.9 CH3
Compound 4 was found to possess a molecular formula of
C20H30O3, as determined by the molecular ion peak at m/z 318.2192
[M]+ in the HREIMS, revealing the compound to be 42 amu less
than that of 3. The 1H and 13C NMR spectroscopic data of 4
indicated its structural similarity to 3, except for the absence of a
1-O-acetyl group in 4 (Table 2). The significant upfield shift of the
doublet of H-1 from δH 4.60 in 3 to δH 4.21 in 4, together with the
loss of 42 amu in 4, suggested that the latter compound is a
deacetylated derivative of 3. The conclusion was supported further
by the HMBC correlation of H-1 (δH 4.21) with C-3 (δC 130.4)
Compound 6 gave a molecular formula of C15H18O on the basis
of the HREIMS (m/z 214.1354 [M]+), a loss of 28 amu when
compared with bupleurynol (12).4,8 The UV spectrum of 6, which
showed absorption maxima at 249, 264, 277, 294, 313, and 334
nm, was similar to that of 12, indicating the presence of a diene-
diyne-ene chromophore.4,8,12 Its IR and 1H and 13C NMR spectro-
scopic data were also similar to those of 12, except for the loss of
two methylenes in compound 6. On the basis of the NMR and mass
data, the structure of 6 was determined as (2Z,8E,10E)-pentadec-
atriene-4,6-diyn-1-ol.
1
and the H-1H COSY correlation from H-1 (δH 4.21) to H-2 (δH
Compound 7 was found to possess the molecular formula
C18H26O2, as inferred from the HREIMS (m/z 274.1920 [M]+). The
UV spectrum exhibited absorption maxima at 210, 238, 251, 264,
and 280 nm, which was typical for an ene-diyne system.12 In
comparison with the NMR data of 13, the absence of a methyl
group at C-17 (δH 0.89, δC 14.1) and the occurrence of an additional
methylene (δH 1.57, δC 32.8) and an oxymethylene (δH 3.64, δC
63.1) suggested that 7 is a C18 alcohol, and the C-18 position was
assigned as a hydroxylmethyl group. Thus, 7 was defined as
(2Z,9Z)-octadecadiene-4,6-diyne-1,18-diol.
5.88). Consequently, compound 4 was deduced to be (2E,4E,9Z)-
1-hydroxyoctadecatrien-6-yn-18-yl acetate.
The molecular formula of compound 5 was established as
C18H28O2 by the HREIMS (m/z 276.2094 [M]+), revealing the
1
compound to be 42 amu less than that of 4. Its H and 13C NMR
spectra (Table 2) were closely related to those of 3 and 4, but
contained no acetyl group signals. Thus, compound 5 was estab-
lished as a deacetylated derivative of 4, namely, (2E,4E,9Z)-
octadecatrien-6-yne-1,18-diol.

Polyacetylenes from Bupleurum longiradiatum
Journal of Natural Products, 2009, Vol. 72, No. 12 2155
Table 2. 1H (600 MHz) and 13C NMR (150 MHz) Data for Compounds 2-5 in CDCl3 (δ in ppm, J in Hz in parentheses)
2
3
4
5
position
δH
δC
δH
δC
δH
δC
δH
δC
1
2
4.62 br d (6.4)
5.80 dt (15.2, 6.4)
64.4 CH2
128.0 CH 5.79 dt (15.3, 6.2)
4.60 br d (6.2)
64.4 CH2
128.0 CH 5.88 dt (15.2, 5.7)
4.21 br d (5.7)
63.1 CH2
133.7 CH 5.88 dt (15.2, 5.7)
4.21 br d (5.7)
63.1 CH2
133.7 CH
3
4
6.29 dd (15.2, 11.0) 133.3 CH 6.29 dd (15.3, 11.0) 133.2 CH 6.28 dd (15.2, 11.0) 130.4 CH 6.28 dd (15.2, 11.0) 130.4 CH
6.50 dd (15.4, 11.0) 139.3 CH 6.50 dd (15.5, 11.0) 139.3 CH 6.52 dd (15.6, 11.0) 139.8 CH 6.52 dd (15.6, 11.0) 139.8 CH
5
6
7
5.64 d (15.4)
113.3 CH 5.63 d (15.5)
79.1 C
92.1 C
113.2 CH 5.60 d (15.6)
79.1 C
92.0 C
112.1 CH 5.60 d (15.6)
79.3 C
91.5 C
112.1 CH
79.3 C
91.5 C
8
9
3.09 br d (6.5)
5.43 dt (10.6, 7.2)
5.49 dt (10.6, 7.2)
2.05 q (7.2)
1.29 m
1.29 m
1.29 m
1.29 m
1.37 m
18.0 CH2
123.6 CH 5.43 dt (10.6, 7.1)
132.2 CH 5.48 dt (10.6, 7.1)
27.2 CH2
29.7 CH2
29.2 CH2
28.9 CH2
31.8 CH2
22.6 CH2
14.1 CH3
3.08 br d (6.6)
18.0 CH2
123.7 CH 5.43 dt (10.6, 7.2)
132.0 CH 5.49 dt (10.6, 7.2)
3.08 br d (6.8)
18.0 CH2
123.8 CH 5.43 dt (10.6, 7.2)
132.0 CH 5.49 dt (10.6, 7.2)
3.08 br d (6.7)
18.0 CH2
123.7 CH
132.1 CH
27.1 CH2
29.5 CH2
29.3 CH2
29.4 CH2
29.2 CH2
25.7 CH2
32.8 CH2
63.1 CH2
10
11
12
13
14
15
16
17
18
OAc-1
2.05 (overlapped)
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
4.05 t (6.8)
27.1 CH2
29.3 CH2
29.2 CH2
29.3 CH2
29.1 CH2
25.9 CH2
28.6 CH2
64.6 CH2
170.7 C
2.08 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
4.05 t (6.8)
27.1 CH2
29.3 CH2
29.1 CH2
29.3 CH2
29.1 CH2
25.8 CH2
28.6 CH2
64.7 CH2
2.05 q (7.2)
1.30 m
1.30 m
1.30 m
1.30 m
1.30 m
1.56 m
3.64 t (6.7)
0.89 t (7.0)
170.7 C
2.08 s
20.9 CH2
2.08 s
2.05 s
20.9 CH2
171.2 C
21.0 CH2
OAc-18
171.3 C
2.05 s
21.0 CH2
Table 3. 1H (600 MHz) and 13C NMR (150 MHz) Data for Compounds 8-11 in CDCl3 (δ in ppm, J in Hz in parentheses)
8
9
10
11
position
δH
δC
δH
4.40 dd (6.5, 1.5)
δC
δH
4.42 dd (6.5, 1.5)
δC
δH
4.41 dd (6.5, 1.5)
δC
1
2
3
4
4.84 dd (6.5, 1.5)
6.14 (overlapped)
5.76 d (11.0)
62.4 CH2
139.5 CH 6.22 (11.0, 6.5)
111.8 CH 5.66 d (11.0)
77.1 C
61.0 CH2
145.2 CH 6.23 dt (11.0, 6.5)
109.5 CH 5.66 d (11.0)
77.8 C
61.1 CH2
145.3 CH 6.22 dt (11.0, 6.5)
109.5 CH 5.66 d (11.0)
77.8 C
61.1 CH2
145.2 CH
109.4 CH
77.8 C
5
80.6 C
79.8 C
79.8 C
79.7 C
6
75.3 C
75.3 C
75.4 C
75.5 C
7
83.3 C
82.9 C
82.8 C
82.7 C
8
5.58 d (15.6)
107.5 CH 5.56 d (15.5)
107.5 CH 5.58 d (15.5)
107.7 CH 5.58 d (15.5)
108.0 CH
9
6.71 dd (15.6, 11.0) 145.5 CH 6.69 dd (15.5, 11.0) 145.4 CH 6.69 dd (15.5, 10.8) 145.1 CH 6.66 dd (15.5, 10.8) 145.0 CH
10
11
12a
12b
13a
13b
14
15
16
17
OAc-1
6.15 (overlapped)
5.91 dt (15.2, 7.1)
2.30 m
2.23 m
1.58 m
1.53 m
3.62 m
1.42 m
1.42 m
129.8 CH 6.14 (15.2, 11.0)
139.7 CH 5.90 dt (15.2, 7.1)
129.8 CH 6.12 dd (15.0, 10.8) 129.9 CH 6.12 dd (15.0, 10.8) 130.2 CH
139.6 CH 5.86 dt (15.0, 7.1)
138.9 CH 5.84 dt (15.0, 7.1)
137.9 CH
26.8 CH2
29.1 CH2
2.28 m
2.20 m
1.56 m
1.51 m
3.62 m
1.42 m
1.42 m
0.92 t (7.0)
29.1 CH2
2.15 m
28.8 CH2
2.50 m
36.0 CH2
36.0 CH2
1.65 m
33.3 CH2
2.50 m
41.5 CH2
71.0 CH
39.8 CH2
18.8 CH2
14.1 CH3
170.7 C
71.0 CH
39.8 CH2
18.8 CH2
14.1 CH3
4.89 m
1.50 m
1.30 m
0.90 t (7.0)
73.5 CH
36.3 CH2
18.5 CH2
13.9 CH3
210.0 C
2.38 m
1.60 m
0.90 t (7.0)
44.8 CH2
17.2 CH2
13.7 CH3
0.93 t (7.0)
2.09 s
20.8 CH3
OAc-14
170.9 C
2.03 s
21.2 CH3
Compound 9 was identified as bupleurotoxin on the basis of
unequivocal assignments of its 1D- and 2D-NMR spectroscopic
data and by comparison with the literature data (including UV, IR,
optical rotation).4 However, the absolute configuration at C-14 of
bupleurotoxin is still unknown. In order to complete the structure
characterization, the absolute configuration of this compound was
determined by application of the modified Mosher ester method.14
Treatment of 9 with (R)-MTPA chloride and (S)-MTPA chloride
afforded the (S)-diester (9a) and (R)-diester (9b), respectively. By
analysis of the ∆δH(S-R) values of the protons neighboring the
oxygenated methane according to the Mosher model (Figure 1),
the assignment of C-14 was the S configuration.
group was presumed to be placed at the C-1 position. This was
confirmed by the HMBC correlation from H-1 (δH 4.84) to the ester
carbonyl (δC 170.7). The absolute configuration of 8 was determined
to be the same as that of 9, because the two compounds displayed
the same optical sign. Therefore, the structure of 8 was elucidated
as (2Z,8E,10E)-14S-hydroxyheptadecatriene-4,6-diyn-1-yl acetate.
In a similar way, the other acetylated derivative of bupleurotoxin
(9) was identified as acetylbupleurotoxin (10) by comparing its
Compound 8 gave a molecular formula of C19H24O3, as shown
by HREIMS at m/z 300.1726 [M]+, 42 amu more than that of
1
bupleurotoxin (9). The H and 13C NMR spectroscopic data of 8
were very similar to those of 9, with differences being due to the
presence of an acetate group [δH 2.09 (3H, s); δC 170.7 (C), 20.8
(CH3)] in 8 (Table 3). Considering the significant downfield shift
of the H-1 doublet from δH 4.40 in 9 to δH 4.84 in 8, the acetate
Figure 1. ∆δ values (in ppm) ) δS - δR obtained for (S)- and
(R)-MTPA esters 9a and 9b.

2156 Journal of Natural Products, 2009, Vol. 72, No. 12
Huang et al.
physical and spectroscopic data with the literature values.4 The
(hexane-EtOAc, 10:1) to give 6 (8 mg, Rf ) 0.28) and 14 (12 mg, Rf
) 0.20). Subfraction FB.6 was purified on a silica gel column
(CHCl3-MeOH, 30:1) to afford 12 (46 mg) and 13 (5 mg). Subfraction
FB.9 was separated by silica gel chromatography, eluting with
hexane-EtOAc (8:1), and then by Sephadex LH-20 chromatography
eluting with CHCl3-MeOH (1:1), to give 5 (24 mg). Fraction C was
subjected to column chromatography on silica gel (hexane-EtOAc,
6:1) and Sephadex LH-20 (CHCl3-MeOH, 1:1) to give 4 (7 mg), 8
(13 mg), 10 (27 mg), and 11 (8 mg). Repeated column chromatography
of fraction D over silica gel (hexane-EtOAc, 2:1; CHCl3-MeOH, 15:
1) gave 7 (5 mg) and 9 (47 mg). Fraction E was chromatographed on
a silica gel column eluting with hexane-EtOAc (1:1), followed by a
Sephadex LH-20 column (MeOH) to give 5 (6 mg).
(2E,4E,8E,10E)-Heptadecatetraen-6-yn-1-yl acetate (1): colorless
oil; UV (MeOH) λmax (log ε) 338 (4.04), 316 (4.01) nm; IR (KBr) νmax
2929, 2858, 2177, 1741, 1639, 1456, 1367, 1234, 1047, 985 cm-1; 1H
and 13C NMR spectroscopic data, see Table 1; EIMS 70 eV m/z 286
[M]+ (15), 244 (8), 226 (10), 175 (10), 169 (19), 159 (38), 156 (34),
155 (100), 145 (34), 144 (31), 141 (27), 133 (29), 129 (36), 117 (36),
115 (94), 107 (27), 91 (64), 79 (24), 77 (11); HREIMS m/z 286.1937
[M]+ (calcd for C19H26O2, 286.1941).
opposite specific rotation values for 9 and 10 ([R]17 +16.3 for 9
D
versus -13.7 for 10) were attributed to the opposite absolute
configuration at C-14. Therefore, the 14R absolute configuration
was proposed for 10.
As far as we know, the 13C NMR spectroscopic data of the known
compounds 9-11 and 13 have not been reported before in the
literature.4,9,10 Herein, we report the complete H NMR and 13C
1
NMR data (Tables 1, 3) on the basis of analysis of their 2D-NMR
spectra (COSY, HMQC, and HMBC).
It must be noted that B. longiradiatum represents a rich source
of polyacetylenes. Natural products of this category have been found
only in B. longiradiatum,4 B. falcatum,11 B. acutifolium,8 B.
salicifolium,15 and B. spinosum13 of the Bupleurum genus, despite
the fact that this genus contains more than 200 species. Compounds
1-5 are the first polyacetylenes with only a single acetylenic bond
isolated from the genus Bupleurum. As major compounds of the
CH2Cl2 extract of B. longiradiatum, bupleurotoxin (9) and acetyl-
bupleurotoxin (10) are claimed to be responsible at least in part
for the toxicity of B. longiradiatum. However, the isolation of these
two compounds has so far been detected only in the title plant.
Thecloseststructuralvariantofbupleurotoxin(9)isoenanthotoxin,16,17
a plant toxin obtained from Oenanthe fistulosa, with the difference
being the configuration of a single double bond (C-2/C-3), with a
Z stereochemistry in bupleurotoxin and E in oenanthotoxin.
Therefore, these polyacetylenes might be viewed as chemotaxo-
nomic markers for B. longiradiatum.
Since some polyacetylenes are reported to possess cytotoxic
activity against cancer cell lines,18 all the isolates were tested for
their cytotoxicity against a human leukemia cell line (HL-60). Only
compounds 8 and 9 were found to be cytotoxic, with IC50 values
of 9.4 and 4.9 µM, respectively. Since all the remaining isolates
were inactive ((IC50 >10 µM)), a hydroxy group on the side chain
appears to enhance the cytotoxicity of these polyacetylenes.
Compounds 1, 2, 6, and 10-14 exhibited IC50 values in the range
11-18 µM.
(2E,4E,9Z)-Heptadecatrien-6-yn-1-yl acetate (2): colorless oil; UV
(MeOH) λmax (log ε) 280 (4.07), 267 (3.88) nm; IR (KBr) νmax 2925,
1
2854, 2212, 1743, 1630, 1457, 1378, 1232, 1047, 980 cm-1; H and
13C NMR spectroscopic data, see Table 2; EIMS 70 eV m/z 288 [M]+
(5), 246 (3), 157 (28), 144 (22), 143 (100), 129 (52), 128 (35), 117
(22), 91 (10), 79 (16), 77 (12); HREIMS m/z 288.2089 [M]+ (calcd
for C19H28O2, 288.2088).
(2E,4E,9Z)-Octadecatrien-6-yne-1,18-diyl diacetate (3): colorless
oil; UV (MeOH) λmax (log ε) 280 (4.05), 267 (3.91) nm; IR (KBr) νmax
2930, 2856, 2189, 1740, 1638, 1437, 1367, 1242, 1043, 980 cm-1; 1H
and 13C NMR spectroscopic data, see Table 2; EIMS 70 eV m/z 360
[M]+ (2), 318, (15), 300 (26), 276 (6), 157 (33), 155 (35), 143(100),
129 (84), 128 (52),117 (38), 115 (34), 91 (60); HREIMS m/z 360.2309
[M]+ (calcd for C22H32O4, 360.2317).
(2E,4E,9Z)-1-Hydroxyoctadecatrien-6-yn-18-yl acetate (4): color-
less oil; UV (MeOH) λmax (log ε) 280 (4.02), 267 (3.88) nm; IR (KBr)
νmax 3429, 2927, 2856, 2212, 1737, 1638, 1463, 1367, 1242, 1043,
980 cm-1; 1H and 13C NMR spectroscopic data, see Table 2; EIMS 70
eV m/z 318 [M]+ (3), 300 (9), 276 (10), 157 (6), 155 (30), 143(83),
129 (88), 128 (59), 115 (52), 91 (100), 79 (59), 77 (30); HREIMS m/z
318.2192 [M]+ (calcd for C20H30O3, 318.2189).
Experimental Section
(2E,4E,9Z)-Octadecatrien-6-yne-1,18-diol (5): colorless oil; UV
(MeOH) λmax (log ε) 280 (3.95), 267 (3.86) nm; IR (KBr) νmax 3289,
3148, 2919, 2852, 2206, 1641, 1467, 1415, 983 cm-1; 1H and 13C NMR
spectroscopic data, see Table 2; EIMS 70 eV m/z 276 [M]+ (11),
143(46), 129 (55), 128 (44), 117 (81), 115 (36), 91 (100), 79 (53), 77
(27); HREIMS m/z 276.2094 [M]+ (calcd for C18H28O2, 276.2098).
(2Z,8E,10E)-Pentadecatriene-4,6-diyn-1-ol (6): colorless oil; UV
(MeOH) λmax (log ε) 334 (3.90), 313 (4.02), 294 (3.91), 277 (3.50)
264 (3.88), 249 (3.92) nm; IR (KBr) νmax 3400, 2956, 2871, 2198, 1642,
General Experimental Procedures. Optical rotations were recorded
using a Perkin-Elmer 341 polarimeter. UV spectra were obtained by a
Shimadzu UV-2550 UV-vis spectrophotometer. IR spectra were
recorded on a Bruker Vector 22 spectrometer with KBr pellets. NMR
spectra were recorded on a Bruker Avance 600 NMR spectrometer in
CDCl3 with TMS as internal standard. EIMS and HREIMS were
acquired on a Thermo DSQ II and a Finnigan MAT 95 mass
spectrometer, respectively. Materials for column chromatography were
silica gel (100-200 mesh; Huiyou Silical Gel Development Co. Ltd.,
Yantai, People’s Republic of China), Sephadex LH-20 (40-70 µm;
Amersham Pharmacia Biotech AB, Uppsala, Sweden), and YMC-gel
ODS-A (50 µm; YMC, Milford, MA). Preparative TLC (0.4-0.5 mm)
was conducted with silica gel precoated glass plates GF254 (Yantai).
Compounds were visualized by exposure to UV light at 254 nm.
Plant Material. The whole plant of B. longiradiatum was collected
from Mao’ershan Town, Shangzhi City, Heilongjiang Province, People’s
Republic of China, in September 2008, and identified by Prof. Han-
ming Zhang, Department of Pharmacognosy, Second Military Medical
University. A voucher specimen (20081002) is kept in the Herbarium
of Second Military Medical University, Shanghai.
Extraction and Isolation. The air-dried and powdered sample of
B. longiradiatum (2.0 kg) was extracted in a Soxhlet apparatus
sequentially with CH2Cl2 (5 L) and ethanol (5 L). The CH2Cl2 extract
(60 g) was separated into five fractions (A-E) by column chromatog-
raphy on silica gel using gradient mixtures of hexane-EtOAc
(100-0%). Fraction A was further chromatographed on silica gel with
hexane-EtOAc (20:1) to give seven subfractions (FA.1-FA.7).
Fraction A.2 was purified by preparative TLC (hexane-EtOAc, 19:1),
yielding compounds 1 (22 mg, Rf ) 0.35) and 2 (4 mg, Rf ) 0.28).
Fraction B was chromatographed using reversed-phase MPLC in a
gradient system of H2O-MeOH (50%-100%) to give nine subfractions
(FB.1-FB.9). Subfraction FB.3 was further purified by preparative TLC
1
1463, 1380, 1182, 1080, 980 cm-1; H and 13C NMR spectroscopic
data, see Table 1; EIMS 70 eV m/z 214 [M]+ (81), 171 (14), 157 (22),
152 (17), 142 (13), 141 (41), 128 (100), 127 (40), 117 (18), 115 (74),
85 (36), 85 (36), 77 (38), 71 (56); HREIMS m/z 214.1354 [M]+ (calcd
for C15H18O, 214.1351).
(2Z,9Z)-Octadecadiene-4,6-diyne-1,18-diol (7): colorless oil; UV
(MeOH) λmax (log ε) 280 (3.74), 264 (3.94), 251 (3.87), 238 (3.53),
210 (4.03) nm; IR (KBr) νmax 3347, 3022, 2927, 2854, 2233, 1685,
1
1637, 1457, 1290, 1029, 732 cm-1; H and 13C NMR spectroscopic
data, see Table 1; EIMS 70 eV m/z 274 [M]+ (15), 173 (16), 159 (59),
145 (43), 141 (60), 131 (41), 129 (66), 128 (59), 117 (59), 115 (73),
106 (43), 91 (100), 77 (29), 68 (58), 55 (38); HREIMS m/z 274.1920
[M]+ (calcd for C18H26O2, 274.1908).
(2Z,8E,10E)-14S-Hydroxyheptadecatriene-4,6-diyn-1-yl acetate
(8): colorless oil; [R]17D +14.3 (c 0.04, MeOH); UV (MeOH) λmax (log
ε) 334 (3.92), 314 (4.05), 295 (3.91), 277 (3.67), 265 (3.90), 249 (3.96)
nm; IR (KBr) νmax 3427, 2931, 2871, 2202, 1740, 1636, 1630, 1440,
1371, 1232, 1022, 980 cm-1; 1H and 13C NMR spectroscopic data, see
Table 3; EIMS 70 eV m/z 300 [M]+ (5), 256 (20), 240 (32), 211 (12),
197 (19), 179 (30), 169 (35), 153 (83), 141 (71), 128 (72), 115 (100),
91 (30); HREIMS m/z 300.1726 [M]+ (calcd for C19H24O3, 300.17242).
MTPA Esters of Bupleurotoxin (9). Bupleurotoxin (9, 1.0 mg) was
dissolved in 0.5 mL of dry pyridine and treated with (R)-MTPA chloride

Polyacetylenes from Bupleurum longiradiatum
Journal of Natural Products, 2009, Vol. 72, No. 12 2157
(10 µL) and N,N-dimethylaminopyridine (DMAP, a spatula tip), then
maintained at room temperature under stirring overnight. After removal
of the solvent, the reaction mixture was purified by preparative TLC
(hexane-EtOAc, 5:1, Rf ) 0.35), affording the (S)-MTPA diester 9a
in a pure state. Using (S)-MTPA chloride, the same procedure afforded
the (R)-MTPA diester 9b in the same yield.
Supporting Information Available: 1D and 2D NMR spectra of
compounds 1-8. This material is available free of charge via the
Internet at http://pubs.acs.org.
References and Notes
1
Bupleurotoxin 1-O-14-O-(S)-MTPA diester (9a): H NMR (600
(1) Pan, S. L. Bupleurum Species-Scientific EValuation and Clinical
Application; Taylor and Trancis Group: Boca Raton, FL, 2006; pp
1-6.
(2) Wang, Y. S. Pharmacology and Applications of Chinese Materia
Medica; Chang, H. M., But, P. P. H., Eds.; World Scientific Publishing:
Singapore, 1987; pp 978-988.
(3) China Pharmacopoeia Committee. Pharmacopoeia of the People’s
Republic of China; Chemical Industry Press: Beijing, 2005; p 198.
(4) Zhao, J. F.; Guo, Y. Z.; Meng, X. S. Yaoxue Xuebao 1987, 22, 507–
511.
MHz, CDCl3) δ 7.46 and 7.34 (MTPA phenyl protons), 6.62 (H-9, dd,
J ) 15.5, 11.0 Hz), 6.07 (H-2, dt, J ) 11.0, 6.5 Hz), 6.03 (H-10, dd,
J ) 15.0, 10.8 Hz), 5.85 (H-11, J ) 15.0, 7.0 Hz), 5.76 (H-3, d, J )
11.0 Hz), 5.52 (H-8, d, J ) 15.5 Hz), 5.04 (H-14, m), 5.01 (H2-1, d, J
) 6.5 Hz), 3.49 (MTPA OCH3, s), 2.12 (H-12a, overlapped), 1.95 (H-
12b, overlapped), 1.72 (H-13a, m), 1.65 (H-13b, m), 1.48 (H2-15, m),
1.48 (H2-16, m), 0.81 (H3-17, d, J ) 7.0 Hz); EIMS m/z 690 [M]+.
1
Bupleurotoxin 1-O-14-O-(R)-MTPA diester (9b): H NMR (600
MHz, CDCl3) δ 7.46 and 7.34 (MTPA phenyl protons), 6.59 (H-9, dd,
J ) 15.5, 11.0 Hz), 6.07 (H-2, dt, J ) 11.0, 6.5 Hz), 5.95 (H-10, dd,
J ) 15.0, 10.8 Hz), 5.77 (H-3, d, J ) 11.0 Hz), 5.70 (H-11, J ) 15.0,
7.0 Hz), 5.49 (H-8, d, J ) 15.5 Hz), 5.04 (H-14, m), 5.01 (H2-1, d, J
) 6.5 Hz), 3.49 (MTPA OCH3, s), 1.97 (H-12a, overlapped), 1.94 (H-
12b, overlapped), 1.61 (H-13a, m), 1.57 (H-13b, m), 1.49 (H2-15, m),
1.49 (H2-16, m), 0.81 (H3-17, d, J ) 7.0 Hz); EIMS m/z 690 [M]+.
Cytotoxicity Assay. Growth inhibiton of all isolates (compounds
1-14) against HL-60 (human leukemia) cells was tested using an
established colorimetric MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide] assay protocol.19 Freshly trypsinized cell
suspensions were seeded in 96-well microtiter plates at densities of
4000-5000 cells per well and allowed to adhere for 24 h before drug
addition. Each cancer cell line was exposed to the tested compounds
with concentrations of 0.01, 0.1, 1, 10, and 100 µg/mL. After 3 days
in culture, attached cells were incubated with MTT (5 mg/mL) and
solubilized in DMSO 4 h later. The optical densities (OD) were read
on an enzyme-labeled detector (Denley MK-2) at a wavelength of 570
nm. The inhibitory rate of cell proliferation was calculated by the
following formula: Growth inhibition (%) ) (ODcontrol - ODtreated)/
ODcontrol × 100%. Doxorubicin was used as positive control and
exhibited an IC50 value of 0.09 µM.
(5) You, Y. J.; Lee, I. S.; Kim, Y.; Bae, K. H.; Ahn, B. Z. Arch. Pharm.
Res. 2002, 25, 600–640.
(6) Huang, H. Q.; Zhang, X.; Lin, M.; Shen, Y. H.; Yan, S. K.; Zhang,
W. D. J. Sep. Sci. 2008, 31, 3190–3201.
(7) Huang, H. Q.; Zhang, X.; Xu, Z. X.; Su, J.; Yan, S. K.; Zhang, W. D.
J. Pharm. Biomed. Anal. 2009, 49, 1048–1055.
(8) Barrero, A. F.; Herrador, M. M.; Akssira, M.; Arteaga, P.; Romera,
J. L. J. Nat. Prod. 1999, 62, 946–948.
(9) Schulte, K. E.; Potter, B. Arch. Pharm. (Weinheim) 1977, 310, 945–
963.
(10) Schulte, K. E.; Wulfhorst, G. Arch. Pharm. (Weinheim) 1977, 310,
285–298.
(11) Morita, M.; Nakajima, K.; Ikeya, Y.; Mitsuhashi, H. Phytochemistry
1991, 30, 1543–1545.
(12) Bohlmann, F.; Burkhardt, T.; Zdero, C. Naturally Occurring Acety-
lenes; London Academic Press: London, 1973; pp 3-26.
(13) Barrero, A. F.; Haidour, A.; Munoz-Dorado, M.; Akssira, M.; Sedqui,
A.; Mansour, I. Phytochemistry 1998, 48, 1237–1240.
(14) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(15) Estevez-Braun, A.; Estevez-Reyes, R.; Moujir, L. M.; Ravelo, A. G.;
Gonzalez, A. G. J. Nat. Prod. 1994, 57, 1178–1182.
(16) Anet, E. F. L. J.; Lythgoe, B.; Silk, M. H.; Trippet, S. J. Chem. Soc.
1953, 62, 309–322.
(17) Appendino, G.; Pollastro, F.; Verotta, L.; Ballero, M.; Romano, A.;
Wyrembek, P.; Szczuraszek, K.; Mozrzymas, J. W.; Taglialatela-
Scafati, O. J. Nat. Prod. 2009, 72, 962–965.
(18) Siddiq, A.; Dembitsky, V. Anticancer Agents Med. Chem. 2008, 8, 132–
170.
Acknowledgment. This research was supported by the Program for
the NCET Foundation, NSFC (30725045), National 863 Program
(2006AA02Z338), “973” Program of China (2007CB507400), Shanghai
Leading Academic Discipline Project (B906), China International
Science and Technology Cooperation Project (2007DFC31030), and
the Scientific Foundation of Shanghai China (07DZ19728, 06DZ19717,
06DZ19005).
(19) Mosmman, T. J. Immunol. Methods 1983, 65, 55–63.
NP900534V