Immunosuppressive sesquiterpene alkaloids from tripterygiunt ivilfordii

Article abstract of DOI:10.1021/np000504a

Nine new sesquiterpene pyridine alkaloids [wilfornines A (1), B (2), C (3), D (4), E (5), F (8), and G (9); wilfordinines I (6) and J (7)] and six known compounds (10-15) were isolated from a clinically used extract (Tji) of Tripterygium ivilfordii. The structures of 1-9 were elucidated by spectroscopic and chemical methods. The inhibitory effects on cytokine production of 1-3 and several related compounds were evaluated. Compounds 10 and 14 showed significant inhibitory effects on cytokine production.

Full text of DOI:10.1021/np000504a

582
J . Nat. Prod. 2001, 64, 582-587
Im m u n osu p p r essive Sesqu iter p en e Alk a loid s fr om Tr ipter ygiu m wilfor d ii
Hongquan Duan,† Yoshihisa Takaishi,*,† Hiroshi Momota,‡ Yasukazu Ohmoto,‡ Takao Taki,‡ Yongfeng J ia,§ and
Duan Li§
Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi 1-78, Tokushima 770-8505, J apan,
Otsuka Pharmaceutical Company, Ltd., Kagasuno, Tokushima 771-0192, J apan, and School of Pharmacy,
Shanghai Medical University, 138 Yi Xue Yuan Road, Shanghai 200032, People’s Republic of China
Received October 27, 2000
Nine new sesquiterpene pyridine alkaloids [wilfornines A (1), B (2), C (3), D (4), E (5), F (8), and G (9);
wilfordinines I (6) and J (7)] and six known compounds (10-15) were isolated from a clinically used
extract (TII) of Tripterygium wilfordii. The structures of 1-9 were elucidated by spectroscopic and chemical
methods. The inhibitory effects on cytokine production of 1-3 and several related compounds were
evaluated. Compounds 10 and 14 showed significant inhibitory effects on cytokine production.
Tripterygium wilfordii Hook f. (Celastraceae) has been
used in traditional Chinese medicine to treat cancer and
also as an insecticide for hundreds of years. Recently, an
extract (the so-called “total multi-glycoside” or “TII extract”)
derived from a water and chloroform partitioning of the
roots of T. wilfordii has been used in the clinical treatment
of rheumatoid arthritis, skin disorders, male-fertility con-
trol, and other inflammatory and autoimmune diseases in
China.1-3 In previous papers, we have reported a number
of immunosuppressive diterpenes and anti-HIV sesquiter-
penes from T. wilfordii.4,5 As a continuation of our studies
on the bioactive principles from the genus Tripterygium,
we have turned our attention to the immunosuppressive
constituents and describe herein the isolation and structure
determination of nine new (1-9) and six known sesqui-
terpene alkaloids (10-15) (Chart 1) from the clinically used
extract of T. wilfordii (TII extract), along with their inhibi-
tory effects on cytokine production.
5.45 (H-7), 5.13 (H-8), and 4.43 (H-11b) correlated with the
acetyl carbonyl carbon signals at δC 167.8, 168.1, 169.6,
169.8, 168.5, and 170.1, respectively. Thus, the six acetyl
groups were assigned at positions C-1, C-2, C-5, C-7, C-8,
and C-11, and the benzoyl group was located at position
C-9′.
Benzoylation of alatusinine (18)8 with benzoyl chloride
and a catalytic amount of 4,4-(dimethylamino)pyridine
afforded a product identical to 1. In the NOESY spectrum
of 1, the proton signal at δH 5.15 (H-11a) correlated with
the signals at δH 6.85 (H-5) and 1.54 (H3-12), and the signal
at δH 5.50 (H-1) with the signals at δH 5.19 (H-2) and 5.13
(H-8), while the signal at δH 5.13 (H-8) correlated with the
signals at δH 5.45 (H-7) and 1.14 (H3-14). Thus, the relative
configurations of the ester groups were determined as 1â,
2â, 5R, 7â, and 8â. Therefore, the structure of 1 was
assigned as 9′-benzoyloxyeuonine.
Wilfornine B (2), C43H49O19N, had five acetyl groups and
1
one benzoyl group from its H NMR spectrum. On the basis
Resu lts a n d Discu ssion
of comparison of its 13C NMR data to those of 1, this
compound could be assigned within the same structural
type. A downfield carbon signal at position C-9′ was
apparent in comparison with 13C NMR data of 18, and a
proton signal at δH 5.25 (H-5) was apparent in a more
upfield position than the same proton in 1 (δH 6.85, H-5).
Compound 2 was therefore deduced as be 5-deacetylwil-
fornine A (1). Acetylation of 2 in the usual way afforded 1.
Thus, wilfornine B (2) was elucidated as 5-deacetyl-9′-
benzoyloxyeuonine.
The powdered TII extract of T. wilfordii was chromato-
graphed repeatedly on Si gel to afford nine new sesqui-
terpene alkaloids, named wilfornines A (1), B (2), C (3), D
(4), E (5), F (8), and G (9) and wilfordinines I (6) and J (7),
as well as six known compounds (10-15).
Wilfornine A (1) was assigned the molecular formula
C45H51O20N from its HRFABMS. Its IR spectrum showed
hydroxyl and ester carbonyl bands (3483 and 1750 cm-1),
and the UV spectrum revealed the presence of an aromatic
ring (228 and 267 nm). The 1H NMR (Table 1) data
revealed the presence of six acetyl, one benzoyl (Bz), and
four sets of methylene groups. In addition, seven methine
protons and a 2,3-substituted pyridine ring were detected.
Due to the presence of seven ester groups and a pyridine
ring, 1 was assumed to be a sesquiterpene alkaloid deriva-
tive, similar to the wilfordate-type sesquiterpenes.6,12 The
13C NMR (Table 2) spectral data of 1 were very similar to
those of wilfordine (16),7 except for the number of acetyl
groups (1, six acetyls, one benzoyl; 16, five acetyls, one
benzoyl) and the downfield shift of the carbon signal
attributed to C-9′ (δC 81.6). In the HMBC spectrum of 1,
the proton signals at δH 5.50 (H-1), 5.19 (H-2), 6.85 (H-5),
Wilfornines C (3) and D (4) were assigned with the same
structural framework as 1 and 2 from their 13C NMR
spectral data. The difference between these compounds was
the nature of the ester groups and their positions. The
HMBC spectral data of 3 indicated that the five acetyl
groups were located at C-1, C-2, C-7, C-8, and C-11, and
one of the benzoyl groups was at C-5. The remaining
benzoyl group was located at C-9′, as in the case of
compounds 1 and 2. For 4, the acetyl groups were assigned
at positions C-1, C-2, C-5, C-7, C-8, and C-11. The furanoyl
group was deduced to be located at C-9′ from the downfield
carbon signal at C-9′. Furanoylation of 18 afforded 4. Thus,
the structures of wilfornine C (3) and D (4) were deter-
mined as O5-benzoyl-O5-deacetyl-9′-benzoyloxyeuonine and
9′-furanoyloxyeuonine, respectively.
* To whom correspondence should be addressed. Tel: 0081-88-6337275.
Fax: 0081-88-6339501. E-mail: takaishi@ph.tokushima-u.ac.jp.
† University of Tokushima.
Wilfornine E (5) had a molecular formula C36H43O18N.
Its 13C NMR spectral data revealed the presence of a
carbonyl carbon at δC 196.7 and different chemical shift
‡ Otsuka Pharmaceutical Company.
§ Shanghai Medical University.
10.1021/np000504a CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/20/2001

Sesquiterpene Alkaloids from Tripterygium
J ournal of Natural Products, 2001, Vol. 64, No. 5 583
Ch a r t 1
values for C-6, C-7, and C-8 than those of compounds 1-4
and 16 (Table 2). In the HMBC spectrum of 5, the proton
signal at δH 6.78 (H-5) correlated with the carbon signals
at δC 52.5 (C-9), 95.5 (C-10), 86.5 (C-13), and 196.0 (C-7),
and the proton signals at δH 5.56 (H-8) and 3.05 (H-6)
correlated with the carbonyl group (δC 196.0). Thus,
compound 5 was characterized as 7-deacetoxy-7-oxoala-
tusinine.
assumed to be an isomeric evoninate-type sesquiterpene
similar to those found in T. hypoglaucum6 and Peritassa
compta.17 Comparison of the 13C NMR spectral data of 6
with those of hypoglaunine C (21)6 indicated that the
pyridyl moiety was a 2-hydroxy-2,3-dimethyl-3-(3′-carboxy-
4′-pyridyl)propanoic acid unit. Furthermore, the proton
signal at δH 4.84 (H-3) correlated with the signal at δC 175.2
(C-11′), and the proton signal at δH 5.42 (H-15a) with the
signal at δC 167.9 (C-12′). These observations indicated that
the pyridyl moiety was linked to the sesquiterpene unit at
positions C-3 and C-15. In the same manner, two benzoyl
groups were located at positions C-2 and C-5, and the four
acetyl groups were assigned at C-1, C-7, C-8, and C-11,
respectively. The stereochemistry of six ester groups was
readily determined by the analysis of the NOESY spectrum
as described for 1. Thus, wilfordinine I (6) was formulated
as O2,O5-dibenzoyl-O2-deacetyl-O5-deacetyl-8′-hydroxyper-
itassine A.
Wilfordinine I (6) was assigned a molecular formula
1
C48H51O19N from HRFABMS. Its H NMR spectrum (Table
1) showed four acetyl groups, two benzoyl groups, two
oxygenated methylene groups, and a secondary methyl
group coupled with a methine. Comparison of the 13C NMR
spectral data of 6 with those of 1-4 indicated that 6
contained a dihydroagarofuran unit linked to a pyridyl
moiety (Table 2). The coupling pattern suggested that the
pyridine unit in 6 is 3,4-substituted [δH 8.99 (s), 8.71 and
7.80 (each 1H, d, J ) 5.4 Hz)]. Thus, compound 6 was

584 J ournal of Natural Products, 2001, Vol. 64, No. 5
Duan et al.

Sesquiterpene Alkaloids from Tripterygium
J ournal of Natural Products, 2001, Vol. 64, No. 5 585
Ta ble 2. 13C NMR Spectral Data of Compounds 1-9 and 16a
carbon
1
2
3
4
5
6
7
8
9
16
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
2′
3′
4′
5′
6′
7′
8′
9′
10′
11′
12′
72.1
69.3
78.4
69.6
73.7
50.7
68.4
71.7
52.0
93.3
60.1
23.3
84.2
17.2
69.7
161.7
125.6
138.2
120.9
152.2
30.6
37.7
81.6
22.7
171.7
167.6
72.1
69.9
78.5
71.9
74.2
52.3
68.8
72.0
50.7
92.8
61.1
24.3
85.4
17.7
71.0
163.1
124.8
138.4
121.4
152.8
31.1
38.4
82.1
22.2
172.0
167.9
72.3
69.5
78.7
69.9
74.9
50.7
68.7
72.2
52.3
93.2
60.4
23.5
84.5
17.3
69.8
161.9
125.9
138.1
121.0
152.4
30.8
38.0
81.9
23.0
172.0
167.8
72.1
69.5
78.6
69.8
73.9
50.9
68.8
71.8
52.2
93.4
60.2
23.2
84.4
17.3
70.1
161.8
125.5
138.5
121.4
152.3
30.6
37.8
81.4
22.3
171.6
167.6
71.9
68.7
76.2
70.2
73.5
62.4
196.0
78.3
52.5
95.5
72.9
68.4
77.9
70.7
74.9
50.7
69.1
70.7
52.3
93.5
60.5
22.7
83.8
18.7
69.8
150.9
127.7
151.5
123.4
152.6
41.8
76.6
16.9
24.2
175.2
167.9
72.7
73.3
75.9
70.7
73.2
50.5
69.7
71.8
51.9
94.0
61.4
23.4
84.5
18.5
70.3
150.9
125.4
156.3
121.6
152.9
33.4
45.7
11.5
10.0
173.6
168.0
75.5
69.8
78.4
70.7
75.1
50.7
69.4
71.3
51.9
94.3
60.5
23.3
84.2
18.6
70.2
165.2
126.1
137.7
121.3
151.6
36.5
46.9
12.0
9.5
73.1
69.8
75.6
70.4
73.7
50.6
68.9
70.5
52.1
94.0
60.3
23.3
84.3
18.6
69.9
165.3
125.0
137.8
121.2
151.6
36.4
45.0
11.9
9.7
73.2
69.3
76.9
69.8
73.6
51.1
69.0
70.8
51.9
94.0
60.6
23.0
85.0
17.9
69.8
164.8
125.5
137.9
120.6
152.2
31.4
38.4
77.7
21.6
172.5
168.0
60.3
23.4
86.5
18.8
70.0
164.8
125.3
138.2
120.9
152.3
31.4
38.7
77.7
27.8
172.3
167.8
174.6
168.7
174.0
168.6
a
CDCl3 was used as solvent, and TMS as internal standard.
Ta ble 3. Inhibitory Effects of Compounds 1-3, 10-17, 19-22,
Wilfordinine J (7), C36H45O17N, contained five acetyl
groups, two secondary methyl groups, and two coupled
methine protons. It also had a 3,4-disubstituted pyridine
ring, as compound 6. The 13C NMR spectral data of 7 were
similar to those of 6, except for the number and nature of
the ester groups and chemical shift values of C-7′, C-8′,
C-9′, and C-10′. The pyridyl unit was assumed to be an
isomeric form of evoninate acid, which already has been
reported from T. hypoglaucum.4 In the HMBC spectrum,
the signal at δH 8.98 (H-2′) correlated with the carbon
signals at δC 125.4 (C-3′), 156.3 (C-4′), 152.9 (C-6′), and
168.0 (C-12′), the signal at δH 1.07 (H-10′) with the signals
at δC 33.4 (C-7′), 45.7 (C-8′), and 173.6 (C-11′), and the
signal at δH 1.37 (H-9′) with the signals at δC 156.3 (C-4′),
33.4 (C-7′), and 45.7 (C-8′). From above observations, the
presence of a 2,3-dimethyl-3(3′-carboxy-4′-pyridyl)propanoic
acid unit was determined. Furthermore, the proton signals
at δH 5.10 (H-2), 6.76 (H-5), 5.53 (H-7), 5.57 (H-8), and 5.11
(H-11a) correlated with the acetyl carbonyl carbon signals
at δC 170.3, 169.9, 170.2, 169.9, and 169.8, respectively.
Therefore, the five acetyl groups were located at C-2, C-5,
C-7, C-8, and C-11. Thus, wilfordinine J (7) was determined
as O1-deacetylperitassine A.
Wilfornine F (8) had a molecular formula of C41H47O17N.
The 1H NMR spectral data revealed four acetyl methyl
groups, two secondary methyls, two coupled methine
protons, and one benzoyl group, as well as a 2,3-substituted
pyridine moiety [δH 8.70 (1H, dd, 4.7, 1.6), 7.26 (1H, dd,
7.8, 4.7), and 8.04 (1H, dd, 7.8, 1.6)]. The 13C NMR spectral
data were similar to those of hyponine D9 (11) except for
the ester groups. In the HMBC spectrum, the proton
signals at δH 5.49 (H-1), 5.58 (H-7), 5.41 (H-8), and 4.73
(H-11a) correlated with the acetyl carbonyl signals at δC
169.5, 170.4, 169.3, and 170.2, respectively. Thus, the
positions of these four acetyl groups were assigned at C-1,
C-7, C-8, and C-11. Further, the proton signal at δH 7.19
(H-5) correlated with the benzoyl carbonyl carbon at δC
166.0, so the benzoyl group was located at C-5. In the
NOESY spectrum, the proton signal at δH 5.34 (H-11b)
correlated with the signals at δH 7.19 (H-5) and 1.60 (H3-
12), the signal at δH 5.49 (H-1) with the signals at δH 4.12
and Prednisolone on Cytokine Productiona
inhibition (%)
compound
TNF-R
IL-1â
IL-8
IL-2
IL-4
IFN-γ
1
2
3
10
11
12
13
14
15
16
17
19
20
21
22
0
-6
-11
76
-9
-9
55
37
62
2
20
-4
-3
-6
49
7
2
-23
-8
97
-15
2
31
84
43
8
60
1
7
-26
64
15
23
22
51
100
45
55
53
100
33
35
50
67
22
57
47
65
50
100
39
100
66
14
11
100
-20
50
77
16
-53
51
77
76
8
7
32
92
13
36
28
99
17
4
71
41
4
20
62
75
2
47
43
55
-6
14
64
53
2
79
64
-28
10
18
68
prednisolone
52
a
For protocols used, see Experimental Section. Concentrations
used: 1-3, 10-17, 19-22, 10 µg/mL; prednisolone, 0.3 µg/mL.
(H-2) and 5.41 (H-8), and the signal at δH 5.41 (H-8) with
the signals at δH 5.58 (H-7) and 1.69 (H3-14). Thus, the
relative configurations of the ester groups were determined
as 1â, 2â, 5R, 7â, and 8â. Therefore, 8 was formulated as
7-(acetyloxy)-O5-benzoyl-O2-deacetyl-O5-deacetyl-7-deoxo-
evonine.
Wilfornine G (9), C42H48O18N2, contained five acetyl
groups and a nicotinoyl group [δH 9.32 (d, 1.5), 8.85 (dd,
1
4.8, 1.5), 8.38 (dt, 7.4, 1.5), 7.48 (dd, 7.4, 4.8)] from its H
NMR spectrum. It had the same skeleton as 8 by compar-
ing the 13C NMR spectral data of these two compounds
(Table 2). The differences between 8 and 9 were the ester
groups and their positions. By using the same strategy as
above (the comparison of 1H, 13C NMR, and 2D NMR
spectral data), the structure of wilfornine G (9) was
determined as 7-(nicotinoyloxy)-7-deoxoevonine.
Several known compounds were identified from their
spectral data upon comparison with values reported in the
literature as ebenifoline E-11 (10),10 hyponine D (11),9

586 J ournal of Natural Products, 2001, Vol. 64, No. 5
Duan et al.
mayteine (12),11 euonymine (13),12 congorinine E-1 (14),13
and triptonine A (15).14
(KBr) νmax 3483, 2361, 1750, 1577, 1442, 1375, 1232, 1135,
1
1052, 755, 716 cm-1; H NMR (CDCl3), see Table 1 and OBz-
9′, δ 7.69 (2H, d, J ) 7.4 Hz, ortho), 7.46 (1H, br t, J ) 7.3 Hz,
para), 7.30 (2H, dd, J ) 7.4, 7.3 Hz, meta); 13C NMR (CDCl3),
see Table 2 and δ 20.1, 167.8 (OAc-1), 21.4, 168.1 (OAc-2), 20.8,
169.6 (OAc-5), 20.9, 169.8 (OAc-7), 20.3, 168.5 (OAc-8), 21.2,
170.1 (OAc-11), 165.6, 129.6, 130.2, 127.9, 132.9 (OBz-9′);
FABMS m/z 926 [M + H]+; HRFABMS m/z 926.3048 [M +
H]+ (calcd for C45H52O20N, 926.3083).
Ben zoyla tion of 18. Compound 18 (10 mg, isolated from
T. wilfordii15) was dissolved in 1 mL of pyridine and a catalytic
amount of 4,4-(dimethylamino)pyridine, and 1 mL of benzoyl
chloride was added to the solution. The reaction mixture was
stirred for 48 h at room temperature under N2. Workup gave
25 mg of residue, which was purified by HPLC (GPC, CHCl3)
to give 7 mg of 1.
In a screen for immunosuppressive agents from the TII
extract of T. wilfrodii, we examined the inhibitory effect
of isolated compounds and other reported compounds6,15
(16, 17, and 19-22, from the same origin) on cytokine
production. The activities of those compounds with an
inhibitory effect are shown in Table 3 (the other tested
compounds were inactive in the test system). Compounds
10 and 14 showed a significant inhibitory effect on cytokine
production from lipopolysaccharide (or phytohemaggluti-
nin)-stimulated human peripheral mononuclear cells com-
pared with the reference compound (prednisolone).16 Com-
pounds 17 and 22 inhibited IL-4, IL-8, and IFN-γ production,
and 19 (isolated from the TII extract) inhibited TNF-R, IL-
1â, and IL-2 production. The immunosuppressive activity
of these known compounds has been determined for the
first time.
Wilfor n in e B (2): amorphous powder; [R]D -17.8° (c 0.8,
MeOH); UV (MeOH) λmax (log ꢀ) 265 (3.60), 228 (4.17) nm; IR
(KBr) νmax 3423, 2927, 2362, 1751, 1453, 1373, 1231, 1101,
1
1047, 715 cm-1; H NMR (CDCl3), see Table 1 and OBz-9′, δ
7.78 (2H, d, J ) 7.3 Hz, ortho), 7.50 (1H, br t, J ) 7.4 Hz,
para), 7.35 (2H, br t, J ) 7.5 Hz, meta); 13C NMR (CDCl3), see
Table 2 and δ 20.1, 168.2 (OAc-1), 21.2, 168.3 (OAc-2), 21.4,
170.0 (OAc-7), 20.4, 168.7 (OAc-8), 21.0, 169.8 (OAc-11), 166.0,
129.6, 130.5, 128.1, 133.2 (OBz-9′); EIMS m/z 883 [M]+ (31),
824 [M - OAc]+ (16), 810 (39), 780 (12), 762 (66), 752 (22),
704 (22), 326 (16), 222 (13), 204 (53), 176 (53), 160 (31), 150
(15), 132 (25), 105 (100), 93 (24), 77 (13), 43 (23); HREIMS
m/z 883.2927 [M]+ (calcd for C43H49O19N, 883.2899).
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Optical rotations
were measured with a J asco DIP-370 digital polarimeter. UV
spectra were run on an UV 2100 UV-vis recording spectrom-
eter (Shimadzu). IR spectra were recorded on a 1720 infrared
Fourier transform spectrometer (Perkin-Elmer). NMR experi-
ments were run on a Bruker ARX-400 instrument using TMS
as internal standard. NMR spectra were recorded at 400 MHz
(1H) and 100 MHz (13C). Mass spectra were obtained on a
J EOL J MSD-300 instrument, matrix: magic/thio12 [the mix-
ture of magic (dithiothreitol-dithioerythritol, 3:1) and thio
(thioglyserol), 1:2]. Column chromatography was performed
using Si gel 60 (Merck). HPLC was carried out as follows: Si
gel HPLC (Hibar RT 250-25, LiChrosorb Si 60), ODS (INERT-
SIL PREP ODS, 20.0 × 250 mm, GL Sciences Inc., Tokyo,
J apan).
P la n t Ma ter ia l. A powdered extract of T. wilfordii (TII) was
purchased in 1997 from the School of Pharmacy, Shanghai
Medical University, Shanghai, People’s Republic of China. This
extract was prepared from the root xylem with water then with
chloroform and by column chromatographic separation (Si gel,
CHCl3-MeOH, 95:5). Samples of TII and the original plant (T.
wilfordii, TW940930) are deposited in the Faculty of Phar-
maceutical Sciences, University of Tokushima, J apan.
Extr a ction a n d Isola tion . The extract (TII, 54 g) prepared
from T. wilfordii was chromatographed over a Si gel column
(1.0 kg, 11 × 90 cm) and eluted with solvents of increasing
polarity [CHCl3-MeOH (99:2, 95:5, 9:1, MeOH)] to give 10
fractions (fractions 1-10). Fraction 3 (0.7 g) was chromato-
graphed by medium-pressure liquid chromatography (MPLC)
on a Si gel column (3 × 35 cm) with a hexane-EtOAc (hexane-
EtOAc, 2:1, 1:2) system to give nine further fractions (fractions
3.1-3.9). Fraction 3.7 was separated by gel-permeation chro-
matography (eluting with MeOH) and then Si gel HPLC to
give 10 (8 mg) and 14 (148 mg). Fraction 4 (7.5 g) was
chromatographed by MPLC on a Si gel column (hexanes-
EtOAc, 3:2, 1:2) to give 15 fractions (fractions 4.1-4.15).
Fraction 4.12 was separated on ODS (MeOH-H2O, 8:2) to give
12 (26 mg), 13 (133 mg), and another major fraction. This
major fraction was further separated on HPLC (Si 60, CHCl3-
MeOH, 99:1) to give 1 (65 mg) and 15 (3 mg). Fraction 4.13
was separated on ODS (MeOH-H2O, 8:2) to give 5 (5 mg), 11
(26 mg), and another five fractions (fractions 4.13.1-4.13.5).
Fraction 4.13.3 was separated by HPLC (Si 60, CHCl3-MeOH,
99:1) to give 4 (8 mg) and 7 (4.5 mg). Fraction 4.13.4 was
separated by HPLC (Si 60, CHCl3-MeOH, 99:1) to give 3 (14
mg) and 8 (4.5 mg). Fraction 5 (16.5 g) was chromatographed
on a Si gel column (6 × 80 cm) by elution with hexanes-EtOAc
(1:1, 1:2, 1:4) to give 12 further fractions (fractions 5.1-5.12).
Fraction 5.9 was separated by ODS (MeOH-H2O, 8:2, then
7:3) to give 2 (23 mg), 6 (5 mg), and 9 (28 mg).
Acetyla tion of 2. Compound 2 (1.5 mg) was treated with
Ac2O (0.3 mL) and C5H5N (0.5 mL) at room temperature
overnight. The reaction mixture on work up gave 1 (1 mg).
Wilfor n in e C (3): amorphous powder; [R]D -50.0° (c 1.1,
MeOH); UV (MeOH) λmax (log ꢀ) 267 (3.74), 231 (4.51) nm; IR
(KBr) νmax 3446, 2923, 2853, 2362, 1752, 1722, 1636, 1453,
1
1373, 1254, 1233, 1133, 1097, 1052, 1004, 904, 715; cm-1; H
NMR (CDCl3), see Table 1 and OBz-5, δ 8.27 (2H, d, J ) 7.9
Hz, ortho), 7.48 (2H, dd, J ) 7.9, 7.4 Hz, meta), 7.59 (1H, t, J
) 7.4 Hz, para), OBz-9′: 7.71 (2H, d, J ) 7.9 Hz, ortho), 7.33
(2H, br t, J ) 7.8 Hz, meta), 7.49 (1H, br t, J ) 7.5 Hz, para);
13C NMR (CDCl3), see Table 2 and δ 20.5, 168.0 (OAc-1), 21.1,
168.3 (OAc-2), 21.5, 170.1 (OAc-7), 20.3, 168.7 (OAc-8), 21.1,
170.1 (OAc-11), 165.7, 129.7, 130.4, 128.9, 133.7 (OBz-5), 165.7,
130.4, 129.4, 133.1, 128.1 (OBz-9′); EIMS m/z 987 [M]+ (58),
866 (95), 822 (19), 754 (6), 675 (4), 616 (3), 326 (11), 253 (5),
176 (18), 105 (100), 28 (59); FABMS m/z 988 [M + H]+;
HRFABMS m/z 988.3203 [M + H]+ (calcd for C50H54O20N,
988.3239).
Wilfor n in e D (4): amorphous powder; [R]D -21.1° (c 0.8,
MeOH); UV (MeOH) λmax (log ꢀ) 263 (3.57), 216 (3.99) nm; IR
(KBr) νmax 3446, 2925, 2853, 2374, 1749, 1635, 1435, 1372,
1318, 1233, 1096, 1047, 1007, 875, 602 cm-1; 1H NMR (CDCl3),
see Table 1 and OFu-9′, δ 6.51 (1H, d, J ) 1.1 Hz), 7.30 (1H,
br s), 7.75 (1H, s); 13C NMR (CDCl3), see Table 2 and δ 20.2,
168.3 (OAc-1), 20.9, 170.2 (OAc-2), 21.5, 170.0 (OAc-5), 21.0,
168.5 (OAc-7), 20.3, 168.9 (OAc-8), 21.3, 170.4 (OAc-11), 162.4,
118.7, 110.2, 143.3, 149.1 (OFu-9′); FABMS m/z 916 [M + H]+;
HRFABMS m/z 916.2858 [M + H]+ (calcd for C43H50O21N,
916.2875).
P r ep a r a tion of 3-F u r oyl Ch lor id e. A mixture of 3-furoic
acid (3 g) and SOCl2 (5 mL) in dry benzene (20 mL) was stirred
for 2 h under reflux. Excess SOCl2 was repeatedly removed
by distillation with benzene at 100 °C. The reaction mixture
was then concentrated to 5 mL under reduce pressure.
Ester ifica tion of 18 w ith 3-F u r oyl Ch lor id e. Compound
18 (10 mg) was dissolved in 5 mL of pyridine, and a catalytic
amount of 4,4-(dimethylamino)pyridine and 1 mL of the
reaction mixture prepared above were added to the solution.
The reaction mixture was stirred for 18 h at room temperature
under N2. The usual workup gave 18 mg of residue, which was
purified by HPLC (GPC, CHCl3) to give 5 mg of 4.
Wilfor n in e E (5): amorphous powder; [R]D +1.1° (c 0.5,
MeOH); UV (MeOH) λmax (log ꢀ) 267 (3.45), 222 (3.82) nm; IR
(KBr) νmax 3454, 2925, 2854, 1752, 1583, 1456, 1374, 1227,
1135, 1088, 1044 cm-1; 1H NMR (CDCl3), see Table 1; 13C NMR
Wilfor n in e A (1): amorphous powder; [R]D -41.9° (c 1.0,
MeOH); UV (MeOH) λmax (log ꢀ) 267 (3.68), 228 (4.27) nm; IR

Sesquiterpene Alkaloids from Tripterygium
J ournal of Natural Products, 2001, Vol. 64, No. 5 587
(CDCl3), see Table 2 and δ 20.5, 169.6 (OAc-1), 21.1, 168.5
(OAc-2), 21.4, 169.3 (OAc-5), 20.2, 169.4 (OAc-8), 20.6, 169.7
(OAc-11); EIMS m/z 777 [M]+ (25), 733 (31), 674 (29), 368 (11),
264 (16), 246 (32), 205 (13), 194 (27), 176 (29), 149 (27), 137
(21), 125 (31), 111 (50), 109 (42), 97 (80), 81 (50), 71 (87), 57
(96), 43 (100); HREIMS m/z 777.2495 [M]+ (calcd for C36H43O18N,
777.2480).
Wilfor d in in e I (6): amorphous powder; [R]D +63.0° (c 0.2,
MeOH); UV (MeOH) λmax (log ꢀ) 262 (3.77), 230 (4.53) nm; IR
(KBr) νmax 3464, 2927, 2855, 2362, 1746, 1599, 1453, 1372,
1313, 1264, 1156, 1097, 1051, 903, 713 cm-1; 1H NMR (CDCl3),
see Table 1 and OBz-2, 8.13 (2H, d, J ) 7.5 Hz, ortho), 7.53
(2H, br t, J ) 7.5 Hz, meta), 7.63 (1H, br t, J ) 7.5 Hz, para),
OBz-5: 8.22 (2H, d, J ) 7.5 Hz, ortho), 7.52 (2H, br t, J ) 7.5
Hz, meta), 7.63 (1H, br t, J ) 7.5 Hz, para); 13C NMR (CDCl3),
see Table 2 and δ 20.5, 169.4 (OAc-1), 21.2, 170.2 (OAc-7), 20.6,
169.1 (OAc-8), 21.4, 170.3 (OAc-11), 164.9, 129.2, 130.1, 128.9,
133.9 (OBz-2), 165.7, 129.2, 130.2, 129.0, 134.0 (OBz-5); EIMS
m/z 945 [M]+ (100), 886 (7), 872 (6), 824 (5), 763 (3), 720 (2),
571 (1), 469 (1), 352 (3), 222 (4), 150 (9), 105 (46), 44 (8);
FABMS m/z 946 [M + H]+; HRFABMS m/z 946.3148 [M +
H]+ (calcd for C48H52O19N, 946.3134).
Wilfor n in e G (9): amorphous powder; [R]D +11.0° (c 0.7,
MeOH); UV (MeOH) λmax (log ꢀ) 260 (3.80), 222 (4.26) nm; IR
(KBr) νmax 3482, 2988, 2360, 1745, 1647, 1590, 1454, 1432,
1371, 1314, 1275, 1232, 1171, 1114, 1059, 1023, 880, 740 cm-1
;
1H NMR (CDCl3), see Table 1 and ONic-7, 9.32 (1H, d, J ) 1.5
Hz), 8.85 (1H, dd, J ) 4.8, 1.5 Hz), 8.38 (1H, dt, J ) 7.5, 1.5
Hz), 7.48 (1H, dd, J ) 7.5, 4.8 Hz); 13C NMR (CDCl3), see Table
2 and δ 20.5, 169.0 (OAc-1), 21.0, 170.0 (OAc-2), 21.7, 169.9
(OAc-5), 20.5, 169.1 (OAc-8), 21.2, 170.5 (OAc-11), 163.7, 124.9,
137.4, 123.7, 154.6, 151.3 (ONic-7); EIMS m/z 868 [M]+ (100),
825 (11), 809 [M - OAc]+ (24), 718 (10), 686 (12), 572 (13),
206 (56), 178 (24), 161 (14), 124 (32), 107 (33), 43 (12); HREIMS
m/z 868.2886 [M]+ (calcd for C42H48O18N2, 868.2902).
Bioa ssa y P r oced u r e. The bioassay was performed as
previously described in ref 4.
Refer en ces a n d Notes
(1) Qian, S. Z. Contraception 1987, 36, 335-345.
(2) Matlin, S. A.; Belenguer, A.; Stacey, V. E.; Qian, S. Z.; Xu, Y.; Zhang,
J . W.; Sanders, J . K. M.; Amor, S. R.; Pearce, C. M. Contraception
1993, 47, 387-400.
(3) Qian, S. Z.; Xu, Y.; Zhang, J . W. Contraception 1995, 51, 121-129.
(4) Duan, H. Q.; Takaishi, Y.; Momota, H.; Ohmoto, Y.; Taki, T.; J ia, Y.
F.; Li, D. J . Nat. Prod. 1999, 62, 1522-1525.
Wilfor d in in e J (7): amorphous powder; [R]D -8.1° (c 0.7,
MeOH); UV (MeOH) λmax (log ꢀ) 263 (3.46), 222 (3.85) nm; IR
(KBr) νmax 3482, 2936, 1747, 1650, 1591, 1456, 1372, 1233,
(5) Duan, H. Q.; Takaishi, Y.; Imakura, Y.; J ia, Y. F.; Li, D.; Cosentino,
L. M.; Lee, K. H. J . Nat. Prod. 2000, 63, 357-361.
(6) Duan, H. Q.; Takaishi, Y. Phytochemistry 1998, 49, 2185-2189.
(7) Han, B. H.; Ryu, J . H.; Han, Y. H.; Park, M. K.; Park, J . H.; Naoki,
H. J . Nat. Prod. 1990, 53, 909-914.
1
1187, 1158, 1120, 1048, 976, 603 cm-1; H NMR (CDCl3), see
Table 1; 13C NMR (CDCl3), see Table 2 and δ 21.1, 170.3 (OAc-
2), 21.7, 169.9 (OAc-5), 21.0, 170.2 (OAc-7), 20.8, 169.9 (OAc-
8), 21.4, 169.8 (OAc-11); EIMS m/z 763 [M]+ (100), 748 (31),
704 [M - OAc]+ (50), 690 (24), 206 (44), 178 (52), 160 (32),
107 (98), 43 (26); HREIMS m/z 763.2682 [M]+ (calcd for
(8) Ishiwata, H.; Shizuri, Y.; Yamada, K. Phytochemistry 1983, 22, 2839-
2841.
(9) Duan, H. Q.; Takaishi, Y. Phytochemistry 1999, 52, 1735-1738.
(10) Itokawa, H.; Shirota, O.; Morita, H.; Takeya, K. Heterocycles 1992,
34, 885-889.
C
36H45O17N, 763.2687).
(11) Han, B. H.; Park, M. K.; Ryu, J . H.; Park, J . H.; Naoki, H.
Phytochemistry 1990, 29, 2303-2307.
Wilfor n in e F (8): amorphous powder; [R]D +18.2° (c 0.4,
(12) Itokawa, H.; Shirota, O.; Morita, H.; Takeya, K.; Iitaka, Y. J . Chem.
Soc., Perkin Trans. 1 1993, 1247-1254.
MeOH); UV (MeOH) λmax (log ꢀ) 264 (3.54), 229 (4.16) nm; IR
(KBr) νmax 3480, 2939, 2361, 1721, 1650, 1541, 1456, 1374,
(13) Shirota, O.; Morita, H.; Takeya, K.; Itokawa, H. Heterocycles 1994,
38, 383-389.
1
1255, 1116, 1057, 787, 715 cm-1; H NMR (CDCl3), see Table
(14) Duan, H. Q.; Takaishi, Y.; Bando, M.; Kido, M.; Imakura, Y.; Lee, K.
H. Tetrahedron Lett. 1999, 40, 2969-2972.
1 and OBz-5, 8.34 (2H, d, J ) 7.4 Hz, ortho), 7.59 (1H, br t, J
) 7.3 Hz, para), 7.50 (2H, br t, J ) 7.5 Hz, mata); 13C NMR
(CDCl3), see Table 2 and δ 21.0, 169.5 (OAc-1), 21.3, 170.4
(OAc-7), 20.7, 169.3 (OAc-8), 21.7, 170.2 (OAc-11), 166.0, 129.8,
130.5, 129.0, 133.7 (OBz-5); EIMS m/z 825 [M]+ (100), 797 (6),
782 (9), 766 [M - OAc]+ (14), 752 (5), 722 (6), 704 (11), 652
(29), 634 (15), 206 (57), 178 (22), 107 (8); HREIMS m/z
825.2834 [M]+ (calcd for C41H47O17N, 825.2844).
(15) Duan, H. Q.; Takaishi, Y.; J ia, Y. F.; Li, D. Chem. Pharm. Bull. 1999,
47, 1664-1667.
(16) Kita, M.; Ohmoto, Y.; Hirai, Y.; Yamaguchi, N.; Imanishi, J . Microbiol.
Immunol. 1992, 36, 507-516.
(17) Klass, J .; Tinto, W. F.; Reynolds, W. F.; Mclean, S. J . Nat. Prod. 1993,
56, 946-948.
NP000504A