Rapulasides A and B: Two novel intermolecular rearranged biiridoid glucosides from the roots of Heracleum rapula

Article abstract of DOI:10.1016/j.tetlet.2005.06.094

Two novel intermolecular rearranged biiridoid glucosides, rapulasides A and B (1 and 2), have been isolated from the roots of Heracleum rapula. Their structures were identified by extensive spectral analysis especially different NMR techniques. NOESY experiment, with the help of Dreiding molecular model, was used to elucidate their relative stereochemistry. Both compounds were tested for their inhibitory effects on rabbit platelet aggregation induced by PAF, ADP, or AA, respectively. Only trends of inhibition were observed for them.

Full text of DOI:10.1016/j.tetlet.2005.06.094

Tetrahedron
Letters
Tetrahedron Letters 46 (2005) 5743–5746
Rapulasides A and B: two novel intermolecular rearranged biiridoid
glucosides from the roots of Heracleum rapula
Weilie Xiao,a,b Shenghong Li,a,* Xuemei Niu,a Yu Zhaoa,b and Handong Suna,*
aState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany,
Chinese Academy of Sciences, Kunming 650204, Yunnan, PR China
bThe Graduate School of the Chinese Academy of Sciences, Beijing 100039, PR China
Received 8 April 2005; revised 9 June 2005; accepted 13 June 2005
Available online 7 July 2005
Abstract—Two novel intermolecular rearranged biiridoid glucosides, rapulasides A and B (1 and 2), have been isolated from the
roots of Heracleum rapula. Their structures were identified by extensive spectral analysis especially different NMR techniques.
NOESY experiment, with the help of Dreiding molecular model, was used to elucidate their relative stereochemistry. Both com-
pounds were tested for their inhibitory effects on rabbit platelet aggregation induced by PAF, ADP, or AA, respectively. Only trends
of inhibition were observed for them.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Heracleum rapula Fr. (Umbelliferae), widely distributed
in Yunnan province of China, is commonly used in
Chinese traditional medicine to dispel wind, remove
dampness, expel cold, relieve pain, dredge all channels
and vessels, promote blood circulation, and relax
muscles and tendons.1 According to the results of phar-
macological experiments, the water-soluble extracts
possessed antibiotic and antiasthmatic effects.2 Previous
phytochemical investigations from our group showed
the presence of a series of coumarins in this plant.1,3,4
Our present search for bioactive compounds from the
water-soluble extracts of its roots resulted in the isola-
tion of two novel rearranged biiridoid glucosides,
namely rapulasides A and B (1 and 2). The structures
of both compounds were identified by extensive NMR
spectroscopic experiments including 1H–1H COSY,
HMQC, HMBC, and NOESY techniques. Both
compounds have been evaluated for their in vitro inhib-
itory activity against rabbit platelet aggregation induced
by PAF, AA, and ADP, respectively. We describe herein
the isolation, structure elucidation, and biological
activity of compounds 1 and 2.
H
H
10'
9'
H
H
H
H
7'
8
8'
O
H
O
O
O
O
10
5' 6'
OMe
H
H
11
MeO
7
H
OHC
3'
4'
11'
O
O
4
6
5
3
9
OMe O
O
OMe O
O
O
1
1'
HO
HO
HO
HO
H
H
HO
O
O
O
O
O
HO
O
OH
OH
OH
OH
HO
HO
HO
HO
HO
HO
1
2
The roots of H. rapula Fr. were collected from Dali
prefecture of Yunnan province in 2003 and verified
by Prof. Zhongwen Lin. The air-dried and powdered
roots (4.0 kg) were extracted with acetone (4 · 15 L)
at room temperature and concentrated in vacuo to give
a crude extract (195 g), which was partitioned between
H2O and EtOAc. The water layer was directly sub-
jected to column chromatography over Diaion 101
macroporous resin (800 g) eluting with H2O, aqueous
MeOH (30%, 40%, and 60%) and MeOH to give five
parts. The 40% aqueous MeOH part (20 g) was
repeatedly chromatographed on silica gel column,
which was further purified with preparative HPLC
(Agilent 1100 HPLC system; Zorbax SB-C-18, Agilent,
Keywords: Heracleum rapula; Umbelliferae; Intermolecular rearranged
biiridoid glucoside; Rapulasides A and B; Anti-platelet aggregation.
*
Corresponding authors. Tel.: +86 871 5223251; fax: +86 871
5216343; e-mail
mail.kib.ac.cn
addresses:
shli@mail.kib.ac.cn;
hdsun@
0040-4039/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2005.06.094

5744
W. Xiao et al. / Tetrahedron Letters 46 (2005) 5743–5746
9.4 mm · 25 cm, MeOH–H2O 35:65) to yield rapula-
sides A (21 mg) and B (18 mg).
H-70 to C-11 and H/C-30 to C/H-10 indicated the link-
ages in the order of C-70–O–C-11 and C-30–O–C-10,
which permitted fragments 1a and 1b to be joined to
get the planar structure of compound 1.
Rapulaside
powder, ½aꢀ
A
(1),5 pale yellow amorphous
23.4
D
ꢁ69.2 (c 0.2, MeOH), showed a quasi-
molecular ion peak at m/z 745 ([MꢁH]ꢁ) in its negative
FAB mass spectrum. The molecular formula of 1 was
revealed as C33H46O19 by HRFABMS data (found
746.2568, calcd 746.2633), corresponding to 11ꢁ of
Sugar analysis of 1 was carried out as follows. A solu-
tion of 1 (12 mg) in 2 M HCl (3 mL) was heated in a
water bath at 70 ꢁC for 6 h. After cooling, the reaction
mixture was neutralized with NaHCO3 and extracted
with CHCl3. We were not able to get the aglycone of 1
unfortunately, because the TLC inspection of the CHCl3
part indicated that there were yields of at least five prod-
ucts, which were not subjected to further isolation and
identification due to the limited amount. However,
through co-TLC with authentic sample using CHCl3–
MeOH–H2O–HOAC (7:3:0.8:1) as a developing system,
glucose was detected in the water layer (Rf = 0.48),
which was further concentrated to dryness and subjected
to a silica gel chromatography eluting with CHCl3–
unsaturation in the molecule. The H and 13C NMR
1
spectra showed the signals of one methoxyl group and
two glucoses and twenty additional carbon signals as
following: four quaternary carbons (including two ester
carbonyl carbons and two olefinic quaternary carbons),
12 methines, three methylenes (including one olefinic
methylene), and one methyl group. The obvious HMBC
cross-peaks from the anomeric protons of two glucoses
at dH 5.40 (d, J = 7.8) and 5.36 (d, J = 7.9) to dC 97.0
(C-1) and 96.5 (C-10) showed that the two sugars were
connected with C-1 and C-10, respectively. Considering
that both C-1 and C-10 were connected with a glucose
moiety and their low-field chemical shift, it is reasonable
to deduce that both C-1 and C-10 were ketal carbons.
Further interpretation of HMBC spectrum showed the
following correlations (Fig. 1): the methine proton at
dH 5.82 (H-1) with C-3 (dC 153.0), C-5 (dC 27.2) and
C-9 (dC 44.8); the methine singlet at dH 7.73 (H-3) with
C-4 (dC 109.3) and C-11 (dC 166.5); the methine proton
at dH 3.68 (H-5) with C-6 (dC 44.6), C-7 (dC 200.9) and
C-11 (dC 166.5); the proton signal at dH 5.66 (H-10) with
C-5 and C-9. The above spectral evidences, along with
the proton spin system H-1/H-9 (/H-10)/H-5/H2–6/H-7
deduced from 1H–1H COSY correlations, led to the
establishment of the partial structure 1a (Fig. 1), which
possessed a substructure of a 7,8-seco-iridoid carbon
skeleton. In addition, HMBC spectrum also showed
the cross-couplings from H2-10 to C-8 and C-90, from
Me-100 to C-70, C-80, and C-90, from H-90 to C-40, from
H-50 to C-30 and C-110, and from methoxyl singlet at dH
3.53 (3H, s) to C-110. Moreover, C-70 and C-30 were de-
duced to be oxygenated due to their low-field chemical
shift at dC 77.1 and 151.5, respectively. These spectral
data, coupled with another proton spin system H-50/
H2-60/H-70/H-80/H3-100/H-90/H-8/H2-10 established by
1H–1H COSY correlations, gave rise to another partial
structure 1b (Fig. 1). Further HMBC correlations of
MeOH (1:1) to give purified D-glucose (4.4 mg):
½aꢀ
18.3
D
+24.5 (c 0.2, H2O).
The relative stereochemistry of 1 was determined using a
NOESY experiment, aided by a Dreiding molecular
model. Stereochemically, H-5 and H-9 were biogeneti-
cally b-oriented and accordingly, CH2-6 was a-oriented.
NOESY correlations of H-1/H-6 and H-10/H-6, and the
fact that no correlation was observed between H-1/H-5
and H-10/H-5, indicated that H-1 and H-10 were a-ori-
ented. The cross-peaks of H-6/H-70 and H-6/Me-100 sug-
gested that H-70 and Me-100 were both in a-orientation
and accordingly H-80 was in b-orientation. The NOESY
correlation of H-80/H-50 indicated that H-50 possessed b-
orientation. The a-orientation of H-90 was established
by the NOESY correlations of H-90/H-70 (Fig. 2).
Rapulaside B (2)6 was obtained as pale yellow amor-
phous powder and has the molecular formula of
1
C35H52O20 as determined by analysis of H, 13C, and
DEPT NMR spectral data, and verified by HRFABMS
(found 792.3015, calcd 792.3052), requiring 10ꢁ of
unsaturation. The 1H NMR spectrum displayed a signal
due to a secondary methyl. The close resemblance of the
NMR spectra (Table 1) to those of 1 suggested 2 to be a
related rearranged biiridoid glucosides. The two com-
pounds are different only because the aldehyde at C-7
in 1 was replaced by a ketal carbon (dC 103.2) in 2
and two extra methoxyl groups were observed in 2. In
addition, these two methoxyl groups were found to be
(1b)
O
O
O (1a)
O
OHC
H
H
H
H
H
OMe O
O
O
O
O
H
HO
HO
HO
H
O
H
CH3
H
H
O
O
O
CHO
H
H
H3CO
OH
H
H
O
OH
H
H
HO
HO
O
O
HO
1H-1H COSY
GlcO
HMBC
Figure 1. Key 1H–1H COSY and HMBC correlations of 1.
H
OGlc
Figure 2. Key NOESY correlations of 1.

W. Xiao et al. / Tetrahedron Letters 46 (2005) 5743–5746
Table 1. 1H and 13C NMR spectral data of 1 and 2 in pyridine-d5
5745
Position
Compound 1
J (Hz)
Compound 2
J (Hz)
b
dH
c
dC
b
c
dH
dC
1
3
4
5
6
5.82
7.73
d, 4.6
97.0 d
153.0 d
109.3 s
27.2 d
44.6 t
5.89
7.72
d, 4.7
97.3 d
152.4 d
111.2 s
29.2 d
32.9 t
s
—
s
—
—
—
3.64–3.72
2.40–2.49
2.81–2.90
9.79
m
3.21–3.29
1.77–1.85
2.27–2.35
4.69
m
m
m
m
t, 4.4
m
t, 5.6
7
200.9 d
134.2 d
44.8 d
120.0 t
166.5 s
96.5 d
151.5 d
112.5 s
31.4 d
39.6 t
103.2 d
135.4 d
44.7 d
119.2 t
166.7 s
96.4 d
151.5 d
112.7 s
32.9 d
39.7 t
8
5.67–5.71
2.86–2.89
5.08
m
5.68–5.72
2.83–2.88
5.10
m
9
10
m
2H, overlapped by H2O
m
2H, overlapped by H2O
—
11
10
—
5.66
—
d, 4.0
—
5.67
d, 3.9
s
—
30
7.64
—
s
—
7.67
—
40
50
3.17–3.25
1.74–1.82
2.31–2.39
5.18–5.24
2.04–2.12
2.23–2.31
0.89
m
3.19–3.27
1.76–1.84
2.37–2.45
5.20–5.26
2.05–2.13
2.24–2.31
0.91
m
60a
m
m
60b
m
m
70
m
77.1 d
39.9 d
46.5 d
13.2 q
167.4 s
—
m
77.0 d
39.9 d
46.6 d
13.2 q
167.5 s
53.2 q
52.2 q
51.1 q
100.8 d
74.9 d
79.1 d
71.5 d
78.6 d
62.7 t
80
m
m
90
m
m
100
3H, d, 6.8
—
—
3H, d, 6.7
—
110
—
—
—
3.27
7-OMe
7-OMe
110-OMe
1-Glc-100
1-Glc-200
1-Glc-300
1-Glc-400
1-Glc-500
1-Glc-600
3H, s
3H, s
s
—
3.53
—
s
—
3.28
3.54
51.0 q
100.7 d
74.9 d
79.1 da1
71.6 d
78.6 da2
62.7 t
5.40
d, 7.8
m
5.39
d, 7.9
m
4.13–4.19
4.22–4.29
4.28
4.13–4.19
4.22–4.28
4.28
m
m
Overlap
Overlap
Overlap
d, 11.0
d, 7.9
m
Overlap
Overlap
Overlap
d, 11.1
d, 7.8
m
4.03
4.40
4.02
4.39
4.56
5.36
4.54
5.33
10-Glc-1000
10-Glc-2000
10-Glc-3000
10-Glc-4000
10-Glc-5000
10-Glc-6000
100.9 d
74.7 d
79.0 da1
71.4 d
78.5 da2
62.7 t
—
100.8 d
74.6 d
79.1 d
71.3 d
78.6 d
62.7 t
—
4.11–4.17
4.20–4.26
4.28
4.10–4.16
4.20–4.26
4.28
m
m
Overlap
Overlap
Overlap
d, 11.0
Overlap
Overlap
Overlap
d, 11.1
4.03
4.40
4.02
4.39
4.56
4.54
a1,a2 Interchangeable.
b Measured at 500 MHz.
c Measured at 125 MHz.
connected to C-7 from the obvious HMBC cross-peaks
from the methoxyl singlets at dH 3.27 (3H, s) and 3.28
(3H, s) to C-7. Sugar analysis of 2 was carried out with
the same method used for 1 and the relative stereochem-
istry was established according to the NOESY experi-
ment and aided by Drieding molecular model as well.
Compound 2 was therefore identified as the 7,7-di-
methyl ether of 1.
Obviously, compounds 1 and 2 were derived from
two iridoid glucoside monomers, loganic acid (3) and
its 7,8-seco-analog (4), through an intermolecular
Table 2. Percentage inhibition of compounds 1–2 on the aggregation of rabbit platelets induced by PAF, AA, and ADP
Compound (10 mg/L)
Aggregation (%)
PAF (7.2 nmol/L)
AA (0.35 lmol/L)
ADP (3 lmol/L)
DMSO
1
60.3 2.9
42.9 3.5b
53.9 4.0
0.6 0.1b
72.6 3.3
69.2 3.6
73.6 2.8
69.5 3.2
68.9 2.5
66.8 2.4
2
BN52021
Aspirin
4.7 0.8b
65.9 5.3
a P < 0.05.
b P < 0.05, as compared with control (t-test). The data were expressed as means SD of four rabbits.

5746
W. Xiao et al. / Tetrahedron Letters 46 (2005) 5743–5746
H
H
H
11'
COOH
11
7
CHO COOH
OH
H
H
H
6'
6
COOH
O
H
4'
3'
4
1
5
9
OHC
7'
5'
3
HO
H
9'
O
+
10
O
O
8'
8
1'
10'
H
OH
O
H
OGlc
OGlc
H
OGlc OGlc
3
4
5
-H2O
H
H
H
H
H
H
O
H
O
O
O
O
methylation
H
O
H
OHC
OHC
O
O
OMe O
O
OH
H
H
OGlc OGlc
OGlc OGlc
1
6
Figure 3. Plausible biogenetic pathway for 1.
rearrangement (to 5) and then dehydration (to 6) and fi-
nally methylation, as shown for 1 in Figure 3. However,
how the rearrangement realized in plants is unknown
yet. Compounds 1 and 2 were evaluated for their in vitro
inhibitory activity against rabbit platelet aggregation
induced by PAF (platelet activating factor), AA (arachi-
donic acid), and ADP (adenosine diphosphate), using
the same bioassay methods as previously described.7
References and notes
1. Sun, H. D.; Lin, Z. W.; Niu, F. D. Acta Bot. Sin. 1978, 20,
244–254.
2. Deng, S. X.; Mo, Y. Q. Zhongcaoyao 1989, 20, 419–421.
3. Niu, X. M.; Li, S. H.; Jiang, B.; Zhao, Q. S.; Sun, H. D.
J. Asian Nat. Prod. Res. 2002, 4, 33–41.
4. Niu, X. M.; Li, S. H.; Wu, L. X.; Li, L.; Gao, L. H.; Sun, H.
D. Planta Med. 2004, 70, 1–4.
23.4
Ginkgolide
B (BN52021) and acetylsalicylic acid
5. Rapulaside A (1): Pale yellow amorphous powder; ½aꢀD
(ASA) were used as positive controls, and 2% PEG.
(polyethylene glycol) was used as contrast. Only the
trends of inhibition were observed for them (Table 2).
ꢁ69.2 (c 0.2, MeOH); UV (MeOH) kmax (lge): 198 (3.97),
235 (4.20), 389 (1.90) nm; IR (KBr) mmax: 3426, 2922, 1701,
1631, 1439, 1385, 1287, 1154, 1074, 768, 575 cmꢁ1; H and
1
13C NMR data see Table 1; negative FABMS m/z 745
[MꢁH]ꢁ; HRFABMS m/z: Found 746.2568, calcd.
746.2633 for C33H46O19.
6. Rapulaside B (2): Pale yellow amorphous powder; ½aꢀD
25.2
ꢁ
Acknowledgements
50.4 (c 0.4, MeOH); UV (MeOH) kmax (lge): 203 (4.10), 234
(4.17), 393 (1.70) nm; IR (KBr) mmax 3424, 2931, 1701, 1633,
This Project was supported by grants from the National
Natural Science Foundation of China (No. 30200349),
the Natural Science Foundation of Yunnan Province
(No. 2002C0017Q), and the Young Academic and Tech-
nical Leader Raising Foundation of Yunnan Province
(No. 2003RC07, awarded to Dr. S. Li).
1440, 1384, 1289, 1155, 1074, 767, 633, 575 cmꢁ1; H and
1
13C NMR data see Table 1; negative FABMS m/z 791
[MꢁH]ꢁ; HRFABMS m/z: Found 792.3015, calcd 792.3052
for C35H52O20.
7. Shen, Z. Q.; Li, L.; Wu, L. O.; Liu, W. P.; Chen, Z. H.
Platelets 1999, 10, 345–348.