Diterpenoid alkaloids from the lateral root of Aconitum carmichaelii

Article abstract of DOI:10.1021/np300225t

Twenty-six new diterpenoid alkaloids, 1-26 (1-4: hetisan-type C ₂₀-diterpenoid alkaloids; 5-26: aconitane C₁₉-diterpenoid alkaloids), and two known analogues, hypaconitine 27 and benzoylmesaconine 28, have been isolated from a water extract of the lateral root of Aconitum carmichaelii. Compounds 7 and 8 are rare examples of conformational isomers obtained from the same material. The conformation and conformational transformation of ring A in the C19- diterpenoid alkaloids are discussed on the basis of NMR data analysis in combination with single-crystal X-ray crystallography of 6 and 27 by anomalous scattering of Cu K7α radiation. In preliminary analgesic and toxicity assays, the isomer with ring A in the chair conformation (8 or 27) was found to be more active than that with ring A in the boat conformation (7 or 27a). In addition, 15, 16, and 19 showed neuroprotective activity.

Full text of DOI:10.1021/np300225t

Article
pubs.acs.org/jnp
Diterpenoid Alkaloids from the Lateral Root of Aconitum carmichaelii
Bingya Jiang, Sheng Lin, Chenggen Zhu, Sujuan Wang, Yanan Wang, Minghua Chen, Jianjun Zhang,
Jinfeng Hu, Naihong Chen, Yongchun Yang, and Jiangong Shi*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
S
* Supporting Information
ABSTRACT: Twenty-six new diterpenoid alkaloids, 1−26
(1−4: hetisan-type C20-diterpenoid alkaloids; 5−26: aconitane
C19-diterpenoid alkaloids), and two known analogues, hypaco-
nitine 27 and benzoylmesaconine 28, have been isolated from
a water extract of the lateral root of Aconitum carmichaelii.
Compounds 7 and 8 are rare examples of conformational
isomers obtained from the same material. The conformation
and conformational transformation of ring A in the C19-
diterpenoid alkaloids are discussed on the basis of NMR data analysis in combination with single-crystal X-ray crystallography of
6 and 27 by anomalous scattering of Cu Kα radiation. In preliminary analgesic and toxicity assays, the isomer with ring A in the
chair conformation (8 or 27) was found to be more active than that with ring A in the boat conformation (7 or 27a). In addition,
15, 16, and 19 showed neuroprotective activity.
he parent and lateral roots of Aconitum carmichaelii Debx.
(Ranunculaceae), which is widely distributed and
the extract. Among the C19-diterpenoid alkaloids, 5 and 8 have
ring A (C-1, C-2, C-3, C-4, C-5, and C-11) in the chair con-
formation, while 6, 7, and 9−26 have ring A in the boat
conformation. Compounds 7 and 8 are conformational isomers.
The boat and chair conformations of ring A in C19-diterpenoid
alkaloids were demonstrated by detailed NMR data and single-
crystal X-ray crystallographic analysis of 6 and 27 by anomalous
scattering of Cu Kα radiation. The boat conformation of ring A
in the C19-diterpenoid alkaloids was speculated to be stabilized
by an intramolecular hydrogen bond between the electron pair
on the N atom and the C-1 or C-3 OH group in ring A.15−17
However, acetylation of 7 and 8 afforded the same 3-mono-
acetate 7a having ring A in the boat conformation. Since there
was no OH group that would form intramolecular hydrogen
bonds in 7a and since ring A in 8 was transformed from the
more stable chair to the less stable boat conformation during
acetylation, we concluded that intramolecular hydrogen
bonding is not the key factor in stabilizing the boat con-
formation of ring A in aconitine alkaloids. As conformational
isomers have different chemical and physical properties,5a,15,17a
the boat and chair conformation of ring A can be denoted by
“A-b” and “A-c”, respectively, in the nomenclature of C19-
diterpenoid alkaloids. In addition, because of the lack of clarity
regarding the C-13 and C-19 configurations in hetisan C20-
diterpenoid alkaloids,18 confusing trivial names are used for
aconitine C20-diterpenoid alkaloids in the literature;2,4,6,7,15−17
hence, R and S representations are used for indicating the
configurations at C-13 and C-19 in 1−4, respectively, and
systematic IUPAC-recommended nomenclature based on the
T
cultivated in southwestern China, are indispensable drugs in
traditional Chinese medicine.1 The lateral roots, named “fu zi”
in Chinese, are used in raw or prepared forms to treat diseases
such as cadianeuria, neuralgia, and rheumatalgia in China,
Japan, and Korea.2,3 Previous chemical and pharmacological
studies have shown that toxic aconitine C19-diterpenoid
alkaloids are the main active constituents of these drugs. Up
to now, more than 35 aconitine alkaloids have been isolated
from different parts of this plant, including aconitine, hypaco-
nitine, mesaconitine, neoline, talatizamine (talatisamine),4
isotalatizidine, karakoline (karacoline), monoacetyltalatizamine,
senbusines A−C,2 fuziline,5 14-acetyltaltizamine, lipoaconitine,
lipohypaconitine, lipomesaconitine, lipodeoxyaconitine, benzoyl-
aconitine, benzoylhypaconitine, benzoylmesaconitine,6 hokbu-
sines A and B,7 beiwutine,8 neojiangyouaconitine,9 aldohypa-
conitine,10 14-O-acetylneoline, foresaconitine, crassicauline,
14-O-cinnamoylneoline, 14-O-anisoylneoline, 14-O-veratroyl-
neoline, lipoforesaconitine, lipoyunanaconitine,3 8-O-cinna-
moylneoline,11 and karakanine.12 In addition, two napelline
C20-diterpenoid alkaloids (songorine5 and songoramine12), a
hetisan C20-diterpenoid alkaloid (ignavine7), and several other
constituents3b have been reported. The complex structures,
interesting chemistry, and noteworthy pharmacological effects
of these alkaloids have been investigated in considerable
detail.13 A water extract of the lateral roots of A. carmichaelii has
been investigated as part of a program to study the chemical
diversity of traditional Chinese medicines and their biological
effects.14 We describe herein the isolation, structure elucidation,
and biological assays of four new C20 (1−4) and 22 new C19
(5−26) diterpenoid alkaloids, together with two known
analogues, hypaconitine 27 and benzoylmesaconine 28, from
Received: March 23, 2012
Published: May 18, 2012
© 2012 American Chemical Society and
American Society of Pharmacognosy
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names of the parent alkaloids “hetisan” and “aconitane”19 is
used in this paper.
the quaternary C-4 was attached to C-3, C-5, C-18, and C-19,
while H2-7/C-8, C-9, C-14, C-15; H-9/C-7, C-8, C-14, C-15;
H-14/C-7, C-8, C-9, C-15; and H2-15/C-7, C-8, C-9, C-14
correlations indicated linkage of the quaternary C-8 with C-7,
C-9, C-14, and C-15. HMBC correlations of H-20/C-6, C-8,
C-13, C-14, C-19, together with their shifts, showed that C-20
was linked to C-14 and via the N atom to C-6 and C-19. H-5/
H-6 and H-11/H-12 coupling correlations were not observed in
1
the H−1H COSY spectrum of 1 because the dihedral angles
for the two pairs of vicinal protons were nearly 90°. However;
the connection between C-5 and C-6 was indicated by HMBC
correlations of H-6/C-10 and H2-7/C-5, while the connection
between C-11 and C-12 was indicated by HMBC correlations
of H-9/C-11, C-12; H-11/C-13, C-16; H-12/C-9, C-11, C-13,
C-14; and H-13/C-11. The above information revealed the
hetisan skeleton in 1.20 Furthermore, the HMBC spectrum
showed correlations of H-1 and H-11 with the carbonyl carbons
of the two acetoxy groups and a correlation of H-2 with the
carbonyl carbon of the benzoyl moiety, indicating that the
acetoxy groups were present at C-1 and C-11 and the
benzoyloxy group at C-2 in 1. The chemical shifts of H-13
and H-19 (Table 1) suggested OH groups at C-13 and C-19.
Therefore, 1 had a planar structure, 1,11-diacetoxy-2-
benzoyloxy-13,19-dihydroxyhetisan. In the nuclear Overhauser
effect (NOE) difference spectrum of 1, irradiation of H-20
enhanced the H-1, H-14, H-19, and H-2′/H-6′ resonances,
while irradiation of H-13 enhanced the H-12 and H-14
resonances, indicating that these protons were oriented on the
same side of the ring system. In addition, irradiation of H-6
enhanced the H-5 and H3-18 resonances, while irradiation of
H-11 enhanced the H-9, H-12, and H-15a resonances,
indicating these protons were oriented on the other side of
the ring system. The coupling constants J1,2 (2.5 Hz), J9,11
(9.0 Hz), and J13,14 (9.0 Hz) were consistent with those of
tadzhacotine,20 the 19-dehydroxy analogue of 1 isolated from
Aconitum zwravschanicun, and its structure was determined by
X-ray crystallographic analysis. These data suggested that 1 had
the same configuration as tadzhacotine, with a 19β-OH group.
Since the absolute configuration of the hetisan nucleus was
repeatedly confirmed by the X-ray crystallographic analysis of
analogues isolated from species of the same genus,21,22 it was
proposed that the absolute configuration of the hetisan nucleus
was retained in 1. Thus, compound 1 was (+)-(13R,19S)-
1β,11α-diacetoxy-2α-benzoyloxy-13,19-dihydroxyhetisan.
The spectroscopic data of compound 2 indicated that it was
an analogue of 1. Comparison of the NMR data of 2 with those
of 1 (Table 1) indicated that the benzoyloxy and acetoxy
groups in 1 were substituted by propionyloxy and 2-methyl-
butyryloxy moieties in 2, respectively, and that the oxymethine
(CH-1) group of the nucleus in 1 was replaced by a methylene
unit (CH2-1) in 2. This was confirmed by the 2D NMR data
analysis of 2. In particular, COSY correlations of H3-5′/H-2′/
H2-3′/H3-4′ and HMBC correlations from H-2′, H2-3′, H3-5′,
and H-13 to C-1′ confirmed the presence of the 2-methyl-
butyryloxy moiety at C-13 in 2. HMBC correlations from H2-2″
and H3-3″ to C-1″, in combination with the H-2 shift, proved
that the propionyloxy moiety was located at C-2. In addition,
the NMR data of 2 indicated the presence of an N-CH3 group
[δH 3.03 (s) and δC 34.6],18 suggesting that 2 was a quaternary-
N base. This was confirmed by HMBC correlations of N-CH3/
C-6, C-19, C-20 and by (+)-HRESIMS, which showed a
pseudomolecular ion peak at m/z 500.3020 (500.3007 calcd for
C29H42NO6). Since trifluoroacetic acid (TFA) was used in the
RESULTS AND DISCUSSION
■
The molecular formula of compound 1 was established as
C31H35NO8 on the basis of high-resolution electrospray ioniza-
tion mass spectroscopy (HRESIMS) and NMR data (Ex-
perimental Section and Table 1). The IR spectrum of 1 showed
absorptions due to the OH (3394 cm−1) and carbonyl (1719
cm−1) functionalities. The NMR data of 1 showed the presence
of a tertiary methyl group, three methylenes, 11 methines (four
of them oxymethines), three quaternary carbons, and an
exocyclic terminal double bond, in addition to a benzoyl and
two acetyl groups. These spectroscopic data suggested that 1
was a C20-diterpenoid alkaloid triester.7 The structure of 1 was
confirmed by 2D NMR data analysis. The proton and
corresponding carbon resonances in the 2D NMR spectra of
1 were assigned by the gradient heteronuclear single quantum
coherence (gHSQC) experiment. The H-1/H-2/H2-3, H-6/H2-7,
H-9/H-11, and H-14/H-13/H-12/H2-17/H2-15 coupling
1
correlations in the H−1H correlation spectroscopy (COSY)
spectrum of 1 revealed three vicinal and one allylic spin
systems. The HMBC spectrum of 1 showed two- and three-
bond correlations: H-1/C-2, C-3, C-5, C-10; H-5/C-9, C-10,
C-20; H-9/C-5, C-10, C-20; and H-20/C-1, C-5, C-9. This
demonstrated that the quaternary C-10 was linked to C-1, C-5,
C-9, and C-20. HMBC correlations of H-5/C-18, C-19; H3-18/
C-3, C-4, C-5, C-19; and H-19/C-3, C-5, C-18 indicated that
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a
Table 1. NMR Spectroscopic Data for Compounds 1−4.
b
c
d
e
1
2
3
4
no.
1a
δH
δC
δH
δC
δH
δC
δH
δC
5.98 d (2.5)
70.3
3.35 brd (16.2)
31.8
3.36 d (16.8)
31.7
3.34 d (16.5)
31.8
1b
2
2.15 dd (16.2, 4.8)
5.21 m
2.15 dd (16.8, 5.4)
5.23 m
2.18 dd (16.5, 5.0)
5.26 m
5.67 m
68.5
33.2
68.7
36.5
68.6
36.4
68.6
36.5
3a
3b
4
2.17 brd (16.0)
2.07 m
2.02 brd (15.6)
1.75 dd (15.6, 4.2)
2.03 brd (15.6)
1.77 dd (15.6, 4.2)
1.97 brd (15.5)
1.80 dd (15.5,4.5)
41.7
55.6
61.6
34.3
40.8
58.7
66.8
32.0
40.6
56.5
69.3
67.4
40.6
56.4
69.4
67.3
5
2.52 brs
2.28 brs
2.41 brs
2.42 brs
3.79 brs
4.19 brs
6
4.44 brs
3.88 brs
3.77 brs
7a
7b
8
2.17 dd (11.0, 5.0)
2.09 m
2.20 dd (15.0, 3.0)
2.05 dd (15.0, 3.0)
4.19 d (3.0)
44.9
51.9
54.2
75.5
49.5
70.3
50.3
33.4
44.7
55.7
52.2
74.0
48.5
73.2
45.7
33.2
52.0
50.0
50.1
73.8
48.7
73.1
44.3
30.3
51.9
50.1
50.2
73.8
48.7
73.2
44.3
30.2
9
2.66 d (9.0)
2.33 d (9.0)
2.68 d (10.2)
2.68 d (9.5)
10
11
12
13
14
15a
15b
16
17a
17b
18
19
20
N-CH3
1′
5.32 d (9.0)
4.35 d (9.0)
2.60 brs
4.29 brd (10.2)
2.61 brs
4.29 d (9.5)
2.61 d (2.5)
5.19 brd (10.0)
3.21 d (10.0)
2.71 d (19.0)
2.18 d (19.0)
2.49 d (2.5)
4.21 brd (9.0)
2.64 d (9.0)
5.13 d (9.6)
3.19 d (9.6)
2.40 d (17.4)
2.26 d (17.4)
5.18 brd (10.2)
3.21 brd (10.2)
2.69 brd (18.0)
2.20 d (18.0)
2.57 brd (17.5)
2.26 brd (17.5)
144.9
109.4
145.6
109.3
145.6
109.4
145.6
109.3
4.87 brs
4.79 brs
1.17 s
4.89 brs
4.74 brs
1.09 s
4.92 brs
4.78 brs
1.07 s
4.92 brs
4.78 brs
1.08 s
22.0
92.5
62.5
22.3
101.0
73.6
34.6
176.0
41.6
26.8
22.1
102.4
72.9
35.5
176.0
41.5
26.9
22.1
102.6
73.0
35.5
176.0
41.5
26.9
5.38 brs
4.99 s
5.89 brs
4.30 s
5.99 brs
4.31 s
5.97 brs
4.27 s
3.03 s
3.06 s
3.08 s
130.8
130.4
129.6
2′
8.05 d (7.5)
7.49 t (7.5)
2.33 heptet (7.2)
1.66 d pentet (13.8,7.2)
1.44 d pentet (13.8,7.2)
0.89 t (7.2)
2.33 heptet (7.2)
1.64 d pentet (13.8,7.2)
1.44 d pentet (13.8,7.2)
0.88 t (7.2)
2.31 heptet (7.5)
1.64 d pentet (14.0,7.5)
1.42 d pentet (14.0,7.5)
0.88 t (7.5)
3′a
3′b
4′
7.65 t (7.5)
7.49 t (7.5)
8.05 d (7.5)
134.2
129.6
130.4
165.5
11.7
17.2
11.7
17.4
11.7
17.4
5′
1.15 d (7.2)
1.15 d (7.2)
1.17d (7.5)
6′
7′
a
1H NMR data (δH) were measured in Me2CO-d6 at 500 MHz for 1 and 4 and at 600 MHz for 2 and 3, respectively. Proton coupling constants (J)
in Hz are given in parentheses. 13C NMR data (δC) were measured in Me2CO-d6 at 125 MHz for 1 and 4 and at 150 MHz for 2 and 3, respectively.
b
The assignments were based on H−1H COSY, HSQC, and HMBC experiments. Data for OAc units in 1: δH 1.90 (3H, s, OAc-1), 2.08 (3H, s,
1
c
OAc-11); δC 171.0, 21.4 (OAc-1); 170.5, 21.2 (OAc-11). Data for propionyl unit in 2: δH 2.58 (1H, dq, J = 16.2, 7.2 Hz, H-2″a), 2.33 (1H, dq, J =
d
16.2, 7.2 Hz, H-2″b), 1.04 (3H, t, J = 7.2 Hz, H-3″); δC 173.9 (C-1″), 28.1 (C-2″), 9.1 (C-3″). Data for propionyl unit in 3: δH 2.59 (1H, dq, J = 16.2,
7.2 Hz, H-2″a), 2.32 (1H, dq, J = 16.2, 7.2 Hz, H-2″b), 1.05 (3H, t, J = 7.2 Hz, H-3″); δC 173.9 (C-1″), 28.1 (C-2″), 9.1 (C-3″). Data for isobutyryl
e
unit in 4: δH 2.72 (1H, dq, J = 7.0 Hz, H-2″), 1.10 (3H, d, J = 7.0 Hz, H-3″), 1.11 (3H, d, J = 7.0 Hz, H-4″); δC 176.7 (C-1″), 34.5 (C-2″), 19.1
(C-3″), 20.0 (C-4″).
HPLC isolation procedure, 2 was expected to be obtained as a
trifluoroacetate, but the absence of trifluoroacetic ion
resonances in the 13C NMR spectrum suggested that 2 was
obtained as a quaternary amine hydroxide. This was supported
by (−)-ESIMS, which exhibited a quasi-molecular ion at m/z
515 [M − H]−. In the NOE difference spectrum of 2,
irradiation of H-2 enhanced the H-3b and H-1b resonances,
and irradiation of H-3b enhanced the H3-18 resonances. H-20
and H-3a resonances were enhanced upon irradiation of H-19.
These enhancements revealed that the 2-propionyloxy and
19-OH groups in 2 were α- and β-oriented, respectively.
Comparison of the coupling constants J9,11 and J13,14 in 2 with
those for 1 (Table 1) suggested that 2 had the same 11α,13R
configuration as 1. Hydrolysis of 2 with 5% NaOH yielded a
Therefore, compound 2 was defined as (−)-(13R,19S)-
11α,19-dihydroxy-N-methyl-13-(S-2-methylbutyryloxy)-2α-
propionyloxyhetisanium hydroxide.
The NMR and IR data of compound 3 were similar to those
of 2 (Table 1 and Experimental Section). Comparison of the
NMR data of 3 with those of 2 indicated that one methylene
unit of the nucleus in 2 was replaced by an oxymethine [δH 4.19
(d, J = 3.0 Hz) and δC 67.4] in 3. In addition, H-5, H-9, H-15a,
C-6, and C-8 in 3 were deshielded by ΔδH +0.13, ΔδH +0.35,
ΔδH +0.29, ΔδC +2.5, and ΔδC +7.3 ppm, respectively. On the
other hand, H-6, C-5, C-9, and C-15 were shielded by
ΔδH −0.11, ΔδC −2.2, ΔδC −5.7, and ΔδC −2.9 ppm, respectively.
This suggested that 3 was a 7-hydroxy derivative of 2, which
was confirmed by HRESIMS and 2D NMR data. In particular,
the COSY correlation of H-6/H-7 and HMBC correlations of
H-7/C-5, C-9, in combination with the shifts of these proton
mixture of 2-methylbutyric acid and propionic acid with [α]20
D
+11.2 (c 0.06, MeOH), indicating a 2′S configuration.
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and carbon resonances, confirmed the OH-7 group in 3. NOE
enhancement of the N-CH3 resonance upon irradiation of H-7
indicated that the OH-7 group was β-oriented. The con-
figuration of the 2-methylbutyryloxy moiety in 3 was proved as
described above. Therefore, compound 3 was determined to be
(−)-(13R,19S)-7β,11α,19-trihydroxy-N-methyl-13-(S-2-methyl-
butyryloxy)-2α-propionyloxyhetisanium hydroxide.
Spectroscopic data showed that compound 4 was a higher
homologue of 3 with an additional CH2 unit (Table 1 and
Experimental Section). Comparison of the NMR data of 3 and
4 demonstrated that the only difference between these
compounds was replacement of the 2-propionyloxy moiety in
3 by a 2-isobutyryloxy moiety in 4. Thus, 4 was assigned as
(+)-(13R,19S)-2α-isobutyryloxy-7β,11α,19-trihydroxy-N-meth-
yl-13-(S-2-methylbutyryloxy)hetisanium hydroxide, which was
confirmed by 2D NMR and NOE difference experiments.
The molecular formula of compound 5 was C34H45NO11 (by
HRESIMS and NMR data). The NMR data indicated four
methylene (one oxymethylene), nine methine (four oxy-
methines), and six quaternary carbons (a carbonyl and two
oxygen-bearing), in addition to a benzoyl, an acetoxy, an N-
ethyl, and four methoxy substituents, suggesting that 5 was a
typical aconitane C19-diterpenoid alkaloid.7,16 Comparison of
the NMR data of 5 with those of 3-deoxyaconitine16 indicated
that the CH2-19 unit in deoxyaconitine was replaced by a
carbonyl group (δC 173.0) in 5, suggesting that 5 was 19-oxo-3-
deoxyaconitine. The 2D NMR and NOE difference experi-
ments confirmed the NMR data and configuration assignments
of 5. In particular, HMBC correlations of C-19 with H2-3, H-5,
H-17, and H2-20 confirmed the 19-oxo group in 5, while
HMBC correlations of H-14/C-1′, OCH3-1/C-1, OCH3-6/C-6,
OCH3-16/C-16, and OCH3-18/C-18 confirmed the locations
of the benzoyloxy and four methoxy groups. The chair
conformation of ring A in 5 was revealed by coupling constants
J1,2a ≈ J1,2b ≈ 7.5 Hz.2,10a,c,12b,15−17 Thus, compound 5 was
assigned as (−)-(A-c)-8β-acetoxy-14α-benzoyloxy-N-ethyl-
13β,15α-dihydroxy-1α,6α,16β,18-tetramethoxy-19-oxo-aconi-
tane; the configuration of the aconitane nucleus was
determined by X-ray crystallographic analysis of several
analogues from Aconitum plants.17
Figure 1. ORTEP diagram of compound 6.
effects rather than the conformational transformation. This
assumption was supported by the similarity between the
splitting patterns and coupling constants for H-1 (dd, J = 7 and
10 Hz) in the triacetates of senbusines A and C in CDCl3 and
pyridine-d6.2
While the spectroscopic data of compound 7 were similar to
those of beiwutine,26 NMR and HRESIMS data indicated
replacement of the OAc group in beiwutine by an OCH3 group
in 7. Thus, 7 was thought to be 8-deacetoxy-8-methoxybeiwu-
tine, which was confirmed by 2D NMR data analysis. HMBC
correlations from OCH3, H-6, H-7, H-9, H-14, and H-15 to C-8
proved the presence of the OCH3-8 group in 7, while NOESY
correlations of H-6/H2-18/H-3/H-5/H-2b/H-1/H-12b/H-14/
H-9/H-6/CH3O-8/H-15/CH3O-16 confirmed that the config-
uration of 7 was identical to that of beiwutine. Similarity of the
splitting patterns and coupling constants of H-1 and H-3 in 6,
7, and beiwutine26 indicated that ring A was in the boat
conformation in these three compounds. Thus, compound 7
was determined to be (−)-(A-b)-14α-benzoyloxy-
3α,10β,13β,15α-tetrahydroxy-1α,6α,8β,16β,18-pentamethoxy-
N-methylaconitane.
Spectroscopic data, including 2D NMR data in CDCl3 and
acetone-d6, indicated that compound 8 had the same planar
structure and configuration as 7. However, comparison of the
NMR data of 8 and 7 in the same solvent, acetone-d6,
demonstrated that in 8 H-1, H-3, H-5, H-7, H-12b, H-17, and
H2-19 were shielded by more than ΔδH −0.2 and that N-CH3,
C-17, C-18, and C-19 were shielded by more than ΔδC −1.7
ppm. On the other hand, H-2b was deshielded by more than
ΔδH +0.5, and C-2, C-5, C-6, C-9, and C-12 were deshielded by
more than ΔδC +1.0 ppm. Particularly, the H-1 and H-3
resonances in 8 were split into doublets of doublets with
coupling constants of about 6 and 8 Hz, respectively. Since the
chemical shifts and coupling constants change because of the
conformational change in ring A of aconitine derivatives,16,17,27
the difference in the NMR data of 8 and 7 suggested that 8 was
the conformational isomer of 7, with ring A in the chair
conformation. This was confirmed by the fact that acetylation
of both 7 and 8 generated the same product, 7a. However, the
splitting patterns and coupling constants for H-1 and H-3 in the
1H NMR spectrum of 7a were consistent with those of 7. This
revealed that ring A in 7a had the boat conformation. However,
the assumption that the boat conformation of this ring in
Compound 6 had a planar structure and configuration
consistent with those of aconifine,23 as demonstrated by
spectroscopic data analysis, including 2D NMR experiments.
However, as compared with the reported data of aconifine, C-1,
C-2, C3, and C-12 in 6 were shielded by ΔδC −1.9, −1.8, −1.3,
and −1.9 ppm, respectively, whereas C-17, C-18, and C-19
were deshielded by ΔδC +3.8, +2.3, and +3.3 ppm. In particular,
the 1H NMR spectrum of 6 showed broad singlets (W1/2 ≈ 6 Hz)
due to H-1 and H-3, while H-3 in aconifine monoacetate was
reported to be a quartet with coupling constants of 10 and 7 Hz
(the corresponding data for aconifine were absent in the
literature). This, combined with the chair conformation of ring
A in aconifine, as determined by X-ray crystallographic
analysis,24 suggested that ring A in 6 was in the boat confor-
mation. The boat conformation was confirmed by single-crystal
X-ray crystallographic analysis of 6 (Figure 1). Thus, com-
pound 6 was confirmed to be (−)-(A-b)-8β-acetoxy-14α-
benzoyloxy-N-ethyl-3α,10β,13β,15α-tetrahydroxy-1α,6α,16β,18-
tetramethoxyaconitane.
Although ring A in aconifine23 and aconine25 was reported to
undergo chair (in CDCl3)-to-boat (in pyridine-d6) trans-
formation, causing changes in the C-2, C-3, and C-4 shifts in
these two solvents, the shift changes should be due to solvent
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aconitine alkaloids is stabilized by intramolecular hydrogen
bonding between the electron pair on the N atom and the
OH-1 or OH-3 group16,17 may not be valid because 7a does not
have any OH group that would be involved in intramolecular
hydrogen bonding. Ring A in 8 underwent chair-to-boat
conformational transformation upon acetylation. Thus, com-
pound 8 was assigned the name (−)-(A-c)-14α-benzoyloxy-
3α,10β,13β,15α-tetrahydroxy-1α,6α,8β,16β,18-pentamethoxy-
N-methylaconitane.
The spectroscopic data of compound 9 demonstrated that it
was a homologue of 7 with an N-CH2CH3 group replacing the
N-CH3 group. This was confirmed by the H-17/C-5, C-6, C-8,
C-10, C-11, C-20 correlations in the HMBC spectrum of 9.
Hence, compound 9 was determined to be (−)-(A-b)-
14α-benzoyloxy-N-ethyl-3α,10β,13β,15α-tetrahydroxy-
1α,6α,8β,16β,18-pentamethoxyaconitane.
The HRESIMS data of compound 10 indicated that it was
another homologue of 7 having the molecular formula
C31H43NO11. Comparison of the NMR data of 10 and 7
(Tables 2 and 5) revealed that the OCH3 group of 7 was
replaced by an OH group in 10. In addition, in the case of 10,
the H-5, H-7, and H-16 resonances were shielded by ΔδH
−0.11, −0.32, and −0.16, respectively. The C-8 and C-16
resonances were shielded by ΔδC −5.1 and −2.0 ppm,
respectively. On the other hand, H-6, H-18b, C-7, and C-15
were deshielded by ΔδH +0.05, ΔδH +0.16, ΔδC +5.8, and ΔδC
+4.8 ppm, respectively. This suggested that 10 was the 8-
hydroxy derivative of 7, which was confirmed by 2D NMR
analysis. Therefore, compound 10 was identified as (−)-(A-b)-
14α-benzoyloxy-3α,8β,10β,13β,15α-pentahydroxy-
1α,6α,16β,18-tetramethoxy-N-methylaconitane.
conformation by X-ray crystallographic analysis.29 Thus,
compound 13 was confirmed to be (−)-(A-b)-8β-acetoxy-
14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-1α,6α,16β,18-tet-
ramethoxyaconitane.
Comparison of the NMR data of 14 with those of 13 (Tables
3 and 5) indicated the absence of acetyl and 15-OH groups in
14. The four methoxy groups were proved to be at C-1, C-6,
C-16, and C-18 by 2D NMR data analysis. COSY correlations
of H-10/H-9/H-14 and H2-15/H-16 and HMBC correlations
of H-9/C-7, C-8, C-12, C-13, C-14 and H-14/C-8, C-9, C-13,
C-16, together with their shifts, confirmed the presence of two
hydroxyl groups at C-8 and C-13. Therefore, compound 14 was
(−)-(A-b)-14α-benzoyloxy-N-ethyl-8β,13β-dihydroxy-
1α,6α,16β,18-tetramethoxyaconitane.
The spectroscopic data of compound 15 revealed that it had
a planar structure and configuration identical to that of benzoyl-
aconitine.30 However, the difference between the NMR data of
15 and benzoylaconitine was identical to that between the
NMR data of 6 and aconifine. This demonstrated that 15 was
the conformational isomer of benzoylaconitine with ring A in
the boat conformation. Therefore, compound 15 was
determined as (−)-(A-b)-14α-benzoyloxy-N-ethyl-3α,8β,13β,
15α-tetrahydroxy-1α,6α,16β,18-tetramethoxyaconitane.
Compound 16 was the conformational isomer of manshur-
itine,31 with ring A in the boat conformation, as indicated by
spectroscopic data and confirmed by 2D NMR experiments.
The difference in the NMR data of 16 and manshuritine was
similar to that in the NMR data of 15 and benzoylaconitine.
Thus, compound 16 was assigned the name (−)-(A-b)-8β,14α-
dibenzoyloxy-N-ethyl-3α,13β,15α-trihydroxy-1α,6α,16β,18-tet-
ramethoxyaconitane.
HRESIMS indicated that the molecular formula of
compound 11 was C34H47NO11. Comparison of the NMR
data of 11 and 7 demonstrated that the OCH3-8, N-CH3, and
hydroxymethine (CHOH-15) units in 7 were replaced by OAc,
N-CH2CH3, and methylene units in 11, respectively. This
suggested that 11 was (−)-(A-b)-8β-acetoxy-14α-benzoyloxy-
N-ethyl-3α,10β,13β-trihydroxy-1α,6α,16β,18-tetramethoxyaco-
nitane, which was supported by 2D NMR and NOE data.
Compound 12 was another derivative of 7, as indicated by
spectroscopic data. The NMR data revealed replacement of the
OCH3-8 group in 7 by an OAc-8 unit in 12, while the OH-3
group in 7 was absent in 12. This indicated that 12 was the
3-dehydroxy-8-acetoxy derivative of 7, which was confirmed by
2D NMR and NOE analyses. In the NOE difference spectrum
of 12, enhancement of the OAc resonance upon irradiation of
H-15 confirmed the 8β-OAc group. Thus, compound 12 was
determined to be (−)-(A-b)-8β-acetoxy-14α-benzoyloxy-
10β,13β,15α-trihydroxy-1α,6α,16β,18-tetramethoxy-N-methyla-
conitane.
The spectroscopic data of compound 17 indicated that it was
the conformational isomer of neojiangyouaconitine, with ring A
in the boat conformation. This was confirmed by 2D NMR and
NOE data analysis, which amended the NMR assignment of
this compound. Therefore, compound 17 was determined as
(−)-(A-b)-14α-benzoyloxy-N-ethyl-13β,15α-dihydroxy-
1α,6α,8β,16β,18-pentamethoxyaconitane.
Compound 18 was a homologue of 14. 1H−1H COSY
correlations of H-6a/H-6b/H-7 and HMBC correlations from
H-7 to C-5, C-8, C-9, C-11, and C-17, together with the shifts
of these proton and carbon resonances, indicated the absence
of any substituent at C-6 in 18. The COSY cross-peak of H-15/
H-16 and HMBC correlations of H-14/C-8, C-9, C-13, C-16
and H-15/C-7, C-8, C-16, in combination with their shifts,
demonstrated the presence of OH groups at C-8, C-13, and
C-15. In addition, the HMBC correlations of H-14/C-7′,
OCH3-1/C-1, OCH3-16/C-16, and OCH3-18/C-18 confirmed
the locations of the OCH3 and benzoyloxy substituents in 18.
Therefore, compound 18 was determined to be (−)-(A-b)-14α-
benzoyloxy-N-ethyl-8β,13β,15α-trihydroxy-1α,16β,18-tri-
methoxyaconitane, whose configuration was supported by the
NOE difference data.
The spectroscopic data of compound 13 demonstrated that it
had a planar structure and configuration identical to that of
3-deoxyaconitine.16,26,28 However, C-1, C-2, C-3, C-5, and C-
18 in 13 were shielded by about ΔδC −4.7, −4.3, −8.6, −7.7,
and −2.7 ppm, respectively, as compared with the reported data
for 3-deoxyaconitine in the same solvent, CDCl3, whereas C-19
was deshielded by about ΔδC +3.5 ppm. The H-1 resonance
The spectroscopic data of 19 indicated that it was an isomer
of 14. The vicinal coupling correlations of H-10/H-9/H-14/H-
1
13 and H-15/H-16, together with their shifts, in the H−1H
COSY spectrum of 19 indicated that OH-13 in 14 was replaced
by OH-15 in 19. This was confirmed by correlations from H-13
and H-9 to C-10 and C-14 in the HMBC spectrum of 19.
HMBC correlations of OCH3-1/C-1, OCH3-8/C-8, OCH3-16/
C-16, and OCH3-18/C-18 confirmed the locations of the
OCH3 groups in 19. HMBC correlations of H-6/C-4, C-5, C-7,
C-8, C-17, along with their shifts, revealed the presence of an
1
appeared as a broad singlet (W1/2 ≈ 6 Hz) in the H NMR
spectrum of 13 (acetone-d6 or CDCl3), while the 1H NMR data
of 3-deoxyaconitine did not include the H-1 resonance.16,26,28
This indicated that 13 was the conformational isomer of
3-deoxyaconitine with ring A in the boat conformation, since
ring A in the latter was confirmed to be in the chair
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OH-6 group. In the NOE difference spectrum of 19, irradiation
of H-1 enhanced the H-10 resonance, and irradiation of H-6
enhanced the H-5, H-7, OCH3-8, and H-9 resonances.
Irradiation of H-14 enhanced the H-9, H-10, and H-13
resonances, while irradiation of H-15 caused enhancement of
the OCH3-16 and H-7 resonances. These enhancements
revealed that these protons were cofacial. Therefore, compound
19 was assigned the name (−)-(A-b)-14α-benzoyloxy-N-ethyl-
6α,15α-dihydroxy-1α,8β,16β,18-tetramethoxyaconitane.
Compound 20 was indicated to be a demethyl analogue of
19 by spectroscopic data. Comparison of the NMR data of 20
and 19 demonstrated that H-7 and C-8 in 20 were shielded by
ΔδH −0.31 and ΔδC −4.4 ppm, respectively. On the other
hand, H-6, H-15, C-7, and C-15 were deshielded by ΔδH +0.08,
ΔδH +0.07, ΔδC +6.8, and ΔδC +4.1 ppm, respectively. This
suggested that the OCH3-8 moiety in 19 was replaced by an
OH-8 group in 20 and was confirmed by the 2D NMR and
NOE difference data for 20. Thus, compound 20 was defined as
(−)-(A-b)-14α-benzoyloxy-N-ethyl-6α,8β,15α-trihydroxy-
1α,16β,18- trimethoxyaconitane.
confirmed to be (−)-(A-b)-14α-cinnamoyloxy-N-ethyl-
1α,8β,15α-trihydroxy-6α,16β,18-trimethoxyaconitane.
Compound 26 was the conformational isomer of penduline
(3,13-dideoxyaconitine),32 with ring A in the boat conforma-
tion, as indicated by spectroscopic data and confirmed by 2D
and NOE experiments. Thus, 26 was determined as (−)-(A-b)-
8β-acetoxy-14α-benzoyloxy-N-ethyl-15α-hydroxy-
1α,6α,16β,18-tetramethoxyaconitane.
The known compounds were identified as hypaconitine 27
and benzoylmesaconine 28 by comparison of their
spectroscopic data with those reported in the literature.4,6
Although aconitane alkaloids with ring A in the
chair2,10a,c,12b,15−17,23,24,26,33 and boat3a,5a,11,12b,15,16,34−36 con-
formations have been reported, the conformation of ring A has
not been clarified. Compounds 7 and 8 are the first examples of
conformational isomers of aconitane alkaloids simultaneously
isolated from the extract of the same plant. Because of the
different physical and chemical properties of the conformational
isomers, the conformation of ring A in aconitane alkaloids
should be defined by structure elucidation and nomenclature.
13C NMR analysis of aconitane alkaloids16 showed that the 13
C
The spectroscopic data of compound 21 indicated that it
differed from 19 only in that an OCH3 group was substituted
by an OCH2CH3 unit. 2D NMR analysis, especially the HMBC
correlation of OCH2CH3/C-8, confirmed that the OCH2CH3
group was at C-8 in 21. Thus, compound 21 was determined to
be (−)-(A-b)-14α-benzoyloxy-8β-ethoxy-N-ethyl-6α,15α-dihy-
droxy-1α,16β,18-trimethoxyaconitane.
chemical shift changes for C-1, C-2, C-3, and C-12 upon
protonation of the nitrogen in pyrodelphine with HCl and
AcOH were due to the conformational change in ring A. This
result prompted us to examine this conformational change,
which possibly occurred during HPLC isolation using a mobile
phase containing TFA, in all the compounds considered.
Hypaconitine 27 and benzoylmesaconine 28 were obtained
without the use of TFA. The chair conformation of ring A in 27
and 28 was indicated by NMR spectra recorded in acetone-d6
(Supporting Information, Table S7), especially by the H-1
resonance at δ 3.10 (dd, J = 10.5 and 6.5 Hz) for 27 and the H-
3 resonance at δ 3.83 (dd, J = 12.5 and 5.0 Hz) for 28. This was
confirmed by X-ray crystallographic analysis of 27 (Supporting
Information, Figure S2).33 Two samples (27a and 28a) were
prepared by passing 27 and 28 through an HPLC column using
30% MeCN in H2O (containing 0.1% TFA) as the mobile
Comparison of the NMR data of compound 22 with those of
20 indicated that the OH-6 group in 22 was absent and that C-
5 and C-7 were shielded by ΔδC −3.8 and −10.3 ppm,
1
respectively. H−1H COSY correlations of H-5/H2-6/H-7 and
HMBC correlations of H2-6/C-4, C-7, C-5, C-8, C-11, C-17
confirmed that 22 was the 6-dehydroxy derivative of 20.
Therefore, compound 22 was assigned as (−)-(A-b)-14α-
benzoyloxy-N-ethyl-8β,15α-dihydroxy-1α,16β,18-trimethoxya-
conitane.
The NMR data of compound 23 showed that there were
only two OCH3 groups and that C-1 was shielded by ΔδC
−10.2 ppm, as compared to those of 22, whereas H-1 and C-2
were deshielded by ΔδH +0.43 and ΔδC +6.6 ppm, respectively.
This revealed that 23 was an analogue of 22, with the OCH3-1
group substituted by an OH-1 group. This was supported by
2D NMR data that amended the NMR assignment of 23.
Therefore, compound 23 was confirmed to be (−)-(A-b)-14α-
benzoyloxy-N-ethyl-1α,8β,15α-trihydroxy-16β,18-dimethoxya-
conitane.
Comparison of the NMR data of compound 24 with those of
23 indicated that the methylene unit (CH2-6) in 23 was
replaced by an OCH3-substituted methine moiety (CH3OCH-
6) in 24. Meanwhile, the H-5, C-5, and C-7 resonances in 24
were deshielded by ΔδH +0.35, ΔδC +3.2, and ΔδC +8.1 ppm,
respectively, and H-7 was shielded by ΔδH −0.16 ppm. This
suggested that 24 was the 6α-methoxy derivative of 23, which
was proved by HMBC correlations of H-6/C-4, C-5, C-7, C-8,
C-17, OCH3 and NOESY correlations of H-6 with H-5, H-7,
and H-9. Thus, 24 was assigned the name (−)-(A-b)-14α-
benzoyloxy-N-ethyl-1α,8β,15α-trihydroxy-6α,16β,18-trimeth-
oxyaconitane.
1
phase. H NMR spectra showed that H-1 in 27a and H-3 in
28a gave broad singlet peaks and resonated at δ 3.70 and 4.30,
respectively. In addition, as compared with those of 27, the C-1,
C-2, C-3, and C- 5 resonances in the case of 27a were shielded
by ΔδC −4.3, −4.7, −7.9, and −5.1 ppm, respectively. This
change was consistent with that observed for pyrodelphine after
protonation of the nitrogen atom16 and with the difference in
the NMR data between 7 and 8. This indicated that ring A in 6,
7, and 9−26 would change to the boat conformation during
HPLC isolation using a mobile phase containing TFA. As
discussed in the structure determination of 8, intramolecular
hydrogen bonding is not the key to the stabilization of the boat
conformation of ring A in aconitane alkaloids. This was further
supported by acetylation of 28 to yield two products, 15-
monoacetate 28b and 3,15-diacetate 28c, in which ring A was
in the boat conformation, as revealed by NMR data. We
speculate that the boat conformation of ring A is stabilized by
protonation of the nitrogen atom16 in the solution state. This
speculation was supported by the X-ray crystallographic analysis
of 6: the molecular ratio was 1:0.5:0.5 for 6/TFA/CH3OH in
the crystal state, and no intramolecular hydrogen bonds were
formed between OH-3 and the nitrogen atom. 13C resonances
of TFA were not observed in the 13C NMR spectra of 6, 7, and
9−26, but the appearance of an exchangeable broad resonance
(around δ 7.5−9.5) with integration less of than one proton in
Compound 25 differed from 24 only in the replacement of
the benzoyl group by a cinnamoyl moiety, as indicated by
HRESIMS and NMR data and confirmed by 2D NMR and
NOE difference experiments. Therefore, compound 25 was
1
the H NMR spectra of 6, 7, 9−22, and 24−26 confirmed the
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a
Table 5. 13C NMR Spectroscopic Data of Compounds 5−8, 7a, and 9−14
b
b
c
b
b
b
d
no.
5
6
7
7a
8
9
10
11
12
13
13
14
1
82.5
26.3
33.8
47.7
49.0
84.5
51.7
90.1
43.4
41.2
49.8
36.3
75.1
79.7
79.6
91.6
60.2
79.2
173.0
41.4
13.2
55.6
57.8
77.9
31.0
70.3
43.7
40.2
82.6
45.4
88.6
53.1
78.6
56.4
47.0
75.4
78.7
80.1
90.3
65.0
77.2
50.4
51.0
11.1
55.3
59.1
78.2
31.1
70.1
44.0
39.0
82.6
43.1
81.7
54.3
79.3
56.8
47.4
76.0
79.3
77.7
93.7
67.7
77.3
52.2
41.9
78.0
28.9
72.4
43.3
39.1
82.3
43.2
81.6
54.1
79.3
56.6
47.6
76.0
79.2
77.5
93.6
68.1
76.9
52.2
42.6
78.1
35.0
69.8
44.1
41.5
84.0
42.4
81.8
56.0
79.4
56.9
48.7
76.2
80.0
78.7
94.9
64.1
75.1
50.5
42.9
78.1
30.9
70.3
43.7
39.6
82.7
43.4
81.7
54.4
79.3
56.7
47.5
76.0
79.3
77.7
94.2
65.3
77.3
50.6
50.5
11.2
55.1
59.1
50.4
62.5
59.2
131.2
130.4
129.3
133.9
129.3
130.4
166.3
78.4
31.0
70.3
44.0
40.2
82.2
48.9
76.6
54.1
79.4
56.3
47.4
75.8
79.5
82.5
91.7
68.1
78.1
52.6
42.2
78.0
30.9
70.2
43.7
40.3
82.8
49.8
82.7
53.8
78.5
57.1
46.6
76.1
78.7
42.2
83.0
66.8
77.2
50.2
50.9
11.0
55.3
59.2
79.0
23.2
27.6
38.6
39.3
82.1
45.1
88.6
53.0
78.6
56.4
47.4
75.4
78.8
80.2
90.0
67.2
78.9
59.2
42.7
81.4
22.2
27.4
39.0
43.0
82.4
45.5
90.9
44.0
39.8
51.0
36.8
74.9
79.4
79.3
91.1
62.8
78.8
58.2
50.7
10.8
55.8
59.3
80.3
22.0
26.6
38.1
41.4
81.9
44.7
90.0
42.9
39.5
50.1
37.7
73.8
78.2
78.4
89.6
60.7
77.5
56.8
50.1
10.0
55.6
58.6
82.1
22.0
27.6
38.9
40.8
81.7
43.5
74.2
45.4
54.8
50.2
36.9
75.6
80.1
43.3
83.7
63.9
79.3
58.9
51.5
10.9
55.9
58.2
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
OCH3-1
55.4
59.1
55.7
59.2
55.7
58.8
55.4
58.3
56.0
59.3
OCH3-6
OCH3-8
50.4
50.4
49.9
OCH3-16
61.3
59.2
61.7
59.3
62.3
62.2
62.2
61.1
59.2
59.6
59.0
61.6
59.1
61.8
58.9
61.6
59.1
59.3
59.1
OCH3-18
59.2
59.3
58.9
1′
2′
3′
4′
5′
6′
7′
130.9
130.3
129.6
134.2
129.6
130.3
166.2
130.6
130.3
129.7
134.5
129.7
130.3
166.1
131.2
130.4
129.3
133.9
129.3
130.4
166.3
131.1
130.4
129.3
133.9
129.3
130.4
166.3
131.7
130.4
129.2
133.6
129.2
130.4
166.5
131.4
130.6
129.2
133.7
129.2
130.6
166.5
130.9
130.4
129.6
134.4
129.6
130.4
166.3
130.6
130.4
129.7
134.4
129.7
130.4
166.1
130.6
130.3
129.7
134.4
129.7
130.3
166.1
129.1
129.6
129.9
133.7
129.9
129.6
165.7
131.5
130.6
129.2
133.8
129.2
130.6
166.5
a
Data (δ) were measured in Me2CO-d6 at 125 MHz for 5−8, 7a, and 9−14. The assignments were based on 1H−1H COSY, gHSQC, HMBC, and
b
NOE experiments. Data of OAc-8: δC 173.0 and 21.5 for 5; 172.9 and 21.3 for 6; 170.0 and 21.3 for 11; 172.9 and 21.4 for 12; and 173.0 and 21.4
for 13. Data of OAc-3 in 7a: δC 170.3 and 21.0. Data (δ) were measured in CDCl3 for 13 at 125 MHz, data of OAc-8: δC 170.4 and 21.2.
c
d
protonation of the nitrogen atom by TFA contamination. This
result was further supported by the 19F NMR spectra of these
compounds, which showed 19F resonances at around δ −76
ppm, indicating the presence of TFA. Since acidification and
saponification are commonly employed in the extraction and
isolation of the aconitane alkaloids,7b,8,12b,15,18,26,30a,32b con-
formational transformation should occur. This, as well as the
effects of the conformational change on biological activity, must
be investigated in detail.
On the basis of the common pharmacological effects of
aconitane alkaloids,13a the analgesic effects of two pairs of
conformational isomers 7/8 and 27/27a were tested. In the
acetic acid-induced writhing test,37 7 and 8 exhibited analgesic
the effects of all the isolates against neurotoxicity induced by
serum deprivation in PC12 cells were investigated by the
methyl thiazolyl tetrazolium (MTT) method.38 The results
showed significant inhibition of MTT reduction by serum
deprivation; at a concentration of 10 μM, 15, 16, and 19
increased cell viability from 55.1 4.2% to 70.8 5.7%, 74.7
4.6%, and 75.8 7.6%, respectively, indicating that they may be
effective in treating neurodegenerative disorders. The isolates
were also assessed for their activities against Fe2+-cystine-
induced rat liver microsomal lipid peroxidation,39 DL-galactos-
amine-induced WB-F344 cell damage,40 and nitric oxide
production in mouse peritoneal macrophages,41 as well as for
their cytotoxicity against several human cancer cell lines,42 but
were found to be inactive at a concentration of 10 μM.
effects with 35
8% and 56
12% reduction of writhes,
respectively, as compared to the blank control, at a dose of
5 mg/kg (ip), while both 27 and 27a caused death of mice.
Subsequent acute toxicity evaluation showed that 27 and 27a
had LD50 values of 2.65 and 4.37 mg/kg (ip), respectively. At a
nontoxic dose of 0.5 mg/kg (ip), 27 and 27a exhibited
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured using a Rudolph Research Autopol III automatic polar-
imeter, and UV spectra were obtained using a Cary 300 spectrometer.
Circular dichroism (CD) spectra were recorded using a JASCO J-815
CD spectrometer. IR spectra were recorded using a Nicolet 5700 FT-
IR microscope (transmission). 1D and 2D NMR spectra were acquired
analgesic effects with 36
9% and 30
11% reduction of
writhes, respectively. This indicated that the isomer with ring A
in the chair conformation (8 or 27) was more active than that
with ring A in the boat conformation (7 or 27a). In addition,
1
at 500 or 600 MHz for H and at 125 or 150 MHz for 13C using
INOVA 500 MHz or SYS 600 MHz spectrometers; Me2CO-d6 and
1154
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Journal of Natural Products
Article
a
Table 6. 13C NMR Spectroscopic Data of Compounds 15−26 and 27a
b
c
d
no.
15
16
17
18
19
20
21
22
23
24
25
26
1
77.9
31.7
70.3
43.8
42.0
82.6
45.4
90.3
53.1
78.7
55.3
47.0
75.4
80.1
78.6
88.6
65.0
77.2
50.4
51.0
11.4
56.3
59.1
80.6
30.1
70.1
44.2
45.5
82.8
44.3
91.5
42.8
40.3
52.2
36.0
75.1
79.4
79.7
90.8
67.3
77.1
51.4
42.8
81.6
22.2
27.3
38.9
42.6
82.6
43.6
83.5
44.9
40.6
51.4
37.4
75.3
79.9
76.5
94.7
63.3
78.9
58.1
50.4
10.9
55.8
59.3
50.4
62.4
59.0
131.1
130.4
129.3
133.9
129.3
130.4
166.3
82.0
22.0
25.2
38.5
39.9
24.6
41.3
78.3
43.7
40.6
50.3
37.2
75.4
80.2
81.6
92.6
63.7
78.4
57.7
50.2
10.8
56.0
81.7
22.0
27.2
38.8
44.2
71.3
44.7
83.4
45.2
43.8
51.6
29.5
38.2
76.5
75.1
93.5
63.6
79.3
58.6
50.2
10.9
55.7
82.0
22.0
27.2
38.7
43.9
71.9
51.5
79.0
45.6
44.2
51.4
29.8
39.7
76.7
79.2
91.1
63.4
79.2
59.0
50.2
10.9
55.7
81.8
22.0
27.2
38.8
44.3
71.3
45.2
83.1
45.2
43.8
51.6
29.9
38.3
76.5
75.7
93.6
63.6
79.2
58.2
50.2
10.9
55.7
82.0
22.0
25.2
38.5
40.0
24.9
41.2
78.4
44.1
43.9
50.5
29.4
39.5
76.6
79.2
91.7
63.6
78.5
57.7
50.1
10.7
56.0
71.8
28.4
25.8
38.5
40.2
25.0
41.3
78.3
44.2
43.9
49.8
30.1
39.3
76.8
79.2
91.6
64.4
78.8
57.3
50.1
10.7
71.8
28.5
27.9
39.0
43.4
82.9
49.4
78.4
45.1
44.0
50.4
30.3
39.4
76.8
79.0
91.4
64.0
79.5
58.5
50.2
10.9
70.9
27.6
27.1
38.1
42.5
82.0
48.4
77.5
44.7
43.1
49.6
29.6
38.3
75.6
78.0
90.5
63.1
78.7
57.7
49.3
10.0
81.3
22.2
27.4
38.9
42.6
82.9
45.5
91.2
44.1
43.1
51.0
30.6
39.5
75.8
76.8
89.8
62.4
78.8
58.1
50.6
10.7
55.7
59.0
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
OCH3-1
55.5
58.9
OCH3-6
58.1
57.3
OCH3-8
50.6
57.4
OCH3-16
61.7
59.3
61.7
59.2
61.3
59.4
57.7
59.2
57.5
59.2
57.6
59.4
57.5
59.4
57.6
59.2
56.7
58.3
58.1
59.2
OCH3-18
59.2
1′
2′
3′
4′
5′
6′
7′
8′
9′
130.6
130.3
129.7
134.5
129.7
130.3
161.9
129.8
129.3
129.1
133.9
129.1
129.3
166.5
131.5
130.6
129.2
133.6
129.2
130.6
166.6
131.5
130.3
129.3
133.8
129.3
130.3
166.1
131.6
130.5
129.2
133.6
129.2
130.5
166.3
131.5
130.3
129.2
133.8
129.2
130.3
166.0
131.6
130.6
129.2
133.6
129.2
130.6
166.3
131.6
130.6
129.2
133.6
129.2
130.6
166.3
131.5
130.6
129.2
133.6
129.2
130.5
166.2
134.7
128.2
128.9
130.2
128.9
128.2
144.6
118.5
130.9
130.2
129.7
134.2
129.7
130.2
165.9
165.6
a
b
Data (δ) were measured in Me2CO-d6 at 125 MHz for 15−19, 21, 22, and 24 and at 150 MHz for 20, 23, 25, and 26, respectively. Data of OBz-8
c
in 16: δC 130.2 (C-1″), 130.3 (C-2″), 130.2 (C-3″), 134.6 (C-4″), 130.2 (C-5″),130.3 (C-6″), and 167.7 (C-7″). Data of OEt-8: δC 58.2 and 15.7 for
21. Data of OAc-8 in 26: δC 173.1 and 21.7.
d
CDCl3 solvent peaks were used as references. ESIMS data were
measured using a Q-Trap LC/MS/MS (Turbo Ionspray Source)
spectrometer. HRESIMS data were measured using an Agilent
Technologies 6520 Accurate Mass Q-TOF LC/MS spectrometer.
Column chromatography (CC) was performed using basified silica gel
(200−300 mesh, Qingdao Marine Chemical Inc. China) and a
Sephadex LH-20 column (Amersham Biosciences, Inc., Shanghai,
China). Preparative thin-layer chromatography (TLC) was performed
on high-performance silica gel preparative TLC plates (HSGF254, glass
precoated, Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai,
China). HPLC separation was performed using an instrument
consisting of a Waters 600 controller, a Waters 600 pump, and a
Waters 2487 dual-wavelength absorbance detector, with a Prevail (250 ×
10 mm i.d.) preparative column packed with C18 (10 μM). TLC was
carried out on glass precoated silica gel GF254 plates. Spots were
observed under UV light or by spraying the developed plates with
Dragendorff’s reagent or a mixture of 5% H2SO4−ethanol, followed by
heating. Unless otherwise noted, all the chemicals were obtained from
commercially available sources and used without further purification.
Plant Material. The lateral root of Aconitum carmichaelii was
collected in June 2009 from the culture field in Jiangyou, Sichuan
Province, People’s Republic of China. Plant identity was verified by Dr.
Yan Ren (Chengdu University of TCM, Sichuan 610075, China). A
voucher specimen (no. ID-S-2383) was deposited at the herbarium of
the Department of Medicinal Plants, Institute of Materia Medica,
Chinese Academy of Medical Sciences and Peking Union Medical
College, Beijing 100050, China.
Extraction and Isolation. The air-dried lateral roots of A.
carmichaelii (50 kg) were powdered and extracted with H2O (3 × 150
L × 6 h) at 40 °C. The H2O extract was concentrated to 120 L under
reduced pressure, subjected to chromatography over a macroporous
adsorbent resin (HPD-110, 19 kg) column (20 × 200 cm), and eluted
successively with H2O (50 L), 30% EtOH (120 L), 50% EtOH (120 L),
and 95% EtOH (100 L) to afford the corresponding fractions A−D.
After removal of the solvent, C (3.5 kg) was chromatographed over
MCI gel CHP 20P with successive elution using H2O (10 L), 30%
EtOH (30 L), 50% EtOH (20 L), and 95% EtOH (10 L) to give
fractions C1−C4. Fraction C3 (200 g) was fractionated via silica gel
CC, with gradient elution [CHCl3/MeOH (100:1−1:4)] to yield C3-
1−C3-7, based on TLC analysis. Fraction C3-2 (10.4 g) was separated
using basified silica gel (pH 8−9) CC eluted with a petroleum ether/
Me2CO/diethylamine (25:1:1 to 4:1:1) mixture to afford C3-2-1−C3-
2-7. Fraction C3-2-2 (3.0 g) was crystallized from MeOH to give 27
(1.2 g). Fraction C3-3 (1.1 g) was subjected to RP HPLC (30%
MeCN in H2O, containing 0.1% TFA) to give C3-3-1−C3-3-6.
Fraction C3-3-2 (250 mg) was separated by PTLC (petroleum ether/
Me2CO/diethylamine, 3:1:1) to yield 22 (13 mg) and 23 (30 mg).
Fraction C3-4 (3.4 g) was subjected to CC over Sephadex LH-20,
1155
dx.doi.org/10.1021/np300225t | J. Nat. Prod. 2012, 75, 1145−1159

Journal of Natural Products
Article
eluted using a CHCl3/MeOH (1:1) mixture, to afford C3-4-1−C3-4-3.
Fraction C3-4-2 (298 mg) was purified by RP HPLC (50% MeCN in
H2O, containing 0.1% TFA) to obtain 1 (20 mg). Fraction C3-4-3 (1.1 g)
was separated by basified silica gel CC (petroleum ether/Me2CO/
diethylamine, 20:1:1 to 5:1:1) to give C3-4-3-1−C3-4-3-6. Fraction
C3-4-3-2 (236 mg) was purified by RP HPLC (30% MeCN in H2O,
containing 0.1% TFA) to yield 6 (10.3 mg) and 7 (50 mg). Fraction
C3-5 (7.8 g) was subjected to RP flash chromatography (30−95%
MeOH in H2O, containing 0.1% TFA) to give C3-5-1−C3-5-5.
Fraction C3-5-2 (3.6 g) was subjected to basified silica gel CC
(petroleum ether/Me2CO/diethylamine, 30:1:1 to 1:1:1) to give C3-
5-2-1−C3-5-2-3. Fraction C3-5-2-2 (3.2 g), using RP HPLC (35%
MeCN in H2O, containing 0.1% TFA), afforded C3-5-2-2-1−C3-5-2-
2-3. Fraction C3-5-2-2-3 was further separated by RP HPLC (55%
MeOH in H2O, containing 0.1% TFA) to yield 14 (15.2 mg), 15 (20.5
mg), 16 (6.7 mg), 17 (7.1 mg), 18 (23.4 mg), and 19 (23.3 mg).
Fraction C3-5-3 (2.4 g) was chromatographed on basified silica gel
(petroleum/Me2CO/diethylamine, 50:1:1, 25:1:1, and 10:1:1 succes-
sively) to give C3-5-3-1−C3-5-3-3. Separation of C3-5-3-2 (716 mg)
by CC over Sephadex LH-20 (petroleum/CHCl3/MeOH, 5:5:1) gave
C3-5-3-2-1 and C3-5-3-2-2, which were further separated by
RP HPLC (45% MeOH in H2O, containing 0.1% TFA) to yield 9
(20.5 mg), 10 (20.3 mg), 11 (6.6 mg), and 12 (4.3 mg). Fraction C3-5-4
(95 mg) was separated by PTLC (petroleum ether/Me2CO/
diethylamine, 3:1:1) and then RP HPLC (50% MeOH in H2O;
0.1% TFA) to yield 5 (13.3 mg). Fraction C3-6 (6.2 g), using RP
MPLC (35% MeCN in H2O; 0.1% TFA), gave C3-6-1−C3-6-4.
Fraction C3-6-2 was subjected to CC over Sephadex LH-20 eluted
with petroleum ether/CHCl3/MeOH (5:3:1) to give C3-6-2-1−C3-6-
2-4. Fraction C3-6-2-1 (611 mg) was separated into C3-6-2-1-1−C3-6-
2-1-3 by PTLC (petroleum ether/Me2CO/diethylamine, 3:1:1) and
then purified by RP HPLC (MeOH in H2O; 0.1% TFA) to afford 13
(5.1 mg), 20 (20.2 mg), and 21 (0.5 mg) from C3-6-2-1-1 (58 mg)
and 24 (89.0 mg), 25 (10.3 mg), and 26 (12.3 mg) from C3-6-2-1-3
(183 mg). Fraction C3-6-3 was separated by basified silica gel CC
eluted with petroleum ether/Me2CO/diethylamine (15:1:1 to 5:1:1)
to give C3-6-3-2−C3-6-3-4. C3-6-3-3 (1.2 g) was subjected to
Sephadex LH-20 CC (CH3Cl−MeOH, 1:1) and then RP HPLC (30%
MeCN in H2O, containing 0.1% TFA) to afford 2 (13.1 mg), 3 (14.6
mg), and 4 (4.3 mg). Fraction C3-6-4 was fractionated by basified
silica gel CC, eluted with petroleum ether/Me2CO/diethylamine
(10:1:1 to 2:1:1), to give C3-6-4-1 and C3-6-4-2. Further separation of
C3-6-4-1 (43 mg) and C3-6-4-2 (386 mg) by PTLC (petroleum ether/
Me2CO/diethylamine, 3:1:1) afforded 8 (10 mg) and 28 (152 mg),
respectively.
NMR (Me2CO-d6, 150 MHz), see Table 1; (+)-ESIMS m/z 516 [M −
OH]+; (+)-HRESIMS m/z 516.2968 [M − OH]+ (calcd for
C29H42NO7, 516.2956).
(+)-(13R,19S)-2α-Isobutyryloxy-7β,11α,19-trihydroxy-N-methyl-
13-(S-2-methylbutyryloxy)hetisanium hydroxide (4): white, amor-
phous powder (MeOH); [α]20D +10.2 (c 0.37, MeOH); UV (MeOH)
λmax (log ε) 202 (3.87) nm; IR (KBr) νmax 3394, 2977, 2924, 2850,
1736 (sh), 1712 (sh),1683, 1464, 1421, 1355, 1307, 1274, 1200, 1141,
1
1076, 1057, 1021, 964, 930, 901, 881, 840, 801, 766, 723 cm−1; H
NMR (Me2CO-d6, 500 MHz) data, see Table 1; 13C NMR (Me2CO-
d6, 125 MHz) data, see Table 1; (+)-ESIMS m/z 530 [M − OH]+;
(+)-HRESIMS m/z 530.3117 [M − OH]+ (calcd for C30H44NO7,
530.3112).
(−)-(A-c)-8β-Acetoxy-14α-benzoyloxy-N-ethyl-13β,15α-dihy-
droxy-1α,6α,16β,18-tetramethoxy-19-oxo-aconitane (5): amor-
phous powder (MeOH); [α]20 −28.4 (c 0.46, MeOH); CD
D
(MeOH) 220 (Δε +0.03), 237 (Δε +0.20); IR (KBr) νmax 3524,
3410, 2934, 2879, 2850, 1720, 1680, 1643, 1454, 1379, 1306, 1278,
1
1208, 1133, 1085, 1055, 987, 961, 927, 900, 843, 803, 725 cm−1; H
NMR (Me2CO-d6, 500 MHz) data, see Table 2; 13C NMR (Me2CO-
d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z 644 [M + H]+;
(+)-HRESIMS m/z 644.3054 [M + H]+ (calcd for C34H46NO11,
644.3065), 666.2876 [M + Na]+ (calcd for C34H45NO11Na, 666.2885).
(−)-(A-b)-8β-Acetoxy-14α-benzoyloxy-N-ethyl-3α,10β,13β,15α-
tetrahydroxy-1α,6α,16β,18-tetramethoxyaconitane (6): colorless
needles (MeOH); [α]20 −7.9 (c 0.53, MeOH); UV (MeOH) λmax
D
(log ε) 230 (3.74), 273 (2.70) nm; CD (MeOH) 215 (Δε +1.43), 222
(Δε +0.96), 234 (Δε +1.58); IR (KBr) νmax 3437, 3066, 2949, 1728,
1
1690, 1494, 1454, 1276, 1199, 1123, 1102, 934, 718 cm−1; H NMR
(Me2CO-d6, 500 MHz) data, see Table 2; 13C NMR (Me2CO-d6, 125
MHz) data, see Table 5; (+)-ESIMS m/z 662 [M + H]+;
(+)-HRESIMS m/z 662.3187 [M + H]+(calcd for C32H46NO11,
662.3171).
X-ray Crystallography of Compound 6. C34H47NO12, M = 661.75,
orthorhombic, P212121, a = 10.296(3) Å, b = 15.228(3) Å, c =
23.631(6) Å, α = β = γ = 90o, V = 3705.0(16) Å3, Z = 4, Dcalcd = 1.448
g cm−3, 6980 reflections independent, 6223 reflections observed (|F|2 ≥
2σ|F|2), R1 = 0.0656, wR2 = 0.1784, S = 1.045.
The data were collected on a Rigaku MicroMax 002+ diffractometer
with Cu Kα radiation by using the ω-scan technique to a maximum 2θ
value of 145.38°. The crystal structures were solved by direct methods
by using SHELXS-97, and all non-hydrogen atoms were refined
anisotropically using the least-squares method. All hydrogen atoms
were positioned by geometrical calculations and difference Fourier
overlapping calculation. The absolute configuration was determined on
the basis of the Flack parameter −0.1(2). Crystallographic data for the
structure of 6 have been deposited with the Cambridge Crystallo-
graphic Data Centre as supplementary publication CCDC 809199.
Copies of these data can be obtained free of charge via www.ccdc.cam.
ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic
Data Centre, 12 Union Road, Cambridge CB21EZ, UK; fax: (+44)
1223-336-033; or e-mail: deposit@ccdc.cam.ac.uk).
(+)-(13R,19S)-1β,11α-Diacetoxy-2α-benzoyloxy-13,19-dihydroxy-
hetisan (1): amorphous powder (MeOH); [α]20 +18.1 (c 0.51,
D
MeOH); UV (MeOH) λmax (log ε) 229 (3.84), 274(2.94) nm; CD
(MeOH) 226 (Δε +3.68), 253 (Δε −0.32); IR (KBr) νmax 3394, 3187,
2921, 2850, 1719, 1692 (sh), 1647, 1469, 1420, 1318, 1205, 1188,
1138, 801, 720, 649 cm−1; 1H NMR (Me2CO-d6, 500 MHz) data, see
Table 1; 13C NMR (Me2CO-d6, 125 MHz) data, see Table 1;
(+)-ESIMS m/z 550 [M + H]+; (+)-HRESIMS m/z 550.2445
[M + H]+ (calcd for C31H36NO8, 550.2435).
(−)-(A-b)-14α-Benzoyloxy-3α,10β,13β,15α-tetrahydroxy-1α,6α,8β,
16β,18-pentamethoxy-N-methylaconitane (7): amorphous powder
(MeOH); [α]20 −5.32 (c 0.14, MeOH); UV (MeOH) λmax (log ε)
(−)-(13R,19S)-11α,19-Dihydroxy-N-methyl-13-(S-2-methylbutyry-
D
230 (4.03), 272 (2.98) nm; CD (MeOH) 217 (Δε +2.37), 231 (Δε
+4.16), 255 (Δε +0.34); IR (KBr) νmax 3361, 3086, 2940, 2832, 1719,
1685, 1603, 1453, 1413, 1317, 1278, 1201, 1108, 1071, 1008, 973, 935,
loxy)-2α-propionyloxyhetisanium hydroxide (2): white, amorphous
powder (MeOH); [α]20 −24.8 (c 0.17, MeOH); UV (MeOH) λmax
D
(log ε) 201 (3.88); IR (KBr) νmax 3413, 2976, 2939, 1738, 1684, 1464,
1421, 1355, 1306, 1275, 1201, 1178, 1142 1097, 1074, 1057, 1021,970,
930, 898, 881, 834, 801, 767, 721 cm−1; 1H NMR (Me2CO-d6,
600 MHz) data, see Table 1; 13C NMR (Me2CO-d6, 150 MHz) data,
see Table 1; (+)-ESIMS m/z 500 [M − OH]+; (−)-ESIMS m/z 515
[M − H]−; (+)-HRESIMS m/z 500.3020 [M − OH]+ (calcd for
C29H42NO6, 500.3007).
1
880, 832, 800, 764, 716, 681 cm−1; H NMR (Me2CO-d6, 500 MHz)
data, see Table 2; 13C NMR (Me2CO-d6, 125 MHz) data, see Table 5;
(+)-ESIMS m/z 620 [M + H]+; (+)-EIMS m/z 619 [M]+., 604 [M −
CH3]+•., 589 [M − 2CH3]+•, 588 [M − CH3O]+•; (+)-HRESIMS m/z
620.3071 [M + H]+ (calcd for C32H46NO11, 620.3065).
(−)-(A-c)-14α-Benzoyloxy-3α,10β,13β,15α-tetrahydroxy-1α,6α,
8β,16β,18-pentamethoxy-N-methylaconitane (8): amorphous pow-
(−)-(13R,19S)-7β,11α,19-Trihydroxy-N-methyl-13-(S-2-methylbu-
tyryloxy)-2α-propionyloxyhetisanium hydroxide (3): white, amor-
phous powder (MeOH); [α]20D −26.1 (c 0.19, MeOH); UV (MeOH)
λmax (log ε) 203 (3.76) nm; IR (KBr) νmax 3394, 3083, 2942, 2852,
1732 (sh), 1678, 1453, 1367, 1320, 1204, 1139, 1052, 956, 845, 802,
der (MeOH); [α]20 −2.07 (c 0.22, MeOH); UV (MeOH) λmax (log
D
ε) 230 (3.82), 272 (2.75) nm; CD (MeOH) 218 (Δε +0.11), 227 (Δε
−0.02), 243 (Δε +0.24); IR (KBr) νmax 3393, 3363, 3190, 2921, 2850,
1678, 1647, 1469, 1421, 1278, 1203, 1135, 1105, 1070, 1045, 973, 801,
1
1
722 cm−1; H NMR (Me2CO-d6, 600 MHz) data, see Table 1; 13C
720, 648 cm−1; H NMR (Me2CO-d6, 500 MHz) data, see Table 2;
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13C NMR (Me2CO-d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z
[M + H]+; (+)-HRESIMS m/z 604.3117 [M + H]+ (calcd for
C32H46NO10, 604.3116).
620 [M + H]+; (+)-EIMS m/z 619 [M]+•, 604 [M − CH3]+•, 589 [M
− 2CH3]+•; (+)-HRESIMS m/z 620.3061 [M + H]+ (calcd for
C32H46NO11, 620.3065).
(−)-(A-b)-8β,14α-Dibenzoyloxy-N-ethyl-3α,13β,15α-trihydroxy-
1α,6α,16β,18-tetramethoxyaconitane (16): amorphous powder
(MeOH); [α]20 −2.11 (c 0.33, MeOH); CD (MeOH) 219 (Δε
D
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-3α,10β,13β,15α-tetrahydroxy-
1
−0.28), 243 (Δε +0.55); H NMR (Me2CO-d6, 500 MHz) data, see
1α,6α,8β,16β,18-pentamethoxyaconitane (9): amorphous powder
Table 3; 13C NMR (Me2CO-d6, 125 MHz) data, see Table 6;
(+)-ESIMS m/z 694 [M + H]+; (+)-HRESIMS m/z 694.3232 [M + H]+
(calcd for C38H48NO11, 694.3222).
(MeOH); [α]20 −13.4 (c 0.30, MeOH); UV (MeOH) λmax (log ε)
D
229 (4.05), 272 (2.90) nm; CD (MeOH) 210 (Δε +0.07), 230 (Δε
+0.08); IR (KBr) νmax 3371, 3091, 2935, 2824, 1713, 1684, 1605,
1492, 1453, 1426, 1400, 1353, 1283, 1250, 1203, 1136, 1111, 1071,
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-13β,15α-dihydroxy-
1
976, 940, 930, 882, 841, 801, 721, 711 cm−1; H NMR (Me2CO-d6,
1α,6α,8β,16β,18-pentamethoxyaconitane (17): amorphous powder
(MeOH); [α]20 −13.9 (c 0.25, MeOH); UV (MeOH) λmax (log ε)
500 MHz) data, see Table 2; 13C NMR (Me2CO-d6, 125 MHz) data,
see Table 5; (+)-ESIMS m/z 634 [M + H]+; (+)-HRESIMS m/z
634.3216 [M + H]+ (calcd for C34H48NO11, 634.3222).
D
230 (3.91), 273 (2.76) nm; CD (MeOH) 218 (Δε +0.10), 228 (Δε +0.25),
251 (Δε −0.22); IR (KBr) νmax 3353, 3071, 2940, 2832, 1717, 1691,
1
1453, 1278, 1200, 1175, 1172, 1076, 978, 800, 717 cm−1; H NMR
(−)-(A-b)-14α-Benzoyloxy-3α,10β,8β,13β,15α-pentahydroxy-
(Me2CO-d6, 500 MHz) data, see Table 3; 13C NMR (Me2CO-d6, 125
MHz) data, see Table 6; (+)-ESIMS m/z 602 [M + H]+; (+)-HRESIMS
m/z 602.3332 [M + H]+ (calcd for C33H48NO9, 602.3324).
1α,6α,16β,18-tetramethoxy-N-methylaconitane (10): amorphous
powder (MeOH); [α]20 −2.34 (c 0.24, MeOH); UV (MeOH) λmax
D
(log ε) 228 (3.99), 272 (3.24) nm; IR (KBr) νmax 3370, 3093, 2922,
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-8β,13β,15α-trihydroxy-
2849, 1711, 1679, 1492, 1425, 1352, 1282, 1250, 1202, 1136, 1111,
1
1092, 1071, 976, 940, 882, 841, 800, 721, 711, 682, 558 cm−1; H
1α,16β,18-trimethoxyaconitane (18): amorphous powder (MeOH);
[α]20 −10.1 (c 0.69, MeOH); UV (MeOH) λmax (log ε) 229 (3.54),
NMR (Me2CO-d6, 500 MHz) data, see Table 2; 13C NMR (Me2CO-
d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z 606 [M + H]+;
(−)-ESIMS m/z 604 [M − H]−; (+)-HRESIMS m/z 606.2909 [M +
H]+ (calcd for C31H44NO11, 606.2909).
D
272 (2.39) nm; CD (MeOH) 214 (Δε +1.68), 232 (Δε +1.18), 254
(Δε −0.10); IR (KBr) νmax 3361, 3199, 3069, 2922, 2851, 1677, 1634,
1468, 1454, 1423, 1322, 1281, 1202, 1180, 1132, 1050, 1027, 835, 801,
1
718 cm−1; H NMR (Me2CO-d6, 500 MHz) data, see Table 3; 13C
(−)-(A-b)-8β-Acetoxy-14α-benzoyloxy-N-ethyl-3α,10β,13β-trihy-
droxy-1α,6α,16β,18-tetramethoxyaconitane (11): amorphous pow-
der (MeOH); [α]20D −3.78 (c 0.55, MeOH); UV (MeOH) λmax (log ε)
228 (3.88), 273 (3.12) nm; CD (MeOH) 217 (Δε +0.34), 232
(Δε +0.73); IR (KBr) νmax 3390, 2937, 2831, 1716, 1681, 1453, 1376,
1280, 1247, 1205, 1137, 1108, 1049, 978, 945, 840, 801, 720 cm−1; 1H
NMR (Me2CO-d6, 500 MHz) data, see Table 2; 13C NMR (Me2CO-
d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z 646 [M + H]+;
(+)-HRESIMS m/z 646.3221 [M + H]+ (calcd for C34H48NO11,
646.3222).
NMR (Me2CO-d6, 125 MHz) data, see Table 6; (+)-ESIMS m/z 558
[M + H]+; (+)-HRESIMS m/z 558.3062 [M + H]+(calcd for
C31H44NO8, 558.3061).
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-6α,15α-dihydroxy-1α,8β,16β,
18-tetramethoxyaconitane (19): amorphous powder (MeOH);
[α]20 −17.3 (c 0.18, MeOH); UV (MeOH) λmax (log ε) 229
D
(4.02), 272 (2.88) nm; CD (MeOH) 218 (Δε +0.19), 232 (Δε
+0.40); IR (KBr) νmax 3394, 3187, 3010, 2921, 2850, 1646, 1469,
1420, 1343, 1300, 1245, 1187, 1119, 721, 646 cm−1; 1H NMR
(Me2CO-d6, 500 MHz) data, see Table 4; 13C NMR (Me2CO-d6, 125
MHz) data, see Table 6; (+)-ESIMS m/z 572 [M + H]+;
(+)-HRESIMS m/z 572.3214 [M + H]+ (calcd for C32H46NO8
572.3218).
(−)-(A-b)-8β-Acetoxy-14α-benzoyloxy-10β,13β,15α-trihydroxy-
1α,6α,16β,18-tetramethoxy-N-methylaconitane (12): amorphous
powder (MeOH); [α]20 −24.3 (c 0.51, MeOH); UV (MeOH) λmax
D
(log ε) 230 (4.09), 272 (2.93) nm; CD (MeOH) 217 (Δε +0.24), 234
(Δε +0.89); IR (KBr) νmax 3486, 3067, 2943, 2831, 1721, 1687, 1454,
1375, 1279, 1247, 1202, 1181, 1112, 1029, 977, 946, 835, 801, 741,
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-6α,8β,15α-trihydroxy-
1α,16β,18-trimethoxyaconitane (20): amorphous powder (MeOH);
[α]20 −6.40 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 228 (3.97),
1
718 cm−1; H NMR (Me2CO-d6, 500 MHz) data, see Table 2; 13C
D
273 (2.83) nm; CD (MeOH) 231 (Δε +0.08), 270 (Δε +0.01); IR
(KBr) νmax 3386, 3083, 2933, 2854, 1679, 1452, 1367, 1318, 1280,
NMR (Me2CO-d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z 632
[M + H]+; (+)-HRESIMS m/z 632.3082 [M + H]+ (calcd for
C33H46NO11, 632.3065).
1
1204, 1137, 1074, 1052, 842, 801, 720 cm−1; H NMR (Me2CO-d6,
600 MHz) data, see Table 4; 13C NMR (Me2CO-d6, 125 MHz) data,
see Table 6; (+)-ESIMS m/z 558 [M + H]+; (+)-HRESIMS m/z
558.3063 [M + H]+ (calcd for C31H44NO8, 558.3061).
(−)-(A-b)-8β-Aacetoxy-14α-benzoyloxy-N-ethyl-13β,15α-dihy-
droxy-1α,6α,16β,18-tetramethoxyaconitane (13): amorphous pow-
der (MeOH); [α]20 −7.40 (c 0.31, MeOH); UV (MeOH) λmax (log ε)
D
(−)-(A-b)-14α-Benzoyloxy-8β-ethoxy-N-ethyl-6α,15α-dihydroxy-
229 (3.71), 272 (2.55) nm; CD (MeOH) 228 (Δε +0.37), 245 (Δε +0.47),
257 (Δε +0.48); IR (KBr) νmax 3487, 3067, 2941, 2831, 1778, 1721,
1692, 1453, 1371, 1280, 1201, 1137, 1113, 1055, 1027, 800, 714 cm−1;
1H NMR (Me2CO-d6, 500 MHz) data, see Table 3; 13C NMR
1α,16β,18-trimethoxyaconitane (21): amorphous powder (MeOH);
[α]20 −12.0 (c 0.42, MeOH); UV (MeOH) λmax (log ε) 232 (4.02),
D
272 (2.91) nm; CD (MeOH) 217 (Δε +0.75), 231 (Δε +1.50), 256
(Δε −0.07); IR (KBr) νmax 3370, 3075, 2936, 2893, 2831, 1692, 1453,
1392, 1280, 1201, 1176, 1126, 1074, 1045, 1008, 954, 830, 800, 718
(Me2CO-d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z 630 [M +
H]+; (+)-HRESIMS m/z 630.3279 [M + H]+(calcd for C34H48NO10,
630.3273).
1
cm−1; H NMR (Me2CO-d6, 500 MHz) data, see Table 4; 13C NMR
(Me2CO-d6, 125 MHz) data, see Table 6; (+)-ESIMS m/z 558 [M + H]+;
(+)-ESIMS m/z 586 [M + H]+; (+)-HRESIMS m/z 586.3383 [M + H]+
(calcd for C33H48NO8, 586.3374).
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-8β,13β-dihydroxy-
1α,6α,16β,18-tetramethoxyaconitane (14): amorphous powder
(MeOH); [α]20 −10.2 (c 0.79, MeOH); UV (MeOH) λmax (log ε)
D
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-8β,15α-dihydroxy-1α,16β,18-
trimethoxyaconitane (22): amorphous powder (MeOH); [α]20
229 (4.01), 273 (3.23) nm; CD (MeOH) 217 (Δε −0.09), 234 (Δε
+0.22), 255 (Δε −0.03); IR (KBr) νmax 3407, 3076, 2943, 2832, 1693,
1453, 1415, 1364, 1323, 1281, 1202, 1118, 1057, 1026, 981, 958, 835,
D
−7.70 (c 0.52, MeOH); UV (MeOH) λmax (log ε) 228 (3.97), 271
(2.91) nm; CD (MeOH) 227 (Δε +0.19), 249 (Δε −0.15); IR (KBr)
νmax 3409, 3071, 2936, 2897, 1679, 1452, 1319, 1279, 1204, 1131,
1
802, 719 cm−1; H NMR (Me2CO-d6, 500 MHz) data, see Table 3;
13C NMR (Me2CO-d6, 125 MHz) data, see Table 5; (+)-ESIMS m/z
1
1095, 1048, 836, 801, 720 cm−1; H NMR (Me2CO-d6, 500 MHz)
572 [M + H]+; (+)-HRESIMS m/z 572.3222 [M + H]+(calcd for
C32H46NO8, 572.3218).
data, see Table 4; 13C NMR (Me2CO-d6, 125 MHz) data, see Table 6;
(+)-ESIMS m/z 542 [M + H]+; (+)-HRESIMS m/z 542.3116 [M + H]+
(calcd for C31H44NO7, 542.3112).
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-3α,8β,13β,15α-tetrahydroxy-
1α,6α,16β,18-tetramethoxyaconitane (15): amorphous powder
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-1α,8β,15α-trihydroxy-16β,18-
(MeOH); [α]20 −16.3 (c 0.10, MeOH); UV (MeOH) λmax (log ε)
dimethoxyaconitane (23): amorphous powder (MeOH); [α]20
D
D
228 (3.85), 273 (3.14) nm; CD (MeOH) 216 (Δε +0.80), 229 (Δε +1.61).
1H NMR (Me2CO-d6, 600 MHz) data, see Table 3; 13C NMR
(CD3COCD3, 150 MHz) data, see Table 6; (+)-ESIMS m/z 604
−11.1 (c 0.22, MeOH); UV (MeOH) λmax (log ε) 229 (3.98), 274
(3.19) nm; CD (MeOH) 226 (Δε +0.33), 239 (Δε +0.17), 255 (Δε
+0.28); IR (KBr) νmax 3314, 2955, 2918, 2849, 1732, 1670, 1539,
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1464, 1397, 1260, 1198, 1100, 1027, 801, 722 cm−1; 1H NMR
(Me2CO-d6, 600 MHz) data, see Table 4; 13C NMR (Me2CO-d6, 150
MHz) data, see Table 6; (+)-ESIMS m/z 528 [M + H]+;
(+)-HRESIMS m/z 528.2958 [M + H]+ (calcd for C30H42NO7,
528.2956).
ASSOCIATED CONTENT
* Supporting Information
Copies of IR, MS, 1D and/or 2D NMR, and CD spectra for
compounds 1−28, 27a, and 28a−c. This can be accessed free
of charge via the Internet at http://pubs.acs.org.
■
S
(−)-(A-b)-14α-Benzoyloxy-N-ethyl-1α,8β,15α-trihydroxy-
6α,16β,18-trimethoxyaconitane (24): amorphous powder (MeOH);
[α]20 −13.0 (c 0.58, MeOH); UV (MeOH) λmax (log ε) 230 (3.83),
AUTHOR INFORMATION
Corresponding Author
*Tel: 86-10-83154789. Fax: 86-10-63017757. E-mail: shijg@
imm.ac.cn.
D
■
272 (2.66) nm; CD (MeOH) 219 (Δε −0.12), 229 (Δε −0.01), 250
(Δε −0.51); IR (KBr) νmax 3359, 3077, 2936, 1692, 1602, 1453, 1279,
1200, 1176, 1113, 1076, 1043, 1002, 956, 891, 829, 800, 718 cm−1; 1H
NMR (Me2CO-d6, 500 MHz) data, see Table 4; 13C NMR (Me2CO-
d6, 125 MHz) data, see Table 6; (+)-ESIMS m/z 558 [M + H]+;
(+)-HRESIMS m/z 558.3070 [M + H]+ (calcd for C31H44NO8,
558.3061).
Notes
The authors declare no competing financial interest.
(−)-(A-b)-14α-Cinnamoyloxy-N-ethyl-1α,8β,15α-trihydroxy-
ACKNOWLEDGMENTS
■
6α,16β,18-trimethoxyaconitane (25): amorphous powder (MeOH);
Financial support from the National Natural Sciences
Foundation of China (NNSFC; grant nos. 30825044 and
20932007), the Program for Changjiang Scholars and
Innovative Research Team in University (PCSIRT, grant no.
IRT1007), and the National Science and Technology Project of
China (no. 2011ZX09307-002-01) is acknowledged.
[α]20 −4.63 (c 1.06, MeOH); UV (MeOH) λmax (log ε) 204 (4.02),
D
277 (3.97) nm; CD (MeOH) 227 (Δε −0.49), 253 (Δε +0.33), 280
(Δε +0.56); IR (KBr) νmax 3373, 3063, 2941, 2828, 1678, 1639, 1451,
1312, 1282, 1202, 1114, 982, 936, 863, 833, 802, 771, 720, 686 cm−1;
1H NMR (Me2CO-d6, 600 MHz) data, see Table 4; 13C NMR
(Me2CO-d6, 150 MHz) data, see Table 6; (+)-ESIMS m/z 584 [M +
H]+; (+)-HRESIMS m/z 584.3223 [M + H]+ (calcd for C33H46NO8,
584.3218).
REFERENCES
■
(−)-(A-b)-8β-acetoxy-14α-benzoyloxy-N-ethyl-15α-hydroxy-
(1) Jiangsu New Medical College. Dictionary of Traditional Chinese
Medicine; Shanghai Science and Technology Publishing House:
Shanghai, 1995; pp 228−232 and 1191−1194.
1α,6α,16β,18-tetramethoxyaconitane (26): amorphous powder
(MeOH); [α]20 −37.9 (c 0.21, MeOH); UV (MeOH) λmax (log ε)
D
228 (3.82), 274 (3.18) nm; 1H NMR (Me2CO-d6, 500 MHz) data, see
Table 4; 13C NMR (Me2CO-d6, 125 MHz) data, see Table 6;
(+)-ESIMS m/z 614 [M + H]+; (+)-HRESIMS m/z 614.3328 [M + H]+
(calcd for C34H48NO9, 614.3324).
(2) Konno, C.; Shirasaka, M.; Hikino, H. J. Nat. Prod. 1982, 45, 128−
133.
(3) (a) Shim, S. H.; Kim, J. S.; Kang, S. S. Chem. Pharm. Bull. 2003,
51, 999−1002. (b) Shim, S. H.; Lee, S. Y.; Kim., J. S.; Son, K. H.;
Kang, S. S. Arch. Pharm. Res. 2005, 28, 1239−1243.
(4) (a) Chen, Y.; Zhu, Y.-L.; Zhu, R.-H. Acta Pharm. Sin. 1965, 12,
435−439. (b) Iwasa, J.; Naruto, S. Yakugaku Zasshi 1966, 86, 585−
590.
(5) (a) Pelletier, S. W.; Mody, N. V.; Varughese, K. I. Heterocycles
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Yunnan. 1982, 4, 73−75.
(6) (a) Kitagawa, I.; Yoshikawa, M.; Chen, Z. L.; Kobayashi, K. Chem.
Pharm. Bull. 1982, 30, 758−761. (b) Kitagawa, I.; Chen, Z. L.;
Yoshihara, M.; Yoshikawa, M. Yakugaku Zasshi 1984, 104, 848−857.
(c) Kitagawa, I.; Chen, Z. L.; Yoshihara, M.; Kobayashi, K.; Yoshikawa,
M.; Ono, N.; Yoshimura, Y. Yakugaku Zasshi 1984, 104, 858−866.
(7) (a) Hikino, H.; Kuroiwa, Y.; Konno, C. J. Nat. Prod. 1983, 46,
178−182. (b) Huang, G. -Y.; Cai, P.; Wang, J.-Z.; Snyder, J. K. J. Nat.
Prod. 1988, 51, 364−366.
(8) Wang, J.-Z.; Han, G.-Y. Acta Pharm. Sin. 1985, 20, 71−73.
(9) Zhang, W.-D.; Han, G.-Y.; Liang, H.-Q. Acta Pharm. Sin. 1992,
27, 670−673.
(10) (a) Wang, X.-K.; Zhao, T.-F.; Lai, S. Chin. Chem. Lett. 1994, 5,
671−672. (b) Wang, X.-K.; Zhao, T.-F.; Lai, S. Chin. Pharm. J. 1995,
30, 716−719. (c) Wang, X.-K.; Zhao, T.-F.; Lai, S. Chin. Pharm. J.
1995, 30, 716−718.
(11) Taki, M.; Niitu, K.; Omiya, Y.; Noguchi, M.; Fukuchi, M.;
Aburada, M.; Okada, M. Planta Med. 2003, 69, 800−803.
(12) (a) Chen, H.-C.; Wang, X.-K.; Zhao, T.-F.; Liao, X. Nat. Prod.
Res. Dev. 2003, 15, 324−325. (b) Xiong, J.; Gu, K.; Tan, N. -H. Nat.
Prod. Res. Dev. 2008, 20, 440−443. (c) Liu, X.-X.; Jian, X.-X.; Cai, X.-
F.; Chao, R.-B.; Chen, Q.-H.; Chen, D.-L.; Wang, X.-L.; Wang, F.-P.
Chem. Pharm. Bull. 2012, 60, 144−149.
(13) (a) Ameri, A. Prog. Neurobiol. 1998, 56, 211−235. (b) Wang, F.
P.; Liang, X. T. In The Alkaloids: Chemistry and Biology; Cordell, G. A.,
Ed.; Elsevier Science: New York, 2002; Vol. 59, pp 1−280. (c) Wang,
F. P.; Chen, Q. H.; Liang, X. T. In The Alkaloids: Chemistry and
Biology; Cordell, G. A., Ed.; Elsevier Science: New York, 2009; Vol. 67,
pp 1−78. (d) Wang, F. P.; Chen, Q. H. In The Alkaloids: Chemistry
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(+)-(A-b)-8β-Acetoxy-14α-benzoyloxy-13β,15α-dihydroxy-
1α,6α,16α,18-tereamethoxy-N-methylaconitane (27a): amorphous
1
powder (MeOH); [α]20 +4.40 (c 0.49, MeOH); H NMR (Me2CO-
D
d6 500 MHz) data, see Supporting Information, Table S7; 13C NMR
(Me2CO-d6 125 MHz) data, see Supporting Information, Table S8.
(−)-(A-b)-14α-Benzoyloxy-3α,8β,10β,13β,15α-pentahydroxy-
1α,6α,16α,18-tereamethoxy-N-methylaconitane (28a): amorphous
1
powder (MeOH); [α]20 −7.5 (c 0.87, MeOH); H NMR (Me2CO-
D
d6, 500 MHz) data, see Supporting Information, Table S7; 13C NMR
(Me2CO-d6 125 MHz) data, see Supporting Information, Table S8.
Hydrolysis of 2−4. Compound 2 (5 mg), 3 (5 mg), or 4 (2 mg)
was dissolved in 95% EtOH (1.2 mL), and NaOH (10 mg) was added.
The solution was stirred at 40 °C for 24 h, acidified with HCl (2 N),
and then evaporated under reduced pressure. The residue was
dissolved in CHCl3/MeOH (1:l, 2 mL) and subjected to CC over
Sephadex LH-20, eluting with CHCl3/MeOH (1:l), to afford a mixture
1
with [α]20 +11.2 (c 0.06, MeOH). The H NMR spectrum of the
D
mixture showed signals corresponding to 2-methylbutyric acid and
propanoic acid.
Acetylation of Compounds 7, 8, and 28. Compound 7 (9.0 mg), 8
(4.5 mg), or 28 (12.0 mg) was dissolved in pyridine (1.0 mL), and
acetic anhydride (0.2 mL) was added. The solution was kept at room
temperature overnight and evaporated to dryness under reduced
pressure. The residue was separated by RP HPLC (40% MeCN/H2O,
containing 0.1% TFA) to give 7a (8 and 4 mg) from 7 and 8 and 28b
(3 mg) and 28c (10 mg) from 28, respectively. 7a: amorphous powder
(MeOH); [α]20 −3.05 (c 0.42, MeOH); 1H NMR (Me2CO-d6
D
500 MHz) data, see Table 2; 13C NMR (Me2CO-d6, 125 MHz)
data, see Table 5; (+)-ESIMS m/z 662 [M + H]+. 28b: amorphous
powder (MeOH); [α]20D −5.4 (c 0.46, MeOH); 1H NMR(Me2CO-d6,
500 MHz) data, see Supporting Information, Table S7; 13C NMR
(Me2CO-d6 125 MHz) data, see Supporting Information, Table S8;
(+)-ESIMS m/z 632 [M + H]+. 28c: amorphous powder (MeOH);
1
[α]20 −8.7 (c 0.57, MeOH); H NMR (Me2CO-d6, 500 MHz) data,
D
see Supporting Information, Table S7; 13C NMR (Me2CO-d6, 125 MHz)
data, see Supporting Information, Table S8; (+)-ESIMS m/z 674
[M + H]+.
1158
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