Structure-analgesic activity relationship studies on the C18- and C19-diterpenoid alkaloids

Article abstract of DOI:10.1248/cpb.57.801

For evaluation of C₁₈- and C₁₉-diterpenoid alkaloids as analgesics, three C₁₉-diterpenoid alkaloids were isolated from the roots of Aconitum hemsleyanum var. circinatum and A. transsecutum; and twenty-five semisynthetic C<

Full text of DOI:10.1248/cpb.57.801

August 2009
Chem. Pharm. Bull. 57(8) 801—807 (2009)
801
Structure–Analgesic Activity Relationship Studies on the C18- and
C19-Diterpenoid Alkaloids
Jian-Li WANG,a Xiang-Li SHEN,b Qiao-Hong CHEN,a Gong QI,c Wei WANG,c and Feng-Peng WANG*,a
a Department of Chemistry of Medicinal Natural Products, West China of Pharmacy, Sichuan University; b West China
c
Second Hospital, Sichuan University; No. 17, Duan 3, Renmin Nan Road, Chengdu 610041, P. R. China: and State Key
Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences; Chinese
Academy of Sciences, Shanghai 201203, P. R. China.
Received January 5, 2009; accepted May 23, 2009; published online May 27, 2009
For evaluation of C18- and C19-diterpenoid alkaloids as analgesics, three C19-diterpenoid alkaloids were iso-
lated from the roots of Aconitum hemsleyanum var. circinatum and A. transsecutum; and twenty-five semisyn-
thetic C18- or C19-diterpenoid alkaloids were prepared from lappaconitine, crassicauline A or yunaconitine. In a
mice acetic acid-induced abdominal constriction assay, four crassicauline A analogs and three yunaconitine
analogs exhibited good analgesic activities with 77.8—94.1% inhibition range in 0.1—10 mg/kg subcutaneous
(s.c.) dose range at the point of 20 min after drug administration. Among them, 8-O-deacetyl-8-O-ethylcrassi-
cauline A (ED50ꢀ0.0972 mg/kg) and 8-O-ethylyunaconitine (ED50ꢀ0.0591 mg/kg) were the most potent analgesics
relative to the reference drugs lappaconitine (ED50ꢀ3.50 mg/kg) and crassicauline A (ED50ꢀ0.0480 mg/kg). Anal-
gesic activity data of these C18- and C19-diterpenoid alkaloids indicate that a tertiary amine in ring A, an acetoxyl
or an ethoxyl group at C-8, an aromatic ester at C-14, and the saturation state of the ring D are important struc-
tural features necessary to the analgesic activity of the C19-diterpenoid alkaloids.
Key words diterpenoid alkaloid; lappaconitine; crassicauline A; yunaconitine; analgesic activity
Aconitum and Delphinium plants have been medicinally semisynthetic alkaloids, as well as three naturally occurring
used for centuries.1,2) The traditional Chinese medicine “Cao- alkaloids, were evaluated in a mice acetic acid-induced ab-
Wu,” the tubers of Aconitum species, has been extensively dominal constriction assay. We describe herein the prepara-
employed for the clinical treatment of pains, rheumatics and tion and structure–analgesic activity relationship (SAR) stud-
neurological disorders.3,4) The pharmacological effects of ies for the C18- and C19-diterpenoid alkaloids.
Aconitum plants are attributed to their characteristic diter-
penoid alkaloids, a group of complex natural products dis- Results and Discussion
playing a lot of interesting chemistry5,6) and biological activi-
Chemistry To further investigate the analgesic activities
ties.1) The analgesic activities of C18- and C19-diterpenoid of C18- and C19-diterpenoid alkaloids, five lappaconitine de-
alkaloids have been extensively investigated since 1981, rivatives (see structures in Fig. 2), seventeen crassicauline A
among which 3-acetylaconitine,7) lappaconitine,8,9) and cras- derivatives (see structures in Fig. 3), and six yunaconitine
sicauline A (bulleyaconitine A)10) have been reported to ex- analogs (see structures in Fig. 4) were synthesized from
hibit marked analgesic activities and have been developed to their parent compounds or isolated from Aconitum plants.
be analgesic drugs clinically used for the treatment of various N-Deethyllappaconitine (1), N-deethyllappaconitine imine
pains in China.12—16) As compared with the known anal- (4) and N-deethyl-5ꢀ-bromolappaconitine imine (5) were
gesics, such as morphine, methadone, etc., all these three al- prepared by treatment of lappaconitine with N-bromosuc-
kaloids induced neither morphine-like tolerance nor physical cinimide (NBS)-HOAc by the similar procedure previously
dependence.
developed by us.22,23) N-Deethyl-N,8,9-triacetyllappaconitine
Although there are a few reports11,13,17—21) on the struc- (2) and N-deethyl-N-acetyllappaconitine (3) were synthe-
ture–analgesic activity relationship studies on the C18- and sized by acetylation of compound 1 with acetic anhydride-
C19-diterpenoid alkaloids, further specific information on the pyridine.
structure features necessary for the analgesic activity of these
8-O-Deacetyl-8-O-ethylcrassicauline A (9) and 8-O-
compounds is still needed. In this paper, twenty-five semi- deacetyl-8-O-isopentylcrassicauline A (10) were prepared
synthetic derivatives were prepared from lappaconitine, cras- from crassicauline A based on the procedure reported in the
sicauline A, and yunaconitine. Analgesic activities of these
Fig. 1. C18- and C19-Diterpenoid Alkaloids that Exhibit Analgesic Activi-
ties
Fig. 2. Lappaconitine Analogues
∗ To whom correspondence should be addressed. e-mail: wfp@scu.edu.cn
© 2009 Pharmaceutical Society of Japan

802
Vol. 57, No. 8
8,14-O-diethylbikhaconine (17) and 14-O-ethylbikhaconine
(18), were obtained by ethylation of hydroxyl in the bikhaco-
nine with ethyl bromide. N-Deethylcrassicauline A (6), N-
deethylcrassicauline A imine (7) and crassicauline A lactam
(8) were achieved by reaction of crassicauline A with N-bro-
mosuccinimide (NBS)-glacial acetic acid based on the meth-
ods developed by us.22,23) Pyrocrassicauline A (19) and 13-t-
butoxycarbonyl-pyrocrassicauline A (20) were prepared by
selective hydrolysis of crassicauline A with dioxane–H2O
(2 : 1) according to Murayama’s method4) followed by reflux-
ing with (Boc)2O in 41% and 35% yields, respectively. 1,16-
Didemethoxy-D15,16-yunaconitine (26), 1-demethoxy-3,13-
diacetyl-14-methane sulfonyl-pyrobikhaconine (27) were
readily prepared according to the method reported in the lit-
erature.25) 1-Demethoxyyunaconitine (24, 70%) and 3-epi-1-
demethoxyyunaconitine (25, 18%) were obtained by reduc-
tion of 1-demethoxy-3-dehydroyunaconitine (31, derived
from yunaconitine,26,27)) with sodium borohydride. Crassi-
cauline A analogues, hemsleyanisine (21) and isohems-
leyanisine (22), as well as yunaconitine analogue 8-O-ethyl-
yunaconitine (23), were isolated from the roots of Aconitum
hemsleyanum var. circinatum and A. transsecutum according
to the procedures described in the literature.27,28)
Fig. 3. Crassicauline A Analogues
Analgesic Activities Pharmacological studies were car-
ried out to assess the in vivo analgesic activities of the above-
mentioned C18- and C19-diterpenoid alkaloids. According to
the one-dose preliminary assay data, the compounds that
showed marked inhibition of writhing movement (ꢁ50%) in-
duced by acetic acid were selected for their ED50 value evalu-
ation. Four most potent analgesics (6, 9, 23, 2826)) were se-
lected for acute toxic examination, based on in vivo
analgesic activity data. All these pharmacological data and
toxic examination data were summarized in Table 1. In a
mice model 0.7% acetic acid-induced abdominal constriction
assay,29) a 10 mg/kg or less subcutaneous (s.c.) dose of N-
deethylcrassicauline A (6), N-deethylcrassicauline imine (7),
8-O-deacetyl-8-O-ethylcrassicauline A (9), hemsleyanisine
(21), 8-O-ethylyunaconitine (23), 1-demethoxyyunaconitine
(24), and 1,16-didemethoxy-8-O-deacetyl-D15,16-yunaconi-
tine (28) exhibited good analgesic activities (78—94% inhi-
bition range) at the point of 20 min after drug administration
(see data in Table 1). Among them, compounds 6, 9, 23, and
28 (ED50ꢂ0.0591—2.58 mg/kg) exhibited superior analgesic
activity compared to the reference drug lappaconitine
(ED50ꢂ3.50 mg/kg); compounds 9 and 23 (ED50ꢂ0.0591,
0.0972 mg/kg) exhibited comparable analgesic activity to the
reference drug crassicauline A (ED50ꢂ0.0480 mg/kg). The
other alkaloids under investigation showed moderate to inac-
tive analgesic activities in the acetic acid-induced abdominal
Fig. 4. Yunaconitine Analogues
literature.24) 8-O-Deacetyl-8-O-benzoylcrassicauline A (11), constriction assay.
8-O-deacetyl-8-O-t-butoxycarbonyloxycrassicauline A (15)
The structure–activity relationship (SAR) data acquired
and 8-O-deacetyl-8-O,13-di-t-butoxycarbonylcrassicauline A for lappaconitine derivatives show that the analogues derived
(16) were synthesized by selective hydrolysis of crassicauline from the modification on ring A of lappaconitine, including
A with dioxane–H2O (2 : 1) according to Murayama’s N-deethyl derivative (1), the amides (2, 3), and the imines (4,
method4) followed by acetylation with benzoyl chloride 5), exhibit significantly decreased analgesic activities (0—
(BzCl) or di-tert-butyl dicarbonate [(Boc)2O]. Three acety- 48% inhibition range at a 10 mg/kg s.c. dose) relative to the
lated bikhaconine derivatives, 8,14-dibenzoylbikhaconine parent compound lappaconitine (ED50ꢂ3.50 mg/kg).
(12), 14-acetylbikhaconine (13) and 14-t-butoxycarbonyl-
The in vivo data acquired for crassicauline A analogues
bikhaconine (14), were produced by acetylation of bikhaco- (9—22) show that analgesic activities can be manipulated by
nine (derived from crassicauline A) with BzCl, Ac2O, and varying the electronic and steric properties of substituents at-
(Boc)2O, respectively. Two ethylated bikhaconine derivatives, tached to C-8. For example, compound 9 possessing an

August 2009
803
Table 1. Antinociceptive Effects of Compounds on Acetic Acid-Induced Table 1). In this subgroup, 8-O-ethylyunaconitine (23) is an
Writhing Test and Preliminary Acute Toxicity in Mice
equipotent analgesics (ED50ꢂ0.0591 mg/kg) relative to cras-
sicauline A (ED50ꢂ0.0480 mg/kg), indicating that the re-
Toxic
dose
Mortalitya)
(died/
Dose
Inhibition
(%)
ED50
placement of C-8 acetoxyl group with ethoxyl group does
not affect the analgesic activities. Similar to crassicauline A
derivatives, D8(15) derivative (27) is inactive. Similarly, D15(16)
derivative (26) possesses less activity (41.2% inhibition at a
10 mg/kg s.c. dose) compared to its parent compound (24,
94.1% inhibition at a 10 mg/kg s.c. dose). Stereochemistry at
C-3 is important for the activity since 3a-hydroxyl analogue
24 exhibits good activity (ED50ꢂ7.98 mg/kg), while its iso-
mer, 3b-hydroxyl analogue 25, is inactive.
In conclusion, the above-mentioned structure–activity
studies show that an N-ethyl substituted tertiary amine in
ring A, an acetoxyl or ethoxyl group at C-8, an aromatic
ester (OBz or OAs) at C-14, and the saturation state of the
ring D are necessary for the manifestation of important anal-
gesic activity of C18- and C19-diterpenoid alkaloids. Also, 3a
or 5-hydroxyl group is helpful for the analgesic activity. The
structure–activity data established in this study constructs the
critical pharmacophore of C18- and C19-diterpenoid alkaloids
as analgesics, which could be beneficial in searching for the
potential analgesics that is equal or more active, but with
lower toxicity, than currently clinical used C18- and C19-alka-
loid-type analgesics.
Compound
(mg/kg)
(mg/kg)
(mg/kg)
tested)
6
7
9
21
23
24
28
0.8
10
0.2
6
90.0
84.2
86.4
77.8
84.0
94.1
94.1
0.411
6.42
4
—
1
5/5
—
0.0972
3.42
2/5
—
—
0.6
—
26
0.1
10
4
0.0591
7.98
3/5
—
2.58
5/5
1
2
3
4
5
8
10
11
12
13
14
15
16
17
18
19
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
—
—
48.0
0.0
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
11.7
0.92
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
10.0
20.0
18.2
25.0
15.0
36.4
16.7
11.1
34.6
20.0
21.7
23.1
19.2
25.0
15.0
11.1
16.7
41.2
22.2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
20
22
25
26
27
—
Experimental
—
General IR spectra were recorded on a Nicolet 200 SXV spectrometer;
mass spectra were obtained with a Finnigan LCQ and Micromass Auto Spec
Ultima-Tof spectrometer; 1H- and 13C-NMR spectra were acquired on a
Bruker AC-E 200 or a Varian INOVA-400/54 spectrometer in CDCl3, with
tetramethylsilane (TMS) as internal standard; Silica GF254 and gel H (10—
40 mm, Qingdao Sea Chemical Factory, China) were used for TLC and CC.
Only key signals for all products in the 1H-NMR spectra were reported. The
starting materials crassicauline A, yunaconitine, and lappaconitine were pur-
chased from Kunming Institute of Botany at the Chinese Academy of Sci-
ences and Lanzhou Pharmaceutical Company in China.
—
—
—
Lappaconitine
Crassicauline A
3.50
0.0480
—
The acetic acid induced writhing data is displayed as percent reduction. a) Dead
mice were counted at 1 h after injection of test samples.
N-Deethyllappaconitine (1) To a solution of lappaconitine (100 mg,
0.17 mmol) in glacial acetic acid (5 ml) was added NBS (910 mg, 0.51
mmol), and the subsequent reaction solution was allowed to stand at room
temperature for 1.5 h. After a general work-up, column chromatography (sil-
ica gel, 3 g, CHCl3–MeOHꢂ94 : 6) of the crude residue gave title compound
1 (92 mg, 95%): 1H-NMR (200 MHz) d: 2.22 (3 H, s, NHCOCH3), 3.30,
3.30, 3.40 (each 3H, s, OCH3ꢃ3), 7.02 (1H, m, H-5ꢀ), 7.50 (1H, m, H-4ꢀ),
7.90 (1H, d, Jꢂ8.0 Hz, H-3ꢀ), 8.66 (1H, d, Jꢂ8.4 Hz, H-6ꢀ), 11.03 (1H, br s,
ethoxyl group at C-8 instead of an acetoxyl group can retain
potential analgesic activity (ED50ꢂ0.0972 mg/kg), while
8,14-diethoxyl analogue 17 is inactive. In contrast, replace-
ment of the acetoxy group at C-8 of crassicauline A with a
bulky alkoxyl (isopenthoxyl) group (10) or bulky ester such
as Boc (15, 16) or BzO (11, 12) abolishes the analgesic ac-
tivity. It is also interesting to note that 14-acetylbikhaconine NHCOCH3). 13C-NMR (50 MHz) d: 82.5 (C-1), 23.7 (C-2), 29.6 (C-3), 83.5
(C-4), 52.3 (C-5), 26.2 (C-6), 44.2 (C-7), 75.9 (C-8), 77.3 (C-9), 36.8 (C-
10), 52.7 (C-11), 24.4 (C-12), 49.2 (C-13), 90.0 (C-14), 44.0 (C-15), 82.3
(C-16), 57.0 (C-17), 50.8 (C-19), 55.8 (C-1ꢄ), 56.0 (C-14ꢄ), 57.8 (C-16ꢄ),
169.0, 25.2 (NHCOCH3), 167.2 (COO), 115.1 (C-1ꢀ), 141.5 (C-2ꢀ), 120.1
(13), 14-t-butoxycarbonyl-bikhaconine (14), and 14-O-ethyl-
bikhaconine (18), possessing a hydroxyl group at C-8 instead
of an acetoxyl group in crassicauline A and a substitute at C-
14 different from crassicauline A, show very weak activity.
Introduction of a double bond at C-8/C-15 via elimination of
C-8 acetoxyl group (19, 20) dramatically diminishes the ac-
tivities. The point of attachment of a p-methoxy-benzoyloxy
(OAs) substituent is a determinant of analgesic activity be-
cause the 14-OAs compound (21), structurally similar to par-
ent compound crassicauline A, shows good analgesic activity
(ED50ꢂ3.42 mg/kg). In contrast, the 16-OAs regioisomer
(22) is inactive. On the other hand, ring A modified deriva-
tives of crassicauline A, N-deethyl derivative (6), imine (7)
and lactam (8), exhibit less activity than their parent com-
pound crassicauline A even though analogues 6 and 7 still
maintain good analgesic activity (ED50ꢂ0.411, 6.42 mg/kg).
The mice acetic acid-induced abdominal constriction assay
data show that yunaconitine subgroup (23—28) exhibits a
(C-3ꢀ), 134.3 (C-4ꢀ), 122.3 (C-5ꢀ), 130.9 (C-6ꢀ). HR-ESI-MS m/z: 557.2847
[MꢅH]ꢅ (Calcd for C30H41N2O8: 557.2857).
N-Deethyl-N,8,9-triacetyllappaconitine (2) and N-Deethyl-N-acetyl-
lappaconitine (3) The acetylated C18-diterpenoid alkaloids 2 and 3 were
synthesized by acetylation of 1 following a general procedure. 2: 89% yield.
1H-NMR (200 MHz) d: 2.03, 2.13, 2.17, 2.21 (each 3H, OCOCH3ꢃ4), 3.23,
3.33, 3.41 (each 3H, s, OCH3ꢃ3), 4.78 (1H, ABq, Jꢂ14.5 Hz, H-19), 4.90
(1H, Jꢂ4.9 Hz, H-14b), 7.03 (1H, m, H-5ꢀ), 7.54 (1H, m, H-4ꢀ), 7.84 (1H, d,
Jꢂ8.4 Hz, H-3ꢀ), 8.66 (1H, d, Jꢂ8.4 Hz, H-6ꢀ), 10.97 (1H, br s, NHAc). 13C-
NMR (50 MHz) d: 81.0 (C-1), 23.3 (C-2), 31.1 (C-3), 88.0 (C-4), 46.4 (C-
5), 26.2 (C-6), 45.9 (C-7), 82.7 (C-8), 83. 9 (C-9), 38.1 (C-10), 50.6 (C-11),
25.2 (C-12), 48.7 (C-13), 82.1 (C-14), 40.1 (C-15), 80.6 (C-16), 57.8
(C-17), 46.6 (C-19), 170.2 (C-21), 25.4 (C-22), 56.5 (C-1ꢄ), 55.7 (C-14ꢄ),
57.5 (C-16ꢄ), 169.2, 169.0, 22.4, 22.3 (OAcꢃ2), 169.2, 23.4 (NHCOCH3),
167.0 (COO), 115.1 (C-1ꢀ), 141.9 (C-2ꢀ), 120.4 (C-3ꢀ), 134.7 (C-4ꢀ), 122.3
(C-5ꢀ), 130.6 (C-6ꢀ). HR-ESI-MS m/z: 705.2967 [MꢅNa]ꢅ (Calcd for
C36H46N2O11Na: 705.2994). 3: 86% yield. 1H-NMR (200 MHz) d: 2.17, 2.22
(each 3H, s, OCOCH3ꢃ2), 3.22, 3.31, 3.34 (each 3H, s, OCH3ꢃ3), 7.01
wide range (low-to-good) of analgesic activities (see data in (1H, t, Jꢂ7.8 Hz, H-5ꢀ), 7.49 (1H, t, Jꢂ8.0 Hz, H-4ꢀ), 7.89 (1H, d, Jꢂ8.4

804
Vol. 57, No. 8
5 : 2, with 1% diethylamine) furnished
8
(50 mg, 4.9%): 1H-NMR
Hz, H-3ꢀ), 8.66 (1H, d, Jꢂ8.4 Hz, H-6ꢀ), 11.03 (1H, br s, NHAc). 13C-NMR
(50 MHz) d: 82.8 (C-1), 24.4 (C-2), 31.1 (C-3), 82.5 (C-4), 53.4 (C-5), 26.2
(C-6), 49.7 (C-7), 75.0 (C-8), 77.8 (C-9), 36.6 (C-10), 50.9 (C-11), 25.2 (C-
12), 53.7 (C-13), 89.7 (C-14), 44.5 (C-15), 80.9 (C-16), 58.6 (C-17), 47.0
(C-19), 169.2 (C-21), 22.3 (C-22), 56.2 (C-1ꢄ), 55.7 (C-14ꢄ), 57.9 (C-16ꢄ),
169.1, 22.5 (NHCOCH3), 167.0 (COO), 115.0 (C-1ꢀ), 141.7 (C-2ꢀ), 120.2
(C-3ꢀ), 134.5 (C-4ꢀ), 122.2 (C-5ꢀ), 130.8 (C-6ꢀ). HR-ESI-MS m/z: 621.2775
[MꢅNa]ꢅ (Calcd for C32H42N2O9Na: 621.2783).
(200 MHz) d: 1.05 (3H, t, Jꢂ7.1 Hz, NCH2CH3), 1.30 (3H, s, OCOCH3),
3.12, 3.28, 3.37, 3.53 (each 3H, s, OCH3ꢃ4), 4.77 (1H, br s, H-14b), 6.89,
7.97 (each 2H, AAꢄBBꢄ system, Jꢂ8.2 Hz, Ar-H). 13C-NMR (50 MHz) d:
84.9 (C-1), 25.5 (C-2), 31.0 (C-3), 41.0 (C-4), 50.7 (C-5), 81.4 (C-6), 50.6
(C-7), 84.0 (C-8), 42.5 (C-9), 45.7 (C-10), 51.3 (C-11), 33.6 (C-12), 75.6
(C-13), 78.5 (C-14), 40.1 (C-15), 83.4 (C-16), 60.1 (C-17), 77.8 (C-18),
124.0 (C-19), 56.1 (C-1ꢄ), 58.7 (C-6ꢄ), 57.1 (C-16ꢄ), 59.5 (C-18ꢄ), 169.7,
21.4 (OAc), 168.0 (COO), 122.3 (C-1ꢀ), 131.6 (C-2ꢀ, 6ꢀ), 113.7 (C-3ꢀ, 5ꢀ),
163.4 (C-4ꢀ), 55.4 (4ꢀ-OCH3). HR-ESI-MS m/z: 658.3230 [MꢅH]ꢅ (Calcd
for C35H48NO11: 658.3227).
8-O-Deacetyl-8-O-ethylcrassicauline A (9) A solution of crassicauline
A (100 mg, 0.17 mmol) in ethanol (5 ml) was kept refluxing for 3 d, and
then the ethanol was removed under reduced pressure. The residue obtained
was purified over silica gel column chromatography employing chloro-
form–methanol (97 : 3) as eluent to give the title compound 9 (84 mg, 89%).
1H-NMR (200 MHz) d: 0.57 (3H, t, Jꢂ6.8 Hz, NCH2CH3), 1.07 (3H, t,
Jꢂ7.0 Hz, OCH2CH3), 3.24, 3.24, 3.28, 3.32 (each 3H, s, OCH3ꢃ4), 3.85
(3H, s, Ar-OCH3), 3.99 (1H, d, Jꢂ3.7 Hz, H-6b), 4.82 (1H, d, Jꢂ5.1 Hz, H-
14b), 6.88, 8.01 (each 2H, AAꢄBBꢄ system, Jꢂ8.7 Hz, Ar-H). 13C-NMR
(50 MHz) d: 85.3 (C-1), 26.3 (C-2), 36.4 (C-3), 38.9 (C-4), 48.9 (C-5),
82.8 (C-6), 48.9 (C-7), 78.0 (C-8), 41.3 (C-9), 46.3 (C-10), 50.4 (C-11),
29.6 (C-12), 75.3 (C-13), 79.0 (C-14), 37.2 (C-15), 84.0 (C-16), 61.3 (C-17),
80.0 (C-18), 53.7 (C-19), 49.1 (C-21), 13.4 (C-22), 56.2 (C-1ꢄ), 58.6 (C-6ꢄ),
58.6 (C-16ꢄ), 58.9 (C-18ꢄ), 166.2 (COO), 123.3 (C-1ꢀ), 131.6 (C-2ꢀ, 6ꢀ),
113.3 (C-3ꢀ, 5ꢀ), 163.0 (C-4ꢀ), 55.7 (4ꢀ-OCH3), 55.7 (OCH2), 15.2
(OCH2CH3). HR-ESI-MS m/z: 630.3635 [MꢅH]ꢅ (Calcd for C35H52NO9:
630.3642).
N-Deethyllappaconitine Imine (4) The imine 4 was obtained in 73%
1
yield by the same synthetic procedure as that of 1. H-NMR (200 MHz) d:
2.18 (3H, s, OCOCH3), 3.22, 3.31, 3.41 (each 3H, s, OCH3ꢃ3), 7.03 (1H,
m, H-5ꢀ), 7.52 (1H, m, H-4ꢀ), 7.96 (1H, d, Jꢂ8.0 Hz, H-3ꢀ), 8.68 (1H, d,
Jꢂ8.4 Hz, H-6ꢀ), 11.0 (1H, br s, NHAc). 13C-NMR (50 MHz) d: 82.3 (C-1),
23.2 (C-2), 27.0 (C-3), 88.6 (C-4), 48.1 (C-5), 26.9 (C-6), 41.3 (C-7), 75.4
(C-8), 88.0 (C-9), 36.8 (C-10), 53.8 (C-11), 26.9 (C-12), 52.4 (C-13),
89.7 (C-14), 44.2 (C-15), 81.3 (C-16), 62.1 (C-17), 159.2 (C-19), 56.2 (C-
1ꢄ), 56.4 (C-14ꢄ), 57.9 (C-16ꢄ), 169.0, 25.5 (NHCOCH3), 167.0 (COO),
115.1 (C-1ꢀ), 141.8 (C-2ꢀ), 120.2 (C-3ꢀ), 134.7 (C-4ꢀ), 122.3 (C-5ꢀ), 130.8
(C-6ꢀ). HR-ESI-MS m/z: 577.2532 [MꢅNa]ꢅ (Calcd for C30H38N2O8Na:
577.2520).
N-Deethyl-5ꢁ-bromolappaconitine Imine (5) A solution of lappaconi-
tine (100 mg, 0.17 mmol) and NBS (246 mg, 1.36 mmol) in glacial acetic
acid (5 ml) was allowed to stand at room temperature overnight. After a gen-
eral work-up, column chromatography (silica gel, 3 g, petroleum ether–ace-
toneꢂ2 : 1) of the crude residue afforded 5 (94 mg, 87%). 1H-NMR
(200 MHz) d: 2.21 (3H, s, NHAc), 3.22, 3.31, 3.34 (each 3H, s, OCH3ꢃ3),
7.67 (1H, dd, Jꢂ10.0, 2.4 Hz, H-4ꢀ), 8.04 (1H, d, Jꢂ2.4 Hz, H-6ꢀ), 8.62 (1H,
d, Jꢂ10.0 Hz, H-3ꢀ), 10.93 (1H, br s, NHAc). 13C-NMR (50 MHz) d: 82.3
(C-1), 22.3 (C-2), 29.6 (C-3), 89.3 (C-4), 52.3 (C-5), 26.8 (C-6), 41.2 (C-7),
75.3 (C-8), 76.5 (C-9), 36.8 (C-10), 53.9 (C-11), 23.2 (C-12), 48.1 (C-13),
89.7 (C-14), 44.2 (C-15), 82.6 (C-16), 62.1 (C-17), 158.8 (C-19), 56.5 (C-
1ꢄ), 56.2 (C-14ꢄ), 57.9 (C-16ꢄ), 169.0, 25.2 (NHCOCH3), 165.8 (COO),
114.0 (C-1ꢀ), 140.8 (C-2ꢀ), 121.9 (C-3ꢀ), 137.4 (C-4ꢀ), 116.6 (C-5ꢀ), 133.1
(C-6ꢀ). HR-ESI-MS m/z: 655.1637 [MꢅNa]ꢅ (Calcd for C30H37N2O8BrNa:
655.3631).
N-Deethylcrassicauline A (6) To a solution of crassicauline A (100 mg,
0.15 mmol) in 5 ml of glacial acetic acid was added NBS (80 mg,
0.45 mmol). The reaction mixture was allowed to stand at room temperature
overnight and basified to ph 10 using 25% NH4OH. The subsequent mixture
was extracted with chloroform. The combined extracts were dried (Na2SO4),
and the solvent was removed in vacuo to afford compound 6 (83 mg, 90%).
1H-NMR (200 MHz) d: 1.34 (3H, s, OCOCH3), 3.16, 3.22, 3.28, 3.53, 3.84
(each 3H, s, OCH3ꢃ5), 3.98 (1H, d, Jꢂ5.4 Hz, H-6b), 4.88 (1H, d,
Jꢂ5.0 Hz, H-14b), 6.88, 7.97 (each 2H, AAꢄBBꢄ system, Jꢂ8.8 Hz, Ar-H).
13C-NMR (50 MHz) d: 83.1 (C-1), 23.4 (C-2), 35.2 (C-3), 39.0 (C-4), 44.1
(C-5), 82.5 (C-6), 53.4 (C-7), 85.5 (C-8), 40.2 (C-9), 43.6 (C-10), 50.3 (C-
11), 29.0 ( C-12), 74.5 (C-13), 78.7 (C-14), 39.7 (C-15), 83.1 (C-16), 55.3
(C-17), 79.9 (C-18), 49.3 (C-19), 57.5 (C-1ꢄ), 58.6 (C-6ꢄ), 57.7 (C-16ꢄ), 59.0
(C-18ꢄ), 169.5, 21.4 (OAc), 165.7 (COO), 122.3 (C-1ꢀ), 131.5 (C-2ꢀ, 6ꢀ),
113.6 (C-3ꢀ, 5ꢀ), 163.3 (C-4ꢀ), 55.3 (4ꢀ-OCH3). HR-ESI-MS m/z: 616.3088
[MꢅH]ꢅ (Calcd for C33H46NO10: 616.3116).
N-Deethylcrassicauline A Imine (7) To a solution of crassicauline A
(1 g, 1.55 mmol) in 25 ml of glacial acetic acid was added NBS (2.2 g,
12.44 mmol), and the reaction was allowed to stand at room temperature
overnight. The reaction mixture was basified to pH 10 using 25% NH4OH,
and the subsequent mixture was extracted with chloroform. The combined
extracts were dried over anhydrous Na2SO4, and the solvent was removed in
vacuo to afford the crude product. Purification of the product by silica gel
(25 g) column chromatography using petroleum ether–acetone (5 : 2, with
1% diethylamine) as eluant furnished 7 (693 mg, 73%): 1H-NMR (200 MHz)
d: 1.27 (3H, s, OCOCH3), 3.04, 3.15, 3.27, 3.51 (each 3H, s, OCH3ꢃ4),
4.87 (1H, d, Jꢂ4.7 Hz, H-14b), 6.87, 7.96 (each 2H, AAꢄBBꢄ system,
Jꢂ8.6 Hz, Ar-H). 13C-NMR (50 MHz) d: 83.5 (C-1), 22.5 (C-2), 35.8 (C-3),
46.5 (C-4), 53.9 (C-5), 82.0 (C-6), 42.8 (C-7), 84.2 (C-8), 40.1 (C-9), 45.7
(C-10), 51.5 (C-11), 27.8 ( C-12), 74.6 (C-13), 78.6 (C-14), 38.6 (C-15),
82.1 (C-16), 61.2 (C-17), 77.8 (C-18), 165.8 (C-19), 55.9 (C-1ꢄ), 58.7 (C-
6ꢄ), 57.1 (C-16ꢄ), 59.0 (C-18ꢄ), 169.5, 21.4 (OAc), 165.8 (COO), 122.4 (C-
1ꢀ), 131.6 (C-2ꢀ, 6ꢀ), 113.7 (C-3ꢀ, 5ꢀ), 163.4 (C-4ꢀ), 55.3 (4ꢀ-OCH3). HR-
ESI-MS m/z: 614.2945 [MꢅH]ꢅ (Calcd for C33H44NO10: 614.2960).
Crassicauline A Lactam (8) To a solution of crassicauline A (1 g,
1.55 mmol) in 25 ml of glacial acetic acid was added NBS (2.2 g,
12.44 mmol), and the reaction solution was allowed to stand at room tem-
perature overnight. After a general work-up, purification of the crude residue
over column chromatography (silica gel, 25 g, petroleum ether–acetoneꢂ
8-Deacetoxy-8-isopentoxycrassicauline A (10) and Pyrocrassicauline
A (19) A solution of crassicauline A (300 mg, 0.45 mmol) in isopentyl al-
cohol (50 ml) was heated at 80 °C for 3 d. After a general work-up, the crude
residue was subjected to column chromatography (silica gel, 10 g, petroleum
ether–acetoneꢂ11 : 2) to give C-8 isopentoxylated analogue 10 (90 mg,
1
30%) and pyrocrassicauline A 19 (126 mg, 41%). 10: H-NMR (200 MHz)
d: 1.09 (3H, t, Jꢂ7.2 Hz, NCH2CH3), 3.26, 3.26, 3.29, 3.54 (each 3H, s,
OCH3ꢃ4), 3.85 (3H, s, 4ꢀ-OCH3), 4.00 (1H, d, Jꢂ6.4 Hz, H-6b), 4.80 (1H,
d, Jꢂ4.9 Hz, H-14b), 6.90, 8.02 (each 2H, AAꢄBBꢄ system, Jꢂ8.8 Hz, Ar-
H). 13C-NMR (50 MHz) d: 85.3 (C-1), 26.3 (C-2), 37.4 (C-3), 39.0 (C-4),
48.2 (C-5), 82.9 (C-6), 49.1 (C-7), 78.0 (C-8), 41.4 (C-9), 46.3 (C-10), 50.6
(C-11), 36.3 (C-12), 75.2 (C-13), 79.1 (C-14), 39.0 (C-15), 84.0 (C-16), 61.3
(C-17), 80.0 (C-18), 53.7 (C-19), 48.9 (C-21), 13.4 (C-22), 56.3 (C-1ꢄ), 58.7
(C-6ꢄ), 58.7 (C-16ꢄ), 58.9 (C-18ꢄ), 166.1 (COO), 123.3 (C-1ꢀ), 131.8 (C-2ꢀ,
6ꢀ), 113.3 (C-3ꢀ, 5ꢀ), 163.1 (C-4ꢀ), 55.3 (4ꢀ-OCH3), 61.3, 30.2, 24.6, 22.6,
22.3 (OCH2CH2CH(CH3)2). HR-ESI-MS m/z 672.4109 [MꢅH]ꢅ (Calcd for
1
C38H58NO9, 672.4106). 19: H-NMR (200 MHz) d: 1.07 (3H, t, Jꢂ7.1 Hz,
NCH2CH3), 3.24, 3.28, 3.30, 3.38 (each 3H, s, OCH3ꢃ4), 3.85 (3H, s, 4ꢀ-
OCH3), 4.20 (1H, d, Jꢂ6.6 Hz, H-6b), 4.94 (1H, d, Jꢂ2.8 Hz, H-14b), 5.54
(1H, d, Jꢂ6.2 Hz, H-15), 6.88, 8.00 (each 2H, AAꢄBBꢄ system, Jꢂ8.8
Hz, Ar-H). 13C-NMR (50 MHz) d: 86.0 (C-1), 25.1 (C-2), 38.4 (C-3),
39.8 (C-4), 48.5 (C-5), 83.5 (C-6), 50.9 (C-7), 146.9 (C-8), 46.6 (C-9),
48.4 (C-10), 51.6 (C-11), 35.3 (C-12), 77.6 (C-13), 78.3 (C-14), 116.0 (C-
15), 83.6 (C-16), 59.2 (C-17), 80.3 (C-18), 54.0 (C-19), 49.8 (C-21), 13.5
(C-22), 56.3 (C-1ꢄ), 58.1 (C-6ꢄ), 57.1 (C-16ꢄ), 59.2 (C-18ꢄ), 167.8 (COO),
122.8 (C-1ꢀ), 131.9 (C-2ꢀ, 6ꢀ), 113.4 (C-3ꢀ,5ꢀ), 163.2 (C-4ꢀ), 55.3 (4ꢀ-
OCH3). HR-ESI-MS m/z: 584.3221 [MꢅH]ꢅ (Calcd for C33H46NO8:
584.3218).
8-Deacetoxyl-8-benzoyloxycrassicauline A (11) A solution of crassi-
cauline A (1 g, 1.55 mmol) in dioxane–H2O (1 : 1) (50 ml) was refluxed
overnight. After a general work-up, residue A (913 mg, 98%) was obtained.
To a solution of residue A (100 mg, 0.16 mmol) in 5 ml of pyridine was
added 4-dimethylaminopyridine (DMAP) (100 mg) and 4.24 mmol of ben-
zoyl chloride, and the resulting reaction mixture was kept refluxing
overnight and cooled to room temperature. After a general work-up, the
crude residue was subjected to column chromatography (silica gel, 3 g,
petroleum ether–acetoneꢂ7 : 1, with 1% of diethylamine) to furnish 11
(50 mg, 43%). 1H-NMR (200 MHz) d: 1.12 (3H, t, Jꢂ6.9 Hz, NCH2CH3),
3.23, 3.24, 3.27, 3.29 (each 3H, s, OCH3ꢃ4), 3.83 (3H, s, 4ꢀ-OCH3), 4.05
(1H, d, Jꢂ6.7 Hz, H-6b), 5.68 (1H, d, Jꢂ4.8 Hz, H-14b), 5.54 (1H, d,
Jꢂ6.2 Hz, H-15), 6.89—8.07 (8H, m, Ar-H). 13C-NMR (50 MHz) d: 85.0
(C-1), 22.6 (C-2), 35.7 (C-3), 39.2 (C-4), 53.1 (C-5), 79.2 (C-6), 53.1 (C-7),
84.1 (C-8), 42.9 (C-9), 46.8 (C-10), 49.5 (C-11), 29.6 (C-12), 73.6 (C-13),
77.1 (C-14), 42.4 (C-15), 82.6 (C-16), 61.8 (C-17), 80.5 (C-18), 54.0 (C-19),
46.8 (C-21), 13.5 (C-22), 56.0 (C-1ꢄ), 57.8 (C-6ꢄ), 57.5 (C-16ꢄ), 59.1 (C-
18ꢄ), 165.6 (COO), 122.4 (C-1ꢀ), 132.0 (C-2ꢀ, 6ꢀ), 113.7 (C-3ꢀ, 5ꢀ), 163.4

August 2009
805
8,14-O-Diethylbikhaconine (17) and 14-O-Ethylbikhaconine (18) To
a solution of bikhaconine (200 mg, 0.42 mmol) in THF (10 ml) was added
NaH (200 mg). The resultant mixture was refluxed for 2 h before adding
ethyl bromide (5 drops). The subsequent reaction mixture was kept refluxing
for an additional 4 h, and cooled to room temperature. After a general work-
up, the crude product was purified by column chromatography (silica gel,
8 g, petroleum ether–acetoneꢂ16 : 10, with 1% diethylamine) to give ethyl-
ated analogues 17 (66 mg, 30%) and 18 (52 mg, 25%). 17: 1H-NMR
(400 MHz) d: 1.07 (3H, t, Jꢂ7.2 Hz, NCH2CH3), 1.13, 1.17 (each 3H, t,
Jꢂ7.0 Hz, OCH2CH3ꢃ2), 3.25, 3.29, 3.30, 3.32 (each 3H, s, OCH3ꢃ4),
4.07 (1H, d, Jꢂ6.6 Hz, H-6b). 13C-NMR (50 MHz) d: 86.1 (C-1), 25.9 (C-
2), 36.2 (C-3), 39.3 (C-4), 50.0 (C-5), 82.2 (C-6), 52.1 (C-7), 82.5 (C-8),
42.2 (C-9), 49.4 (C-10), 50.0 (C-11), 35.2 (C-12), 73.4 (C-13), 80.0 (C-14),
41.4 (C-15), 84.0 (C-16), 62.47 (C-17), 80.7 (C-18), 53.7 (C-19), 49.3 (C-
21), 13.6 (C-22), 56.1 (C-1’), 57.2 (C-6ꢄ), 57.0 (C-18ꢄ), 65.7, 58.6, 16.1,
15.5 (OCH2CH3ꢃ2). HR-ESI-MS m/z: 524.3607 [MꢅH]ꢅ (Calcd for
(C-4ꢀ), 55.3 (OCH3-4ꢀ), 165.6 (COO), 122.4 (C-1ꢆ), 129.8 (C-2ꢆ, 6ꢆ), 128.1
(C-3ꢆ, 5ꢆ), 132.6 (C-4ꢆ). HR-ESI-MS m/z: 706.3557 [MꢅH]ꢅ (Calcd for
C40H52NO10: 706.3586).
8,14-Dibenzoylbikhaconine (12) Crassicauline A (500 mg, 0.75 mmol)
was added to a solution of NaOH in methanol (20 ml), the subsequent reac-
tion solution was heated at 50 °C for 30 min. The methanol was removed to
give a residue, to which were added DMAP (300 mg), pyridine (10 ml), and
benzoyl chloride (20 of drops, 8.48 mmol). The subsequent reaction mixture
was allowed to stand at room temperature overnight. After a general work-
up, the crude residue was purified over column chromatography (silica gel,
10 g, petroleum ether–acetoneꢂ3 : 2) to yield 12 (47 mg, 13%). 1H-NMR
(200 MHz) d: 1.12 (3H, t, Jꢂ7.2 Hz, NCH2CH3), 3.24, 3.25, 3.28, 3.30
(each 3H, s, OCH3ꢃ4), 4.06 (1H, d, Jꢂ6.7 Hz, H-6b), 5.73 (1H, d,
Jꢂ4.9 Hz, H-14b), 7.26—8.12 (10H, m, Ar-H). 13C-NMR (50 MHz) d: 85.1
(C-1), 26.0 (C-2), 35.8 (C-3), 39.3 (C-4), 49.6 (C-5), 79.2 (C-6), 53.3 (C-7),
84.3 (C-8), 43.0 (C-9), 46.8 (C-10), 50.4 (C-11), 35.0 (C-12), 73.6 (C-13),
77.4 (C-14), 42.4 (C-15), 82.6 (C-16), 61.8 (C-17), 80.6 (C-18), 54.0 (C-19),
49.1 (C-21), 13.6 (C-22), 56.0 (C-1ꢄ), 57.8 (C-6ꢄ), 57.5 (C-16ꢄ), 59.1 (C-
18ꢄ), 166.0, 165.6 (COOꢃ2), 130.2, 130.7 [(C-1ꢀ)ꢃ2)], 129.8, 129.9 [(C-
2ꢀ)ꢃ2)], 128.2, 128.5 [(C-3ꢀ, 5ꢀ)ꢃ2)], 132.6, 133.0 [(C-4ꢀ)ꢃ2)]. HR-ESI-
MS m/z: 676.3457 [MꢅH]ꢅ (Calcd for C39H50NO9: 676.3480).
1
C29H50NO7: 524.3582). 18: H-NMR (200 MHz) d: 1.05 (3H, t, Jꢂ7.2 Hz,
NCH2CH3), 1.14 (3H, t, Jꢂ7.0 Hz, OCH2CH3), 3.20, 3.27, 3.28, 3.36 (each
3H, s, OCH3ꢃ4), 4.04 (1H, d, Jꢂ6.8 Hz, H-6b). 13C-NMR (50 MHz) d: 85.4
(C-1), 26.2 (C-2), 37.3 (C-3), 39.1 (C-4), 49.9 (C-5), 82.9 (C-6), 52.3 (C-7),
76.3 (C-8), 41.6 (C-9), 49.4 (C-10), 50.0 (C-11), 34.9 (C-12), 73.9 (C-13),
82.4 (C-14), 42.4 (C-15), 85.3 (C-16), 62.1 (C-17), 80.7 (C-18), 53.7 (C-19),
49.1 (C-21), 13.5 (C-22), 56.1 (C-1ꢄ), 57.8 (C-6ꢄ), 57.3 (C-16ꢄ), 59.1 (C-
18ꢄ), 66.2, 15.8 (OCH2CH3). HR-ESI-MS m/z: 496.3289 [MꢅH]ꢅ (Calcd
for C27H46NO7: 496.3269).
14-Acetylbikhaconine (13) and 14-t-butoxycarbonylbikhaconine (14)
Bikhaconine analogue 13 (92 mg) was obtained in 86% yield by acetylation
of bikhaconine (100 mg, 0.21 mmol) with acetic anhydride (1 ml) in 5 ml of
pyridine. Similarly, bikhaconine analogue 14 (38 mg) was prepared in 32%
yield by treatment of bikhaconine (100 mg, 0.21 mmol) with (Boc)2O
(93 mg, 0.42 mmol) and DMAP (100 mg) in 10 ml of pyridine. 13: 1H-NMR
(200 MHz) d: 1.08 (3H, t, Jꢂ7.1 Hz, NCH2CH3), 2.08 (3H, s, OCOCH3),
3.23, 3.30, 3.30, 3.36 (each 3H, s, OCH3ꢃ4), 4.02 (1H, d, Jꢂ6.8 Hz, H-6b),
4.91 (1H, d, Jꢂ4.9 Hz, H-14b). 13C-NMR (50 MHz) d: 85.5 (C-1), 25.9 (C-
2), 37.3 (C-3), 39.3 (C-4), 50.2 (C-5), 82.7 (C-6), 53.2 (C-7), 85.0 (C-8),
42.4 (C-9), 48.9 (C-10), 50.3 (C-11), 35.0 (C-12), 73.5 (C-13), 81.2 (C-14),
41.5 (C-15), 83.6 (C-16), 62.1 (C-17), 80.6 (C-18), 53.8 (C-19), 49.4 (C-21),
13.6 (C-22), 56.2 (C-1ꢄ), 58.6 (C-6ꢄ), 57.4 (C-16ꢄ), 59.2 (C-18ꢄ), 171.8, 21.2
(OAc). HR-ESI-MS m/z: 510.3042 [MꢅH]ꢅ (Calcd for C27H44NO8:
13-t-Butoxycarbonyl-pyrocrassicauline A (20) To a solution of the
residue A (200 mg, 0.32 mmol) in 5 ml of pyridine were added (Boc)2O
(300 mg, 1.83 mmol) and DMAP (200 mg, 0.32 mmol), and the resultant re-
action solution was kept refluxing overnight. After a general work-up, the
residue obtained was subjected to column chromatography (silica gel, 8 g,
petroleum–acetoneꢂ11 : 1) to produce acetylated product 20 (76 mg, 35%):
1H-NMR (200 MHz) d: 1.05 (3H, t, Jꢂ6.9 Hz, NCH2CH3), 1.46 (9H, s,
CH3)3C–OCO), 3.21, 3.21, 3.28, 3.49 (each 3H, s, OCH3ꢃ4), 3.82 (3H, s,
4ꢀ-OCH3), 4.29 (1H, d, Jꢂ6.6 Hz, H-6b), 5.24 (1H, d, Jꢂ2.5 Hz, H-14b),
5.54 (1H, d, Jꢂ6.6 Hz, H-15), 6.87, 8.07 (each 2H, AAꢄBBꢄ system,
Jꢂ8.8 Hz, Ar-H). 13C-NMR (50 MHz) d: 85.9 (C-1), 25.1 (C-2), 37.2 (C-3),
39.8 (C-4), 48.1 (C-5), 80.0 (C-6), 50.9 (C-7), 145.2 (C-8), 44.8 (C-9), 48.0
(C-10), 51.7 (C-11), 35.3 ( C-12), 81.9 (C-13), 77.4 (C-14), 116.6 (C-15),
83.5 (C-16), 74.7 (C-17), 80.3 (C-18), 54.2 (C-19), 49.6 (C-21), 13.5 (C-22),
56.0 (C-1ꢄ), 58.1 (C-6ꢄ), 57.6 (C-16ꢄ), 59.1 (C-18ꢄ), 166.4 (COO), 123.2 (C-
1ꢀ), 132.3 (C-2ꢀ, 6ꢀ), 113.3 (C-3ꢀ, 5ꢀ), 163.2 (C-4ꢀ), 55.3 (4ꢀ-OCH3), 152.6 s,
85.4 s, 27.7 q [(CH3)3C–OCO]. HR-ESI-MS m/z: 684.3722 [MꢅH]ꢅ (Calcd
for C38H54NO10: 684.3742).
1
510.3061). 14: H-NMR (200 MHz) d: 1.08 (3H, t, Jꢂ7.2 Hz, NCH2CH3),
1.47 (9H, s, (CH3)3C-OCO), 3.23, 3.29, 3.30, 3.34 (each 3H, s, OCH3ꢃ4),
4.03 (1H, d, Jꢂ6.4 Hz, H-6b), 4.70 (1H, d, Jꢂ4.6 Hz, H-14b). 13C-NMR
(50 MHz) d: 85.6 (C-1), 29.6 (C-2), 37.5 (C-3), 37.6 (C-4), 50.1 (C-5), 82.3
(C-6), 52.8 (C-7), 82.4 (C-8), 42.2 (C-9), 49.7 (C-10), 49.2 (C-11), 35.1 (C-
12), 73.1 (C-13), 82.3 (C-14), 41.0 (C-15), 83.8 (C-16), 62.1 (C-17), 80.6
(C-18), 53.6 (C-19), 49.2 (C-21), 13.6 (C-22), 56.2 (C-1ꢄ), 58.5 (C-6ꢄ), 57.3
(C-14ꢄ), 59.1 (C-16ꢄ), 154.0 s, 76.8 s, 27.6 q [(CH3)3C–OCO]. HR-ESI-MS
m/z: 568.3478 [MꢅH]ꢅ (Calcd for C30H50NO9: 568.3480).
1-Demethoxyyunaconitine (24) To a solution of 1-demethoxy-3-dehy-
droyunnaconitine (31)26) (2.812 g, 4.485 mmol) in methanol (90 ml) was
added NaBH4 (544 mg, 14.3 mmol), and the reaction was allowed to proceed
with stirring at room temperature for 1 h. After removal of methanol, the
residue was diluted with water (5 ml), and the subsequent suspension was
extracted with chloroform (80 mlꢃ3). The combined extracts were dried
over anhydrous sodium sulfate, and the organic solvent was removed under
reduced pressure. The residue obtained was purified over silica gel (75 g)
column chromatography eluting with petroleum ether–acetone (5 : 1) to give
compound 24 (1.961 g, 70%): 1H-NMR (400 MHz) d: 1.17 (1H, dd, Jꢂ14.0,
6.0 Hz, H-1b), 1.87—1.92 (1H, m, H-1a), 1.70—1.73 (1H, m, H-2b),
1.64—1.70 (1H, m, H-2a), 3.73 (1H, dd, Jꢂ12.0, 5.2 Hz, H-3b), 1.99 (1H,
d, Jꢂ6.4 Hz, H-5), 4.00 (1H, d, Jꢂ6.8 Hz, H-6b), 2.97 (1H, s, H-7), 2.84
(1H, dd, Jꢂ7.2, 5.6 Hz, H-9), 2.11 (1H, dd, Jꢂ14.0, 12.8 Hz, H-10), 1.55
(1H, dd, Jꢂ14.4, 5.6 Hz, H-12), 1.87—1.92 (1H, m, H-12), 4.88 (1H, d,
Jꢂ5.2 Hz, H-14b), 2.40—2.43 (1H, m, H-15), 3.02 (1H, dd, Jꢂ15.6, 8.8 Hz,
H-15), 3.32 (1H, m, H-16), 2.54 (1H, s, H-17), 3.59, 3.79 (each 1H, ABq,
Jꢂ9.2 Hz, H2-18), 2.25, 2.95 (each 1H, ABq, Jꢂ11.6 Hz, H-19), 1.92—
1.94, 2.45—2.50 (each 1H, m, H2-21), 1.10 (3H, t, Jꢂ7.2 Hz, NCH2CH3),
3.15 (3H, s, OCH3-6), 3.54 (3H, s, OCH3-16), 3.30 (3H, s, OCH3-18), 1.34
(3H, s, OAc-8), 7.99, 6.92 (each 2H, AAꢄBBꢄ system, Jꢂ12.0 Hz, H-2ꢀ, 6ꢀ,
H2-3ꢀ, 5ꢀ), 3.86 (3H, s, OCH3-4ꢀ). 13C-NMR (100 MHz) d: 28.9 (C-1), 28.9
(C-2), 73.8 (C-3), 43.0 (C-4), 48.6 (C-5), 83.1 (C-6), 48.3 (C-7), 85.9 (C-8),
44.0 (C-9), 40.4 (C-10), 45.8 (C-11), 36.9 (C-12), 74.5 (C-13), 78.5 (C-14),
40.0 (C-15), 83.7 (C-16), 63.8 (C-17), 76.4 (C-18), 50.8 (C-19), 48.6 (C-21),
13.2 (C-22), 57.4 (C-6ꢄ), 58.7 (C-16ꢄ), 58.9 (C-18ꢄ), 169.6, 21.4 (OCOCH3-
8), 165.6 (COO), 122.3 (C-1ꢀ), 131.4 (C-2ꢀ, 6ꢀ), 113.6 (C-3ꢀ, 5ꢀ), 163.3 (C-
4ꢀ), 55.2 (OCH3-4ꢀ). HR-ESI-MS m/z: 630.3297 [MꢅH]ꢅ (Calcd for
C34H48NO10: 630.3273).
8-O-Deacetyl-8-O-t-butoxycarbonylcrassicauline A (15) and 8-O-
Deacetyl-8-O,13-di-t-butoxy-carbonylcrassicauline A (16) To a solution
of residue A (100 mg, 0.33 mmol) in dichloromethane (10 ml) were added
DMAP (200 mg) and (Boc)2O (177 mg, 0.81 mmol), and the reaction was al-
lowed to stand at room temperature overnight. After a general work-up, the
crude residue was purified by column chromatography (silica gel, 8 g, petro-
leum ether–acetoneꢂ9 : 1) to yield 15 (90 mg, 34%) and 16 (118 mg, 51%).
1
15: H-NMR (200 MHz) d: 1.07 (3H, t, Jꢂ6.9 Hz, NCH2CH3), 1.11 (9H, s,
(CH3)3C–OCO), 3.17, 3.24, 3.27, 3.49 (each 3H, s, OCH3ꢃ4), 3.84 (3H, s,
4ꢀ-OCH3), 4.00 (1H, d, Jꢂ6.8 Hz, H-6b), 4.89 (1H, d, Jꢂ5.0 Hz, H-14b),
6.89, 8.01 (each 2H, AAꢄBBꢄ system, Jꢂ8.8 Hz, Ar-H). 13C-NMR (50 MHz)
d: 84.9 (C-1), 26.3 (C-2), 36.0 (C-3), 39.1 (C-4), 48.9 (C-5), 82.8 (C-6),
49.7 (C-7), 85.3 (C-8), 41.2 (C-9), 45.4 (C-10), 50.1 (C-11), 34.7 (C-12),
75.2 (C-13), 78.5 (C-14), 39.2 (C-15), 83.8 (C-16), 61.9 (C-17), 80.5 (C-18),
53.8 (C-19), 49.1 (C-21), 13.4 (C-22), 57.5 (C-1ꢄ), 58.8 (C-6ꢄ), 57.5 (C-16ꢄ),
59.1 (C-18ꢄ), 166.2 (COO), 123.2 (C-1ꢀ), 131.9 (C-2ꢀ, 6ꢀ), 113.5 (C-3ꢀ, 5ꢀ),
163.2 (C-4ꢀ), 55.3 (4ꢀ-OCH3), 151.8 s, 80.6 s, 27.4 q [(CH3)3C–OCO]. HR-
ESI-MS m/z: 702.3816 [MꢅH]ꢅ (Calcd for C38H56NO11: 702.3848). 16: 1H-
NMR (200 MHz) d: 1.05 (3H, t, Jꢂ7.2 Hz, NCH2CH3), 1.06, 1.46 (each 9H,
s, (CH3)3C–OCOꢃ2), 3.16, 3.23, 3.26, 3.42 (each 3H, s, OCH3ꢃ4), 3.83
(3H, s, 4ꢀ-OCH3), 3.98 (1H, d, Jꢂ6.6 Hz, H-6b), 5.08 (1H, d, Jꢂ5.2 Hz, H-
14b), 6.88, 8.02 (each 2H, AAꢄBBꢄ system, Jꢂ8.8 Hz, Ar-H). 13C-NMR
(50 MHz) d: 84.8 (C-1), 26.2 (C-2), 35.6 (C-3), 39.1 (C-4), 48.8 (C-5), 80.2
(C-6), 49.4 (C-7), 85.2 (C-8), 41.4 (C-9), 43.9 (C-10), 50.2 (C-11), 34.7 (C-
12), 76.6 (C-13), 76.5 (C-14), 39.4 (C-15), 82.8 (C-16), 61.3 (C-17), 80.5
(C-18), 53.9 (C-19), 48.9 (C-21), 13.5 (C-22), 55.8 (C-1ꢄ), 58.1 (C-6ꢄ), 57.5
(C-16ꢄ), 59.1 (C-18ꢄ), 165.9 (COO), 123.2 (C-1ꢀ), 132.1 (C-2ꢀ, 6ꢀ), 113.4
(C-3ꢀ, 5ꢀ), 163.1 (C-4ꢀ), 55.3 (4ꢀ-OCH3), 152.4 s, 151.7 s, 82.5 s, 81.7 s,
27.7 q, 27.3 q [(CH3)3C–OCOꢃ2]. HR-ESI-MS m/z: 802.4340 [MꢅH]ꢅ
(Calcd for C43H64NO13: 802.4372).
3-Epi-1-demethoxyyunaconitine (25) To a solution of 1-demethoxy-3-
dehydroyunaconitine (31,26) 2.812 g, 4.485 mmol) in methanol (90 ml) was

806
Vol. 57, No. 8
added gradually NaBH4 (544 mg, 14.3 mmol), and the reaction mixture was Regulations of Experimental Animal Administration issued by the State
kept stirring at room temperature for 1 h. Removal of solvent gave a residue, Committee of Science and Technology of the People’s Republic of China on
which was diluted with water (50 ml). The resulting suspension was ex- November 14, 1988 and followed the guidelines of the Animal Care and Use
tracted with chloroform (80 mlꢃ3), the combined extracts were dried over Committee of Shanghai Institute of Materia Medica.
anhydrous sodium sulfate, and the solvent was removed under reduced pres-
sure. The residue obtained was subjected to silica gel (75 g) column chro-
Acetic Acid Writhing Test The writhing test in mice was performed
according to Koster’s method (1959).29) Mice were injected intraperitoneally
matography eluting with petroleum ether–acetone (5 : 1) to give the title with 0.1 ml/10 g body weight of 0.7% acetic acid in normal saline 20 min
1
compound 25 (525 mg, 18%): H-NMR (400 MHz) d: 1.64—1.68 (1H, m, after s.c. administration of the test samples or the vehicle. The mice were
H-1), 1.41—1.48 (1H, m, H-2a), 2.43—2.51 (1H, m, H-2b), 3.76 (1H, t, then kept individually for observation and the total number of abdominal
Jꢂ2.4 Hz, H-3a), 2.69 (1H, d, Jꢂ6.8 Hz, H-5), 4.08 (1H, d, Jꢂ12.0 Hz, H-
contractions (writhing movements) occurring between 5 and 15 min after the
6b), 2.95 (1H, s, H-7), 2.85 (1H, dd, Jꢂ6.8, 5.6 Hz, H-9), 2.11 (1H, dd, acetic acid injection was recorded. The data represent average of the total
Jꢂ13.0, 12.4 Hz, H-10), 1.56 (1H, dd, Jꢂ14.0, 4.8 Hz, H-12), 1.99—2.05 number of writhes observed. The analgesic activity was expressed as per-
(1H, m, H-12), 4.89 (1H, d, Jꢂ4.2 Hz, H-14b), 2.37—2.42 (1H, m, H-15), centage change from writhing controls.
3.02 (1H, dd, Jꢂ15.2, 8.8 Hz, H-15), 3.30 (1H, hidden, H-16), 2.53 (1H, s,
Acute Toxicity Five female mice for each group subcutaneously in-
H-17), 3.18, 4.03 (each 1H, ABq, Jꢂ8.4 Hz, H2-18), 2.23 (1H, ABq, jected with different doses of compounds were observed for 1 h and kept
Jꢂ11.2 Hz, H-19), 2.47—2.53 (2H, m, H2-21), 1.09 (3H, t, Jꢂ7.2 Hz,
NCH2CH3), 3.17 (3H, s, OCH3-6), 3.54 (3H, s, OCH3-16), 3.32 (3H, s,
OCH3-18), 1.36 (3H, s, OCOCH3-8), 7.99, 6.92 (each 2H, AAꢄBBꢄ system,
Jꢂ12.0 Hz, H-2ꢀ, 6ꢀ, H-3ꢀ, 5ꢀ), 3.86 (3H, s, OCH3-4ꢀ). 13C-NMR (100 MHz)
d: 25.8 (C-1), 27.7 (C-2), 78.1 (C-3), 42.3 (C-4), 42.6 (C-5), 83.3 (C-6),
48.2 (C-7), 85.7 (C-8), 44.2 (C-9), 40.5 (C-10), 45.9 (C-11), 36.8 ( C-12),
74.7 (C-13), 78.6 (C-14), 40.1 (C-15), 83.9 (C-16), 64.3 (C-17), 81.2 (C-18),
50.8 (C-19), 48.7 (C-21), 13.2 (C-22), 57.5 (C-6ꢄ), 58.7 (C-16ꢄ), 58.9 (C-
alive for 24 h; the morbidity or mortality, if happens, was recorded for each
group at the end of observation period. Four of the most analgesics (6, 9, 23,
28) were selected for acute toxic examination, based on in vivo analgesic ac-
tion data. The mice showed toxic symptom such as eructation, shiver and
salivary in 20 min after administrating the test samples at a dose of 10 times
of their ED50 value. The mortality was 5/5, 2/5, 3/5, 5/5, respectively, at 1 h
after injection of the compounds.
For calculating the ED50 values, a linear regression was made based on
18ꢄ), 169.5, 21.5 (OAc-8), 165.7 (COO), 122.5 (C-1ꢀ), 131.5 (C-2ꢀ, 6ꢀ), at least four dosages for each compound (with the high efficacy over 80%,
113.6 (C-3ꢀ, 5ꢀ), 163.3 (OCH3-4ꢀ), 55.3 (OCH3-4ꢀ). HR-ESI-MS m/z: and low efficacy less than 20%), without providing the 95% confidence
630.3271 [MꢅH]ꢅ (Calcd for C34H48NO10: 630.3273).
limits.
1,16-Didemethoxy-D15,16-yunaconitine (26) To a solution of compound
2926) (70 mg, 0.10 mmol) in MeOH (3 ml) was added p-TsOH (31 mg) and
Acknowledgements We are grateful to the National Natural Science
acetic acid. The reaction was allowed to proceed with stirring at room tem- Foundation of China (No. 30672526) for financial support of this research.
perature for 4 h. Basification of the reaction mixture with 10% NaOH to pH
12, the subsequent mixture was extracted with chloroform (10 mlꢃ3), the
combined extracts was dried over anhydrous Na2SO4, and the solvent was re-
moved in vacuo. The residue obtained was purified by column chromatogra-
phy over silica gel (2.0 g) eluting with petroleum ether–acetone (8 : 2) to af-
ford compound 26 (37 mg, 64%). 1H-NMR (200 MHz) d: 1.04 (3H, t,
Jꢂ7.0 Hz, NCH2CH3), 1.39 (3H, s, OCOCH3-8), 3.13, 3.29, 3.83 (each 3H,
s, OCH3ꢃ3), 4.14 (1H, d, Jꢂ6.6 Hz, H-6b), 4.91 (1H, d, Jꢂ3.4 Hz, H-14b),
5.98 (1H, dd, Jꢂ10.0, 1.4 Hz, H-15), 6.54 (1H, dd, Jꢂ10.0, 1.4 Hz, H-16),
6.87, 7.91 (each 2H, AAꢄBBꢄ system, d, Jꢂ8.8 Hz, H-3ꢀ, 5ꢀ, and H-2ꢀ, 6ꢀ).
13C-NMR (50 MHz) d: 28.8 (C-1), 29.3 (C-2), 74.7 (C-3), 43.2 (C-4), 48.9
(C-5), 82.5 (C-6), 44.7 (C-7), 83.7 (C-8), 43.5 (C-9), 41.8 (C-10), 45.6 (C-
11), 39.1 ( C-12), 75.8 (C-13), 77.8 (C-14), 126.3 (C-15), 136.0 (C-16), 65.2
(C-17), 77.3 (C-18), 47.2 (C-19), 48.9 (C-21), 13.3 (C-22), 57.1 (C-6ꢄ), 59.1
(C-18ꢄ), 169.5, 21.7 (OCOCH3), 166.4 (COO), 122.3 (C-1ꢀ), 131.6 (C-2ꢀ,
6ꢀ), 113.6 (C-3ꢀ, 5ꢀ), 163.4 (OCH3-4ꢀ), 55.3 (OCH3-4ꢀ). HR-ESI-MS m/z:
598 [MꢅH]ꢅ (100). HR-ESI-MS m/z: 598.3001 [MꢅH]ꢅ (Calcd for
C33H44NO9: 598.3011).
References
1) Benn M. H., Jacyno J. M., “Alkaloids: Chemical and Biological Per-
spective,” Vol. 1, ed. by Pelletier S. W., John Wiley and Sons, New
York, 1983, pp. 153—210.
2) Bisset N. G., J. Ethnopharmacol., 4, 247—336 (1981).
3) Huang T. K., “Handbook of Compostion and Pharmacological Action
of Commonly-Used Traditional Chinese Medicine,” Yi-Yao Science &
Technology Press, Beijing, 1994, pp. 923—926, 1367—1372.
4) Mori T., Ohsawa T., Murayama M., Bando H., Wada K., Amiya T.,
Heterocycles, 29, 873—885 (1989).
5) Wang F. P., Liang X. T., “The Alkaloids: Chemistry and Pharmacol-
ogy,” Vol. 42, ed. by Cordell G. A., Academic Press, New York, 1992,
pp. 157—247.
6) Wang F. P., Liang X. T., “The Alkaloids: Chemistry and Biology,” Vol.
59, ed. by Cordell G. A., Academic Press, New York, 2001, pp. 1—
280.
7) Tang X. C., Feng J., Acta Pharmacol. Sin., 2, 82—84 (1981).
8) Wei B. Y., Kong X. C., Zhao Z. Y., Wang H. C., Zhu Z. H., Bull. Chin.
Mater. Med., 2, 28—30 (1981).
9) Tang X. C., Zhu M. Y., Feng J., Wang Y. E., Acta Pharm. Sin., 18,
579—584 (1983).
1-Demethoxy-3,13-diacetoxy-14-methanesulfonyl-pyrobikhaconine
(27) Compound 3026) (2.6 g, 3.95 mmol) was placed in 500 ml of round
flask and heated under vacuum (0.5 mmHg) to give a residue, which was
chromatographed over silica gel H (55 g) using cyclohexane–acetone (10 : 1)
1
as eluent to yield the title compound 27 (1.4 g, 60%). H-NMR (400 MHz)
10) Tang X. C., Liu X. J., Lu W. H., Wang M. D., Li A. C., Acta Pharm.
Sin., 21, 886—891 (1986).
d: 1.11 (3H, t, Jꢂ7.2 Hz, NCH2CH3), 2.05, 2.10 (each 3H, s, OCOCH3ꢃ2),
3.15 (3H, s, OSO2CH3), 3.24, 3.32, 3.37 (each 3H, s, OMeꢃ3), 4.88 11) Zhang H. L., Yang Y. R., Wang Y., Zheng P., Wang H. C., Lao A. N.,
(1H, dd, Jꢂ12.4, 5.2 Hz, H-3a), 4.94 (1H, d, Jꢂ3.6 Hz, H-14), 5.54 (1H,
d, Jꢂ6.4 Hz, H-15). HR-ESI-MS m/z: 598.2664 [MꢅH]ꢅ (Calcd for 12) Tang X. C., Liu X. J., Feng J., Zhu M. Y., Li A. L., Acta Pharmacol.
C29H44NO10: 598.2680). Its structure was also confirmed by comparison Sin., 7, 413—418 (1986).
with the authenticated sample in TLC plate using different solvent systems 13) Liu J. H., Zhu Y. X., Tang X. C., Acta Pharmacol. Sin., 8, 301—305
Acta Pharmacol. Sin., 15, 176—180 (1994).
(petroleum ether–acetoneꢂ2 : 1, CHCl3–MeOHꢂ98 : 2, CHCl3–acetoneꢂ
(1987).
9 : 1).
14) Guo X., Tang X. C., Acta Pharmacol. Sin., 10, 504—507 (1989).
Analgesic Activity Assay in a Mice Model Acetic Acid-Induced 15) Guo X., Tang X. C., Acta Pharmacol. Sin., 11, 107—112 (1990).
Writhing Test: Material and Methods. Preparation of Test Samples 16) Tang X. C., New Drug & Clinic., 5, 120—121 (1986).
for Bioassay All test samples were given subcutaneously to test animals in 17) Yu C. X., Huang L. Y., Tang X. C., J. Fujian Med. College, 30, 10—12
0.1 ml/10 g body weight after dissolving in DMSO or 0.1 M HCl followed by
diluting to certain concentration with distilled water. The animals of control 18) Zhou Y. P., Liu W. H., Zeng Y. G., Chen D. H., Li H. Y., Song W. L.,
group received the same experimental handling as those of the testing Acta Pharm. Sin., 19, 641—646 (1984).
groups except that the drug treatment was replaced with appropriate vol- 19) Mori T., Murayama M., Bando H., Kawahara N., Chem. Pharm. Bull.,
umes of the dosing vehicle. 39, 379—383 (1991).
Animals Female Kunming strain mice (20ꢇ2 g) were supplied by 20) Manners G. D., Panter K. E., Pelletier S. W., J. Nat. Prod., 58, 863—
Shanghai Experimental Animal Center, Chinese Academy of Sciences. All 869 (1995).
animals were kept under a controlled light/dark cycle and temperature 21) Song D. J., Lu M. W., Yan W. M., Chen D. C., Han S. G., Bull.
(20ꢇ2 °C), with free access to food and water. The animals were left for 2 d Phamarcol. Sin., 3, 157—161 (1987).
for acclimatization to animal room conditions. A minimum of six animals 22) Wang F. P., Li Z. B., Yang J. S., Li B. G., Chin. Chem. Lett., 10, 453—
were used in each group. Experiments were carried out under principles of 456 (1999).
(1996).

August 2009
807
23) Chen Q. H., Xu L., Wang F. P., Tetrahedron, 58, 9431—9444 (2002).
24) Desai H. K., Joshi B. S., Ross S. A., Pelletier S. W., J. Nat. Prod., 52,
720—725 (1989).
27) Gao F., Chen Q. H., Wang F. P., J. Nat. Prod., 70, 876—879 (2007).
28) Chen D. L., Jian X. X., Chen Q. H., Wang F. P., Acta Chim. Sin., 61,
901—906 (2003).
25) Keith L. H., Pelletier S. W., J. Org. Chem., 33, 2497—2499 (1968).
26) Shen X.-L., “Synthetic Research of Taxol Analogues. VII. Syntheses
of Taxol Analogues through Chemical Conversion from C19-Diter-
penoid Alkaloids,” ph. D. Dissertation, Sichuan University, 2004, pp.
9, 43—47.
29) Koster R., Anderson M., De Beer E. J., Fed. Proc., 18, 412—418
(1959).
30) Suzuki Y., Oyama T., Ishige A., Isono T., Asami A., Ikeda Y., Noguchi
M., Omiya Y., Planta Med., 60, 391—394 (1994).