Minor constituents of Spigelia anthelmia and their cardiac activities

Article abstract of DOI:10.1016/S0031-9422(01)00020-6

A more detailed phytochemical analysis of extracts of the aerial parts of Spigelia anthelmia L. (Loganiaceae) yielded 20 structurally related new compounds besides spiganthine and ryanodine. Structure elucidation was achieved mainly by spectroscopic methods. The compounds were tested on their cardiac and on their insect antifeedant activities.

Full text of DOI:10.1016/S0031-9422(01)00020-6

Phytochemistry 57 (2001) 285–296
www.elsevier.com/locate/phytochem
Minor constituents of Spigelia anthelmia and their cardiac activities$
Harald Hubner a,*, Wolfgang Vierling b, Wolfgang Brandt b,
Melchior Reiter b, Hans Achenbach a
aDepartment of Pharmaceutical Chemistry, University of Erlangen, D-91052 Erlangen, Germany
bInstitute of Pharmacology and Toxicology, Technical University, D-80802 Munchen, Germany
¨
Received 16 August 2000;received in revised form 21 December 2000
Abstract
A more detailed phytochemical analysis of extracts of the aerial parts of Spigelia anthelmia L. (Loganiaceae) yielded 20 structu-
rally related new compounds besides spiganthine and ryanodine. Structure elucidation was achieved mainly by spectroscopic
methods. The compounds were tested on their cardiac and on their insect antifeedant activities. # 2001 Elsevier Science Ltd. All
rights reserved.
Keywords: Spigelia anthelmia;Loganiaceae;Spiganthine;20-Deoxyspiganthine;8 a-Hydroxyspiganthine;20-Norspiganthine-5-carboxylic acid;
Ryanodine;10- epi-Ryanodine;8,9-Dehydro-10- epi-ryanodine;20-Hydroxyryanodine;Cardioactivity;Insect antifeedant activity
1. Introduction
LH 201 and silica gel, respectively. The separation
procedures were monitored by the characteristic colour
reaction of the spiganthine- and ryanodine-type com-
pounds with anisaldehyde on TLC plates.
Further separation and isolation of pure constituents
was achieved by HPLC on reversed phase silica gel.
These procedures yielded constituents 1–17 from the
CHCl3-extracts and the more polar constituents 18–22
from the EtOAc-extracts (Table 1).
Stimulated by the bioassay-monitored isolation of the
cardioactive spiganthine from extracts of Spigelia
anthelmia (Achenbach et al., 1995), we were interested
in a more detailed phytochemical analysis of the plant.
Major target of this investigation was the isolation of
minor constituents, their structure elucidation and bio-
logical tests in order to gain structure–activity correla-
tions.
The spectroscopic investigations, and particularly
FAB–MS (negative ions) revealed a number of differ-
ently oxygenated but structurally related C20-structures
with the common feature of a pentacyclic C14O-skeleton
carrying a pyrrolecarboxylic ester group, which is typi-
cal for spiganthine (Achenbach et al., 1995) and ryano-
dine (Wiesner et al., 1967).
2. Results and discussion
An extract from 2 kg dried aerial parts of S. anthelmia
(‘‘Urtinktur’’ prepared with EtOH/water according to
the prescription of the German Homoeopathic Phar-
macopoeia) was diluted with water and repeatedly
extracted with chloroform and then EtOAc followed by
chromatography of the dried extracts on Sephadex
1
In the H NMR and 13C NMR, all signals appeared
well separated and –– supported by the results of our
earlier studies (Achenbach et al., 1995) — could easily
be assigned to the individual positions. Correlation
spectroscopy and NOE-measurements corroborated the
assignments (Tables 2 and 3).
The structures of the spiganthine-type compounds 1–
3 and 18 are easily recognized by a double doublet in
the range of ꢀ 3.23–3.86, which belongs to H-4 and
causes a splitting of the typical low-field signal of H-3 at
d 5.6–5.8 (see Table 2).
$
Part 91 in the series ‘Constituents of Tropical Medicinal Plants’.
For part 90 see Nkunya et al., 2000.
* Corresponding author. Fax: +49-9131-8522585.
E-mail addresses: Huebner@pharmazie.uni-erlangen.de (H. Hub-
ner), achenbach@pharmazie.uni-erlangen.de (H. Achenbach).
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(01)00020-6

286
H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨

H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
287
These results were in accordance with the reaction of
ryanodine under aqueous acidic conditions (Kelly et al.,
1951).
In contrast to the spiganthines, constituents 4–17 and
19–22 exhibited ‘‘only’’ a singulet signal for H-3 at
considerably lower field (ꢀ 5.60–5.70 ppm), which indi-
cated a hydroxy-substituted quarternary C-4. Further
NMR-studies revealed 4 as ryanodine (Wiesner et al.,
1967) and 19 as its 20-hydroxy derivative.
The remaining isolated constituents were mainly
characterized by typical differences of the resonance
signal of H-10: whereas this signal in 1–4, 18 and 19
always appeared in the ꢀ 3.69–3.80 ppm range as a doublet
(Jꢀ10 Hz) indicating a trans-diaxial relationship, it was
observed in all other compounds as a singulet, ‘‘broad’’
singulet or a doublet with only a small coupling constant
of 1 Hz at ꢀ 3.66–4.66 ppm. These spectral features sug-
gested ‘‘loss’’ of the neighbouring H-9 and/or an equator-
ial position of H-10 (=10-epi-configuration). Conse-
quently, NOE measurements were performed and corro-
borated an equatorial H-10 by the significant interaction
observed between H-10 and CH3-17 for constituents 5–17
and 20–22. The results gave evidence that these com-
pounds belong to the rare 10-epi-ryanodine series.
The less polar compound 2 carried the methyl group
CH3-20 at C-5 instead of the hydroxymethyl in spi-
ganthine (1). In comparison to 1, the more polar 3 was
additionally hydroxylated at C-8. The equatorial con-
figuration of this hydroxyl resulted from the coupling
constants of the neighbouring protons and from NOE
studies. Within the spiganthine-group, 18 with a free
carboxyl at C-5 represented the most polar compound.
On treatment with diazomethane, 18 promptly formed
the methyl ester.
Under hydrolytic conditions, 1 underwent saponifica-
tion giving spiganthol (23) and 2-pyrrolecarboxylic acid;
in addition, anhydrospiganthol (24) was formed.
Table 1
Compounds isolated from the ‘‘Urtinktur’’ of Spigelia anthelmia
Compound class
Compound
Fraction
Contenta (%ꢁ10ꢂ3
)
Spiganthines
Spiganthine (1)
CHCl3
CHCl3
CHCl3
EtOAc
7.7
0.5
0.3
6.7
20-Deoxyspiganthine (2)
8a-Hydroxyspiganthine (3)
20-Norspiganthine-5-carboxylic acid (18)
Ryanodines
(a) Ryanodine-type
Ryanodine (4)
20-Hydroxyryanodine (19)
CHCl3
EtOAc
2.7
6.3
(b) 10-epi-Ryanodines with a 9-OH group
9-Hydroxy-10-epi-ryanodine (5)
9-Hydroxy-9-epi-10-epi-ryanodine (6)
CHCl3
CHCl3
CHCl3
EtOAc
1.2
13.3
1.0
(13S)-9,18-Dihydroxy-9-epi-10-epi-ryanodine (7)
9,20-Dihydroxy-9-epi-10-epi-ryanodine (20)
1.0
(c) 10-epi-Ryanodines with a ꢀ8,9-double bond
8,9-Dehydro-10-epi-ryanodine (8)
CHCl3
CHCl3
CHCl3
CHCl3
EtOAc
EtOAc
2.0
2.0
1.5
5.0
0.5
1.5
(13S)-8,9-Dehydro-18-hydroxy-10-epi-ryanodine (9)
8,9-Dehydro-20-hydroxy-10-epi-ryanodine (10)
8,9-Dehydro-21-hydroxy-10-epi-ryanodine (11)
(13S)-8,9-Dehydro-18,21-dihydroxy-10-epi-ryanodine (21)
8,9-Dehydro-20,21-dihydroxy-10-epi-ryanodine (22)
(d) 10-epi-Ryanodines with an epoxy group
8a,9a-Epoxy-10-epi-ryanodine (12)
8b,9b-Epoxy-10-epi-ryanodine (13)
(13S)-8a,9a-Epoxy-18-hydroxy-10-epi-ryanodine (14)
6-Deoxy-6b,9b-epoxy-8b-hydroxy-10-epi-ryanodine (15)
8a,9a-Epoxy-20-hydroxy-10-epi-ryanodine (16)
8a,9a-Epoxy-21-hydroxy-10-epi-ryanodine (17)
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
CHCl3
4.5
1.3
1.2
1.2
0.7
1.3
a
Amount of residue after evaporation of the ‘‘Urtinktur’’=100%.

Table 2
1H NMR data of the isolated natural compounds 1–22 and their derivatives 23 and 24 [ꢀ (ppm), multiplicity and J (Hz);solvent CD 3OD]
Proton
H-3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5.78 d
5.65 d
5.76 d
5.63 s
5.67 s
5.67 s
5.63 s
5.69 s
5.65 s
5.69 s
5.69 s
5.65 s
5.62 s
5.60 s
5.68 s
5.66 s
5.65 s
5.78 d
5.67 s
5.67 s
5.65 s
5.70 s
4.46
5.14
8.5
8.5
8.5
8.5
d 8
dq 8, 2
H-4
3.55 dd
3.23 dd
3.53 dd
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.86 dd
–
–
–
–
3.31–3.36 3.04 dd
8, 2
8.5, 2
8.5, 2
8.5, 2
8.5, 2
m
H-7a
2.10 ddd 2.04 ddd 2.06 dd
2.10 ddd
12.5,
2.20 ddd 2.08 ddd 2.08 ddd 2.58 brd 2.58 brd 2.77 brd 2.64 brd 2.40
2.44
2.40 d
1.81 dd 2.59
13, 4.5 d 15
2.43
2.15–2.25 2.08–2.30 2.20–2.30 2.64 brd 2.83 brd 2.07–2.16 1.50–1.60
18 18
12, 12,
13.5,
12, 6
12, 11
13,
12, 12,
13,
17
17
17
17
d 15
dd15, 2 14.5
d 15
m
m
m
m
m
6
12, 5
13, 4.5
4
12.5, 5
H-7b
H-8a
H-8b
1.35–1.41 1.22 ddd 1.82 dd
1.26 ddd
1.35-1.42 1.39 ddd 1.37–1.43 1.92 brdd 1.92 brdd 2.22–2.30 2.00 brdd 1.82 dd 1.93 dd 1.83 dd
2.04 dd 2.20 dd
1.82 dd 1.21 brd 1.65 ddd 1.81 ddd 2.00 dd
15, 6 12 13, 4 ,3 14, 5, 4 18, 5
2.34 brdd 1.28–1.36 1.64–1.70
18, 5
m
13.5,
12, 5
12.5, 5, 2.5
m
12, 4, 4
m
17, 5.5
17, 5.5
m
17, 5
15, 6
15, 2
14.5, 6
13, 10
15, 6
m
m
4.5, 4
1.50–1.64 1.45–1.58
–
1.40–1.57
1.54 ddd 1.59 ddd 1.59 ddd 5.47
5.47 brd 5.46 brd 5.77 brd
5.5
–
3.36 dd
–
–
–
–
1.49–1.61 1.43–4.55 1.57 ddd 5.77 brd 5.76 brd 1.49–1.60 1.54–1.64
14, 4, 4
m
m
m
14, 4.5,
2.5
13, 4, 4
12.5,
brd
5
5
2, 2
m
m
5
5
m
m
5, 4.5
5.5
1.50–1.64 1.45–1.58 3.68 ddd 1.40–1.57
2.00 ddd 1.95 ddd 1.95 ddd
–
–
–
–
3.08 dd
–
3.09 dd
3.84 dd 3.05 dd
3.23 dd 1.49–1.61 1.43–4.55 1.96 ddd
6, 1 14, 14, 5
–
–
1.49–1.60 1.54–1.64
m
m
11,
m
14, 13,
4.5
13, 12, 4 13, 12.5,
5
6, 1
6, 1
10, 4.5 6, 1
m
m
m
m
10, 5
H-9
1.76–1.86 1.77-1.87 1.70–1.80 1.79–1.89
–
–
–
–
–
–
–
–
–
–
–
–
–
1.73–1.87 1.78–1.86
–
–
–
1.77–1.85 1.74–1.83
m
m
m
m
m
m
m
m
H-10
H-13
3.69
3.75
3.73
3.79
3.66 s
3.67 s
3.67 s
3.93 brs 3.94 brs 3.92 brs 4.21 brs 4.00
d 1
4.14 s
4.02
4.66 s
4.00
4.30
3.73
3.80
3.68 brs 4.22 brs 4.21 brs 3.69
d 10
3.92
d 10
d 10
d 10.5
d 10
d 1
d 1
d 1
d 10
d 10
d 10
2.36 qq
2.41 qq
2.36 qq
2.27 qq
2.19–2.29 2.24 qq
2.15–2.23 2.26 qq
2.17–2.26 2.21–2.29 2.27 qq
2.23 qq 2.23 qq 2.14–2.24 2.21 qq 2.18–2.28 2.24 qq 2.31 qq
2.20–2.30 2.18–2.27 2.18–2.26 2.21–2.31 2.15–2.25 2.70–2.80
6.5, 6.5
6.5, 6.5
7, 7
6.5, 6.5
m
6.5, 6.5
m
6.5, 6.5
m
m
7, 7
6.5, 6.5 6.5, 6.5
m
7, 7
m
7, 7
6.5, 6.5
m
m
m
m
m
m
H-14A 2.21
2.35
2.23
2.56
2.63
2.60
2.54
2.66
2.60
2.58
2.68
2.58
2.64
2.52
2.51
2.50
2.59
2.66
2.50
2.53
2.62
2.59
2.21
3.35
d 13
d 13.5
d 13.5
d 13.5
d 14
d 13.5
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 13
d 19.5
H-14B 1.73 dd
1.77 dd
1.73 dd
1.93
1.96
1.87
1.88
1.94
1.95
2.30
1.95
1.86
1.91
1.87
1.61
2.21
1.87
2.24 dd
2.24
2.22
1.96
2.31
1.60 dd
2.39 dd
13, 2
13.5, 2
13.5, 2
d 13.5
d 14
d 13.5
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
d 14
14, 2
d 14
d 14
d 14
d 14
13, 2
19.5, 2
CH3-17 1.38 s
1.36 s
1.39 s
1.39 s
1.36 s
1.35 s
1.37 s
1.37 s
1.41 s
1.38 s
1.39 s
1.34 s
1.36 s
1.38 s
1.41 s
1.34 s
1.35 s
1.36 s
1.39 s
1.35 s
1.42 s
1.39 s
1.32 s
1.77
d
2
CH3-18 1.16
1.16
1.16
1.11
1.09
1.09
–
1.10
–
1.10
1.10
1.08
1.09
–
1.09
1.09
1.09
1.14
1.12
1.10
–
1.11
1.12
1.12
d 6.5
d 6.5
d 7
d 6.5
d 6.5
d 6.5
d 6.5
d 6.5
d 7
d 6.5
d 6.5
d 7
d 7
d 7
d 6.5
d 6.5
d 6.5
d 6.5
d 6.5
d 7
or
CH2-18
–
–
–
–
–
–
3.93 dd
10.5, 5
3.84 dd
10.5, 4
–
3.96 dd
10.5, 4
–
–
–
–
3.97 dd
10.5, 5
3.81 dd
10.5, 4
–
–
–
–
–
–
3.97 dd
10.5, 5.5
3.83 dd
10.5, 4
–
–
–
3.83 dd
10.5, 4.5
CH3-19 0.81
0.84
0.81
0.76
0.75
0.75
0.90
0.75
0.92
0.75
0.76
0.74
0.73
0.91
0.73
0.73
0.74
0.77
0.75
0.73
0.93
0.75
1.02
1.18
d 6.5
d 6.5
d 7
d 6.5
d 6.5
d 6.5
d 7
d 6.5
d 6.5
d 6.5
d 7
d 6.5
d 6.5
d 6.5
d 7
d 7
d 7
d 6.5
d 6.5
d 6.5
d 7
d 6.5
d 6.5
d 7
CH3-20
–
0.77 s
–
0.89 s
0.93 s
0.91 s
0.91 s
0.94 s
0.95 s
–
0.96 s
0.90 s
0.92 s
0.91 s
0.94 s
–
0.91 s
–
–
–
0.97 s
–
–
–
or
(continued on next page)

H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
289
Further 1H and 13C NMR-studies revealed the structure
of 9-hydroxy-10-epi-ryanodine (5) (NOE between CH3-
21 and Hax-8) and of the hydroxylated 9-epi-10-epi-rya-
nodines 6, 7 and 20, which carried the CH3-21 methyl
group in the axial position. Major argument for this
configuration at C-9 comes from the observed NOE
between CH3-21 and Hax-7.
The depicted stereochemistry at C-13 in 7 is also
based on NOE-studies, which revealed strong effects
from CH3-17 to CH2-18, from CH3-19 to H-3, and from
H-13 to H-14. With regard to the established absolute
configuration of ryanodine, these NMR-observations
confirmed the 13S-configuration for 7.
Among the 10-epi-ryanodines, the common structural
feature of constituents 8–11, 21 and 22 was an unsa-
turation within the cyclohexane ring system. 1H and 13
C
NMR easily determined its ꢀ8,9-position and also
recognized the presence and positions of additional
hydroxyl groups. The configuration at the steric center
C-13 in 9 and 21 was established as in 7.
Catalytic hydrogenation of 11 saturated the double
bond and simultaneously deoxygenated the allylic OH-
group at C-21 to give 9-epi-10-epi-ryanodine in about
70% yield.
Treatment of 11 with 3-chloroperoxybenzoic acid
(Waterhouse et al., 1987) produced, among others, the
two expected stereoisomeric epoxides, one of which
proved to be identical with the isolated constituent 17.
Assignment of structure 17 and its corresponding ste-
reoisomer was deduced from characteristic coupling
differences in the H-7/H-8 spin-system of their 1H
NMR: in 17, H-8 appears as a doublet (J7eq,8ꢀ6 Hz)
with J7ax,8ꢀ0 Hz, whereas in the stereoisomeric epoxide
obtained only by epoxidation of 11 coupling constants
of J7ax,8ꢀJ7eq,8ꢀ2 Hz were observed for H-8. On the
basis of these data, the structure with the a-orientation
of the oxirane ring has to be attributed to 17.
This result was valuable for the structure elucidation
of constituents 12–14 and 16, which belonged to the a-
series of epoxides with the only exemption of 13.
A small amount of the epoxide 12 was subjected to a
hydrolytic ring-opening reaction using oxalic acid. This
reaction resulted in the ––almost quantitative––conver-
sion of 12 to an isomeric product identical with the iso-
lated constituent 15.
A very characteristic feature in the 13C NMR of 15
was a pronounced downfield-shift of the resonance of
the C-6 from ꢀ 84.3 ppm (in 12 and all other hitherto
mentioned constituents) to ꢀ 96 ppm in 15. Extensive
NMR-studies established structure 15 and rationalized
the in vitro conversion of 12 to 15: protonation of the
oxirane oxygen facilitates the nucleophilic attack of C-9
by the axial hydroxyl at C-6. This process might occur
as a concerted reaction and brings about 15.
The colour reaction with anisaldehyde/sulfuric acid
was used as a spot test on TLC plates in the course of

Table 3
13C NMR data of the isolated compounds 1–22 and their derivatives 23 and 24 [d (ppm);solvent CD OD]
3
Carbon
1
2
3
4
5
6
7
8
9
10
11
13
14
66.1
15
66.2
16
66.0
17
66.0
18
65.1
19
66.0
20
66.3
21
66.6
22
66.6
23
65.1
24
1
65.3
86.1
85.0
52.6
51.3
84.9
28.0
29.8
35.3
72.5
86.8
97.9
31.1
37.1
64.7
86.6
85.2
57.7
48.3
84.3
26.4
29.9
35.4
72.9
86.8
97.8
31.1
41.5
65.5
86.8
84.8
52.6
51.1
84.0
37.4
73.1
43.4
70.6
86.2
97.5
31.1
37.1
65.9
84.4
91.0
92.4
49.5
86.6
26.8
29.3
35.4
72.8
87.4
96.7
30.9
41.8
66.3
84.0
90.5
92.4
49.5
85.7
24.2
31.6
74.9
76.8
83.3
96.8
30.8
41.2
66.2
84.1
90.3
92.5
49.1
85.5
25.8
32.3
73.1
78.8
82.6
96.9
30.8
41.2
66.4
84.7
90.3
92.5
49.0
85.4
25.9
66.4
84.4
90.6
92.3
49.1
84.0
31.5
66.6
85.0
90.4
92.2
49.6
83.8
31.5
66.5
66.5
66.6
151.5a
134.4a
75.1
58.1
50.9
83.5
27.5
29.4
35.2
72.7
96.0a
93.0a
28.8
35.6
173.5
11.8
19.5a
22.0a
64.9
18.7
–
2
84.3
90.3
92.7
54.0
83.6
34.4
84.4
90.6
92.3
49.2
84.4
31.4
84.4
89.9
92.5
49.0
84.4
28.5
63.4
61.4
75.1
81.0
96.6
30.8
41.3
84.9
90.4
92.1
49.0
83.8
29.7
59.0
61.2
73.8
81.2
84.5
89.9
90.6
46.7
96.0
32.1
75.9
92.2
77.9
88.2
84.2
90.4
92.6
53.3
83.8
32.5
59.2
60.8
73.6
81.2
98.2
30.8
37.1
84.3
90.7
92.2
49.0
84.3
29.4
55.3
63.4
70.8
80.7
98.3
30.8
41.8
87.0
83.2
57.7
56.8
83.6
28.1
30.0
35.2
72.8
87.7
97.1
30.8
37.4
84.3
90.7
92.8
54.0
86.5
29.5
29.6
35.2
72.8
87.5
96.7
30.9
37.2
84.0
90.3
93.0
53.7
85.3
28.6
85.0
90.4
92.2
49.3
84.2
31.5
84.3
90.4
92.8
54.1
84.0
34.4
87.2
83.3
57.3
52.3
84.4
27.6
29.9
35.3
72.7
86.7
97.4
30.9
40.0
3
4
5
6
7
8
32.3 122.7 122.7 123.7 124.0
73.1 134.6 134.5 133.7 138.5
32.4 124.0 125.2
72.8 138.5 137.7
9
10
11
12
13
14
15
17
18
19
20
21
22
23
24
25
26
78.9
82.8
96.9
37.1
41.6
74.6
81.5
97.1
30.8
41.6
74.2
81.7
97.0
37.1
41.9
74.0
81.7
96.9
30.8
36.9
70.4
81.2
97.0
30.8
41.6
78.8
82.8
96.9
30.8
36.5
70.4
81.4
96.9
37.1
41.9
70.2
81.4
96.8
30.9
37.0
98.2 101.0
37.0
42.0
30.9
40.9
103.6 103.6 103.6 102.9 104.6 103.2 103.2 103.3 103.2 103.5 103.3 103.9 103.9 104.6 104.1 104.0 103.4 103.1 103.4 103.2 103.5 103.9
9.7
19.6
19.0
64.0
18.9
9.6
19.5
19.1
16.7
18.9
9.8
19.6
19.0
64.0
15.2
10.2
19.5
18.9
12.6
19.0
11.1
19.3
18.7
11.9
26.5
11.2
19.4
18.7
12.1
27.7
10.9
65.8
13.5
12.2
27.7
11.2
19.4
18.7
12.1
21.0
10.9
65.8
13.5
12.1
20.9
11.3
19.5
18.7
59.8
20.9
11.2
19.4
18.7
12.1
64.3
11.2
19.3
18.6
12.0
22.5
11.0
65.8
13.5
12.0
19.8
11.0
19.3
18.6
13.1
14.7
11.3
19.5
18.7
59.5
19.8
11.3
19.3
18.7
9.6
19.5
18.8
10.3
19.7
18.9
60.4
19.0
11.2
19.6
18.7
59.5
27.6
10.9
65.8
13.5
12.1
64.3
11.2
19.5
18.7
59.8
64.3
9.8
19.7a
19.5a
64.3
18.9
–
12.0 176.6
63.4 18.8
161.8 162.0 161.7 161.9 161.6 161.7 161.5 161.7 161.5 161.5 161.7 161.4 161.5 161.4 161.5 161.7 161.8 161.7 161.6 161.5 161.5
123.1 123.6 123.4 123.4 123.3 123.3 123.3 123.4 123.3 123.3 123.4 123.4 123.2 123.4 123.2 123.3 123.5 123.3 123.3 123.3 123.3
116.9 116.8 116.9 117.0 117.0 116.9 117.0 116.9 117.0 117.2 116.9 116.8 117.1 116.9 117.3 117.0 117.0 117.3 117.3 117.0 117.2
110.9 110.9 111.0 111.0 111.0 110.9 111.0 110.9 111.0 111.1 110.9 110.9 111.0 111.9 111.1 111.0 110.7 111.0 111.1 111.0 111.1
125.2 125.3 125.5 125.6 125.7 125.6 125.6 125.6 125.6 125.7 125.6 125.5 125.7 125.5 125.7 125.6 124.9 125.6 125.7 125.7 125.7
–
–
–
–
–
–
–
–
a
Similar values might be interchanged.

H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
291
our isolation work: all isolated compounds gave this
rather sensitive reaction, which, in addition, permitted a
general distinction between ryanodines/epi-ryanodines
and spiganthines, respectively. In contrast to the ryano-
dines/epi-ryanodines, which produced a yellow-ochre
colour, the spiganthines gave
a red-purple spot.
Obviously, the colour reaction did not depend on the
presence of the 2-pyrrolecarboxylic ester group, since
compounds 23 and 24 also reacted positively.
Despite all the precautions taken during our isolation
and structure elucidation work (avoidance of higher
temperatures, storage under nitrogen at low tempera-
tures, absence of acidic or alkaline media, no excessive
light etc.) some of the isolated compounds might be
artefacts or partly artefacts: 9-hydroxylated compounds
might have been transferred into the corresponding
Fig. 1. Effect of 8,9-dehydro-10-epi-ryanodine (8) on the contraction
of the guinea-pig papillary muscle. Increasing concentrations (nmol/l)
of 8 caused a remarkable prolongation of the latency time between the
stimulus (S) and the beginning of the contraction;additionally, the
force of contraction (mN) decreases. (Stimulation frequency: 1 Hz).
Ordinate: force in mN;abscissa: time in msec.
8,9-dehydro compounds (e.g.
5
or 6!8; 7!9).
Proton catalyzed rearrangement during work-up might
have converted 12 to 15. Oxygenating enzymes might
be responsible for the generation of some of the
epoxides.
However, considering the amounts isolated of the
individual constituents and the various distinct grades
of hydroxylation we regard most of the described com-
pounds original natural products.
Among these, only spiganthine, ryanodine and 9-
hydroxy-10-epi-ryanodine (5) (Jefferies et al., 1992) are
already known compounds, whereas the others repre-
sent new natural products.
20–22) were 100–1000 times higher than the value of
ryanodine (4).
For the most active substances 1, 2, 4 and 8, the fol-
lowing structure–activity relationships were revealed:
omission of the hydroxy group from position C-4 (4!2)
or C-20 (1!2) with an otherwise unchanged structure
caused only slight attenuation of the biological activity;
and transfer of the hydroxyl from C-4 to C-20 (4!1)
did also not significantly influence the activity. Further-
more, compound 8, which represents the least hydro-
xylated substance in the 8,9-dehydro-series, is similarly
active as 4, from which it structurally differs by
unsaturation (ꢀ8,9) and epimerisation at C-10. But 10-
epimerisation combined with 8,9-epoxidation (4!12,
13) reduced the cardiac activity considerably to less than
10%, and the presence of the two hydroxy groups at C-
4 and C-20 (1!19 or 4!19) led to an even more dra-
matic reduction of activity. These results suggested an
essential influence of the polarity of ryanodine/spi-
ganthine-type compounds for their cardiac activity.
As a general guide line we summarize our results: the
already known compounds spiganthine (1) and ryano-
dine (4) exhibit highest cardiac activity, which is not
much influenced by neither removal of their hydroxy
group at C-4 or C-20 nor by introduction of an 8,9-
double bond together with 10-epimerization. In con-
trast, 8,9-epoxidation and, particularly, introduction
of additional hydroxy groups causes loss of cardiac
activity.
3. Biological activities
Ryanodine and spiganthine are known for their car-
diac (Jenden and Fairhurst, 1969;Penefsky, 1974;Vier-
ling, 1988;Achenbach et al., 1995;Sutko et al., 1997)
and insecticidal (Jefferies et al., 1992;Gonza lez-Coloma
et al., 1996, 1999) activities. The availability of quite a
number of new substances with structural variations
arouse the interest to start structure–activity studies.
3.1. Cardiac activity
These tests were carried out as described earlier
(Achenbach et al., 1995). Each of the tested compounds
induced a delay in the development of cardiac contrac-
tions in a similar way as spiganthine (1) and ryanodine
(4) but with divergent potencies. Fig. 1 shows a repre-
sentative experiment using 8, a substance about equally
potent as 1 and 4.
The concentration-versus-activity relationships of 12
of the most effective compounds are presented in Fig. 2,
and the corresponding EC50 values are compiled in
Table 4, where they have been divided in roughly three
groups: strong, medium and weak activity. The EC50
values of the other tested compounds (3, 5, 14–18 and
3.2. Insect antifeedant and insecticidal activity
Major efforts have been invested to study the anti-
feedant and insecticidal potential of ryanodine (4) and
structurally related compounds (Wiesner et al., 1967;

292
H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
Fig. 2. Concentration–activity relationship of the 12 most active compounds isolated from Spigelia anthelmia. Ordinate: prolongation of the latency
time (ꢀtL) in % of the maximal prolongation of each muscle group. (Shown are mean values ꢄ S.E.M.) Abscissa: concentration of the test com-
pounds in mmol/l (logarithmic scale). The depicted curves were fitted to the mean values using a logistic function.
Table 4
nodine (Waterhouse et al., 1987), which might inhibit
the biological decomposition of 12 (Gonzalez-Coloma
et al., 1999).
EC50 values (nmol/l) of the most active compounds isolated from
Spigelia anthelmia determined by fitting a logistic function to the mean
values of the concentration–activity curves presented in Fig. 2
Substance
Number of experiments (n)
EC50 (nmol/l)
4. Experimental
4
8
5
9
8
6
4
4
3
3
6
3
6
3
14
17
4.1. General
1
25
54
2
7
390
440
540
770
1100
1500
1800
1900
TLC was done on ready-made plates using S-
1=CHCl3:MeOH:H2O (80:20:1). Detection: UV and
anisaldehyde reagent (Stahl and Kaltenbach, 1961).
Unless stated otherwise, IR were run as dry films on
NaCl;[ a]d, UV, and CD measurements in MeOH at
20ꢃ. 1H NMR at 360 MHz and 13C NMR at 90 MHz in
CD3OD with TMS as int. standard. MS were recorded
by FABMS (negative ions) in glycerol (xenon, 8 kV).
Sephadex1 LH 20 was used for CC, Si gel 60 for MPLC
(at ca 3 bar);HPLC was done on Nucleosil 1 RP18 with
the solvent systems S-2 = MeOH:H2O (43:57), S-3 =
MeOH:H2O (30:70) or S-4 = MeOH:H2O (20:80).
10
13
12
19
9
6
11
Waterhouse et al., 1987;Jefferies et al., 1992;Gonza lez-
Coloma et al., 1996). A newer investigation also inclu-
ded spiganthine (1) and a number of our isolated rya-
nodine derivatives in these studies. The results have
been published very recently (Gonzalez-Coloma et al.,
1999) and demonstrated significant antifeedant activity
against some test beetles for spiganthine, ryanodine, the
b-epoxide 13 and, particularly, 9-hydroxy-10-epi-ryano-
dine (5). In addition, considerable insecticidal activities
were observed for spiganthine, ryanodine, and the a-
epoxide 12. These effects were especially remarkable for
12 in the presence of piperonyl butoxide, an agent
known as a synergist for the insecticidal action of rya-
4.2. Extraction and chromatography
The exctract of 2 kg dried aerial parts of Spigelia
anthelmia (purchased from Muggenburg, Hamburg,
Germany, batch-no. 2917) was prepared with 20 l
EtOH:H2O (86:14) according to the German Homoeo-
pathic Pharmacopoeia. Evapn. yielded 260 g residue,
which was diluted in 6 l MeOH:H2O (1:3) and subse-
quently extracted with CHCl3 (3ꢁ5 l) and EtOAc (3ꢁ3

H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
293
l). Evapn of the solvents afforded 75 g CHCl3-I, 1.8 g
CHCl3-II, 1.2 g CHCl3-III, 8.2 g EtOAc-I, 2.3 g EtOAc-
II and 1.2 g EtOAc-III. Repeated CC of CHCl3-II,
CHCl3-III, and EtOAc-I was done on Sephadex LH 20
with MeOH followed by MPLC on Si gel using the
solvent system CHCl3:MeOH (from 95:5 to 8:2).
Finally, HPLC with S-2 or S-3 yielded pure spi-
ganthines/ryanodines as described in Table 1. The
separation procedures were monitored by the char-
acteristic colour reaction of these compounds with ani-
saldehyde reagent.
57.5 min (S-2). [a]d +12ꢃ (c 0.1). IR vmax cmꢂ1: 3400,
2925, 1695. UV lmax nm (log "): 265 (3.88). CD lmax nm
1
(ꢁ): 205 (+2165), 225 (ꢂ2801), 295 (ꢂ515). H NMR:
Table 2. 13C NMR: Table 3. FABMS m/z (rel. int.): 476
[M-H]ꢂ (50), 365 [M-111-H]ꢂ (60), 281 (100).
4.9. 8ꢁ-Hydroxyspiganthine (3)
Crystals from CHCl3:Me2CO (3:1) (1 mg), mp 255–
257ꢃ. TLC: Rf 0.24;anisaldehyde: red-purple. HPLC: Rt
17.5 min (S-2). [a]d +26ꢃ (c 0.1). IR vmax cmꢂ1: 3410,
2930, 1700. UV lmax nm (log "): 265 (4.18). CD lmax nm
(ꢁ): 205 (+4170), 225 (ꢂ3650), 244 (+390), 265
(ꢂ576). 1H NMR: Table 2. 13C NMR: Table 3. FABMS
m/z (rel. int.): 508 [M-H]ꢂ (80), 397 [M-111-H]ꢂ (50),
281 (100).
4.3. Spiganthine (1)
Crystals from CHCl3:Me2CO (3:1) (23 mg), mp 159ꢃ.
TLC: Rf 0.46;anisaldehyde: red-purple. HPLC: Rt 35.0
min (S-2). [a]d +35ꢃ (c 0.5). CD lmax nm (ꢁ): 205
(+5875), 225 (ꢂ4000), 245 (+570), 265 (ꢂ95), 280
(+275). 1H NMR: Table 2. 13C NMR: Table 3. FABMS
m/z (rel. int.): 492 [M-H]ꢂ (100), 381 [M-111-H]ꢂ (40).
All other physicochemical properties were also identical
with an authentic sample (Achenbach et al., 1995).
4.10. 20-Norspiganthine-5-carboxylic acid (18)
Crystals from CHCl3:MeOH (1:1) (20 mg), mp
>300ꢃ. TLC: Rf 0.09;anisaldehyde: red-purple. HPLC:
Rt 5.0 min (S-2), Rt 7.5 min (S-4). [a]d +15ꢃ (c 0.4). IR
KBr
max
v
cmꢂ1: 3400, 2970, 1680. UV lmax nm (log "): 265
4.4. Hydrolysis of spiganthine (1)
(4.11). CD lmax nm (ꢁ): 205 (+55), 230 (ꢂ3170), 265
(+1995). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 506 [M-H]ꢂ (100).
A solution of 7.5 mg of 1 in 5 ml aqueous KOH (1%)
was stirred for 90 min at 60ꢃC. After neutralization with
2 n H2SO4, the aqueous phase was extracted with
EtOAc, the organic extract evapd and the residue pur-
ified by CC on silica gel using S-1. This yielded com-
pounds 23 (2.5 mg), 24 (2.5 mg) and 2-pyrrolecarboxylic
acid (1 mg).
4.11. Methyl ester of 18
Compound 18 (7 mg) was dissolved in 1 ml MeOH
and treated with 3 ml diazomethane/Et2O at room
temp. for 5 h. After evapn the residue was purified by
HPLC on RP-18 using S-2 to obtain 1 mg of the methyl
ester of 18 (amorphous). TLC: Rf 0.51;anisaldehyde:
red-purple. HPLC: Rt 34.0 min (S-2). [a]d +18ꢃ (c 0.1).
1H NMR: ꢀ 0.78 (3H, d, J = 6.5 Hz, Me-19), 1.00 (3H,
d, J = 6.5 Hz, Me-21), 1.14 (3H, d, J = 6.5 Hz, Me-18),
1.15–1.22 (1 H, m, H-7b), 1.36 (3H, s, Me-17), 1.45–1.56
2H, m, H-8b, H-8a), 1.74–1.87 (1H, m, H-9), 2.23 (1H,
ddd, J1 = 13, J2=11, J3= 6 Hz, H-7a), 2.26–2.36 (1H,
m , H-13) overlapped by 2.32 (1H, d, J = 13 Hz, H-
14B), 2.61 (1H, d, J = 14Hz, H-14A), 3.10 (3H, s,
CO2Me), 3.75 (1H, d, J = 10, H-10), 3.94 (1H, dd,
J1 = 8,5 J2 = 2 Hz, H-4), 5.76 (1H, d, J = 8.5 Hz,
H-3), 6.19 (1H, dd, J1 = 4, J2 = 2.5 Hz, H-25), 6.72
(1H, dd, J1 = 4, J2 = 1.5 Hz, H-24), 6.99 (1H, dd, J1 =
2.5, J2 = 1.5 Hz, H-26). FABMS m/z (rel. int.): 520
[M-H]ꢂ (100).
4.5. Spiganthol (23)
Amorphous. TLC: Rf 0.31;anisaldehyde: pink. 1H
NMR: Table 2. 13C NMR: Table 3. FABMS m/z (rel.
int.): 399 [M-H]ꢂ (80), 367 [M-32-H]ꢂ (90), 281 (100).
4.6. Anhydrospiganthol (24)
Amorphous. TLC: Rf 0.44;anisaldehyde: pink. 1H
NMR: Table 2. 13C NMR: Table 3. FABMS m/z (rel.
int.): 381 [M-H]ꢂ (100).
4.7. 2-Pyrrolecarboxylic acid
Amorphous. TLC: Rf 0.27;anisaldehyde: yellow. UV
l
max nm (log e): 260 (3.54). 1H NMR: ꢀ 6.12 (1H, dd, J1
= 4, J2 = 2.5 Hz, H-4), 6.76 (1H, dd, J1 = 4, J2 = 1.5
Hz, H-3), 6.85 (1H, dd, J1 = 2.5, J2 = 1.5 Hz, H-5).
4.12. Ryanodine (4)
Crystals from CHCl3:Me2CO (3:1) (8 mg), mp 180ꢃ.
TLC: Rf 0.46;anisaldehyde: yellow-ochre. HPLC: Rt
57.5 min (S-2). [a]d +9ꢃ (c 0.7). IR vmax cmꢂ1: 3305,
2930, 1695. UV lmax nm (log "): 265 (4.15). CD lmax nm
(ꢁ): 205 (+4495), 230 (ꢂ4915), 245 (ꢂ510), 260
4.8. 20-Deoxyspiganthine (2)
Crystals from CHCl3:Me2CO (1:1) (1.5 mg), mp 144–
147ꢃ. TLC: Rf 0.48;anisaldehyde: red-purple. HPLC: Rt

294
H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
(ꢂ2095). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 492 [M-H]ꢂ (100), 381 [M-111-
H]ꢂ (70). All other physicochemical properties were also
identical with an authentic sample purchased from Cal-
biochem1 Biochemicals.
(ꢁ): 205 (+5030), 230 (ꢂ3730), 245 (ꢂ410), 265
(ꢂ2350). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 524 [M-H]ꢂ (100), 413 [M-111-
H]ꢂ (20).
4.18. 8,9-Dehydro-10-epi-ryanodine (8)
4.13. 20-Hydroxyryanodine (19)
Crystals from CHCl3:Me2CO (1:1) (6 mg), mp 165.
TLC: Rf 0.56;anisaldehyde: yellow-ochre. HPLC: Rt
62.0 min (S-2). [a]d +20 (c 0.1). IR vmax cmꢂ1: 3365,
2930, 1700. UV lmax nm (log "): 265 (4.15). CD lmax nm
(ꢁ): 210 (+2205), 230 (ꢂ4370), 245 (ꢂ385), 265
(ꢂ1920). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 490 [M-H]ꢂ (100), 379 [M-111-
H]ꢂ (40).
Crystals from CHCl3:MeOH (3:1) (19 mg), mp 188ꢃ.
TLC: Rf 0.33;anisaldehyde: yellow-ochre. HPLC: Rt
18.5 min (S-2). [a]d +8ꢃ (c 1.0). IR vmax cmꢂ1: 3305,
2955, 1695. UV lmax nm (log "): 265 (4.32). CD lmax nm
(ꢁ): 205 (+5815), 230 (ꢂ5040), 245 (ꢂ765), 265
(ꢂ2565). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 508 [M-H]ꢂ (100), 397 [M-111-
H]ꢂ (40).
4.19. (13S)-8,9-Dehydro-18-hydroxy-10-epi-ryanodine
(9)
4.14. 9-Hydroxy-10-epi-ryanodine (5)
Crystals from CHCl3:Me2CO (3:1) (3.5 mg), mp 169ꢃ.
TLC: Rf 0.51;anisaldehyde: yellow-ochre. HPLC: Rt
71.0 min (S-2). [a]d +2ꢃ (c 0.1). IR vmax cmꢂ1: 3305,
2930, 1695. UV lmax nm (log e): 265 (4.06). CD lmax nm
(ꢁ): 205 (+6435), 230 (ꢂ7670), 245 (ꢂ460), 265
(ꢂ3200). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 508 [M-H]ꢂ (100), 397 [M-111-
H]ꢂ (40).
Crystals from CHCl3:Me2CO (3:1) (6 mg), mp 168ꢃ.
TLC: Rf 0.46;anisaldehyde: yellow-ochre. HPLC: Rt
26.0 min (S-2). [a]d +11ꢃ (c 0.2). IR vmax cmꢂ1: 3390,
2920, 1695. UV lmax nm (log "): 265 (4.07). CD lmax nm
(ꢁ): 210 (ꢂ355), 230 (ꢂ3730), 245 (ꢂ445), 265 (ꢂ2375).
1NMR: Table 2. 13C NMR: Table 3. FABMS m/z (rel.
int.): 506 [M-H]ꢂ (60), 395 [M-111-H]ꢂ (60), 281 (100).
4.20. 8,9-Dehydro-20-hydroxy-10-epi-ryanodine (10)
4.15. 9-Hydroxy-9-epi-10-epi-ryanodine (6)
Crystals from CHCl3:Me2CO (3:1) (4.5 mg), mp 148ꢃ.
TLC: Rf 0.40;anisaldehyde: yellow-ochre. HPLC: Rt
23.0 min (S-2). [a]d +14ꢃ (c 0.2). IR vmax cmꢂ1: 3350,
2920, 1700. UV lmax nm (log "): 265 (4.18). CD lmax nm
(ꢁ): 210 (+1615), 230 (ꢂ2355), 245 (ꢂ355), 265
(ꢂ1680). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 506 [M-H]ꢂ (100), 395 [M-111-
H]ꢂ (60).
Crystals from CHCl3:Me2CO (3:1) (40 mg), mp 178ꢃ.
TLC: Rf 0.40;anisaldehyde: yellow-ochre. HPLC: Rt
KBr
max
60.0 min (S-2). [a]d +11ꢃ (c 1.0). IR v
cmꢂ1: 3340,
2935, 1690. UV lmax nm (log "): 265 (4.33). CD lmax nm
(ꢁ): 205 (+4915), 230 (ꢂ5465), 245 (ꢂ325), 265
(ꢂ2415). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 508 [M-H]ꢂ (100), 397 [M-111-
H]ꢂ (50).
4.21. 8,9-Dehydro-21-hydroxy-10-epi-ryanodine (11)
4.16. (13S)-9,18-Dihydroxy-9-epi-10-epi-ryanodine (7)
Crystals from CHCl3:Me2CO (3:1) (15 mg), mp 178ꢃ.
TLC: Rf 0.35;anisaldehyde: yellow-ochre. HPLC: Rt
33.0 min (S-2). [a]d +25ꢃ (c 1.0). IR vmax cmꢂ1: 3365,
2935, 1700. UV lmax nm (log "): 265 (4.71). CD lmax nm
(ꢁ): 205 (+3070), 230 (ꢂ4175), 245 (ꢂ165), 265 (ꢂ1565).
1H NMR: Table 2. 13C NMR: Table 3. FABMS m/z (rel.
int.): 506 [M-H]ꢂ (100), 395 [M-111-H]ꢂ (50).
Crystals from CHCl3:Me2CO (3:1) (3 mg), mp 190ꢃ.
TLC: Rf 0.28;anisaldehyde: yellow-ochre. HPLC: Rt
24.0 min (S-2). [a]d +10ꢃ (c 0.35). IR vmax cmꢂ1: 3305,
2925, 1695. UV lmax nm (log "): 265 (4.17). CD lmax nm
(ꢁ): 210 (+2025), 230 (ꢂ3940), 245 (ꢂ470), 270
(ꢂ4720). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 524 [M-H]ꢂ (100), 413 [M-111-
H]ꢂ (90).
4.22. 9-Epi-10-epi-ryanodine from 11
4.17. 9,20-Dihydroxy-9-epi-10-epi-ryanodine (20)
The solution of 4.5 mg 11 in 4 ml EtOH was stirred in
the presence of palladium (10% on carbon) (25 mg)
under hydrogen for 6 h at room temp. The reaction
mixture was evapd and the residue purified by CC on
silica gel using S-1 to obtain 3 mg of 9-epi-10-epi-
ryanodine (amorphous). TLC: Rf 0.56;anisaldehyde:
Crystals from CHCl3:MeOH (3:1) (3 mg), mp 178ꢃ.
TLC: Rf 0.28;anisaldehyde: yellow-ochre. HPLC: Rt
20.0 min (S-2). [a]d +4ꢃ (c 0.1). IR vmax cmꢂ1: 3390,
2930, 1695. UV lmax nm (log "): 265 (4.26). CD lmax nm

H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
295
yellow-ochre. [a]d +14ꢃ (c 0.3). UV lmax nm (log "):
4.26. (13S)-8,9-Dehydro-18,21-dihydroxy-10-
epi-ryanodine (21)
265 (4.25). CD lmax nm (ꢁ): 200 (+4760), 230 (ꢂ4040),
1
245 (ꢂ180), 255 (ꢂ595), 265 (+890). H NMR: ꢀ 0.74
(3H, d, J = 6.5 Hz, Me-19), 0.90 (3H, s, Me-20), 1.08
(3H, d, J = 6.5 Hz, Me-18), 1.14 (3H, s, Me-21), 1.27–
1.43 (5H, m, H-7b, H-8a) including 1.34 (3H, s, Me-17),
1.89 (1H, d, J = 14 Hz, H-14B), 1.95–2.08 (2H, m, H-9,
H-8b), 2.11–2.28 (2H, m, H-7a, H-13), 2.58 (1H, d, J =
14 Hz, H-14A), 3.78 (1H, d, J = 2.5 Hz, H-10), 5.65
(1H, s, H-3), 6.24 (1H, dd, J1 = 4, J2 = 2.5 Hz, H-25),
6.87 (1H, dd, J1 = 4, J2 = 1.5 Hz, H-24), 7.03 (1H, dd,
J1 = 2.5, J2 = 1.5 Hz, H-26). FABMS m/z (rel. int.):
492 [M-H]ꢂ (100), 381 [M-111-H]ꢂ (90).
Crystals from CHCl3:MeOH (1:1) (1.5 mg), mp 162ꢃ.
TLC: Rf 0.26;anisaldehyde: yellow-ochre. HPLC: Rt 14.5
min (S-2). [a]d +7ꢃ (c 0.2). IR vmax cmꢂ1: 3390, 2930,
1695. UV lmax nm (log "): 265 (4.56). CD lmax nm (ꢁ):
1
210 (+1450), 230 (ꢂ2580), 245 (ꢄ0), 265 (ꢂ1185). H
NMR: Table 2. 13C NMR: Table 3. FABMS m/z (rel.
int.): 522 [M-H]ꢂ (100), 411 [M-111-H]ꢂ (50).
4.27. 8,9-Dehydro-20,21-dihydroxy-10-epi-ryanodine (22)
Crystals from CHCl3:MeOH (1:1) (4.5 mg), mp 173ꢃ.
TLC: Rf 0.20;anisaldehyde: yellow-ochre. HPLC: Rt
16.0 min (S-2). [a]d +6ꢃ (c 0.2). IR vmax cmꢂ1: 3365,
2930, 1695. UV lmax nm (log "): 265 (4.08). CD lmax nm
(ꢁ): 205 (+2275), 230 (ꢂ3630), 245 (ꢂ300), 265
(ꢂ1730). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 522 [M-H]ꢂ (100), 411 [M-111-H]ꢂ
(20).
4.23. Epoxydation of 11
Compound 11 (7 mg) was epoxydized with 3-chloro-
peroxybenzoic acid (20 mg) according to Waterhouse
et al. (1987). Purification of the reaction mixture by CC
on silica gel using S-1 and subsequent HPLC on RP-18
(S-2) yielded the two epoxydation products EP-1 (17)
and EP-2 (iso-17).
4.28. 8ꢁ,9ꢁ-Epoxy-10-epi-ryanodine (12)
4.24. EP-1
Crystals from CHCl3:Me2CO (3:1) (13.5 mg), mp
196ꢃ. TLC: Rf 0.54;anisaldehyde: yellow-ochre. HPLC:
Rt 42.0 min (S-2). [a]d +13ꢃ (c 0.4). IR vmax cmꢂ1: 3305,
2935, 1705. UV lmax nm (log "): 265 (4.11). CD lmax nm
(ꢁ): 205 (+5035), 230 (ꢂ4520), 245 (ꢂ410), 265
(ꢂ2455). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 506 [M-H]ꢂ (100), 395 [M-H-
111]ꢂ (30).
Amorphous (0.8 mg). TLC: Rf 0.30;anisaldehyde:
yellow-ochre. [a]d +10ꢃ (c 0.05). All other physico-
chemical properties were also identical with the isolated
natural compound 17.
4.24.1. 8ꢁ,9ꢁ-Epoxy-21-hydroxy-10-epi-ryanodine (17)
(=EP-1)
Crystals from CHCl3:Me2CO (3:1) (4 mg), mp 193ꢃ.
TLC: Rf 0.30;anisaldehyde: yellow-ochre. HPLC: Rt 30.0
min (S-2). [a]d +13ꢃ (c 0.2). IR vmax cmꢂ1: 3365, 2935,
1700. UV lmax nm (log "): 265 (4.14). CD lmax nm (ꢁ):
4.29. Acid-catalyzed isomerization of 12
Aqueous oxalic acid (1%) (3 ml) was added to a
solution of 12 (4 mg) in Me2CO (3 ml) and stirred for
60 h at room temp. After evapn of the organic
solvent, the aqueous residue was diluted with 5 ml H2O
and extracted with 3ꢁ10 ml EtOAc. Evapn and CC of
the extract on silica gel using S-1 yielded 3.5 mg of the
pure product. Crystals from CHCl3:Me2CO (3:1),
mp 172ꢃ. TLC: Rf 0.38;anisaldehyde: yellow-ochre.
HPLC: Rt 41.5 min (S-2). [a]d ꢂ10ꢃ (c 0.2). All spec-
troscopic data were also identical with those of genuine
15.
1
205 (+4160), 230 (ꢂ4780), 245 (ꢂ290), 265 (ꢂ2060). H
NMR: Table 2. 13C NMR: Table 3. FABMS m/z (rel.
int.): 522 [M-H]ꢂ (100), 411 [M-111-H]ꢂ (50).
4.25. EP-2 (=8ꢂ,9ꢂ-Epoxy-21-hydroxy-10-epi-
ryanodine)
Amorphous (1 mg). TLC: Rf 0.30;anisaldehyde: yel-
low-ochre. HPLC: Rt 36.0 min (S-2). [a]d +20ꢃ (c 0.05).
1H NMR: ꢀ 0.73 (3H, d, J = 6.5 Hz, Me-19), 0.93 (3H,
s, Me-20), 1.09 (3H, d, J = 6.5 Hz, Me-18), 1.37 (3H, s,
Me-17), 1.91 (1H, d, J = 14 Hz, H-14B), 1.98 (1H, dd,
J1 = 15, J2 = 2 Hz, H-7b), 2.17–2.28 (1H, m, H-13),
2.42 (1H, dd, J1 = 15, J2 = 2 Hz, H-7a), 2.64 (1H, d, J
= 14 Hz, H-14A), 3.55 (1H, dd, J1 = J2 = 2 Hz, H-8),
3.71 (2H, s, H-21A, H-21B), 4.29 (1H, s, H-10), 5.63 (1H,
s, H-3), 6.24 (1H, dd, J1 = 4, J2 = 2.5 Hz, H-25), 6.86
(1H, dd, J1 = 4, J2 = 1.5 Hz, H-24), 7.03 (1H, dd, J1 =
2.5, J2 = 1.5 Hz, H-26). FABMS m/z (rel. int.): 522 [M-
H]ꢂ (100), 411 [M-111-H]ꢂ (20).
4.30. 6-Deoxy-6ꢂ,9ꢂ-epoxy-8ꢁ-hydroxy-10-
epi-ryanodine (15)
Crystals from CHCl3:Me2CO (3:1) (6 mg), mp 172ꢃ.
TLC: Rf 0.38;anisaldehyde: yellow-ochre. HPLC: Rt
41.5 min (S-2). [a]d ꢂ8ꢃ (c 0.2). IR vmax cmꢂ1: 3400,
2930, 1695. UV lmax nm (log "): 265 (3.93). CD lmax nm
(ꢁ): 205 (+1685), 230 (ꢂ3265), 245 (ꢂ400), 265
1
(ꢂ2400), 295 (+230). H NMR: Table 2. 13C NMR:

296
H. Hubner et al. / Phytochemistry 57 (2001) 285–296
¨
Table 3. FABMS m/z (rel. int.): 506 [M-H]ꢂ (80), 395
[M-111-H]ꢂ (60), 275 (100).
Acknowledgements
Thanks are due to the Deutsche Forschungsgemein-
schaft and to the Fonds der Chemischen Industrie for
financial support.
4.31. 8ꢂ,9ꢂ-Epoxy-10-epi-ryanodine (13)
Crystals from CHCl3:Me2CO (3:1) (4 mg), mp 212ꢃ.
TLC: Rf 0.51;anisaldehyde: yellow-ochre. HPLC: Rt
References
50.0 min (S-2). [a]d +7ꢃ (c 0.2). IR vmax cmꢂ1
:
3390, 2915, 1595. UV lmax nm (log "): 265 (4.03). CD
lmax nm (ꢁ): 205 (+1705), 230 (ꢂ3015), 245 (ꢄ0), 265
(ꢂ1245). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 506 [M-H]ꢂ (100), 395 [M-111-
H]ꢂ (10).
Achenbach, H., Hubner, H., Vierling, W., Brandt, W., Reiter, M.,
1995. Spiganthine, the cardioactive principle of Spigelia anthelmia.
Journal of Natural Products 58, 1092–1096.
Gonzalez-Coloma, A., Gutierrez, C., Hubner, H., Achenbach, H.,
Terrero, D., Fraga, B.M., 1999. Selective insect antifeedant and
toxic action of ryanoid diterpenes. Journal of Agricultural and
Food Chemistry 47, 4419–4424.
Gonzalez-Coloma, A., Terrero, D., Perales, A., Escoubas, P., Fraga,
B.M., 1996. Insect antifeedant ryanodane diterpenes from Persea
indica. Journal of Agricultural and Food Chemistry 44, 296–300.
Jenden, D.J., Fairhurst, A.S., 1969. The pharmacology of ryanodine.
Pharmacological Reviews 21, 1–25.
4.32. (13S)-8ꢁ,9ꢁ-Epoxy-18-hydroxy-10-epi-ryanodine
(14)
Crystals from CHCl3:Me2CO (3:1) (3.5 mg), mp 200ꢃ.
TLC: Rf 0.39;anisaldehyde: yellow-ochre. HPLC: Rt
17.0 min (S-2). [a]d +7ꢃ (c 0.2). IR vmax cmꢂ1: 3370,
2930, 1695. UV lmax nm (log "): 265 3.87). CD lmax nm
(ꢁ): 205 (+1015), 230 (ꢂ2365), 245 (ꢂ360), 265
(ꢂ1210). 1H NMR: Table 2. 13C NMR: Table 3.
FABMS m/z (rel. int.): 522 [M-H]ꢂ (100), 411 [M-111-
H]ꢂ (30).
Jefferies, P.R., Toia, R.F., Brannigan, B., Pessah, I.N., Casida, J.E.,
1992. Ryania insecticide: analysis and biological activity of 10 natural
ryanoids. Journal of Agricultural and Food Chemistry 40, 142–146.
Kelly, R.B., Whittigham, D.J., Wiesner, K., 1951. The structure of
ryanodine. Canadian Journal of Chemistry 29, 905–910.
Nkunya, M.H.H., Jonker, S.A., Makangara, J.J., Waibel, R., Achen-
bach, H., 2000. Aporphinoid alkaloids and other constituents from
Lettowianthus stellatus. Phytochemistry 53, 1067–1073.
Penefsky, Z.J., 1974. Studies on mechanism of inhibition of cardiac
muscle contractile tension by ryanodine. Mechanical response.
Pflugers Archiv 347, 173–184.
4.33. 8ꢁ,9ꢁ-Epoxy-20-hydroxy-10-epi-ryanodine (16)
Stahl, E., Kaltenbach, U., 1961. Thin-layer chromatography. VI.
Trace analysis of sugar mixtures on layers of Kieselgur G. Journal
of Chromatography 5, 351–355.
Crystals from CHCl3:Me2CO (3:1) (2 mg), mp 208ꢃ.
TLC: Rf 0.37;anisaldehyde: yellow-ochre. HPLC: Rt
19.0 min (S-2). [a]d +8ꢃ (c 0.1). IR vmax cmꢂ1: 3365,
2930, 1700. UV lmax nm (log "): 265 (4.12). CD lmax nm
(ꢁ): 205 (+3730), 230 (ꢂ3530), 245 (ꢂ480), 265
(ꢂ2030).
Sutko, J.L., Airey, J.A., Welch, W., Ruest, L., 1997. The pharmacol-
ogy of ryanodine and related compounds. Pharmacological Reviews
49, 53–98.
Vierling, W., 1988. Ryanodine in mammalian heart ventricular muscle:
indication for the induction of calcium leakage from the sarco-
plasmic reticulum. European Journal of Pharmacology 145, 329–334.
Waterhouse, A.L., Pessah, I.N., Francini, A.O., Casida, J.E., 1987.
Structural aspects of ryanodine action and selectivity. Journal of
Medicinal Chemistry 30, 710–716.
4.34. Pharmacological experiments
Pharmacological tests were performed as described
previously (Achenbach et al., 1995).
Wiesner, K., Valenta, Z., Findlay, J.A., 1967. The structure of ryano-
dine. Tetrahedron Letters, 221–225.