Catharsitoxins from the Chinese remedy qiung laug

Article abstract of DOI:10.1016/S0040-4039(01)01468-X

Novel imidazole compounds, catharsitoxins A, B, C, D, E and F, were isolated from the Chinese remedy qiung laug. Their structures were determined by 2D NMR. To confirm their structures and to ensure an adequate supply for further biological studies, catharsitoxins A and D were synthesized.

Full text of DOI:10.1016/S0040-4039(01)01468-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 7079–7081
Catharsitoxins from the Chinese remedy qiung laug
Kiyotake Suenaga,a Hiroki Shimogawa,b Satoru Nakagawab and Daisuke Uemurab,*
aResearch Center for Materials Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan
bDepartment of Chemistry, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan
Received 5 July 2001; revised 8 August 2001; accepted 9 August 2001
Abstract—Novel imidazole compounds, catharsitoxins A, B, C, D, E and F, were isolated from the Chinese remedy qiung laug.
Their structures were determined by 2D NMR. To confirm their structures and to ensure an adequate supply for further biological
studies, catharsitoxins A and D were synthesized. © 2001 Elsevier Science Ltd. All rights reserved.
The Chinese remedy qiung laug is prepared by scalding
the dung beetle Catharsius molossus L. to death in
boiling water and drying with a charcoal fire. While it
has been used in China for more than 500 years to treat
spasmodic contractions, it carries a risk: it contains
poison,1 and pregnant women must be particularly
careful. However, the toxic components of this
medicine have not yet been clarified. We have obtained
novel imidazole compounds, catharsitoxins A, B, C, D,
E and F, by monitoring acute toxicity against mice. We
report here the isolation and structures of these
compounds.
TFA, iii. Develosil ODS-HG-5, 15% MeOH containing
0.1% TFA) guided by acute toxicity against mice to
give catharsitoxin A (1, 1.5 mg) as a colorless oil. The
ethyl acetate layer was extracted with 1% aqueous
AcOH, and the aqueous layer was basified (pH 10) with
NH4OH and re-extracted with EtOAc. The resulting
alkaloid fraction was purified by chromatography by
monitoring acute toxicity against mice to give catharsi-
toxins B (2, 0.5 mg), C (3, 0.9 mg), D (4, 1.2 mg), E (5,
1.1 mg), and F (6, 2.3 mg) as colorless oils, respectively.
The molecular formula of 1 was found to be C10H16N2
by HRFABMS (m/z 165.1400, calcd for C10H17N2 [M+
1
The Chinese remedy qiung laug (10 kg) was crushed
and extracted with 75% ethanol for 2 days. The extract
was filtered, concentrated and partitioned between
EtOAc and H2O. Both layers were toxic against mice.
The aqueous layer was chromatographed on TSK
G-3000S polystyrene gel (50% EtOH), DEAE Sephadex
A-25 (0.02 M pH 6.9 phosphate buffer), CM Sephadex
C-25 (0.2 M pH 6.9 phosphate buffer), and DEAE
Sephadex A-25 (0.02 M pH 6.9 phosphate buffer)
again, and reversed-phase HPLC (i. Develosil C8-10,
40% MeOH containing 0.1% trifluoroacetic acid (TFA);
ii. Develosil 300C4-HG-5, 20% MeOH containing 0.1%
H]+ 165.1392). The H NMR spectrum of 1 showed the
presence of a methyl group and an ethyl group (Table
1). An analysis of the COSY spectrum of 1 allowed for
the construction of four contiguous methylenes (C5–
C8). The presence of an imidazole ring was suggested
by characteristic carbon NMR data (lC 142.5, 130.5,
123.2). The HMBC correlations shown in Table 1
indicated connectivity among the partial structures
described above. Although two carbons, C2 and C3,
were exchangeable, they were assigned by comparison
of their 13C chemical shifts with those of 5,6,7,8-tetra-
hydroimidazo[1,2-a]pyridine (lC2 118.3, lC3 135.6).2
* Corresponding author. Tel./fax: +81-52-789-3654; e-mail: uemura@chem3.chem.nagoya-u.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01468-X

7080
K. Suenaga et al. / Tetrahedron Letters 42 (2001) 7079–7081
Table 1. NMR data for catharsitoxin A (1) in CD3ODa
(Scheme 1). The synthesis of 1 was started from d-
valerolactam. d-Valerolactam was converted into imi-
noether 7 with Meerwein reagent,4 which was alkylated
with 3-chloro-2-pentanone (10% in two steps).5
Cyclization6 of amide 8 gave catharsitoxin A (1) in 56%
yield. Catharsitoxin D (4) was synthesized from 2-
aminoacetophenone. Acylation of 2-aminoacetophe-
none gave amide 9 (73%), which was cyclized to give
catharsitoxin D (4) in 85% yield. Synthetic catharsitox-
ins A (1) and D (4) were identical to natural 1 and 4 in
Position
1Hb
13Cc
HMBC (1H“13C)
2
3
5
123.2
130.5
43.2
4.02 (dd, J=5.9, 5.9
Hz, 2H)
C-8a
6
7
8
2.09 (m, 2H)
1.98 (m, 2H)
2.94 (dd, J=6.3, 6.3
Hz, 2H)
21.3
18.6
22.7
C-8a, 7
1
all respects including spectroscopic data (IR, H and
13C NMR, MS) and acute toxicity against mice (LD99=
100 mg/kg for 1, 50 mg for 4).
8a
9
142.5
15.3
2.67 (q, J=7.6 Hz,
2H)
1.18 (t, J=7.6 Hz,
3H)
C-2, 3, 10
C-3, 9
10
11
13.0
8.7
In conclusion, we isolated catharsitoxins A, B, C, D, E
and F, novel imidazole compounds from the Chinese
remedy qiung laug, which has been used in China for
more than 500 years to treat spasmodic contractions.
Structurally related alkaloids containing an imidazole
ring7 such as tolazoline and pilocarpine8 have been used
for clinical drugs. Further biological studies on cathar-
sitoxins are currently in progress.
2.24 (s, 3H)
C-2, 3
a One drop of TFA was added to the solvent.
b Recorded at 800 MHz.
c Recorded at 200 MHz.
Thus, the structure of catharsitoxin A was determined
to be as shown in formula 1.
Table 2. NMR data for catharsitoxin D (4) in CD3ODa
The structure of catharsitoxin D (4) was determined as
follows. The molecular formula of 4 was found to be
C13H16N2 by HRFABMS (m/z 201.1396, calcd for
C13H17N2 [M+H]+ 201.1392). The presence of an
isobutyl group and a phenyl group was indicated by the
1H NMR and COSY spectra (Table 2). Characteristic
carbon NMR data (lC 148.0, 133.5, 114.2) and HMBC
correlations from an aromatic singlet proton (l 7.79) to
C2 and C5 suggested the presence of a 2,5-disubstituted
imidazole ring. Furthermore, the HMBC correlations,
H10/C-2 and H7/C5, allowed for connection of the
above partial structures, which established that the
structure of catharsitoxin D was as shown in formula 4.
The structures of catharsitoxins B (2), C (3), E (5), and
F (6) were determined by 2D NMR in the same manner
as described above for 1 and 4.3
Position
1Hb
13Cc
HMBC (1H“13C)
2
4
5
6
7
148.0
114.2
133.5
126.8
125.2
7.79 (s, 1H)
C-2, 5
7.70 (dd, J=1.4, 7.6
Hz, 2H)
7.53 (dd, J=7.6, 7.6
Hz, 2H)
7.47 (t, J=7.6 Hz,
1H)
2.89 (d, J=7.3 Hz,
2H)
C-5, 6, 8
C-7, 9
8
129.1
129.3
34.1
9
C-8
10
C-2, 11, 12
11
12
2.19 (m, 1H)
1.04 (d, J=6.5 Hz,
6H)
28.4
21.1
C-10, 12
C-10, 11
a One drop of TFA was added to the solvent.
b Recorded at 800 MHz.
To confirm the structures of catharsitoxins and to
ensure an adequate supply for further biological stud-
ies, catharsitoxins A (1) and D (4) were synthesized
c Recorded at 200 MHz.
Scheme 1. (a) Me3OBF4, i-Pr2NEt, CH2Cl2, rt, 2 h; (b) 2-chloropentanone, KBr, DMF, 110°C, 24 h, 10% in two steps; (c)
AcONH4, AcOH, reflux, 8 h, 56%; (d) isovaleric acid, Et3N, EDCI·HCl, CH2Cl2, rt, 2 h, 73%; (e) AcONH4, AcOH, reflux, 5 h,
85%.

K. Suenaga et al. / Tetrahedron Letters 42 (2001) 7079–7081
7081
Acknowledgements
Compound 3: 1H NMR (800 MHz, CD3OD containing
one drop of TFA) l 7.12 (s, 1H), 4.09 (dd, J=5.9, 5.9 Hz,
2H), 2.97 (dd, J=6.3, 6.3 Hz, 2H), 2.97 (m, 1H), 2.09 (m,
2H), 2.01 (m, 2H), 1.31 (d, J=7.2 Hz, 6H); HRFABMS
m/z 165.1395, calcd for C10H17N2 (M+H)+ 165.1392.
Compound 5: 1H NMR (800 MHz, CD3OD containing
one drop of TFA) l 7.79 (s, 1H), 7.70 (dd, J=8.0, 1.4 Hz,
2H), 7.53 (dd, J=7.6, 7.6 Hz, 2H), 7.47 (t, J=7.6 Hz, 1H),
3.19 (m, 1H), 1.86 (m, 2H), 1.47 (d, J=9.6 Hz, 3H), 0.96
(t, J=7.2 Hz, 3H); HRFABMS m/z 201.1412, calcd for
C13H17N2 (M+H)+ 201.1392. This compound was found to
be racemic by chiral HPLC analysis.
This work was supported in part by Grants-in-Aid for
Scientific Research (No. 11558079) and Scientific
Research on Priority Areas (A) (No. 12045235) from
the Ministry of Education, Culture, Sports, Science,
and Technology, Japan. We are indebted to Wako Pure
Chemical Industries Ltd., and Banyu Pharmaceutical
Co., Ltd. for their financial support. We thank Profes-
sors R. Noyori and M. Kitamura (COE, Nagoya Uni-
versity) for performing the NMR experiments.
Compound 6: 1H NMR (800 MHz, CD3OD containing
one drop of TFA) l 7.79 (s, 1H), 3.02 (m, 1H), 2.79 (d,
J=6.3 Hz, 1H), 2.10 (m, 1H), 1.32 (d, J=7.4 Hz, 6H),
0.98 (d, J=6.5 Hz, 6H); HRFABMS m/z 167.1553, calcd
for C10H18N2 (M+H)+ 167.1548.
References
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3. Compound 2: 1H NMR (800 MHz, CD3OD containing
one drop of TFA) l 3.92 (dd, J=5.9, 5.9 Hz, 2H), 2.96
(dd, J=6.3, 6.3 Hz, 2H), 2.66 (q, J=7.6 Hz, 2H), 2.24 (s,
3H), 2.10 (m, 2H), 2.00 (m, 2H), 1.23 (t, J=7.6 Hz, 3H);
HRFABMS m/z 165.1415, calcd for C10H17N2 (M+H)+
165.1392.