Cantharimide dimers from the Chinese blister beetle, Mylabris phalerate PALLAS

Article abstract of DOI:10.1248/cpb.55.92

Five cantharidin-related compounds were isolated from the Chinese blister beetle, Mylabris phalerate PALLAS (Meloidae). Their structures were determined based on spectroscopic and chemical evidence. Three of them were identified as cantharimide dimers, which consist of two units of cantharimide combined with a tri-, tetra-, or penta-methylene group.

Full text of DOI:10.1248/cpb.55.92

9
2
Chem. Pharm. Bull. 55(1) 92—94 (2007)
Vol. 55, No. 1
Cantharimide Dimers from the Chinese Blister Beetle, Mylabris phalerate
PALLAS
Takafumi NAKATANI, Katsuaki JINPO, and Naoki NODA*
Faculty of Pharmaceutical Sciences, Setsunan University; 45–1 Nagaotoge-cho, Hirakata, Osaka 573–0101, Japan.
Received September 13, 2006; accepted October 10, 2006
Five cantharidin-related compounds were isolated from the Chinese blister beetle, Mylabris phalerate PAL-
LAS (Meloidae). Their structures were determined based on spectroscopic and chemical evidence. Three of them
were identified as cantharimide dimers, which consist of two units of cantharimide combined with a tri-, tetra-,
or penta-methylene group.
Key words Chinese blister beetle; Mylabris phalerate; cantharimide dimer; Meloidae; cantharidin
The Chinese blister beetle (Mylabris phalerate PALLAS) has the appearance of unequivalent methylene signals (d 3.66,
H
1
3
been used in traditional Chinese medicine for the treatment 3.93, each 1H, d, Jꢁ11.0 Hz). In the C-NMR spectrum,
1
—3)
of cancer.
Among the constituents of this insect, can- signals of methine (C-3, C-6), methylene (C-4, C-5), and car-
4,5)
tharidin is a well-known active principle. Recently, Chi- bonyl (C-7, C-8) were observed separately. From these find-
nese researchers have reported the isolation of two fatty acid ings, compound 2 was considered to be an asymmetric can-
6)
amides from this drug. Our previous study revealed that this tharimide, and one of the two tertiary methyl groups in 1 is
beetle contains so-called cantharimides, in which the anhy- replaced by a hydroxymethyl group. The heteronuclear mul-
dride oxygen atoms in cantharidin are replaced by the basic tiple-bond correlation (HMBC) spectrum showed diagnostic
7)
amino acid moieties. We have continued to study the con- correlations between H -1/C-2, C-6, and C-7, and H -2/C-1,
2
3
stituents of this insect and have isolated five cantharidin-re- C-3, and C-8. Compound 2 showed specific rotation, [a]D
lated compounds. Three of them are cantharimide dimers, ꢂꢃ1° (cꢁ0.3, MeOH) and no CD maximum, indicating that
1
which consist of two units of cantharimide combined with a it is a racemate. The H-NMR spectrum of its (ꢀ)-MTPA de-
tri-, tetra-, and penta-methylene group. This paper deals with rivative exhibited two sets of signals due to optical isomers in
the isolation and structural elucidation of these compounds.
a ratio of ca. 1 : 1. These observations revealed that 2 was
The H O-eluted fraction (fr. III) described in the previous a racemate, (1R*,2R*,3S*,6R*)-1-hydroxymethyl-2-methyl-
2
7)
paper was chromatographed over silica gel to give fractions 3,6-epoxycyclohexane-1,2-dicarboximide (Fig. 1).
—3. Each of fr. 1—3 was further purified by HPLC to give The positive ion FAB-MS of 3 exhibited an [MꢄH] ion
compounds 1 and 2. The lower layer obtained by the treat- peak at m/z 431, and its mass was approximately two-fold
ꢄ
1
13
ment of the MeOH extract with CHCl –MeOH–H O was greater than those of compounds 1 and 2. The C-NMR
3
2
separated by the method described in the experimental sec- spectrum showed two methylene signals (d 25.3, 36.1), to-
C
tion to give three compounds 3, 4, and 5 together with re- gether with the five typical signals due to a cantharimide unit
1
markable amounts of cantharidin.
(d 12.6, 23.6, 53.8, 83.3, 181.1). In the H-NMR spectrum,
C
The molecular formula, C H NO , of compound 1 was two new methylene signals were observed at d 1.91 (quin-
established by high-resolution (HR) FAB-MS (m/z 194.0813 tet, H -2ꢅ) and d 3.48 (t, H -1ꢅ, 3ꢅ) in a ratio of 1 : 2. Based
1
0
12
3
H
2
H
2
ꢀ
1
[
MꢀH] ). The H-NMR spectrum of 1 exhibited characteris- on the above information combined with its molecular
tic signals due to a cantharidin skeleton, i.e., two signals each weight, compound 3 was considered to be a dimer, which
of methyl (6H, d 1.14), methylene (2H, dH 1.63; 2H, d consists of two units of a cantharimide moiety combined
H
H
1
3
1
.87), and methine protons (2H, dH 4.48). The C-NMR with a trimethylene group. Diagnostically important frag-
spectrum showed, in contrast to that of cantharidin, a remark- ment ion peaks at m/z 194 and 236 in its FAB-MS support
able downfield shift (7.0 ppm) of C-7 and C-8, while the the above suggestion (Fig. 2).
other signals remain almost unshifted. These findings indi-
Confirmation of the structure was achieved by the synthe-
cate that 1 is a cantharimide, in which the anhydride O atom sis of compound 3. Treatment of cantharidin with 1,3-di-
8)
in cantharidin is replaced by NH. Compound 1 has been aminopropane at 180 °C for 5 h gave a product that was
9)
13
1
synthesized, although no comparable spectroscopic data shown to be identical to 3 by the C- and H-NMR spectro-
have been reported.
Treatment of cantharidin with 28% ammonium hydroxide
at 180 °C gave a product of which the spectroscopic data
were identical with those of 1; the above suggestion was thus
confirmed, and the structure of compound 1 was character-
ized as (1S,2R,3S,6R)-1,2-dimethyl-3,6-epoxycyclohexane-
1
,2-dicarboximide (Fig. 1).
The HR-FAB-MS of 2 exhibited an [MꢀH] ion peak at
ꢀ
m/z 210.0764, consistent with the molecular formula
1
C H NO . The H-NMR spectrum of 2, when compared
1
0
12
4
with that of 1, showed the absence of one methyl group and Fig. 1. Structures of Compounds 1—5
∗
To whom correspondence should be addressed. e-mail: noda@pharm.setsunan.ac.jp
© 2007 Pharmaceutical Society of Japan

January 2007
93
scopic data (Chart 1). Based on the results obtained above, unclear. It seems likely that the cantharimides isolated in this
the structure of compound was characterized as study correspond to the above complex. The biological activ-
3
bis[(1S,2R,3S,6R)-1,2-dimethyl-3,6-epoxycyclohexane-1,2- ities of these compounds will be examined in future investi-
dicarboximido]-trimethylene (Fig. 1). Compound 3 has been gations.
10)
synthesized by Chinese researchers, but its isolation from
natural sources has not previously been reported.
Experimental
1
13
H- and C-NMR spectra were recorded on a JEOL ECA 600SN and a
JEOL JNM GX400 spectrometer, respectively, using tetramethylsilane
The positive-ion FAB-MS of compound 4 gave an
ꢄ
[
MꢄH] ion peak at m/z 445 which was 14 mass units more (TMS) as an internal reference. FAB-MS, including high-resolution MS,
1
3
than that of 3. The C-NMR spectrum exhibited the same were recorded on a JEOL JMS-700T spectrometer (accelerating voltage,
seven signals as those of 3, but in its H-NMR spectrum, un-
1
5 kV; matrix, glycerin; collision gas, Xe). Column chromatography was car-
ried out on Diaion HP-20 (Mitsubishi Chemical Co.), Sephadex LH-20
like 3, two methylene signals appeared at d 1.55 (H -2ꢅ, 3ꢅ)
H
2
(Amersham Pharmacia Biotech AB), Merck silica gel (230—400 mesh, art.
and 3.48 (H -1ꢅ, 4ꢅ) in a ratio of 1 : 1. These findings, to-
2
9385), and Bio-beads S-X2 (Bio-Rad) columns. Preparative HPLC was con-
gether with the significant fragment ion peaks at m/z 194 and ducted on Mightysil RP-18 GP Aqua (5 mm, 20ꢆ250 mm, Kanto Chemical
m/z 250 in its FAB-MS (Fig. 2), suggest that 4 is an analogue Co., Inc.), and Inertsil ODS-3 Jet (5 mm, 10ꢆ50 mm, GL Sciences) on a
JASCO 880-PU equipped with a JASCO 830-RI unit.
Material The commercial crude drug “Mylabris,” M. phalerata PALLAS
was purchased from Matsuura Yakugyo Co. (lot no. HS000126C). A
voucher specimen was deposited in the Faculty of Pharmaceutical Sciences,
of 3 with a different alkyl chain; the trimethylene group in 3
is replaced by a tetramethylene group in 4. Treatment of can-
tharidin with 1,4-diaminobutane under the same conditions
1
13
as described for 3 gave a product of which the H- and C- Setsunan University.
NMR spectra were good accordance with those of 4 (Chart
Isolation of Compounds 1—5 Whole bodies of Mylabris (3.4 kg) were
extracted with MeOH (4 l) at room temperature. The solvent was removed in
vacuo to give an extract (450.0 g). This was shaken with CHCl3–
1
). Therefore 4 has the structure shown in Fig. 1.
Compound 5 exhibited a [MꢄH] ion peak at m/z 457
ꢄ
MeOH–H O (1 : 1 : 1) to give an upper and a lower layer. The upper layer
2
which was 28 mass units more than that of 3. By analyses of
was concentrated to give an extract, fr. A (200 g). It was shaken with n-
the NMR spectroscopic data, 5 was found to be another can- BuOH–H O to give an n-BuOH and an H O layer. The n-BuOH and H O
2
2
2
layers were concentrated to give fr. I (30.8 g) and fr. II (175.0 g), respec-
tively. Fr. II was subjected to Diaion HP-20 column chromatography using
tharimide dimer that differed from 3 in the linkage alkyl
chain. The trimethylene group in 3 was replaced by a pen-
tamethylene residue. The structure was confirmed by con-
H O and MeOH successively to give fr. III (155.9 g) and fr. IV (16.6 g). Part
2
(45 g) of fr. III was placed on a silica gel column and eluted successively
densation of cantharidin with 1,5-diaminopentane, which with CHCl –MeOH (10 : 1→8 : 2)→CHCl –MeOH–H O (7 : 3 : 0.5→
3
3
2
yielded 5 (Chart 1). Thus the structure of 5 was deduced to 6 : 4 : 1) to give five fractions, fr. 1—5. Among them, the selected fr. 3 (3.6 g)
was separated by HPLC with Mightysil RP-18 GP Aqua using
MeOH–H O (2 : 8) as an eluent to give compounds 1 (2.6 mg) and 2
a
be bis[(1S,2R,3S,6R)-1,2-dimethyl-3,6-epoxycyclohexane-
2
1,2-dicarboximido]-pentamethylene (Fig. 1).
(
3.2 mg). The lower layer was concentrated to give fr. B (250 g). Part (101 g)
of fr. B was subjected to silica gel column chromatography using n-
In conclusion, three cantharimide dimers together with two
cantharimides were isolated. All of them are the first exam- hexane–AcOEt (2 : 1) to give cantharidin (394 mg) (positive to 0.1%
ples of naturally occurring cantharimides isolated from an in- bromocresol green in isopropanol with NaOH until appearance of blue
11)
color. Part (28 g) of fr. B was partitioned between NaOH 1 M and Et O.
sect. In 1967, during the isolation of cantharidin from an-
other blister beetle, Epicauta pestifera, Walter and Cole re-
ported that cantharidin exists partly free and partly combined
2
The ether-soluble fraction was concentrated in vacuo to give an oily fraction
(7.0 g). This was applied to a Bio-beads S-X2 column using AcOEt as an
eluent to give fr. 6—8 together with cantharidin (8.4 mg). Fr. 7 was further
11)
in the insect. However, the structures of the latter are as yet separated by HPLC with an Inertsil ODS-3 Jet column (ꢆ2) using
MeOH–H O (1 : 1) as a mobile phase to give compounds 3 (14.0 mg), 4
2
(
16.6 mg), and 5 (6.4 mg).
1S,2R,3S,6R)-1,2-Dimethyl-3,6-epoxycyclohexane-1,2-dicarboximide
1): Colorless needles from EtOH, mp 205—206 °C. HR-FAB-MS m/z:
(
(
1
6
ꢀ
1
94.0813 [MꢀH] (Calcd for C H NO : 194.0817). H-NMR (CD OD,
10 12 3 3
1
3
00 MHz) d: see Table 1. C-NMR (CD OD, 150 MHz) d: see Table 2.
3
(
1R*,2R*,3S*,6R*)-1-Hydroxymethyl-2-methyl-3,6-epoxycyclohexane-
1
2
6
,2-dicarboximide (2): White powder, mp 195—197 °C. FAB-MS m/z:
ꢀ
1
10.0764 [MꢀH] (Calcd for C H NO : 210.0766). H-NMR (CD OD,
1
0
12
4
3
1
3
00 MHz) d: see Table 1. C-NMR (CD OD, 150 MHz) d: see Table 2.
3
Bis[(1S,2R,3S,6R)-1,2-dimethyl-3,6-epoxycyclohexane-1,2-dicarbox-
imido]-trimethylene (3): White powder, mp 156—157 °C. HR-FAB-MS m/z:
ꢄ
1
4
31.2190 [MꢄH] (Calcd for C H N O : 431.2182). H-NMR (CDCl3,
23 31 2 6
1
3
600 MHz) d: see Table 1. C-NMR (CDCl , 150 MHz) d: see Table 2.
3
Bis[(1S,2R,3S,6R)-1,2-dimethyl-3,6-epoxycyclohexane-1,2-dicarbox-
imido]-tetramethylene (4): White powder, mp 246—247 °C. HR-FAB-MS
ꢄ
1
m/z: 445.2343 [MꢄH] (Calcd for C H N O : 445.239). H-NMR (CDCl ,
2
4
33
2
6
3
1
3
6
00 MHz) d: see Table 1. C-NMR (CDCl , 150 MHz) d: see Table 2.
Bis[(1S,2R,3S,6R)-1,2-dimethyl-3,6-epoxycyclohexane-1,2-dicarbox-
3
Fig. 2. Fragmentation Pattern of Compounds 3 and 4
Chart 1

9
4
Vol. 55, No. 1
1
a,b)
Table 1. H-NMR Data for 1—5
Position
1
2
3
4
5
3
4
5
6
4.48 (2H, dd, 2.1, 1.2)
1.63 (1H, m), 1.87 (1H, m)
1.63 (1H, m), 1.87 (1H, m)
4.48 (1H, dd, 2.1, 1.2)
1.14 (3H, s)
4.47 (1H, d, 4.0)
4.57 (2H, dd, 2.2, 0.8)
1.68 (2H, m), 1.78 (2H, m)
1.68 (2H, m), 1.78 (2H, m)
4.57 (2H, dd, 2.2, 0.8)
1.15 (6H, s)
4.54 (2H, dd, 2.4, 0.6)
1.68 (2H, m), 1.79 (2H, m)
1.68 (2H, m), 1.79 (2H, m)
4.54 (2H, dd, 2.4, 0.6)
1.13 (6H, s)
4.55 (2H, m)
1.65 (2H, m)
1.68 (2H, m), 1.79 (2H, m)
1.68 (2H, m), 1.79 (2H, m)
4.55 (2H, m)
1.65 (1H, m), 1.93 (1H, m)
4.50 (1H, d, 3.2)
1
2
1
1
2
3
4
5
-CH3
1.13 (6H, s)
-CH3
1.14 (3H, s)
1.30 (3H, s)
1.15 (6H, s)
1.13 (6H, s)
1.13 (6H, s)
-CH OH
3.66, 3.93 (each 1H, d, 11.0)
2
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
3.48 (2H, t, 7.2)
3.51 (2H, m)
1.55 (2H, m)
1.55 (2H, m)
3.51 (2H, m)
3.47 (2H, t, 7.2)
1.58 (2H, quint., 7.2)
1.26 (2H, m)
1.91 (2H, quint., 7.2)
3.48 (2H, t, 7.2)
1.58 (2H, quint., 7.2)
3.47 (2H, t, 7.2)
a) 1 and 2 were dissolved in CD OD and 3—5 were dissolved in CDCl . b) Chemical shift values are given in ppm, and J values in parentheses are given in Hz.
3
3
1
3
a)
Table 2.
Position
C-NMR Data for 1—5
which was identical to compound 3.
Preparation of 4 A mixture of cantharidin (19.6 mg), 1,4-diaminobu-
tane (4.4 mg), and triethylamine (0.014 ml) was dissolved in MeOH (3 ml)
and heated at 180 °C for 24 h and purified in the same manner as describe
for 3 to give 4 (19.7 mg, 89%).
Preparation of 5 A mixture of cantharidin (38.9 mg), 1,5-diaminopen-
tane (10 mg), and triethylamine (0.027 ml) was dissolved in MeOH (3 ml)
and heated at 180 °C for 24 h and purified in the same manner as describe
for 3 to give 5 (39.2 mg, 86%).
1
2
3
4
5
1
2
3
4
5
6
7
8
56.5
56.5
85.1
24.5
24.5
85.1
184.9
184.9
12.6
12.6
63.0
55.9
85.3
24.6
24.9
83.1
182.8
184.9
53.8
53.8
83.3
23.6
23.6
83.3
181.0
181.0
12.6
12.6
53.8
53.8
83.5
23.7
23.7
83.5
181.4
181.4
12.6
12.6
53.8
53.8
83.6
23.7
23.7
83.6
181.4
181.4
12.7
b)
b)
Acknowledgments The authors wish to thank Dr. Masatoshi Nishi and
Mr. Shoji Inoue of our university for measurements of the NMR spectra and
MS.
1
2
1
1
2
3
4
5
-CH3
-CH3
12.3
59.2
12.7
-CH OH
References
2
ꢅ
ꢅ
ꢅ
ꢅ
ꢅ
36.1
25.3
36.1
38.3
24.4
24.4
38.3
38.9
27.1
23.7
27.1
38.9
1) Wang G.-S., J. Ethnoparmacol., 26, 147—162 (1989).
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Science and Technologic Publishers and Shougakukan, Tokyo, 1985,
pp. 2196—2198.
3) Oak W. W., Ditunno J. F., Magnani T., Levery H. A., Mills L. C., Arch.
Intern. Med., 105, 106—114 (1960).
a) 1 and 2 were dissolved in CD OD and 3—5 were dissolved in CDCl . b) Inter-
changeable within a column.
3
3
4) Robiquet M., Ann. Chim., 76, 302—321 (1810).
5
)
)
Stork G., Van Tamelen E. E., Friedman L. J., Burgstahler A. W., J. Am.
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T.-S., Park H.-Y., Bioorg. Med. Chem. Lett., 14, 4277—4280 (2004).
imido]-pentamethylene (5): White powder, mp 148—149 °C. HR-FAB-MS
m/z: 459.2487 [MꢄH] (Calcd for C H N O : 459.2495). H-NMR
6
ꢄ
1
2
5
35
2
6
1
3
(
CDCl , 600 MHz) d: see Table 1. C-NMR (CDCl , 150 MHz) d: see Table
3
3
7) Nakatani T., Konishi T., Miyahara K., Noda N., Chem. Pharm. Bull.,
2, 807—809 (2004).
2
.
5
Preparation of 1 Cantharidin (50 mg) was dissolved in 28% ammonium
8)
Pretsh E., Clerc T., Seibl J., Simon W., “Tabellen zur
Strukturaufklärung organisher Verbindungen mit spectroskopishen
Methoden,” ed. by Nakanishi K., Kajiwara M., Tsutsumi K., Kodan-
sha, Shougakukan, Tokyo, 1982, p. C180.
hydroxide (2 ml) and heated at 180 °C for 2 h. After removal of the solvent,
the reaction mixture was recrystallized from EtOH to give 1 (45.7 mg, 91%).
Preparation of 3 A mixture of cantharidin (37.6 mg), 1,3-damino-
propane (7.1 mg), and triethylamine (0.027 ml) was dissolved in MeOH
3 ml) and heated at 180 °C for 24 h in a sealed tube. After being cooled to
room temperature, the reaction mixture was concentrated, and the residue
was column chromatographed on silica gel to give a product (35 mg, 85%),
9)
Walter W. G., J. Pharm. Sci., 78, 66—67 (1989).
(
1
1
0) Liu J., Zhang B., Sun J., Yaoxue Xuebao, 18, 752—759 (1983).
1) Walter W. G., Cole J. F., J. Pharm. Sci., 56, 174—176 (1967).