July 2004
809
Experimental
(400 MHz, CD3OD) d: 1.16 (3H, s, H3-8ꢁ), 1.19 (3H, s, H3-9ꢁ), 1.19—1.29
(2H, m, H2-4), 1.35—1.55 (2H, m, H2-5), 1.42 (9H, s, Boc CH3ꢃ3), 1.67
(2H, m, Hb-5ꢁ, 6ꢁ), 1.91 (2H, m, Ha-5ꢁ, 6ꢁ), 2.13 (2H, m, H2-3), 2.99 (2H, m,
H2-6), 4.48 (1H, m, H-2), 4.50 (2H, br s, H-4ꢁ, 7ꢁ). 13C-NMR (100 MHz,
CD3OD) d: 12.6 (C-8ꢁ), 12.8 (C-9ꢁ), 24.6 (C-5ꢁ), 24.6 (C-4), 24.8 (C-6ꢁ),
28.8ꢃ3 (Boc CH3), 29.0 (C-5), 30.2 (C-3), 41.1 (C-6), 55.0 (C-3ꢁa), 55.1
(C-7ꢁa), 56.5 (C-2), 79.7 (Boc C-(CH3)3), 85.1ꢃ2 (C-4ꢁ, 7ꢁ), 158.2 (Boc
CO), 176.5 (C-1), 183.1 (C-1ꢁ), 183.2 (C-3ꢁ).
Melting points (mp) were determined on Yanaco MP-S3 apparatus and are
uncorrected. Optical rotations were measured at 25 °C with a JASCO DIP-
140 polarimeter. H- and 13C-NMR spectra were recorded on a JEOL JNM
1
GX400 and a GE Omega 600 spectrometers, respectively, using tetramethyl-
silane (TMS) as an internal reference. ESI-MS, including high-resolution
MS, were recorded on a JEOL JMS-700T spectrometer. TLC was carried
out on silica gel precoated Al sheets (Merck art. 5554). Spots were visual-
ized with 5% H2SO4 in MeOH or 0.3% ninhydrin in BuOH (by heating).
Column chromatography was carried out on Diaion HP-20 (Mitsubishi
Chemical Co.), Sephadex LH-20 (Amersham Pharmacia Biotech AB),
Merck silica gel (230—400 mesh, art. 9385), and Cosmosil 75C18-OPN
(Nacalai Tesque, Inc.) colums. Preparative HPLC was conducted on
Mightysil RP-18 GP Aqua (5 mm, 20ꢃ250 mm, Kanto Chemical Co., Inc.)
on a JASCO 880-PU equipped with a JASCO 830-RI.
Deprotection of 1a A solution of 1a (20.1 mg, 0.047 mmol) in trifluo-
roacetic acid (3 ml) was stirred at room temperature for 30 min and then
evaporated in vacuo. The residue was dissolved in water, and the solution
was neutralized with 1 M NaOH. After removal of the solvent, the reaction
mixture was subjected to Diaion HP-20 column chromatography using H2O
and MeOH successively to give an MeOH eluate. The MeOH eluate was
concentrated in vacuo to yield a pale yellow oil (18.1 mg), and this was sub-
jected to silica gel column chromatography (CHCl3–MeOH–H2O, 6 : 4 : 1) to
give 1b (16 mg, 100%) as a white powder. [a]D ꢀ5.4° (cꢂ0.50, MeOH).
Material The commercial crude drug “Mylabris,” M. phalerata PALL.
was purchased from Matsuura Yakugyo Co. (lot no. HS000126C). A
voucher specimen was deposited in the Faculty of Pharmaceutical Sciences,
Setsunan University.
Preparation of Nd-Boc-L-ornitine A solution of Nd-Boc-Na-CBZ-
nithine (Kokusan Chemical Works, 500 mg, 1.37 mmol) was shaken with 10
L-or-
%
Isolation of Compounds, 1—3 Whole bodies (3.4 kg) of Mylabris were
extracted with MeOH (4 l) at room temperature. The solvent was removed in
vacuo to give a extract (450.0 g). This was shaken with CHCl3–MeOH–H2O
(1 : 1 : 1) to give an upper and a lower layer. After removal of the solvent, the
upper layer was shaken with n-BuOH–H2O to give an n-BuOH and a H2O
layer. The n-BuOH and H2O layers were concentrated to give fr. I (30.8 g)
and fr. II (175.0 g), respectively. Fraction II was subjected to Diaion HP20
column chromatography using H2O and MeOH successively to give fr. III
palladium carbon (50 mg) in EtOH (10 ml) for 48 h. The catalyst was re-
moved by filtration, and the filtrate was concentrated in vacuo to give a prod-
uct. This product was subjected to silica gel chromatography (CHCl3–
MeOH–H2O, 7 : 3 : 0.5) to give Nd-Boc-L-ornithine (274.7 mg, 86.4%) as a
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white powder. H-NMR (400 MHz, CD3OD) d: 1.45 (9H, s, Boc CH3ꢃ3),
1.58 (2H, m, H2-4), 1.80 (1H, m, Ha-3), 1.89 (1H, m, Hb-3), 3.11 (2H, m,
H2-5), 3.52 (1H, dd, Jꢂ5.2, 7.2 Hz, H-2). 13C-NMR (100 MHz, CD3OD) d:
26.8 (C-4), 28.7ꢃ3 (Boc CH3), 29.6 (C-3), 40.7 (C-5), 55.7 (C-2), 79.8 (Boc
C-(CH3)3), 158.3 (Boc CO), 174.1 (C-1).
(155.9 g) and fr. IV (16.6 g). Fraction IV was chromatographed on a Sephadex
Preparation of 2a A mixture of cantharidin (60 mg, 0.31 mmol), Nd-
Boc-L-ornithine (80 mg, 0.34 mmol), and triethylamine (0.06 ml) was dis-
solved in toluene (5 ml) and treated in the same manner as described for the
preparation of 1a to give 2a (116.8 mg, 92%) as a white powder. [a]D ꢀ2.6°
(cꢂ2.0, MeOH). IR (KBr) cmꢀ1: 3386, 2978, 1699, 1404, 1169, 996. 1H-
NMR (400 MHz, CDCl3ꢄCD3OD, 1 : 1) d: 1.16 (3H, s, H3-8ꢁ), 1.18 (3H, s,
H3-9ꢁ), 1.38 (2H, m, H2-4), 1.42 (9H, s, Boc CH3ꢃ3), 1.71 (2H, m, Hb-5ꢁ,
6ꢁ), 1.85 (2H, m, Ha-5ꢁ, 6ꢁ), 2.10 (2H, m, H2-3), 3.06 (2H, t, Jꢂ6.4 Hz, H2-
5), 4.50 (1H, dd, Jꢂ4.8, 10.8 Hz, H-2), 4.59 (2H, br s, H-4ꢁ, 7ꢁ). 13C-NMR
(100 MHz, CDCl3ꢄCD3OD) d: 12.3 (C-8ꢁ), 12.5 (C-9ꢁ), 23.7 (C-5ꢁ), 23.8
(C-6ꢁ), 25.6 (C-4), 26.7 (C-3), 28.4ꢃ3 (Boc CH3), 39.8 (C-5), 53.8 (C-3ꢁa),
53.9 (C-7ꢁa), 54.5 (C-2), 79.1 (Boc C-(CH3)3), 83.8 (C-4ꢁ), 83.9 (C-7ꢁ),
156.4 (Boc CO), 174.9 (C-1), 181.6 (C-1ꢁ), 181.7 (C-3ꢁ).
LH-20 column (MeOH) to give fr. 1 (5.5 g), fr. 2 (5.4 g), and fr. 3 (2.3 g).
Part of fr. 2 (2.7 g) was successively chromatographed on silica gel (CHCl3–
MeOH–H2O, 8 : 2 : 0.1Æ7 : 3 : 0.5Æ6 : 4 : 1ÆMeOH) to yield four fractions,
fr. 4 (543 mg), fr. 5 (819 mg), fr. 6 (651 mg), and fr. 7 (276 mg). Fraction 6
was chromatographed on silica gel (CHCl3–MeOH–H2O, 6 : 4 : 1), to give fr.
8 (90 mg), fr. 9 (158 mg), fr. 10 (373 mg), and fr. 11 (25 mg). Fraction 10
was subjected to preparative HPLC with Mightysil RP-18 GP Aqua (size,
20ꢃ250 mm; 5 mm) using 10% CH3CN to give 1 (64.0 mg), 2 (9.1 mg), 3
(24.6 mg), and fr. 12 (250 mg). Fraction 9 was subjected to preparative
HPLC with Mightysil RP-18 GP Aqua (size, 2ꢃ250 mm; 5 mm) using 10%
CH3CN to give 3 (32.6 mg).
(2S)-6-Amino-2-[(3aR*,4S*,7R*,7aS*)-3a,7a-dimethyl-1,3-dioxo-4,7-
epoxyoctahydroisoindol-2-yl]-hexanoic Acid (1): Powder, mp 159.0—
162.0 °C. [a]D ꢀ23.3° (cꢂ0.30, MeOH). HR-ESI-MS m/z: 323.1607 (Calcd
for C16H23N2O5: 323.1607) [MꢀH]ꢀ. IR (KBr) cmꢀ1: 2980, 1697, 1644,
1588, 1404, 1358, 1236, 993, 817, 675. 1H- and 13C-NMR d: see Table 1.
(2S)-5-Amino-2-[(3aR*,4S*,7R*,7aS*)-3a,7a-dimethyl-1,3-dioxo-4,7-
epoxyoctahydroisoindol-2-yl]-pentanoic Acid (2): Powder, mp 157.0—160.0
°C. [a]D ꢀ26.9° (cꢂ0.26, MeOH–H2O, 1 : 1). HR-ESI-MS m/z: 309.1450
(Calcd for C15H21N2O5: 309.1451) [MꢀH]ꢀ. IR (KBr) cmꢀ1: 2979, 1701,
1583, 1404, 1235, 990, 903, 812. 1H- and 13C-NMR d: see Table 1.
Deprotection of 2a A solution of 2a (100 mg, 0.244 mmol) in trifluo-
roacetic acid (5 ml) was treated in the same manner as described for the
preparation of 1b to give 2b (58.4 mg, 77%) as a white powder. [a]D ꢀ3.8°
(cꢂ1.0, MeOH–H2O, 1 : 1).
Acknowledgments The authors are grateful to Dr. Masatoshi Nishi and
Mr. Shoji Inoue of this university for NMR and MS measurements.
(2S)-2-[(3aR*,4S*,7R*,7aS*)-3a,7a-Dimethyl-1,3-dioxo-4,7-epoxy-oc-
tahydroisoindol-2-yl]-5-guanidino Pentanoic Acid (3): Powder, mp 195.0—
197.0 °C. [a]D ꢀ21.1° (cꢂ2.2, MeOH–H2O, 1 : 1). HR-ESI-MS m/z:
References
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.
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Intern. Med., 105, 106—114 (1960).
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Chem. Soc., 75, 384—392 (1953).
6) Noda N., Kubota S., Miyata Y., Miyahara K., Chem. Pharm. Bull., 48,
1749—1752 (2000).
351.1666 (Calcd for C16H23N4O5: 351.1667) [MꢀH]ꢀ. IR (KBr) cmꢀ1
:
1
3373, 2978, 1767, 1693, 1628, 1461, 1402, 1359. H- and 13C-NMR d: see
Table 1.
Preparation of 1a A mixture of cantharidin (Sigma, 20 mg, 0.102
mmol), Ne-Boc-L-lysine (Aldrich, 25 mg, 0.102 mmol), and triethylamine
(0.02 ml) was dissolved in toluene (5 ml) and heated at 200 °C for 48 h in a
sealed tube. After being cooled to room temperature, the reaction mixture
was diluted with EtOAc (50 ml) and extracted with 10% NaHCO3 (3ꢃ50
ml). The aqueous layer was acidified with HCl and extracted with EtOAc
(3ꢃ50 ml). The organic layer was washed with H2O and brine, dried over
MgSO4, and then concentrated in vacuo to give a product (21 mg, pale yel-
low oil). This product was subjected to silica gel column chromatography
(CHCl3–MeOH, 4 : 1) to give 1a (20.1 mg, 47%) as a colorless solid. [a]D
7) Noda N., Yashiki Y., Nakatani T., Miyahara K., Du X.-M., Chem.
Pharm. Bull., 49, 930—931 (2001).
8) McCluskey A., Walkom C., Bowyer M. C., Ackland S. P., Gardiner E.,
Sakoff J. A., Bioorg. Med. Chem. Lett., 11, 2941—2946 (2001).
1
ꢀ1.5° (cꢂ1.0, MeOH). IR (KBr) cmꢀ1: 1695, 1598, 1549, 1384. H-NMR