G. Bringmann, D. Menche, R. Brun, T. Msuta, B. Abegaz
FULL PAPER
(lg ε) ϭ 206 (1.27), 225 (1.58), 254 (0.96), 287 (0.94), 431 (0.36) synthesis,[17,21] was dissolved in dry THF (2 mL), treated at 0 °C
nm. CD: ∆ε280 ϭ ϩ2.25, ∆ε235 ϭ Ϫ2.59, ∆ε220 ϭ Ϫ 4.16 (EtOH). with an LiPPh2 solution (0.6 mL, 364 µmol) as prepared above,
1
IR (KBr): ν ϭ 3435, 2924, 2852, 1682, 1629, 1205, 720 cmϪ1. H
and stirred at 70 °C for 3 h. After the mixture had cooled, H2O
˜
NMR (400 MHz, CD3COCD3): δ ϭ 2.12 (s, 3 H, ArCH3), 2.68 (s, (1 mL) and 2 HCl (1 mL) were added, and the mixture was thor-
3 H, ArCOCH3), 3.68 (s, 3 H, ArOCH3), 6.41 (s, 1 H, 5Ј-H), 7.29 oughly extracted with EtOAc. Drying (MgSO4) of the combined
(dd, J ϭ 7.6, 1.0 Hz, 1 H, 7-H), 7.30 (s, 1 H, 2-H), 7.53 (dd, J ϭ organic phases, evaporation of the solvent, and purification by
7.6, 1.0 Hz, 1 H, 5-H), 7.74 (dd, J ϭ 7.6, 7.6 Hz, 1 H, 6-H), 12.0 HPLC with a preparative LC 25 mm module with two Nova-Pak
(s, 1 H), 12.0Ϫ12.3 (s, 2 H), 12.5 (s, 1 H). 13C NMR (101 MHz, C-18 column segments (25 ϫ 100 mm each) from Waters (Esch-
CD3COCD3): δ ϭ 21.0 (ArCH3), 33.1 (COCH3), 56.1 (OCH3), 92.1 born, Germany) and a 510 pump (Waters, Eschborn, Germany), a
(C-5Ј), 106.1 (C-3Ј), 108.2 (C-1Ј), 115.7 (C-13), 116.5 (C-12), 120.1
flow of 4.0 mL minϪ1, and UV detection at 254 nm (solvent:
(C-5), 124.0 (C-7), 125.2 (C-2), 129.4 (C-4), 132.9 (C-14), 135.7 (C- MeOH/H2O, 75:25, acidified with 0.1% trifluoroacetic acid), af-
11), 138.1 (C-6), 153.0 (C-3), 161.5 (C-8), 162.4 (C-1), 163.0, 163.6, forded (P)-6 (19.2 mg, 44.2 µmol, 72%) as an amorphous red solid,
163.7 (C-2Ј/C-4Ј/C-6Ј), 182.7 (C-10), 194.1 (C-9), 204.3 (Ar- fully identical in all respects to the material obtained above.
COCH3). The 13C assignments were achieved by HMQC and
Plasmodium falciparum and Cytotoxicity Assay: Antiplasmodial ac-
HMBC experiments. MS (EI, 70 eV): m/z (%) ϭ 434 [M]ϩ (100),
tivity was determined with the K1 strain (resistant to chloroquine
419 [M Ϫ CH3]ϩ (46), 403 [M Ϫ OCH3]ϩ (31), 392 [M Ϫ COCH2]ϩ
and pyrimethamine) of P. falciparum. A modification of the [3H]-
(27), 361 [392 Ϫ OCH3]ϩ (16). HRMS: 434.1000 (C24H18O8: calcd.
hypoxanthine incorporation assay[29] was used.[30] Briefly, infected
human red blood cells were exposed to serial drug dilutions in mic-
434.1001). The known natural products knipholone (4), knipholone
anthrone, 6Ј-O-methylknipholone, and 4Ј-O-demethylknipholone
were identified in the crude extract by HPLC coelution analysis,
rotiter plates for 48 h at 37 °C in a gas mixture with reduced oxygen
and elevated CO2. [3H]-Hypoxanthine was added to each well, and
with the previously synthesized compounds as reference mat-
after further incubation for 24 h, the wells were harvested on glass
erials.[17]
fiber filters and counted in a liquid scintillation counter. The IC50
value was calculated from the sigmoidal inhibition curve. The as-
says were run in duplicate and repeated at least once. Cytotoxicity
was tested against rat skeletal muscle myoblast (L-6) cells.[31]
Reductive Cleavage of 6: Na2S2O4 (3 mg, 17 µmol) was added to a
solution of 6 (2.0 mg, 4.6 µmol) in 5% NaOH (1 mL), and the
reaction mixture was stirred for 1 h at 70 °C. The solution was
acidified and thoroughly extracted with EtOAc. Evaporation of the
solvent from the dried (MgSO4) organic phases and purification by
flash chromatography on silica gel (CH2Cl2) afforded the anthra-
quinone 2 (0.7 mg, 2.8 µmol, 61%) and 2,6-dihydroxy-4-methoxy-
acetophenone (7, 0.5 mg, 2.7 µmol, 60%). These cleavage products
were identical to authentic chrysophanol (Fluka) and to synthetic
7, as obtained by cleavage from xanthoxylline (10, see below), by
Acknowledgments
This work was supported by the Deutsche Forschungsgemeinschaft
(SFB 347), the Fonds der Chemischen Industrie, the DAAD (fel-
lowship to D. M.), and the UNDP/World Bank/WHO Special Pro-
gram. Particular thanks are due to Christian Scheurer for carrying
out the in vitro assays. Technical support by F. Meyer is gratefully
acknowledged. B. M. A. is thankful to the University of Botswana
for a useful grant and T. M. is grateful to DAAD-NAPRECA for
a fellowship. We also thank G. Pole for the identification of the
plant material.
1
TLC and H NMR.
4,6-Dihydroxy-2-methoxyacetophenone (11): BBr3 (4.00 mL,
4.00 mmol, 1 in CH2Cl2) was added under argon to a cooled (0
°C) solution of 10[27] (400 mg, 2.04 mmol) in dry CH2Cl2 (30 mL).
After this had stirred for 3 h at room temp.,[28] MeOH was added,
the solvent was evaporated in vacuo, and the residue was purified
by flash chromatography on silica gel with CH2Cl2/MeOH as the
eluent (100:0 up to 100:3). Crystallization from EtOH yielded 11
(215 mg, 1.18 mmol, 58%); m.p. 204 °C (ref.[25] 204Ϫ205 °C). The
spectroscopic data were identical to those reported in ref.[18]
[1] [1a]
C. Reid in Plants of Southern Africa: Names and Distribu-
tion (Eds.: T. H. Arnold, B. C. de Wet), National Botanical
[1b]
Institute, Pretoria, 1993, p. 133.
R. A. Dyer, The Flowering
2,6-Dihydroxy-4-methoxyacetophenone (7): A solution of HPPh2
(0.5 mL, 538 mg, 2.89 mmol) in dry THF (3 mL) in a flame-dried
flask was treated dropwise, at 0 °C under argon, with a cold solu-
tion of n-butyllithium in hexane (2.5 , 1.27 mL, 3.18 mmol). Stir-
ring was continued, and the red solution was allowed to warm to
room temp. over a period of 20 min. A solution of 10[27] (100 mg,
649 µmol) in dry THF (5 mL) was treated at 0 °C under argon with
an LiPPh2 solution (2.4 mL, 1.43 mmol LiPPh2), prepared accord-
ing to the procedure given above, and stirred at 50 °C for 1 h. H2O
(3 mL) and 2 aqueous HCl (3 mL) were added, and the mixture
was thoroughly extracted with EtOAc. Drying (MgSO4) of the
combined organic phases, evaporation of the solvent, purification
by flash chromatography on silica gel with CH2Cl2/MeOH (100:0
up to 100:3) as the eluent, and crystallization from EtOH/H2O af-
forded 7 (87.4 mg, 480 µmol, 74%); m.p. 139Ϫ140 °C (ref.[13]
142Ϫ143 °C). The spectroscopic data were identical to those re-
ported in ref.[13]
[1c]
Plants of Africa 1961, 34, Plate 1350.
M. Blundell, Collins
Guide to the Wild Flowers of East Africa, Collins, London,
1987, p. 421.
For a review on the use of Bulbine species in traditional medi-
cine, see: J. M. Watt, M. G. Breyer-Brandwijk, The Medicinal
and Poisonous Plants of Southern and Eastern Africa, 2nd ed.,
Livingstone, Edinburgh, 1962.
M. Bezabih, B. M. Abegaz, K. Dufall, K. Croft, T. Skinner-
Adams, T. M. E. Davis, Planta Med. 2001, 67, 340Ϫ344.
M. Koyama, K. Takahashi, T. C. Chou, Z. Darzynkiewicz, J.
Kapuscinski, T. R. Kelly, K. A. Watanabe, J. Med. Chem. 1989,
32, 1594Ϫ1599.
[2]
[3]
[4]
[5]
D. E. Zembower, C. M. Kam, J. C. Powers, L. H. Zalkow, J.
Med. Chem. 1992, 35, 1597Ϫ1605.
[6] [6a]
M. C. B. van Rheede van Oudtshoorn, Planta Med. 1963,
[6b]
11, 332Ϫ337.
B.-E. van Wyk, A. Yenesew, E. Dagne,
Biochem. Syst. Ecol. 1995, 23, 277Ϫ281. [6c] E. Dagne, A. Yene-
sew, Pure Appl. Chem. 1994, 66, 2395Ϫ2398.
[7]
[8]
G. Bringmann, D. Menche, M. Bezabih, B. M. Abegaz, R. Ka-
minsky, Planta Med. 1999, 65, 757Ϫ758.
E. Dagne, W. Steglich, Phytochemistry 1984, 23, 1729Ϫ1731.
Bulbine-Knipholone (6). ؊ By Regioselective O-Demethylation of
(P)-9: Compound (P)-9 (27.5 mg, 61.4 µmol), as obtained by total
1110
Eur. J. Org. Chem. 2002, 1107Ϫ1111