852 Journal of Natural Products, 2005, Vol. 68, No. 6
Itoh et al.
6′-O-(3,4,5-Trimethoxybenzoyl)glomeratose A (7): white
powder; [R]28D -20° (c 1.0, MeOH); UV (MeOH) λmax (log ꢀ) 214
(4.61), 272 (4.11), 303 (4.23) nm; IR (KBr) νmax 3385, 1719,
(DMSO)], and 2 units of topo II. The reaction mixtures were
incubated at 37 °C for 30 min and terminated by adding 2 µL
of loading buffer consisting of 5% sarkosyl, 0.0025% bromophe-
nol blue, and 25% glycerol. The mixtures were subjected to
1% agarose gel electrophoresis in TAE (Tris-acetate-EDTA)
running buffer. The agarose gel was stained with ethidium
bromide (EtBr), and DNA was visualized on a UV transillu-
minator. Relaxation activity of topo I was analyzed in the same
manner described above except that the reaction mixtures
contained 10 mM Tris-HCl (pH 7.9), pBR322 DNA (0.25 µg),
1 mM EDTA, 150 mM NaCl, 0.1% bovine serum albumin
(BSA), 0.1 mM spermidine, 5% glycerol, and 2 units of topo I.
One unit was defined as the amount of enzyme capable of
relaxing 0.25 µg of DNA in 15 min at 37 °C.
Cell Culture and Measurement of Cell Viability. A
human promyelocytic leukemia cell line, HL-60, was obtained
from the Health Science Research Bank (Osaka, Japan). The
cells were cultured in RPMI 1640 medium supplemented with
10% fetal bovine serum, penicillin (100 units/mL), and strep-
tomycin (100 µg/mL) at 37 °C in a humid atmosphere of 5%
CO2/95% air. For the cell growth assay, HL-60 cells were plated
at 3 × 105 cells into each well of 96-well microplates with
various concentrations of the alkaloids. These compounds were
dissolved in phosphate-buffered saline (PBS) at a concentration
of 10 mM as a stock solution. The stock solutions were diluted
to the appropriate final concentrations with growth medium
just before use. The cell viability was determined by MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide) assay.25
1506, 1128, 1057 cm-1 1H NMR (CD3OD, 500 MHz) δ 3.43
;
(1H, t, J ) 10.0 Hz, H-4′), 3.46 (1H, dd, J ) 10.0, 3.5 Hz, H-2′),
3.60 (1H, d, J ) 12.0 Hz, H-1), 3.66 (1H, d, J ) 12.0 Hz, H-1),
3.69 (1H, dd, J ) 12.0, 3.0 Hz, H-6), 3.69 (1H, t, J ) 10.0 Hz,
H-3′), 3.76 (1H, dd, J ) 12.0, 6.0 Hz, H-6), 3.78 (3H, s, OMe-
4′′), 3.81 (3H, s, OMe-4′′′), 3.85 (6H, s, OMe-3′′′, 5′′′), 3.86 (6H,
s, OMe-3′′, 5′′), 3.90 (1H, ddd, J ) 8.0, 6.0, 3.0 Hz, H-5), 4.26
(1H, ddd, J ) 10.0, 5.0, 2.0 Hz, H-5′), 4.32 (1H, t, J ) 8.0 Hz,
H-4), 4.46 (1H, dd, J ) 12.0, 5.0 Hz, H-6′), 4.73 (1H, dd, J )
12.0, 2.0 Hz, H-6′), 5.47 (1H, d, J ) 8.0 Hz, H-3), 5.50 (1H, d,
J ) 3.5 Hz, H-1′), 6.55 (1H, d, J ) 16.0 Hz, H-R), 6.95 (2H, s,
H-2′′, 6′′), 7.32 (2H, s, H-2′′′, 6′′′), 7.71 (1H, d, J ) 16.0 Hz,
H-â); 13C NMR (CD3OD, 125 MHz) δ 56.8 (OMe-3′′, 5′′, 3′′′,
5′′′), 61.2 (OMe-4′′, 4′′′), 63.2 (C-6), 65.7 (C-1), 65.8 (C-6′), 71.9
(C-4′), 72.5 (C-5′), 73.2 (C-2′), 74.1 (C-4), 74.8 (C-3′), 79.7 (C-
3), 84.2 (C-5), 93.3 (C-1′), 105.0 (C-2), 106.9 (C-2′′, 6′′), 108.2
(C-2′′′, 6"′), 117.8 (C-R), 126.4 (C-1′′′), 131.4 (C-1′′), 141.4 (C-
4′′), 143.7 (C-4′′′), 147.3 (C-â), 154.4 (C-3′′′, 5′′′), 154.8 (C-3′′,
5′′), 167.6 (CO of benzoyl), 167.7 (CO of cinnamoyl); NOESY,
H-1/H-3; H-2′′, 6′′/OMe-3′′, 5′′; HMBC, H-1fC-2, 3; H-3fC-4,
CO; H-4fC-6; H-1′fC-2, 3′, 5′; H-6′fC-4′, CO; H-2′′, 6′′fC-
4′′, â; OMe-3′′, 5′′fC-3′′, 5′′; OMe-4′′fC-4′′; H-âfC-1′′, 2′′, 6′′,
R; H-RfC-1′′, CO; H-2′′′, 6′′′fC-1′′′, 4′′′, CO; OMe-3′′′, 5′′′fC-
3′′′, 5′′′; OMe-4′′′fC-4′′′; SIMS m/z 755 [M - H]-, 535, 237,
211; HRSIMS m/z 755.2400 (calcd for C34H43O19, 755.2400).
Methylation of Ajmaline and Isoajmaline. A solution
of ajmaline (10.0 mg) and methyl iodide (3.82 µL) in MeCN
(0.5 mL) was refluxed for 1 h. The concentrated reaction
mixture was purified by preparative TLC (CHCl3-MeOH-
NH4OH, 70:30:3) and then treated with an anion-exchange
Acknowledgment. We thank Dr. M. Sugiura (Kobe Phar-
maceutical University) for the NMR spectra and Dr. K. Saiki
(Kobe Pharmaceutical University) for the MS measurements.
This research was supported financially by the Kobe Phar-
maceutical University Collaboration Fund.
resin (AG 1-X4) to afford 1 (9.8 mg): [R]30 +109° (c 0.23,
D
1
MeOH); SIMS m/z 341 [M]+. UV, IR, H NMR, and 13C NMR
spectroscopic data were identical to those of 1 from R.
serpentina.
References and Notes
(1) Sto¨ckigt, J. In The Alkaloids, Vol. 47; Cordell, G. A., Ed.; Academic
Press: New York, 1995; Chapter 2.
(2) Neuss, N. In Indole and Biogenetically Related Alkaloids; Phillipson,
J. D.; Zenk, M. H., Eds.; Academic Press: New York, 1980; Chapter
17.
In a similar manner, isoajmaline (3.2 mg) gave 2 (2.2 mg):
[R]28 +74° (c 0.22, MeOH); SIMS m/z 341 [M]+. UV, IR, and
D
1H NMR spectroscopic data were identical to those of 2 from
R. serpentina.
(3) Jokela, R.; Lounasmaa, M. Planta Med. 1996, 62, 577-579.
(4) Majumdar, S. P.; Poisson, J.; Potier, P. Phytochemistry 1973, 12,
1167-1169.
Methylation of 4 and 5. A methanolic solution (0.25 mL)
of 4 (5.1 mg) was methylated with (trimethylsilyl)diazo-
methane and purified by preparative TLC (CHCl3-MeOH-
(5) Djerassi, C.; Fishmen, J.; Gorman, M.; Kutney, J. P.; Pakrashi, S. C.
J. Am. Chem. Soc. 1957, 79, 1217-1222.
NH4OH, 90:9:1) to afford yohimbine (1.8 mg): [R]29 +77° (c
D
0.18, pyridine); EIMS m/z 354 [M]+. UV, IR, 1H NMR, and 13
C
(6) Schu¨bel, H.; Treiber, A.; Sto¨ckigt, J. Helv. Chim. Acta 1984, 67, 2078-
2081.
NMR spectroscopic data were identical to those of authentic
yohimbine.
(7) Clivio, P.; Richard, B.; Deverre, J.-R.; Sevenet, T.; Zeches, M.; Olivier,
L. L. Phytochemistry 1991, 30, 3785-3792.
In a similar manner, 5 (10.0 mg) gave isorauhimbine (3.6
(8) Feng, X. Z.; Kan, C.; Potier, P.; Kan, S.-K.; Lounasmaa, M. Planta
Med. 1982, 44, 212-214.
mg): [R]29 -78° (c 0.36, pyridine); EIMS m/z 354 [M]+. UV,
D
(9) Atta-ur-Rahman; Malik, S.; Habit-ur-Rehman. Phytochemistry 1986,
25, 1731-1733.
1
IR, H NMR, and 13C NMR spectroscopic data were identical
to those of authentic isorauhimbine.
(10) Wenkert, E.; Chang, C.-J.; Chawla, H. P. S.; Cochran, D. W.;
Hagaman, E. W.; King, J. C.; Orito, K. J. Am. Chem. Soc. 1976, 98,
3645-3655.
Acid Hydrolysis of Compound 7. Compound 7 (1 mg) was
heated at 95 °C with dioxane (0.5 mL) and 5% H2SO4 (0.5 mL)
for 1 h. After neutralization with Amberlite IRA-400 (OH-
form), the reaction mixture was concentrated and the residue
was passed through a Sep-Pak C18 cartridge with H2O. The
eluate was concentrated and the residue was treated with
L-cysteine methyl ester hydrochloride (1 mg) in pyridine (0.125
mL) at 60 °C for 1 h. The solution was then treated with N,O-
bis(trimethylsilyl)trifluoroacetamide (0.05 mL) at 60 °C for 1
h. The supernatant was applied to GLC; GLC conditions:
column, Supelco SPB-1, 30 m × 0.25 mm; column temperature,
230 °C; N2 flow rate, 0.8 mL/min; tR of derivatives, D-glucose
12.9 min, L-glucose 13.4 min, D-fructose 11.1 min, L-fructose
10.8 min. D-Glucose and D-fructose were detected from 7.
DNA Topoisomerase Assays. Recombinant human DNA
topoisomerase I and II (topo I and II) (2 units/µL, each) were
purchased from TopoGen, Inc. (Columbus, OH). Relaxation
activity of topo II was determined by detecting the conversion
of supercoiled plasmid DNA to its relaxed form.24 The topo II
reaction was performed in 20 µL of reaction mixture containing
50 mM Tris-HCl buffer (pH 8.0), 120 mM KCl, 10 mM MgCl2,
0.5 mM ATP, 0.5 mM dithiothreitol, supercoiled pUC19 DNA
(0.25 µg), 2 µL of inhibitor solution [10% dimethyl sulfoxide
(11) Nakamoto, K.; Otsuka, H.; Yamasaki, K. Phytochemistry 1988, 27,
1856-1858.
(12) Muhammad, I.; Dunbar, D. C.; Khan, R. A.; Ganzera, M.; Khan, I. A.
Phytochemistry 2001, 57, 781-785.
(13) Calis, I.; Sticher, O. Phytochemistry 1984, 23, 2539-2540.
(14) Achenbach, H.; Lo¨wel, M.; Waigbel, R.; Gupta, M.; Solis, P. Planta
Med. 1992, 58, 270-272.
(15) Zhang, D.; Miyase, T.; Kuroyamagi, M.; Umehara, K.; Noguchi, H.
Phytochemistry 1998, 47, 45-52.
(16) Levin, R. H.; Lallemand, J.-Y.; Roberts, J. D. J. Org. Chem. 1973,
38, 1983-1986.
(17) Batista, C. V. F.; Schripsema, J.; Verpoorte, R.; Rech, S. B.; Henriques,
A. T. Phytochemistry 1996, 41, 969-973.
(18) Danieli, B.; Lesma, G.; Palmisano, G.; Ricca, G. S. Tetrahedron 1984,
40, 5255-5261.
(19) Stoll, A.; Hofmann, A. Helv. Chim. Acta 1953, 36, 1143-1147.
(20) Hofmann, A. Helv. Chim. Acta 1954, 37, 849-865.
(21) Ikeshiro, Y.; Tomita, Y. Planta Med. 1984, 50, 485-488.
(22) Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1986, 34, 1843-
1845.
(23) Mizushina, Y.; Akihisa, T.; Ukiya, M.; Murakami, C.; Kuriyama, I.;
Xu, X.; Yoshida, H.; Sakaguchi, K. Cancer Sci. 2004, 95, 354-360.
(24) Spitzner, J. R.; Chung, I. K.; Muller, M. T. Nucleic Acids Res. 1990,
18, 1-11.
(25) Mosmann, T. J. Immunol. Methods 1983, 65, 55-63.
NP058007N