1014 Journal of Natural Products, 2010, Vol. 73, No. 5
Notes
voucher specimen is deposited at Jard´ın Bota´nico Joaquin Antonio
Uribe, Medellin, Colombia (voucher number JAUM-50622).
concentrations in the range 3.1-100 µg/mL were evaluated for each
compound dissolved in DMSO. After 72 h of incubation, 20 µL of
MTT (5 mg/mL) was added to each well. Plates were further incubated
for 4 h. The enzymatic reaction was stopped by addition of 100 µL/
well of 50% 2-propanol-10% sodium dodecyl sulfate solution. Optical
density at 570 nm was measured using an ELISA plate reader (Bio
Rad). Parasites treated with DMSO or in the absence of treatment but
maintained under the same conditions were used as controls. Ampho-
tericin B was used as a reference drug. Two independent experiments
were conducted in triplicate, and results were expressed as EC50 and
calculated by Probit analysis.
Extraction and Isolation. Powdered leaves (1.0 kg) of G. pana-
mensis were extracted successively with petroleum ether, EtOAc, and
MeOH (10 L each) in a percolator at room temperature and concentrated
in vacuo to give the corresponding extract (8, 34, and 44 g, respectively).
The ethyl acetate extract was subjected to silica gel column chroma-
tography (5 × 80 cm) eluting with a step gradient of n-hexane-ethyl
acetate (100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80,
10:90, 0:100, each 500 mL), to obtain 10 fractions (F1-F10) collected
on the basis of their TLC profiles. Fractions F4 and F5 were recognized
as the most interesting ones, due to the appearance of blue spots after
spraying with anisaldehyde reagent. Compounds 2 (30 mg) and 3 (38
mg) were isolated from F4, and compounds 1 (35 mg) and 5 (12 mg)
from F5, by preparative TLC using CH2Cl2-EtOAc (4:1), except for
compound 2, for which an n-hexane-ethyl acetate (4:1) mixture was
employed. Compound 4 appeared during purification of phebalosin (1).
7-{[(2R*)-3,3-Dimethyloxiran-2-yl]methoxy}-8-[(2R*,3R*)-3-iso-
propenyloxiran-2-yl]-2H-chromen-2-one (1): white, amorphous pow-
der; [R]D -40 (23 °C, c 0.25, CHCl3); UV (MeOH) λmax (log ε) 214
Cytotoxicity of compounds 1-4 against human U937 (CRL-1593.2)
cells and evaluation of leishmanicidal activity of compound 2 on intra-
cellular amastigotes were performed following previously reported
methods.22,23
Acknowledgment. We thank Mr. A. Cogollo for collection and
identification of plant material, Ms. D. Mun˜oz for collaboration with
bioassays, Dr. W. Quin˜ones for NMR measurements, and Dr. S. Hindle
for editorial assistance. This research was supported financially by
Universidad de Antioquia (Programa de Sostenibilidad).
1
(2.5), 251 (1.8), 322 (2.3) nm; IR (KBr) νmax 2924, 1735 cm-1; H
NMR (CDCl3, 400 MHz) δ 1.38 (3H, s, 3′-CH3), 1.40 (3H, s, 3′-CH3),
1.85 (3H, s, 1′′′-CH3), 3.18 (1H, m, H-2′), 3.92 (1H, m, H-3′′), 4.02
(1H, dd, J ) 10.9, 2.1 Hz, H-2′′), 4.17-4.34 (2H, m, -OCH2-), 5.08
(1H, s, H-2′′′), 5.29 (1H, d, J ) 7.8 Hz, H-2′′′), 6.27 (1H, d, J ) 9.8
Hz, H-3), 6.93 (1H, d, J ) 8.7 Hz, H-6), 7.40 (1H, d, J ) 8.7 Hz,
H-5), 7.61 (1H, d, J ) 9.8 Hz, H-4); 13C NMR (CDCl3, 100 MHz) δ
17.3 (1′′′-CH3), 19.1 (3′-CH3), 24.5 (3′-CH3), 51.6 (C2′′), 58.3 (C3′),
60.7 (C3′′), 61.0 (C2′), 68.5 (-OCH2-), 108.9 (C6), 113.2 (C4a), 113.3
(C8), 113.6 (C2′′′), 113.7 (C3), 128.8 (C5), 141.3 (C1′′′), 143.3 (C4),
154.2 (C8a), 160.2 (C2), 160.5 (C7); HRTOFESIMS m/z 351.1194
(calcd for C19H20O5Na, 351.1208).
7-Methoxy-8-(4-methyl-3-furyl)-2H-chromen-2-one (2): white,
amorphous powder; UV (MeOH) λmax (log ε) 208 (2.2), 260 (1.4), 325
(1.9) nm; IR (KBr) νmax 2900, 1728 cm-1; 1H NMR (CDCl3, 300 MHz)
δ 1.89 (3H, d, J ) 0.5 Hz, 4′-CH3), 3.88 (3H, s, -OCH3), 6.25 (1H, d, J
) 10.0 Hz, H-3), 6.94 (1H, d, J ) 8.5 Hz, H-6), 7.34 (1H, brs, H-5′), 7.45
(1H, d, J ) 8.5 Hz, H-5), 7.48 (1H, d, J ) 1.2 Hz, H-2′), 7.67 (1H, d, J
) 10.0 Hz, H-4); 13C NMR (CDCl3, 75 MHz) δ 8.8 (4′-CH3), 56.2
(-OCH3), 107.5 (C6), 109.9 (C8), 113.1 (C4a), 113.3 (C3), 115.7 (C3′),
121.0 (C4′), 128.0 (C5), 139.7 (C5′), 142.2 (C2′), 143.6 (C4), 152.9 (C8a),
160.3 (C7), 161.0 (C2); APCIMS m/z 257 [M + H]+ (100).
Supporting Information Available: NMR spectra of compounds
1 and 2, calculated geometries, and predicted NOESY correlations of
all diastereoisomers of compound 1, and table of antileishmanial activity
against axenic amastigote forms of L. panamensis for all compounds
and extracts tested. This material is available free of charge via the
References and Notes
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(19) Imai, F.; Kinoshita, T.; Itai, A.; Sankawa, U. Chem. Pharm. Bull. 1986,
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2-(7-Methoxy-2-oxo-2H-chromen-8-yl)-3-methylbut-2-enal, Mur-
ralongin (4). Phebalosin (3, 5.0 mg), dissolved in CH2Cl2 (0.5 mL),
was absorbed in silica gel (50 mg) and submitted to column chroma-
tography (Pasteur pipet, silica gel) using CH2Cl2 as eluent to give 4.7
mg (94%) of murralongin (4) as a white powder: UV (MeOH) λmax
(log ε) 205 (1.8), 241 (1.4), 325 (1.5) nm; IR (KBr) νmax 1733, 1656,
1
1606 cm-1; H NMR (CDCl3, 300 MHz) δ 1.78 (3H, s, -CH3), 2.42
(3H, s, -CH3), 3.86 (3H, s, -OCH3), 6.22 (1H, d, J ) 9.6 Hz, H-3),
6.90 (1H, d, J ) 8.7 Hz, H-6), 7.44 (1H, d, J ) 8.7 Hz, H-5), 7.68
(1H, d, J ) 9.6 Hz, H-4), 10.20 (1H, s, -CHO); 13C NMR (CDCl3, 75
MHz) δ 19.8 (-CH3), 24.9 (C4′), 56.2 (-OCH3), 107.6 (C6), 113.0
(C3), 113.1 (C4a), 113.2 (C8), 128.6 (C5), 129.2 (C2′), 143.6 (C4),
152.5 (C8a), 159.7 (C3′), 160.1 (C7), 161.8 (C2), 188.9 (C1′); APCIMS
m/z 259 [M + H]+ (100).
Computational Methods. Initial equilibrium geometries for all
diastereoisomers of compound 1 were calculated using an AM1
semiempirical method starting from a MMFF minimal energy con-
former. These geometries were used as a starting point for refinement
using a RB3LYP/6-311G(d) calculation. Single-point energy and NMR
(NOESY) spectra were obtained using the same RB3LYP/6-311G(d)
method. All calculations were performed within Spartan’08 (Spartan’08
Wavefunction, Inc., Irvine, CA) using default settings. See details of
results in the Supporting Information).
Bioassays. To estimate the 50% effective concentrations (EC50) of
compounds 1-4 in axenic amastigote forms of L. panamensis (M/
HOM//87/UA140 epirGFP) strain, the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) enzymatic micromethod was
used.22 Briefly, axenic amastigotes (1 × 106 parasites/mL) were cultured
at 32 °C with test compounds in Schneider’s Drosophila medium (pH
5.2, Sigma) containing 20% heat-inactivated fetal calf serum. Six
(23) Varela, M. R. E.; Mun˜os, D. L.; Robledo, S. M.; Kolli, B. K; Dutta,
S.; Chang, K. P.; Muskus, C. Exp. Parasitol. 2009, 122, 134–139.
NP100146Y