Notes
Journal of Natural Products, 2005, Vol. 68, No. 6 961
A voucher specimen (Bye and Linares 31018) has been
deposited in the National Herbarium (MEXU), Instituto de
Biolog´ıa, UNAM.
ω-Acetoxy-2-methoxy-4-methylacetophenone. (1a) In
a 50 mL round-bottomed flask was mixed glacial acetic acid
(0.20 mL, 4.3 mmol) in dry THF (7 mL) at room temperature.
To this solution was added DBU (0.65 mL, 4.3 mmol) under
vigorous stirring. After 10 min, ω-bromoketone 7 (0.2 g, 2.88
mmol) dissolved in dry THF (2 mL) was added to the mixture,
which was then stirred for 2.5 h.21 After removal of the solvent
in vacuo, the residue was dissolved in CH2Cl2 (20 mL) and
washed consecutively with saturated Na2CO3 solution (2 × 10
mL) and water (2 × 10 mL). The organic fraction was dried
over MgSO4, and the solvent was removed in vacuo to give a
crude product that was purified by open column chromatog-
raphy [silica gel, hexane-EtOAc (8:2)]. The desired product
1a (0.48 g, 76% yield) was obtained as a colorless oil: IR (film)
Extraction and Isolation. The air-dried plant material
(1.6 kg) was ground into a powder and extracted by maceration
with CH2Cl2-MeOH (1:1) at room temperature. After filtra-
tion, the extract was evaporated under reduced pressure to
yield 350 g of a green residue, which was subjected to column
chromatography over silica gel (818 g) and eluted with a
gradient of hexane-CH2Cl2 (50:50 f 0:100) and CH2Cl2-
MeOH (99:1 f 50:50). Fractions of 1 L each were collected
and pooled based on TLC profiles to yield 12 major fractions
(F1-F12). According, to the Petri dish bioassay fractions F3-
F6 concentrated the phytotoxic activity (IC50 ) 63.09, 52.78,
45.39, and 47.21 µg/mL, respectively). From inactive primary
fraction F2 (5 g, eluted with hexane-CH2Cl2, 2:3) 49 mg of
â-carotene spontaneously crystallized. From primary active
fraction F3 (4 g, eluted with hexane-CH2Cl2, 3:7) 456 mg of
euparine spontaneously crystallized. From active fraction F4
(16.7 g, eluted with hexane-CH2Cl2, 1:4) precipitated 147 mg
of â-sitosterol. The mother liquors of F4 (16 g) were further
chromatographed on a silica gel (269 g) column, eluting with
a gradient of hexane-CH2Cl2 (10:0 f 1:9) and CH2Cl2-MeOH
(10:1 f 5:5) to afford eight secondary fractions (F4-1-F4-8).
According to the bioautographic assay the strongest activity
was in secondary fractions, namely, F4-4 and F4-5. Fraction
F4-4 (2.5 g, eluted with hexane-CH2Cl2, 7:3) was further
resolved on a silica gel (80 g) column, eluting with mixtures
of hexane-CH2Cl2 (10:0 f 0:10) and CH2Cl2-MeOH (10:0 f
0:10) to give 11 tertiary fractions (F4-4-1-F4-4-11). The only
active tertiary fraction was F4-4-8 [342 mg, eluted with CH2-
Cl2]. The latter fraction was further purified by column
chromatography on silica gel (30 g), eluting with hexane-CH2-
Cl2, 1:1, to afford 8 mg of compound 1. Column chromatogra-
phy of the active fraction F4-5 (1.9 g, eluted with hexane-
CH2Cl2, 6:4) on silica gel (55 g) using CH2Cl2 as mobile phase
yielded 72 mg of compound 2.
2′-(2′′-Hydroxy-4′′-methylphenyl)-2′-oxoethyl acetate
(1): colorless crystalline needles, mp 87 °C; UV (CH2Cl2) λmax
(log ꢀ) 328 (3.78), 263 (3.30) nm; IR νmax (KBr) 3500, 2920, 1747,
1657, 1508, 1276, 1228, 1205, 1076, 998 cm-1; 1H NMR (CDCl3)
δ 2.24 (3H, s, H-2), 2.36 (3H, s, Ar-CH3), 5.33 (2H, s, H-1′),
6.73 (1H, ddq, J ) 8.1, 1.5, 0.6 Hz, H-5′′), 6.83 (1H, dq, J )
1.5, 0.6 Hz, H-3′′), 7.49 (1H, d, J ) 8.4 Hz, H-6′′), 11.6 (1H, s,
OH); 13C NMR (CDCl3) δ 20 (C-2), 22 (Ar-CH3), 64 (C-1′), 119
(C-3′′), 115 (C-4′′), 121 (C-5′′), 128 (C-6′′), 149 (C-1′′), 162
(C-2′′), 170 (C-1), 196 (C-2′); EIMS m/z 208 [M]+ (33), 166
(48), 149 (18), 135 (100), 119 (6), 107 (21), 91 (3), 77 (21), 53-
(5), 43 (15); HREIMS m/z 208.2149 (calcd for C11H12O4,
208.2159).
3-Methylphenyl Acetate (3), 2-Hydroxy-4-methyl-
acetophenone (4), 2-Methoxy-4-methylacetophenone (5),
2-Benzyloxy-4-methylacetophenone (6), and 2-Methoxy-
ω-bromo-4-methylacetophenone (7). Compounds 3-7 were
prepared as previously described10-15,19,20 (see Supporting
Information).
1
νmax 1735, 1678, 1605, 1404, 1236, 1172, 815 cm-1; H NMR
(CDCl3) δ 2.21 (3H, s, H-2), 2.38 (3H, s, Ar-CH3), 3.93 (3H, s,
-OCH3), 5.22 (2H, s, H-1′), 6.78 (1H, br d, J ) 8.1 Hz, H-5′′),
6.85 (1H, br d, J ) 1.2 Hz, H-3′′), 7.85 (1H, d, J ) 8.4 Hz,
H-6′′); 13C NMR (CDCl3) δ 20 (C-2), 22 (Ar-CH3), 55 (-OCH3),
70 (C-1′), 112 (C-3′′), 113 (C-4′′), 122 (C-5′′), 131 (C-6′′), 146
(C-1′′), 160 (C-2′′), 171 (C-1), 192 (C-2′); EIMS m/z 222 [M]+
(15), 207 (19), 197 (2), 195 (1), 161(4), 135 (4), 134 (5), 91
(100).
ω-Acetoxy-2-Benzyloxy-4-methylacetophenone. (9) In
a 50 mL round-bottomed flask was mixed glacial acetic acid
(0.40 mL, 7.03 mmol) in dry THF (10 mL) at room tempera-
ture. To this solution was added DBU (1.05 mL, 7.03 mmol)
under vigorous stirring. After 10 min, ω-bromoketone 8 (1.5
g, 4.68 mmol) dissolved in dry THF (2 mL) was added to the
mixture, which was then stirred for 2.5 h. After removal of
the solvent in vacuo, the residue was dissolved in CH2Cl2 (20
mL) and washed consecutively with saturated Na2CO3 solution
(2 × 10 mL) and water (2 × 10 mL).21 The organic fraction
was dried over MgSO4, and the solvent was removed in vacuo
to give a crude product that was purified by open column
chromatography [silica gel, hexane-EtOAc (8:2)]. The desired
product 1a (1.15 g, 82.45% yield) was obtained as a white
solid: mp 73 °C; λmax (log ꢀ) 253 (4.14), 308 (3.7) nm; IR (KBr)
1
νmax 1735, 1678, 1605, 1404, 1236, 1172, 815 cm-1; H NMR
(CDCl3) δ 2.17 (3H, s, H-2), 2.39 (3H, s, Ar-CH3), 5.15 (2H, s,
CH2), 5.16 (2H, s, CH2), 6.86 (1H, br d, J ) 1.2 Hz, H-5′′), 6.82
(1H, br d, J ) 7.8 Hz, H-3′′), 7.47-7.26 (5H, m, H-2′′′-H-6′′′),
7.87 (1H, d, J ) 8.1 Hz, H-6′′); 13C NMR (CDCl3) δ 20.6 (-CH3),
21. 9 (-CH3), 70.1 (CH2), 70.87 (CH2), 113.2 (C-5′′), 122.2 (C-
4′′), 122.3 (C-3′′), 127.9, 128.5, 128.9 (C-2′′′-C-6′′′), 131.2 (C-
6′′), 135.7 (C-1′′′), 146.2 (C-1′′), 158.8 (C-2′′), 170.5 (-CO), 192.2
(-CO); EIMS m/z 298 [M]+ (2), 256 (2), 238 (30), 225 (92), 221
(2), 197 (2), 195 (1), 161 (4), 135 (4), 134 (5), 91 (100), 65 (5),
43 (5), 39 (2).
2′-(2′′-Hydroxy-4′′-methylphenyl)-2′-oxoethyl acetate
(1). ω-Acetoxyacetophenone (9) (1.0 g, 3.3 mmol) in EtOAc (100
mL) and Pd/C (10%, 0.1 g) were placed in a hydrogenation flask
under a stream of anhydrous N2 gas; the flask was then
pressurized with H2 (60 bar) and shaken at 60 °C for 5 h (TLC
control).15 When the reaction was over, the catalyst was
removed by filtration over Celite and the solvent evaporated
under reduced pressure to yield 1, which was purified by open
column chromatography [silica gel, hexane-EtOAc (8:2)] to
yield 564 mg (57%). The physical and spectroscopy data were
identical to those of the natural product.
2-Benzyloxy-ω-bromo-4-methylacetophenone (8). Com-
pound 6 (2.0 g, 8.33 mmol) and anhydrous benzene (15 mL)
were mixed; then the system was cooled to 0 °C. To this
solution was added dropwise, under vigorous stirring, bromine
(0.43 mL, 8.33 mmol) dissolved in benzene (10 mL).20 The
reaction mixture was stirred at room temperature for 4 h and
then evaporated to dryness to give a solid that was purified
by open column chromatography [silica gel, hexane-EtOAc
(97:3)]. Compound 8 (0.98 g 40% yield) was obtained as a white
solid: mp 84 °C; IR (KBr) νmax 1676, 1601, 1494, 1418, 1267,
Phytogrowth-Inhibitory Bioassays. The phytogrowth-
inhibitory activity of the crude extract, fractions, and pure
compounds was evaluated on seeds of Amaranthus hypochon-
driacus, Echinochloa crus-galli, and Medicago sativa using a
Petri dish bioassay.2,3 In addition, a bioautographic phyto-
growth-inhibitory bioassay2,3 was employed to guide secondary
fractionation. The seeds of the weeds were purchased from
Mercado de Tulyehualco, Mexico City, Mexico. The results
were analyzed by ANOVA (p < 0.05), and IC50 values were
calculated by probit analysis based on percent of radicle
1
1180, 1119, 991 cm-1; H NMR (CDCl3) δ 2.40 (3H, s, CH3),
4.51 (2H, s, -CH2Br), 5.17 (2H, s, Ar-CH2O), 6.86 (1H, br d,
H-3′), 6.87 (1H, br d, J ) 7.2 Hz, H-1′), 7.45-7.39 (5H, m,
H-2′′-H-6′′), 7.76 (1H, d, J ) 8.4 Hz, H-6′); 13C NMR (CDCl3)
δ 22 (-CH3), 37.6 (-CH2Br), 70.9 (ArCH2O), 113. 3 (C-5′), 122.3
(C-4′), 122.5 (C-3′), 127.8, 128.5, 128.8 (C-2′′-C-6′′), 131.8 (C-
6′), 135.7 (C-1′′), 146.0 (C-1′), 158.0 (C-2′), 193 (-CO); FABMS
m/z 319, 321 [M + H]+ (10, 8), 239 (15), 149 (40), 91 (100), 69
(34), 43 (44).
growth. Samples were evaluated at 10, 100, and 1000 µg mL-1
.
The bioassays were performed at 28 °C. 2,4-D was used as
positive control (IC50 values for A. hypochondriacus, E. crus-
galli, and M. sativa were 4 × 10-5, 2 × 10-4, and 0.3 × 10-5
M, respectively).