Phytotoxins from Hofmeisteria schaffneri: Isolation and synthesis of 2′-(2″-hydroxy-4″-methylphenyl)-2′-oxoethyl acetate

Article abstract of DOI:10.1021/np0501278

Activity-directed fractionation of a CH₂Cl₂-MeOH (1:1) extract of Hofmeisteria schaffneri led to the isolation of a new phytotoxin characterized as 2′-(2″-hydroxy-4″-methylphenyl)- 2′-oxoethyl acetate and designated the trivial name of hofmeisterin (1). In addition, the known compounds β-carotene, euparin, and 3′,4′,4a′,9a′-tetrahydro-6,7′- dimethylspiro[benzofuran-3(2H),2′-pyrano[2,3-b]benzofuran]-2, 4a′-diol (2) were obtained. The identification of the isolates was accomplished by spectroscopic methods. The structure of 1 was unequivocally confirmed by synthesis. The methyl derivative 1a was also synthesized following the same strategy. Compounds 1 and 2 inhibited radicle growth of Amaranthus hypochondriacus (IC₅₀ = 3.2 × 10^-4 and 1.2 × 10^-5 M, respectively) and significantly inhibited activation of the calmodulin (CaM)-dependent enzyme cAMP phosphodiesterase (PDE) with IC ₅₀ values of 4.4 and 4.22 μM, respectively.

Full text of DOI:10.1021/np0501278

J. Nat. Prod. 2005, 68, 959-962
959
Phytotoxins from Hofmeisteria schaffneri: Isolation and Synthesis of
2′-(2′′-Hydroxy-4′′-methylphenyl)-2′-oxoethyl Acetate1
Araceli Pe´rez-Va´squez,† Adelfo Reyes, Edelmira Linares,§ Robert Bye,§ and Rachel Mata*,†
Departamento de Farmacia, Facultad de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Me´xico DF 04510, Me´xico,
Jardı´n Bota´nico, Instituto de Biologı´a, Universidad Nacional Auto´noma de Me´xico, Me´xico DF 04510, Me´xico, and Facultad de
Estudios Superiores Zaragoza, Batalla del 5 de Mayo Esquina Fuerte de Loreto, Ejercito de Oriente, Me´xico DF 09230, Me´xico
Received April 13, 2005
Activity-directed fractionation of a CH2Cl2-MeOH (1:1) extract of Hofmeisteria schaffneri led to the
isolation of a new phytotoxin characterized as 2′-(2′′-hydroxy-4′′-methylphenyl)-2′-oxoethyl acetate and
designated the trivial name of hofmeisterin (1). In addition, the known compounds â-carotene, euparin,
and 3′,4′,4a′,9a′-tetrahydro-6,7′-dimethylspiro[benzofuran-3(2H),2′-pyrano[2,3-b]benzofuran]-2,4a′-diol (2)
were obtained. The identification of the isolates was accomplished by spectroscopic methods. The structure
of 1 was unequivocally confirmed by synthesis. The methyl derivative 1a was also synthesized following
the same strategy. Compounds 1 and 2 inhibited radicle growth of Amaranthus hypochondriacus (IC50
)
3.2 × 10-4 and 1.2 × 10-5 M, respectively) and significantly inhibited activation of the calmodulin (CaM)-
dependent enzyme cAMP phosphodiesterase (PDE) with IC50 values of 4.4 and 4.22 µM, respectively.
As part of our systematic search of potential herbicidal
agents from Mexican biodiversity,2-4 we have selected
Hofmeisteria schaffneri (A. Gray) R. M. King & H. Robinson
(Asteraceae) for bioassay-guided fractionation on the basis
of its significant phytotoxic activity against Amaranthus
hypochondriacus and Echinochloa crus-galli. H. schaffneri,
a rare perennial medicinal herb known as “ambar”, grows
naturally in the oak and pine-oak forested mountains of
the central Mexican states of Jalisco and San Luis Potosi
and is cultivated in home gardens in the valley of Mexico.
The decoction of the fresh, leafy stems and flowers is used
for treating skin wounds, fevers, and gastrointestinal
ailments.5 Herein, we describe the isolation, structure
elucidation, and biological activity of the major phytotoxins
from H. shaffneri including their effect on the regulatory
protein calmodulin (CaM) as a possible target of phytotoxic
action. The synthesis of 2′-(2′′-hydroxy-4′′-methylphenyl)-
2′-oxoethyl acetate (1), a novel phytotoxin isolated during
the course of the present investigation, is also reported.
A crude CH2Cl2-MeOH (1:1) extract prepared from the
plant H. schaffneri showed an inhibitory effect on radicle
growth of Amaranthus hypochondriacus (IC50 ) 52.5 µg/
mL) and was accordingly subjected to bioassay-guided
fractionation. Extensive chromatography of the active
extract resulted in the isolation of 2′-(2′′-hydroxy-4′′-
methylphenyl)-2′-oxoethyl acetate (1), a novel phytotoxin
that was given the trivial name of hofmeisterin. The known
compounds euparin,6 â-carotene,7,8 and 3′,4′,4a′,9a′-tet-
rahydro-6,7′-dimethylspiro[benzofuran-3(2H),2′-pyrano[2,3-
b]benzofuran]-2,4a′-diol (2)9 were also isolated. The prop-
erties of the known compounds, including IR, 1H NMR, and
13C NMR data, were identical to those previously reported
in the literature.
ring; a methyl group attached to an aromatic ring; and an
acetyl moiety, a chelated hydroxyl, a conjugated ketone,
and a methylene group attached to an oxygenated func-
tionality. Disposition of substituents on the aromatic ring
was determined by analysis of the HMBC and NOESY
spectra. Thus, the HMBC correlations of the methyl group
at δC 22 (Ar-CH3) with H-3′′ and H-5′′ as well as that of
the ketone-carbonyl carbon (C-2′) with H-6′′ were consistent
with the placement of the methyl and ketone groups at C-4′′
and C-1′, respectively. Since the hydroxyl group was
chelated, it was readily placed at C-2′′, i.e., ortho to the
ketone functionality. Furthermore, HMBC correlations of
C-1 and C-2′ with H-1′ were in agreement with the acetoxyl
and ketone carbonyls being attached to the C-1′ methylene.
The NOESY interactions OH/H-3′′, H-3′′/Ar-CH3, H-5′′/Ar-
CH3, H-1′/H-2, and H-6′′/H-1′ provided additional support
for these proposals.
To verify the structure of 1 and to obtain additional
amounts for biological studies, its synthesis was under-
taken. The synthetic path to phytotoxin 1 is outlined in
Scheme 1. The general strategy was based on a Fries
rearrangement of a suitable acetyl derivative of m-cresol
(3).10,11 The synthesis started from m-cresol, which was
treated with Ac2O to furnish 3,12 which was subjected to
Compound 1 was assigned the molecular formula
C11H12O4 (HRMS). The IR spectrum showed bands for
phenolic (3500 cm-1), ester (1747 cm-1), and conjugated
ketone (1657 cm-1) functionalities. The NMR spectra were
consistent with the presence of a trisubstituted benzene
* To whom correspondence should be addressed. Tel: (525)-55-622-5289.
Fax: (525)-55-622-5329. E-mail: rachel@servidor.unam.mx.
† Departamento de Farmacia, Universidad Nacional Auto´noma de Me´xico.
§ Jard´ın Bota´nico, Universidad Nacional Auto´noma de Me´xico.
FES-Zaragoza.
10.1021/np0501278 CCC: $30.25
© 2005 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/09/2005

960 Journal of Natural Products, 2005, Vol. 68, No. 6
Notes
Scheme 1a
a Conditions: (i) Ac2O; (ii) AlCl3,CS2,180 °C; (iii) NaOH, (CH3)2 SO4, ice-water bath; (iv) benzyl bromide, K2CO3, acetone reflux, 5 days; (v) Br2, benzene,
rt, 4 h; (vi) AcOH, DBU, THF, rt, 2.5 h; (vii) H2, 10% Pd-C, 60 psi, 60 °C, 5 h.
Fries rearrangement at 180 °C to generate 2-hydroxy-4-
methylacetophenone (4).11 The free hydroxyl group of 4 was
protected with benzyl bromide to yield 2-benzyloxy-4-
methylacetophenone (6),13 which upon R-bromination with
Br2 rendered 2-benzyloxy-ω-bromo-4-methylacetophenone
(8); the protection of the phenolic group facilitated the
R-ketone halogenation reaction. The displacement of bro-
mide from 8 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)-
CH3COOH produced ω-acetoxy-2-methoxy-4-methylace-
tophenone (9, 17% of yield), which was catalytically
hydrogenated with palladium-charcoal (10% Pd-C) to
afford 1 with an overall yield of 11.8%. The methyl
derivative 1a was also synthesized (Scheme 1) using the
same approach; in this case the intermediate 4 was
subjected to methylation with (Me)2SO4 to yield 2-methoxy-
4-methylacetophenone (5),14 which upon treatment with
Br2 rendered 2-methoxy-ω-bromo-4-methylacetophenone
(7);15 finally, treatment of 7 with acetic acid/DBU afforded
1a (15% of yield). The structures of all the synthetic
compounds were elucidated mainly on the basis of their
1H and 13C NMR spectra as well as MS data.
The phytotoxic effects of 1 and 2 were determined using
a Petri dish bioassay.2,3 Both natural products inhibited
radical growth of Amaranthus hypochondriacus (IC50 ) 3.8
× 10-4 and 1.2 × 10-5 M, respectively). The dimer was 10-
fold more active than the northymol derivative 1. Echin-
ocloa crus-galli was also sensitive to the treatments with
compound 1. Among the synthetic compounds only the
methyl derivative 5 interfered (IC50 ) 3.2 × 10-4 M) with
the growth of seedlings of Amaranthus. However, none of
the tested compounds affected significantly radical growth
of Medicago sativa.
agents that inhibit the activity of CaM should have an
effect on the development of the plant as previously
described.3,4,17 To study if the phytotoxins affected the
enzyme regulatory properties of CaM in vitro, their effect
on the activity of PDE1 was investigated. PDE1 catalyzes
the hydrolysis of cyclic nucleotides to nucleotide mono-
phosphates.3,4 Since PDE1 is widely used as a tool to
discover CaM inhibitors, and in general to demonstrate the
activity of CaM in biochemical studies, we assessed the
effect of 1 and 2 on this enzyme. The PDE reaction was
coupled to the 5′-nucleotidase reaction, and the amount of
inorganic phosphate released represented the activity of
PDE1. Bovine-brain CaM was used as an activator of the
enzyme.3,4 Natural products 1 and 2 inhibited activation
of PDE1 in the presence of CaM with IC50 values of 4.4 (
0.46 and 4.2 ( 0.5 µM, respectively. Their effect was
comparable to chlorpromazine (IC50 ) 6.8 ( 1.9 µM), a well-
known CaM inhibitor used as positive control.
Thymol derivatives have been described previously from
other Asteraceae such as Eupatorium fortunei Turcz.18 and
Arnica sachalinensis (Regl.) A. Gray.9 However, northymol
derivatives as 1 are uncommon.
Experimental Section
General Experimental Procedures. Melting point de-
terminations were determined using a Fisher-Johns apparatus
and are uncorrected. Optical rotations were recorded on a
JASCO DIP 360 digital polarimeter. IR spectra were obtained
using KBr disks on a Perkin-Elmer 599B spectrophotometer.
UV spectra were recorded on a Shimadzu 160 UV spectrometer
in MeOH solution. NMR spectra including COSY, NOESY,
HMBC, and HMQC experiments were recorded in CDCl3 on a
Varian Unity Plus 500 spectrometer or on a Bruker DMX500
spectrometer at 500 MHz (1H) or 125 MHz (13C) NMR, using
tetramethylsilane (TMS) as an internal standard. MS were
obtained on a JEOL JMS-AX505HA mass spectrometer.
Column chromatography: silica gel 60 (70-230 mesh, Merck).
TLC: Si gel 60 F254 (Merck). Solvents and chemicals were of
analytical grade and purchased from Aldrich Company.
Plant Material. The whole plant of H. schaffneri was
collected from cultivated plants near Ozumba, State of Mexico.
The inhibitory effects of 1 and 2 on CaM were also
performed to check the relationship between phytotocity
and CaM inhibition. This target was selected considering
that CaM is a major Ca2+-binding protein that influences
a number of important plant growth events through the
activation of CaM-dependent enzymes, such as cAMP
phosphodiesterase (PDE-1), NAD-kinase, protein phos-
phatase, and nitric oxide (NO) synthase.16 Therefore,

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).

962 Journal of Natural Products, 2005, Vol. 68, No. 6
Notes
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M. C. J. Agric. Food Chem. 2003, 51, 4559-4562.
(5) King, R. M. Rhodora 1967, 69, 352-371.
CaM-PDE1 Assay. The CaM-PDE1 assay was performed
in a 96-well plate as previously described;4 the assay is based
on the quantification of the amount of inorganic phosphate
released from the hydrolysis of AMP by the action of a
5′-nucleotidase from Crotalus atrox venom (Sigma). The AMP
is generated from cAMP by the action of PDE1. Chlorprom-
azine was used as a positive control.
(6) Castan˜eda, P.; Go´mez, L.; Mata; R.; Lotina Hennsen, B.; Anaya, A.
L.; Bye, R. J. Nat. Prod. 1996, 56, 323-326.
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M. H. Proc. Natl. Acad. Sci. 1997, 94, 10600-10605.
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1999, 55, 2997-3006.
Acknowledgment. This work was supported by grants
from CONACyT and DGAPA-UNAM. We thank R. del Villar,
M. Gutierrez, and G. Duarte-Lisci for recording IR, UV, NMR,
and mass spectra. Special thanks are due to M. Macias, P.
Lozano, M. Trejo, F. Basurto, I. Rivero, and L. Acevedo for
their valuable technical assistance. A.R. acknowledges the
fellowship granted by DGAPA-UNAM for a sabbatical stay at
Facultad de Qu´ımica, UNAM. A.P. acknowledges the scholar-
ship awarded by CONACyT to carry out graduate studies.
(10) Voguel, A. I. Practical Organic Chemistry; Longmans: London; 1966;
p 1188.
(11) Paul, S.; Gupta, M. Synthesis 2004, 1789-1792.
(12) Bartoli, G.; Bosco, M.; Marcantoni, E.; Massacesi, M.; Rinaldi, S.;
Sambri, L. Tetrahedron Lett. 2001, 57, 791-804.
(13) Litkei, G.; Patonay, T.; Bognar, R.; Khilja, V.; Aitmambetov, A.; Turov,
A.; Babichev, F. Pharmazie 1984, 39, 741-744.
(14) Smith, K.; El-Hiti, G. A.; Jayne, A. J.; Butters, M. Org. Biomol. Chem.
2003, 1, 1560-1564.
(15) Ishikawa, H.; Ihara, S.; Masumizu, T.; Hirayama, K.; Kajikawa, K.
Jpn. Kokai Tokki Koho. JP 04297433 (Nov. 21, 1992).
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Supporting Information Available: This material is available
free of charge via the Internet at http://pubs.acs.org.
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References and Notes
(1) This work represents part of the Ph.D. thesis of A. Pe´rez-Va´squez.
(2) Valencia-Islas, N.; Paul, R. N.; Shier, W. T.; Mata, R.; Abbas, H.
Phytochemistry 2002, 61, 141-148.
(3) Mata, R.; Rivero-Cruz, I.; Macias, M.; Pe´rez, O.; Bye, R.; Linares, E.;
Timmermann, B. N. Phytochemistry 2003, 64, 285-291.
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