Phytotoxic compounds from Prionosciadium watsoni

Article abstract of DOI:10.1021/np010448t

Bioassay-guided fractionation of a phytotoxic extract of Prionosciadium watsoni led to the isolation of three new pyranocoumarins and two pyranochromones. The new compounds were characterized as propionic acid (9R,10R)-9-acetoxy-8,8-dimethyl-9-10-dihydro-2H,8H-benzo[1,2-b:3,4-b ′]dipyran-2-one-10-yl ester (1), isobutyric acid (9R,10R)-9-hydroxy-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4. b′]-dipyran-2-one-10-yl ester (2), isobutyric acid (9R)-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b′]dipyran-2 -one-9-yl ester (10), 2-methylbut-(2Z)-enoic acid (3R)-5-methoxy-3,4-dihydro-2,2,8-trimethyl-6-oxo-2H, 6H-benzo[1,2.b:5,4-b′]dipyran-3-yl ester (11), and isobutyric acid (3R)-5-methoxy-3,4-dihydro-2,2,8-trimethyl-6-oxo-2H,6H-benzo[1,2-b:5, 4-b′]dipyran-3-yl ester (12) by spectroscopic and chemical methods. The stereochemistry at the stereogenic centers was established by applying the Mosher ester methodology. The structures of 1 and 2 were corroborated by single-crystal X-ray diffraction studies. The phytotoxic activity of the isolated compounds was assessed on Amaranthus hypochondriacus, Echinochloa crus-galli, and Lemna pausicostata. The phytotoxins also modified the electrophoretic mobility of calmodulin from both bovine-brain and spinach.

Full text of DOI:10.1021/np010448t

828
J . Nat. Prod. 2002, 65, 828-834
P h ytotoxic Com p ou n d s fr om P r ion oscia d iu m wa tson i1
Norma Valencia-Islas,† Hamed Abbas,‡ Robert Bye, Rube´n Toscano,§ and Rachel Mata*,†
Departamento de Farmacia, Facultad de Quı´mica, Universidad Nacional Auto´noma de Me´xico, Me´xico, D.F. 04510, Me´xico,
Crop Genetics & Production Research Unit, USDA/ ARS, Stoneville, Mississippi 38776, J ard´ın Bota´nico, Instituto de Biologı´a,
Universidad Nacional Auto´noma de Me´xico, Me´xico, D.F. 04510, Me´xico, and Laboratorio de Rayos X, Instituto de Qu´ımica,
Universidad Nacional Auto´noma de Me´xico, Me´xico, D.F. 04510, Me´xico
Received September 28, 2001
Bioassay-guided fractionation of a phytotoxic extract of Prionosciadium watsoni led to the isolation of
three new pyranocoumarins and two pyranochromones. The new compounds were characterized as
propionic acid (9R,10R)-9-acetoxy-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b′]dipyran-2-one-10-
yl ester (1), isobutyric acid (9R,10R)-9-hydroxy-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b′]-
dipyran-2-one-10-yl ester (2), isobutyric acid (9R)-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-
b′]dipyran-2-one-9-yl ester (10), 2-methylbut-(2Z)-enoic acid (3R)-5-methoxy-3,4-dihydro-2,2,8-trimethyl-
6-oxo-2H,6H-benzo[1,2-b:5,4-b′]dipyran-3-yl ester (11), and isobutyric acid (3R)- 5-methoxy-3,4-dihydro-
2,2,8-trimethyl-6-oxo-2H,6H-benzo[1,2-b:5,4-b′]dipyran-3-yl ester (12) by spectroscopic and chemical
methods. The stereochemistry at the stereogenic centers was established by applying the Mosher ester
methodology. The structures of 1 and 2 were corroborated by single-crystal X-ray diffraction studies.
The phytotoxic activity of the isolated compounds was assessed on Amaranthus hypochondriacus,
Echinochloa crus-galli, and Lemna pausicostata. The phytotoxins also modified the electrophoretic mobility
of calmodulin from both bovine-brain and spinach.
Prionosciadium watsoni Coulter & Rose ex S.Watson
(Umbelliferae), commonly known as “flor de ocotillo” and
“hierba del oso”, is a medicinal plant found in moist
habitats of the pine-oak forest from Chihuahua to Hidalgo,
Mexico. A tea prepared from the roots of this species is
drunk to alleviate grastrointestinal pains, while the fruits
are employed to treat diabetes, fever, and other diseases.2
During the course of our search for potential herbicidal
agents from Mexican plants, P. watsoni was selected for
activity-guided fractionation on the basis of its significant
phytotoxicity against Amaranthus hypochondriacus L.,
Echinochloa crus-galli (L.) Beuv., and Lemna pausicostata
L. (duckweed). We report herein the isolation and structure
elucidation of the major phytotoxins from P. watsoni.
Resu lts a n d Discu ssion
A CH2Cl2-MeOH (1:1) extract of the aerial parts of P.
watsoni provoked notable inhibition of radicle elongation
of seedlings of A. hypochondriacus and E. crus-galli (Table
1) when tested by the Petri dish germination and radicle
elongation bioassay.3 In addition, after 72 h in duckweed
culture4 the extract caused growth inhibition, chlorophyll
reduction, and electrolytic leakage (Table 2). Bioassay-
guided fractionation of the active extract, using the Petri
dish test to monitor phytotoxicity, led to the isolation of
phytotoxins 1-12. Compounds 1, 2, 10, 11, and 12 are
novel natural products, and their structures were deter-
mined by means of spectroscopic, spectrometric, and chemi-
cal methods as propionic acid (9R,10R)-9-acetoxy-8,8-
dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b′]dipyran-2-
one-10-yl ester (1), isobutyric acid (9R,10R)-9-hydroxy-8,8-
dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b′]dipyran-2-
one-10-yl ester (2), isobutyric acid (9R)-8,8-dimethyl-9,10-
dihydro-2H,8H-benzo[1,2-b:3,4-b′]dipyran-2-one-9-yl ester
(10), 2-methylbut-(2Z)-enoic acid (3R)-5-methoxy-3,4-dihy-
dro-2,2,8-trimethyl-6-oxo-2H,6H-benzo[1,2-b:5,4-b′]dipyran-
3-yl ester (11), and isobutyric acid (3R)-5-methoxy-3,4-di-
hydro-2,2,8-trimethyl-6-oxo-2H,6H-benzo[1,2-b:5,4-b′]di-
pyran-3-yl ester (12). In addition, the known compounds
quianhucoumarin A
(3),5 2-methylbut-(2Z)-enoic acid
* To whom correspondence should be addressed. Tel: (525) 622-5289.
Fax: (525) 622-5329. E-mail: rachel@servidor.unam.mx.
† Departamento de de Farmacia, Universidad Nacional Auto´noma de
Me´xico.
J ard´ın Bota´nico, Universidad Nacional Auto´noma de Me´xico.
‡ Crop Genetics & Production Research Unit, USDA/ARS.
§ Laboratorio de Rayos X, Universidad Nacional Auto´noma de Me´xico.
10.1021/np010448t CCC: $22.00
© 2002 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/11/2002

Phytotoxic Compounds from Prionosciadium
J ournal of Natural Products, 2002, Vol. 65, No. 6 829
Ta ble 1. Phytogrowth-Inhibitory Activity of the
CH2Cl2-MeOH (1:1) Extract, Isolated Compounds, and
Chemical Derivatives from P. watsoni on Radicle Elongation
(IC50, M) of A. hypochondriacus and E. crus-galli
Ta ble 2. Phytotoxicity of the Extract and Compounds 9 and 10
in Lemna pausicostata Culturesa
conductivity
leakage
(µΩ/cm)
growth
inhibition
(%)
chlorophyll
reduction (%)
sample
concn
treatment
A. hypochondriacus
E. crus-galli
extract 25 µg mL-1
50 µg mL-1
-57.00 ( 14.8
91.50 ( 39.9
3.01 ( 2.9
7.79 ( 3.8
0.00 ( 12.7
0.00 ( 0.0
extracta
1
2
4
6
7
0.68
2.12
3.96 × 10-4
1.89 × 10-4
2.62 × 10-4
4.02 × 10-4
NDc
4.39 × 10-4
5.95 × 10-4
1.55 × 10-4
1.62 × 10-4
NDc
100µg mL-1 262.50 ( 12.1 100.00 ( 0.0
36.74 ( 11.7
91.51 ( 6.5
5.94 ( 4.9
200 µg mL-1 262.50 ( 24.7 100.00 ( 0.0
9
12 µM
25 µM
50 µM
100 µM
25 µM
50 µM
100 µM
200 µM
-77.00 ( 42.4
-30.50 ( 31.1
59.50 ( 7.1
100.00 ( 9.2
124.20 ( 21.1
100.00 ( 1.4
86.00 ( 2.1
15.33 ( 11.4
37.51 ( 14.2
47.59 ( 5.7
100.00 ( 0.0
19.59 ( 8.3
23.52 ( 13.8 54.30 ( 2.1
58.81 ( 8.3
4.37 ( 4.3
12.82 ( 10.8
40.32 ( 3.3
20.00 ( 16.8
8
9
3.61 × 104
2.60 × 10-4
2.37 × 10-4
1.43 × 10-4
7.41 × 10-5
3.25 × 10-4
1.08 × 10-4
3.44 × 10-4
3.83 × 10-4
1.80 × 10-4
4.39 × 10-4
8.63 × 10-4
1.69 × 10-4
2.64 × 10-5
1.48 × 10-5
6.19 × 10-6
8.08 × 10-4
2.96 × 10-4
4.61 × 10-4
>2.20 × 10-4
10
10
11
12
1a
1b
9a
11a
2,4-Db
84.92 ( 4.0
87.38 ( 0.0
260.00 ( 13.4 100.00 ( 0.0
a
Results are the means of three replicates ( standard deviation.
The compounds exhibited significantly (Student’s unpaired t-test)
higher phytotoxicity than the control (culture medium; conductiv-
ity leakage, 0.00 ( 26.2 µΩ/cm; growth inhibition, 0.00 ( 2.7%;
chlorophyll reduction, 0.00 ( 4.2%).
Expressed in µg mL-1
.
b Positive control (2,4-dichlorophenoxy-
a
acetic acid). c Not determined because of the paucity of material.
preferential loss of the ester moiety attached to C-9.11,12
The isobutyrate residue in compound 2 was located at C-10
considering the chemical shift values for C-9/H-9 and C-10/
H-10 as well as the C-9/H-11 and H-12 correlations
observed in the HMBC spectrum. The absolute configura-
tion at C-9 and C-10 was established by applying the
Mosher ester methodology13 on (+)-cis-methylkhellactone
(1a ). The latter compound was obtained by acid hydrolysis
(HCl/MeOH) of 1 and 2. Analysis of the ∆S-R data (Table
4) for (+)-cis-methylkhellactone-(S)-MTPA (2a ) and (+)-
cis-methylkhellactone-(R)-MTPA (2b) derivatives showed
that the absolute stereochemistry at C-9 was R. Thereafter,
the absolute configuration at C-10 was automatically
assigned as R because of the cis relationship between H-9
and H-10.
(9R,10R)-10-hydroxy-8,8-dimethyl-9,10-dihydro-2H,8H-ben-
zo[1,2-b:3,4-b′]dipyran-2-one-9-yl ester (4),6 2-methylbut-
(2E)-enoic acid (9R,10R)-10-hydroxy-8,8-dimethyl-9,10-
dihydro-2H,8H-benzo[1,2-b:3,4-b′]dipyran-2-one-9-yl ester
(5),7 seravshanin (6),7 quianhucoumarin D (7),5 (+)-cis-
khellactone (8),8,9 and jatamansin (9)10 were obtained and
identified by comparison of their published spectroscopic
data.
The structures of the novel coumarins 1 and 2 and that
of the known compound 7 were confirmed by X-ray analysis
(Figures 1-3). In all cases, the crystal structures clearly
show the syn disposition of the substituents at C-9 and
C-10. The pyranocoumarin skeleton of the molecules is
essentially planar. The pyran ring adopts a half-chair
conformation with C-8 and C-9 on opposite sides of the ring
plane. In addition, both carbonyl oxygen atoms point
toward the same direction and the ester group attached to
C-10 displays some orientational disorder. Crystal packing
of compounds 1 and 7 is governed by van der Waal forces,
which are responsible for the crystal cohesion. For com-
pound 2 the intramolecular hydrogen bonds between the
hydroxy group (O-3) at C-9 and the coumarin oxygen atom
(O-2) arrange the molecules in tetramers.
Compound 10 has a molecular formula of C18H20O5 as
inferred by EIMS, 13C NMR, and elemental analysis. The
1H NMR spectrum was similar to that of jatamansin (9)10
and other lomatin analogues.10 The most obvious differ-
ences between the NMR spectra of compounds 9 and 10
resulted from the presence of the signals for an isobutyroyl
moiety in 10 instead of the resonances for the angeloyl unit
in 9. The absolute configuration at C-9 was also ac-
complished by applying the Mosher ester metodology13 on
(+)-lomatin (9a ), obtained by prolonged treatment of 10
with boiling methanolic KOH. Analysis of the ∆S-R data
(Table 4) for the (+)-lomatin-(S)-MTPA (9b) and (+)-
lomatin-(R)-MTPA (9c) derivatives showed that the abso-
lute stereochemistry at C-9 was R. In accordance with the
above discussion 10 was characterized as isobutyric acid
(9R)-8,8-dimethyl-9,10-dihydro-2H,8H-benzo[1,2-b:3,4-b′]-
dipyran-2-one-9-yl ester.
Compounds 1 and 2 were isolated as colorless plates and
needles, respectively. The molecular formulas were deduced
as C19H20O7 and C18H20O6 by elemental analysis and 13C
NMR. Detailed analysis of the NMR (Table 3) spectra
indicated that both compounds were cis-khellactone es-
ters.8,9 Compared to cis-khellactone8,9 the NMR spectra of
1 showed additional resonances for one acetate and one
propionate. Compound 2 differed from cis-khellactone in
the presence of an isobutyroyl moiety. In the case of
compound 1 the mass spectral fragmentation pattern
suggested the position of the acetyl and propionyl units at
C-9 and C-10, respectively, since the highest mass ion
observed in the EIMS was at m/z 300 and corresponded to
the loss of acetic acid from the molecular ion. In this regard,
it is well known that khellactone diesters undergo a

830 J ournal of Natural Products, 2002, Vol. 65, No. 6
Ta ble 3. NMR Data for Compounds 1, 2, and 10a
Valencia-Islas et al.
1
2
10
b
b
b
position
δH (m, J in Hz)
δC
δH (m, J in Hz)
δC
δH (m, J in Hz)
δC
2
3
4
4a
5
6
6a
8
159.8
113.2
143.2
112.6
129.1
114.4
156.7
77.4
159.9
113.0
143.3
112.5
129.3
114.5
156.9
78.6
161.2
112.6
143.8
112.2
126.7
114.3
153.4
76.6
6.23 (d, 9.5)
7.59 (d, 9.5)
6.23 (d, 9.6)
7.59 (d, 9.6)
6.23 (d, 9.50)
7.63 (d, 9.50)
7.35 (d, 8.5)
6.80 (d, 8.5)
7.36 (d, 8.5)
6.81 (d, 8.5)
7.26 (d, 8.8)
6.79 (d, 8.8)
9
5.31 (d, 5.0)
6.55 (d, 5.0)
70.1
60.8
4.05 (d, 4.8)
6.38 (d, 4.8)
71.7
63.4
5.11 (t, 5.5)
3.21 (dd, 5.5, 17.6)
2.92 (dd, 5.5, 17.6)
69.2
23.0
10
10′
10a
10b
11
12
-OH
13
14
15
16
16′
17
107.0
154.0
22.7
107.6
154.2
22.3
107.2
156.3
22.4
1.44 (s)
1.41 (s)
1.48 (s)
1.42 (s)
2.37 (brs)
1.36 (s)
1.38 (s)
24.8
20.7
24.8
170.0
20.7
173.3
27.5
178.5
34.3
18.8
18.9
176.2
34.0
18.8
19.0
2.09 (s)
2.68 (hept, 6.5)
1.26 (d, 6.5)
1.23 (d, 6.5)
2.55 (hept, 7.00)
1.17 (dd, 7.00)
1.14 (dd, 7.00)
2.43 (dq, 7.5,16.0)
2.38 (dq, 7.5,16.0)
1.20 (dd, 7.5)
9.1
a
b
Spectra recorded in CDCl3 (1H, 500 MHz and 13C, 125 MHz); δ in ppm. Assigned by HMQC, HMBC, and DEPT.
Ta ble 4. Partial 1H NMR Data of the (S)- and (R)-Mosher Esters of 1a and 9a a
1a
9a
(R)-MTPA
carbinol
confign.
carbinol
confign.
proton
(S)-MTPA
(R)-MTPA
∆δS-R
(S)-MTPA
∆δS-R
9
10
5.306
4.963
5.346
4.882
R
5.246
3.311
3.065
1.323
1.273
5.245
3.266
2.981
1.378
1.364
R
+0.081
+0.045
+0.84
-0.055
-0.091
11b
12b
1.401
1.347
1.494
1.399
-0.093
-0.052
a
b
Spectra recorded in CDCl3 (300 MHz). Interchangeable signals.
F igu r e 2. X-ray crystal structure for 2.
NMR spectra (Table 5) of 11 and 12 showed signals due to
the presence of two carbonyl groups, a methoxy functional-
ity, five methyl groups, and two methines in the aromatic
region. The main differences between the NMR spectra
(Table 5) of compounds 11 and 12 resulted in the signals
due to the ester moiety at C-3. In 12 these signals were
consistent with the presence of an isobutyrate, while in 11
with an angelate. Cursory inspection of the HMBC spectra
of 11 and 12, which displays the correlations summarized
F igu r e 1. X-ray crystal structure for 1.
The molecular formulas of compounds 11 and 12 were
determined as C21H24O6 and C20H24O6, respectively, by MS,
NMR, and elemental analysis. Their NMR (Table 5), UV,
and IR spectra indicated that these compounds are linear
pyranochromones related to (-)-hamaudol,14 a linear pyra-
nochromone possessing a 3(S)-hydroxy functionality. The

Phytotoxic Compounds from Prionosciadium
J ournal of Natural Products, 2002, Vol. 65, No. 6 831
and Ca2+-ATPase. Therefore, CaM and/or the enzymes it
regulates could be important targets in vivo for the
phytotoxic action of many phytotoxins.17 In this context, it
has been previously described that some phytotoxins such
as ophiobolin A interact with CaM, altering its enzyme
activator properties.18 The interaction of ophiobolin A with
CaM has been demonstrated by electrophoresis, by fluo-
rescence experiment, and by evaluating its capability of
inhibiting the activation of cAMP-phosphodiesterase.18
Concerning the first experiment, it is well known that the
interaction of any compound with Ca2+-CaM complex alters
its conformation and changes its electrophoretic mobility;
however, it does not reveal if the tested compound modifies
the enzyme activator properties of CaM.15,18 Figure 4 shows
that all compounds tested (0.033 µg mL-1) modify the
electrophoretic mobility of both proteins. Thus, these
compounds interact with calmodulin and, as ophiobolin A,
might exert their phytotoxic action by interfering with
calmodulin-regulated processes in vivo.
In conclusion, the results of the present investigation
revealed that P. watsoni contains phytotoxins that interact
with Ca2+-calmodulin. These compounds might be useful
leads for the development of new herbicide agents. Further
work is in progress in order to evaluate if this interaction
affects calmodulin function.
F igu r e 3. X-ray crystal structure for 7.
in Table 5, confirmed the pyranochromone skeleton as well
as the position of the methoxy, methyl, and ester groups
in both molecules. Upon alkaline hydrolysis (KOH/MeOH),
11 and 12 yielded (+)-5-methoxyhamaudol (11a ), which is
also a new analogue of (-)-hamaudol. The NMR spectra of
11a (see Experimental Section) differ from those of hamau-
dol14 in the presence of the signal for the methoxyl group
at C-5. The sign of the optical rotation of 11a is opposite
that of hamaudol, suggesting that the stereochemistry at
C-3 of 11 and 12 is the opposite that of (-)-hamaudol. The
application of the advanced Mosher ester methodology13
on 11a corroborated this proposal since analysis of the ∆S-R
data (Table 6) for the (+)-5-methoxyhamaudol-(S)-MTPA
(11b) and (+)-5-methoxyhamaudol-(R)-MTPA (11c) deriva-
tives clearly indicated that the absolute stereochemistry
at C-3 was R.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. Melting points were
determined on a Fisher-J ohns apparatus and are uncorrected.
IR spectra were measured on a Perkin-Elmer 599 spectrom-
eter. UV spectra were obtained on a Shimadzu 160 UV
spectrometer in MeOH solution. Optical rotations were taken
on a J ASCO DIP-360 polarimeter. NMR spectra, including
COSY spectra, NOESY, HMQC, and HMBC experiments were
obtained on a Varian UNITY PLUS 500 spectrometer in CDCl3
or MeOD at either 300 or 500 MHz (1H), or 75 or 125 MHz
(13C), or on a Bruker DMX500 at 500 MHz (1H) or 125 MHz
(13C) using tetramethylsilane (TMS) as an internal standard.
EIMS data were recorded on a J EOL J MS-AX505HA mass
spectrometer, at an ionization energy of 70 eV. HPLC was
carried out with a Waters UV photodiode array detector (900)
set at 190-350 nm, using a preparative silica gel column (µ-
porasil 10 Å, 100 mm i.d. × 300 mm). Control of the equipment,
data acquisition, processing, and management of chromato-
graphic information were performed by the Millenium 2001
software program (Waters). Open column chromatography: Si
gel 60 (70-230 mesh, Merck). Analytical and preparative
TLC: Si gel 60 F254 (Merck).
The phytotoxic activity of the isolates and chemical
derivatives 1a , 1b, 9a , and 11a was determined on
germination and radicle elongation of A. hypochondriacus
and E. crus-galli seedlings using 2,4-D (2,4-dichlorophen-
oxyacetic acid) as the positive control. Table 1 summarizes
the phytotoxic effect (expresed as IC50 values) of the tested
compounds. In general, the compounds inhibited radicle
growth in a concentration-dependent manner. Compounds
11, 12, and 1a were the most active and showed high
selectivity against E. crus-galli seeds. Of all the isolates,
only 9 and 10 showed significant phytotoxicity on duckweed
at concentrations of 100 and 200 µM (Table 2). These
compounds caused growth inhibition, chlorophyll reduction,
and electrolytic leakage. Apparently, during the course of
this study it was not possible to isolate the active compound
responsible for the marked phytotoxicity on duckweed
because the extract was more active than compounds 9 and
10. On the other hand, it is possible that the compounds
present in the extract exerted a synergistic effect on
duckweed.
P la n t Ma ter ia l. The aerial parts of P. watsoni were
collected on February 12, 1999, at San Luis Potos´ı, Mexico
(Sierra Alvarez, Municipio Villa de Zaragoza), and identified
by Dr. Robert Bye, Instituto de Biolog´ıa UNAM. A voucher
specimen (R. BYE et al. 26911) is preserved at the Ethnobo-
tanical Collection of the National Herbarium (MEXU), Insti-
tuto de Biolog´ıa, UNAM.
Extr a ction a n d Isola tion of 1-12. The air-dried and
ground aerial parts of P. watsoni (1.8 Kg) were extracted with
CH2Cl2-MeOH (1:1). The resulting extract (345.2 g) was
subjected to open Si gel (1200 g) column cromatography and
eluted with a gradient of hexane-EtOAc (1:0f 0:1) and
EtOAc-MeOH (9:1f1:1) mixtures. Altogether, 49 fractions
were collected and combined according to their TLC patterns
to yield eight primary fractions (F1-F8). Bioactivities in the
Petri dish germination and radicle elongation bioassay showed
three active pools: F2 (160.4 g, IC50 ) 53.3 µg mL-1 for A.
hypochondriacus; IC50 ) 1114.5 µg mL-1 for E. crus-galli), F4
Continuing with our search of potential herbicide agents
that could interact with calmodulin (CaM), the phytotoxic
compounds were further evaluated for their ability to bind
spinach and bovine brain CaM.15,16 CaM is an intracellular
receptor for Ca2+ in eukaryotes which modulates enzymatic
response to calcium. This protein plays a leading role in
plant signal transduction during germination and plant
growth, modulating the activity of several important plant
enzymes such as NAD-kinase, gluatamate decarboxilase,
(144 g, IC50 ) 241.6 µg mL-1 for A. hypochondriacus; IC50
)
56.1 µg mL-1 for E. crus-galli), and F5 (12.7 g, IC50 ) 56.2 µg
mL-1 for A. hypochondriacus; IC50 ) 174.7 µg mL-1 for E. crus-
galli). From the active fraction F4, eluted with hexane-EtOAc,
7:3, precipitated a mixture of 1, 6, and 7 (35 g). Part of this

832 J ournal of Natural Products, 2002, Vol. 65, No. 6
Ta ble 5. NMR Data for Pyranochromones 11 and 12a
Valencia-Islas et al.
11
12
b
b
position
δH (m, J in Hz)
δC
HMBC (C f H)
δH (m, J in Hz)
δC
HMBC (C f H)
2
3
81.9
89.3
27.7
4,10,11
4,11,10
81.6
89.0
27.6
4,10,11
4,10,11
5.03 (dd, 7.0)
3.33 (dd, 7.0, 9.5)
3.27 (dd, 7.0, 9.5)
5.01 (dd, 7.0)
3.30 (dd, 7.0, 9.6)
3.23 (dd, 7.0, 9.6)
4R
4â
4a
5
5a
6
7
8
8a
9
116.7
155.8
111.9
177.1
111.4
163.3
159.9
93.7
4,9
-OCH3,4
9
116.7
155.7
111.5
177.1
111.4
163.3
159.9
93.7
4,9
-OCH3,4
9
5.99 (q, 0.5)
6.52 (s)
-CH3
7,-CH3
9,-CH3
5.99 (q, 0.75)
6.53 (s)
9,-CH3
7,-CH3
9,-CH3
9a
10
11
12
13
14
15
16
-OCH3
-CH3
164.4
21.4
22.1
167.1
128.5
137.7
15.6
20.6
61.1
19.7
4,9
164.4
21.1
22.0
167.2
34.9
18.8
4,9
1.62 (s)
1.61 (s)
1.56 (s)
1.53 (s)
15
13,14,15
14,15
13
15,16
15,16
16
2.41 (hept, 7.0)
1.06 (d, 7.0)
1.05 (d, 7.0)
5.98 (ddq, 1.5, 7.5, 14.5)
1.89 (dq, 1.5, 7.5)
1.68 (q, 1.5, 7.0)
5.93 (s)
18.8
13
15
3.94 (s)
2.28 (d, 0.75)
61.1
19.8
2.27 (d, 0.5)
7
7
a
b
Spectra recorded in CDCl3 (1H, 500 MHz and 13C, 125 MHz); δ in ppm. Assigned by HMQC, HMBC, and DEPT.
Ta ble 6. Partial 1H NMR Data of the (S)- and (R)-Mosher
Esters of 11a a
carbinol
proton
(S)-MTPA
(R)-MTPA
∆δS-R
confign.
3
4.867
3.079
3.253
1.664
1.661
4.095
3.067
3.247
1.734
1.725
R
4R
4â
10
11
+0.012
+0.006
-0.070
-0.060
a
Spectra recorded in CDCl3 (300 MHz).
mixture (200 mg) was resolved by extensive preparative TLC
(hexane-EtOAc, 3:1) to yield 1 (60 mg), 6 (130 mg), and 7 (1
mg). The mother liquors from phytotoxic fraction F4 were
rechromatographed on a Si gel (734 g) column eluting with a
gradient of hexane-EtOAc (1:0 f 0:1) to yield 19 secondary
fractions (FI-FXIX). According to the Petri dish germination
and radicle elongation bioassay, the phytotoxic activity was
concentrated in secondary fractions F4VI (6.07 g) and F4IX (8.6
g). Secondary fraction F4VI, eluted with hexane-EtOAc, 9:1,
was chromatographed on a Si gel (81 g) column eluting with
a gradient of hexane-EtOAc (1:0 f 0:1) to afford 10 tertiary
fractions. The phytotoxic activity was concentrated in fraction
F4VI-6 (4.9 g). Extensive TLC (hexane-EtOAc, 3:1) of active
fraction F4VI-6 (200 mg), eluted with hexane-EtOAc, 9:1,
yielded 9 (50 mg) and 10 (12 mg). Secondary active fraction
F
4IX, eluted with hexane-EtOAc, 8:2, was further chromato-
graphed on another Si gel (151.0 g) column eluting with a
gradient of hexane-EtOAc (1:0 f 0:1) and EtOAc-MeOH (1:
1) to yield nine tertiary fractions (F4-IX-1-F4-IX-9). The phy-
totoxic activity was concentrated in fraction F4-IX-5 (10.89 g),
eluted with hexane-EtOAc, 7:3. The latter fraction was
rechromatographed on a Si gel (217.8 g) column eluting with
F igu r e 4. SDS-PAGE of bovine (a) and spinach (c) calmodulins after
treatment with isolates and chemical derivatives. Electrophoresis of
2 µg samples of calmodulins in the presence of 1 mM CaCl2. Pretreat-
ments of the calmodulin samples, for 1.5 h at 30 °C in the presence of
CaCl2: A, no additions; B, 0.033 µg/mL (2S,3S)-5-hydroxy-6,8,10-
trimethoxy-2,3-dimethyl-4H-dihydronaphtho[2,3-b]pyran-4-one; C, 1;
D, 2; E, 4, F, 6; G, 9; H, 10; I, 11; J , 12; K, 1a ; L, 1b; M, 9a ; N, 11a .
In all cases 0.033 µg/mL of treatment (1-11a ) was applied.
a
gradient of CH2Cl2-MeOH (1:0 f 1:1) to give seven
quaternary fractions (F4-IX-5-I-F4-IX-5-VII). The phytotoxic
activity was concentrated in fraction F4-IX-5-V (4.9 g), eluted
with CH2Cl2-MeOH, 99.95:0.05. Extensive HPLC purification
of the active quaternary fraction F4-IX-5-V (100 mg) on a
normal-phase Si gel column [10 mL min-1, Hex-i-PrOH-
MeOH (95:2.5:2.5), λ 230 nm] yielded 2 (15 mg), 3 (5 mg), 4 (5
mg), 5 (5 mg), and additional amounts of 1 (20 mg); retention
times: 18.6, 19.1, 26.3, 27.4, and 29.5 min, respectively. Active
fraction F5, eluted with hexane-EtOAc, 6:4, was further
resolved by column chromatograpy on Si gel (182 g) eluting
with hexane-EtOAc (1:0 f 0:1) and EtOAc-MeOH (9:1 f 1:1)
F5-XXIII), and the Petri dish bioassay indicated that the
phytotoxic activity was in secondary fractions F5-XII (607.4 mg)
and F5-XIII (1.5 g). Further purification of active fracion F5-XII
(eluted with hexane-EtOAc, 6:4) by HPLC [10 mL min-1
,
mixtures. This process yielded 23 secondary fractions (F5-I
-
Hex-i-PrOH-MeOH (95:2.5:2.5), λ 230 nm] yielded 8 (20 mg),

Phytotoxic Compounds from Prionosciadium
J ournal of Natural Products, 2002, Vol. 65, No. 6 833
retention time 25.2 min. Extensive HPLC purification of the
spectroscopic and spectrometric data, including UV, IR, and
1HNMR, were the same as those previously described.10
P r ep a r a tion of (+)-5-Meth oxyh a m a u d ol (11a ). A solu-
tion of 11 (10 mg) or 12 (5 mg) in 3 mL of MeOH was treated
as described for 9 and 10. The reaction mixtures were purified
by column chromatography on Si gel [2 g, EtOAc] to give 11a
(5 and 2 mg respectively), mp 137-138 °C; [R]20 +560 (c 1 mg
active secondary fraction F5-XIII (200 g), eluted with hexane-
EtOAc, 6:4, on a normal-phase Si gel column [10 mL min-1
,
Hex-i-PrOH-MeOH (90:5.0:5.0), λ 230 nm] yielded 11 (60 mg)
and 12 (10 mg); retention times: 20.2 and 20.9 min, respec-
tively.
P r op ion ic a cid (9R,10R)-9-a cetoxy-8,8-d im eth yl-9,10-
d ih yd r o-2H,8H-ben zo[1,2-b:3,4-b′]d ip yr a n -2-on e-10-yl es-
ter (1): colorless plates, mp 208 °C; [R]20 +18 (c 1 mg mL-1
CHCl3); UV (MeOH) λmax (log ꢀ) 321 (4.23) nm; IR νmax (KBr)
3097, 2980, 1746, 1605, 1225, 1024 cm-1; 1H NMR (CDCl3, 500
MHz) and 13C NMR (CDCl3, 125 MHz), see Table 3; EIMS m/z
300 [M+ - AcOH (29)], 285 (8), 261 (6), 244 (30), 229 (100), 57
(21), 43 (15). Anal. Calcd for C19H20O7: C 63.33, H 5.59.
Found: C 63.39, H 5.53.
1
mL-1 CHCl3); H NMR (CDCl3, 300 MHz) δ 6.02 (1H, q, J )
0.75 Hz, H-3), 6.61 (1H, s, H-6), 4.75 (1H, dd, J ) 4.5, 8.24
Hz, H-3′), 3.34 (1H, dd, J ) 4.25, 8.24 Hz, H-4′), 1.29 (3H, s,
H-5′), 1.23 (3H, s, H-5′), 2.32 (1H, d, J ) 0.75 Hz, CH3), 3.92
(1H, s, -OCH3); 13C NMR (CDCl3, 125 MHz) δ 179.80 (C-2),
166.58 (C-4), 166.43 (C-7), 161.44 (C-5), 156.96 (C-8a), 118.47
(C-8), 111.54 (C-4a), 111.49 (C-3), 92.65 (C-2′), 72.25 (C-3′),
61.04 (-OCH3), 28.72 (C-4′), 25.4 (C-5′), 25.31 (C-6′), 19.71
(-CH3); EIMS m/z 290 [M+(100)], 272 (18), 257 (23), 243 (13),
231 (88), 213 (87), 201 (57), 189 (32), 186 (24), 174 (11), 160
(8), 132 (8), 59 (39), 43 (9). Anal. Calcd for C16H18O5: C 66.20,
H 6.25. Found: C 66.25, H 6.24.
Mosh er Ester s of Com p ou n d s 1a , 9a , a n d 11a . 1a (3.0
mg), 9a (3.0 mg), or 11a (3.0 mg) was dissolved in CH2Cl2 (1
mL) and treated with (S)- or (R)-MTPA (18 mg), DCC (18 mg),
and 4-DMAP (4 mg). The mixtures were stirred at room
temperature (25 °C) for 3 h and then poured into ice-water.
The resulting mixtures were then extracted with CHCl3; the
organic phases were successively washed with 5% aqueous
HCl, saturated NaHCO3, and water and then dried over Na2-
SO4 and filtered. Evaporation of the solvent under reduced
pressure afforded residues which were purified by chroma-
tography on Si gel [2 g, hexane-EtOAc (7:3)] for 1a and 9a .
In the case of Mosher esters of compound 11a the column was
eluted with EtOAc to yield the corresponding (S)- and (R)-
MTPA esters.
X-r a y Cr ysta llogr a p h ic An a lysis of Com p ou n d s 1, 2,
a n d 7.20 Molecular structures of compounds 1, 2, and 7 were
analyzed by X-ray diffraction methods following very similar
procedures. For each sample, crystals were mounted, in air,
on glass fibers. Accurate cell parameters were determined by
refinement from the setting of 25 reflections and diffraction
intensities measured at 293 K using an ω-θ scan method on
a Siemens P4/PC diffractometer equipped with graphite-
monochromated radiation. The intensities of three standard
reflections, recorded every 100 collected reflections, showed
no changes. All data sets were corrected for Lorentz-polariza-
tion effects, but no absorption corrections were applied. The
structure of each compound was determined by direct methods
(SIR92)21 and refined by full-matrix least-squares methods
using SHELXL97.22 Hydrogen atoms attached to C atoms were
set to ride on the parent C atoms, and for those bonded to O
atoms their positional parameters were refined; an isotropic
temperature factor 1.2 times the Ueq of the parent atom was
used. The non-hydrogen atoms were refined with anisotropic
thermal parameters.
Isobu tyr ic a cid (9R,10R)-9-h yd r oxy-8,8-d im eth yl-9,10-
d ih yd r o-2H,8H-ben zo[1,2-b:3,4-b′]d ip yr a n -2-on e-10-yl es-
ter (2): colorless needles, mp 134 °C; [R]20 +151 (c 1 mg mL-1
CHCl3); UV (MeOH)) λmax (log ꢀ) 323 (4.27) nm; IR νmax (KBr)
3475, 2977, 2926, 1730, 1607, 1234, 1108 cm-1
;
1H NMR
(CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz), see Table
3; EIMS m/z 332 [M+(16)], 314 [M+ - H2O(5)], 299 (17), 261
(18), 244 (13), 229 (100), 215 (9), 191 (27), 175 (11), 71 (24), 43
(29). Anal. Calcd for C18H20O6: C 65.05, H 6.07. Found: C
65.00, H 6.09.
Isobu tyr ic a cid (9R)-8,8-d im eth yl-9,10-d ih yd r o-2H,8H-
ben zo[1,2-b:3,4-b′]d ip yr a n -2-on e-9-yl ester (10): vitreous
colorless solid, mp 108-110 °C; [R]20 +20 (c 1 mg mL-1 CHCl3);
UV (MeOH) λmax (log ꢀ) 324 (3.9) nm; IR νmax (film) 2979, 2944,
1
1732, 1606, 1144, 1113; H NMR (CDCl3, 500 MHz) and 13C
NMR (CDCl3, 125 MHz), see Table 3 EIMS m/z 316 [M+(19)],
246 (3), 228 (4), 213 (100), 83 (7), 71 (17), 55 (6), 43 (29). Anal.
Calcd for C18H20O5: C 68.34, H 6.37. Found: C 68.38, H 6.30.
2-Meth ylbu t-(2Z)-en oic a cid (3R)-5-m eth oxy-3,4-d ih y-
d r o-2,2,8-tr im eth yl-6-oxo-2H,6H-ben zo[1,2-b:5,4-b′]d ip y-
r a n -3-yl ester (11): yellow oil; [R]20 +340 (c 1 mg mL-1
CHCl3); UV (MeOH) λmax (log ꢀ) 214 (4.50), 285 (4.12) nm; IR
ν
max (film) 2981, 2987, 1714, 1660, 1621, 1470, 1238, 1141 cm-1
;
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz),
see Table 5; EIMS m/z 372 [M+(25)], 289 (2), 272 (23), 257
(100), 83 (30). Anal. Calcd for C21H24O6: C 67.73, H 6.50.
Found: C 67.78, H 6.49.
Isobu tyr ic a cid -(3R)- 5-m eth oxy-3,4-d ih yd r o-2,2,8-tr i-
m eth yl-6-oxo-2H,6H-ben zo[1,2-b:5,4-b′]d ip yr a n -3-yl ester
(12): vitreous solid, mp 140-141 °C; [R]20 +560 (c 1 mg mL-1
CHCl3); UV (MeOH) λmax (log ꢀ) 213 (4.6), 284 (4.10) nm; IR
ν
max (KBr) 2972, 2926, 1731, 1655, 1617, 1468, 1378, 1143 cm-1
.
1H NMR (CDCl3, 500 MHz) and 13C NMR (CDCl3, 125 MHz),
see Table 5; EIMS m/z 360 [M+(29)], 273 (27), 257 (100), 231
(55), 229 (51), 71 (8), 43 (13). Anal. Calcd for C20H24O6: C 66.65,
H 6.71. Found: C 66.62, H 6.75.
P r ep a r a tion of (+)-cis-Meth ylk ella cton e (1a ) a n d (-)-
tr a n s-Meth ylk h ella cton e (1b). A solution of 1 (50 mg) in 3
mL of MeOH was mixed with 3.5 mL of 1 N methanolic HCl
and refluxed for 22 h. The reaction mixture was allowed to
stand for 45 min and adjusted to pH 8 with a NaHCO3 (10%
aqueous) solution, concentrated in vacuo to remove the MeOH,
and extracted with CH2Cl2. The CH2Cl2 layers were combined,
washed with water, and dried over Na2SO4. The reaction
mixture was chromatographed on preparative TLC [hexane-
EtOAc (7:3)] to yield 1a (10 mg) and 1b (15 mg). 1a : mp 118-
119 °C, [R]20 +690 (c 1 mg mL1 CHCl3); 1b: mp 157-158 °C;
[R]20 -336 (c 1 mg/mL CHCl3). The spectroscopic and spectro-
metric data of 1a and 1b, including UV, IR, 1HNMR, and
13CNMR, were identical to those previously described.19
P r ep a r a tion of (+)-Lom a tin (9a ). To a solution of 9 or
10 (10 mg each) in 2 mL of MeOH was added 2 mL of 1 N
methanolic KOH, and the mixture was refluxed for 3.5 h. The
reaction mixture was diluted with water, concentrated in vacuo
to remove the organic solvent, acidified with 2 N sulfuric acid
(2 mL), and extracted with EtOAc. The combined EtOAc layers
were succesively washed with 10% aqueous NaHCO3 solution
and water and dried over Na2SO4. The resulting residues were
purified by preparative TLC [hexane-EtOAc (1:1)] to give 9a
(5 mg), mp 184-185 °C; [R]20 +210 (c 1 mg mL-1 CHCl3). The
Cr ysta l Da ta for Com p ou n d 1: C19H20O7, Mr ) 360.35,
colorless plates with dimensions 0.32 × 0.26 × 0.06 mm,
orthorhombic. The space group is P212121 with unit cell
parameters (at 25 °C) a ) 8.5950(4), b ) 10.394(1), c ) 20.015-
(1) Å, V ) 1788.07(19) Å3, Z ) 4, F(000) ) 752, dcalcd ) 1.331
g cm-3, µ/mm-1 ) 0.861.
Cr ysta l Da ta for Com p ou n d 2: C18H20O6, Mr ) 332.34,
colorless needle with dimensions 0.60 × 0.12 × 0.10 mm,
monoclinic. The space group is C2 with unit cell parameters
(at 25 °C) a ) 29.101(3), b ) 9.010(1), c ) 14.373(1) Å, and V
) 3535.2(6) Å3, Z ) 8, F(000) ) 71408, dcalcd ) 1.249 g cm-3
,
µ/mm-1 ) 0.782.
Cr ysta l Da ta for Com p ou n d 7: C18H20O7, Mr ) 346.32,
colorless prism with dimensions 0.60 × 0.32 × 0.10 mm,
orthorombic. The space group is P212121 with unit cell param-
eters (at 25 °C) a ) 8.631(1), b ) 10.403(2), c ) 20.111(5) Å,
and V ) 1805.7(6) Å3, Z ) 4, F(000) ) 728, dcalcd ) 1.274 g
cm-3, µ/mm-1 ) 0.099.
Bioa ssa y Mea su r in g P la n t Gr ow th In h ibition . The
phytogrowth inhibitory activity on the extract and pure
compounds on seedlings of A. hypochondriacus and E. crus-
galli was evaluated using the Petri dish radicle elongation and

834 J ournal of Natural Products, 2002, Vol. 65, No. 6
Valencia-Islas et al.
germination bioassay.3 The results were analyzed by ANOVA
(p < 0.05), and IC50 values were calculated by Probit analysis
based on percent of radicle growth or germination inhibition.
Su p p or tin g In for m a tion Ava ila ble: Experimental crystallo-
graphic details, tables of atomic coordinates and equivalent isotropic
displacement parameters, and hydrogen bond schemes for compounds
1, 2, and 7. This material is available free of charge via the Internet
at http://pubs.acs.org.
The extract was evaluated at 1, 10, 100, and 100 µg mL-1
.
The fractions and pure compounds were tested at 10, 50, 100,
and 200 µg mL-1. 2,4-D was used as the positive control. The
bioassays were performed at 28 °C.
Refer en ces a n d Notes
Du ck w eed Bioa ssa y. The duckweed assay was carried out
essentially as described in detail by Tanaka et al.4 The extract
at concentrations of 10, 100, and 1000 µg mL-1 and the pure
compounds at concentrations of 25, 50, 100, and 200 µM in
half-strength Hunter’s medium22 were added to 10 duckweed
colonies of three fronds each. In the experiment 3 mL of media
was used and the test was run for 3 days. Electrolyte leakage
was determined by a conductivity meter at the beginning and
during 72 h. Chlorophyll was determined based on fresh
weight by first removing duckweed from bathing media,
blotting with paper towels, and extracting with 5 mL of DMSO
by soaking overnight in the dark. The total chlorophyll (a and
b) was determined by ultraviolet spectroscopy according to
Hiscox and Israelstam.24 Growth inhibition was measured by
determining duckweed fresh weight at the beginning and the
end of the experiment.
Eva lu a tion of th e In ter a ction of Isola ted Com p ou n d s
a n d Der iva tives w ith Sp in a ch a n d Bovin e Br a in Ca lm -
od u lin s. The interaction of the isolated compounds and
derivatives with both spinach and brain calmodulins (Sigma)
was performed through denaturing homogeneous electro-
phoresis (SDS-PAGE). SDS-PAGE was performed according
to previously described procedures using 15% polyacrylamide
gels.15,16 The interaction of the phytotoxins with both calm-
odulins was evaluated by observing the difference in the
electrophoretic mobility in the presence of Ca2+. Each electro-
phoretic run was done by trplicate, and the compound (2S,3S)-
5-hydroxy-6,8,10-trimethoxy-2,3-dimethyl-4H-dihydronaphtho-
[2,3-b]pyran-4-one15,16 was used as the positive control.
(1) This work represents part of the Ph.D. Thesis submitted to the
graduate school of Chemistry UNAM, Mexico, by N.V.
(2) Aguilar, A., J .; Camacho, S.; Chino, P.; J a´cquez, P.; Lo´pez, M. Herbario
Medicinal del Instituto Mexicano del Seguro Social; Instituto Mexi-
cano del Seguro Social, 1994; p 253.
(3) Mata, R.; Mac´ıas, M.; Rojas, S.; Lotina-Hensen, B.; Toscano, R.;
Anaya, A. Phytochemistry 1998, 49, 441-449.
(4) Tanaka, T.; Abbas, H.; Duke, S. Phytochemistry 1993, 33, 779-785.
(5) Murray, R. Nat. Prod. Rep. 1995, 477-505.
(6) Bolhman, F.; Bhaskar, R.; Grenz, M. Tetrahedron Lett. 1968, 3947-
3950.
(7) Swager, T.; Cardellina, J ., II. Phytochemistry 1985, 24, 805-813.
(8) Lemmich, J .; Alsted-Pedersen, P.; Nielsen, B. Tetrahedron Lett. 1969,
39, 3365-3366.
(9) Nielsen, E.; Larsen, P.; Lemmich, J . Acta Chem. Scand. 1971, 25,
529-533.
(10) Shanbhag, S.; Mesta, C.; Maheshwari, M.; Paknikar, S.; Bhata-
charyya, S. Tetrahedron 1964, 20, 2605-2615.
(11) Shaath, N.; Soine, T.; Shipchandler, M. J . Pharm. Sci. 1976, 65,
1028-1033.
(12) Lingyi, K.; Xian, L.; Zhida, M.; Yang, L.; Qitai, Z.; Tingru, Z.
Yaokedaxue Xuebao 1996, 27, 136-138.
(13) Ontani, I.; Kusumi, T.; Kakisawa, H. J . Am. Chem. Soc. 1991, 1113,
4092-4096.
(14) Nitta, A. Yakugaku Zasshi 1965, 85 (1), 55-61.
(15) Mac´ıas, M.; Ulloa, M.; Gamboa, A.; Mata, R. J . Nat. Prod. 2000, 63,
757-761, and references therein.
(16) Mac´ıas, M.; Gamboa, A.; Ulloa, M.; Toscano, R. A.; Mata, R. Phy-
tochemistry 2001, 58, 751-758.
(17) Lee, S. H.; J ohnsn, J . D.; Walsh, M. P.; Van Leirop, J . E., Sutherland,
C.; Xu, A.; Snedden, W. A.; Kosk-Kosicka, D.; Fomm, H.; Narayanan,
N.; Cho, M. J . Biochem. J . 2000, 350, 299-306.
(18) Au, T. K.; Chick, W. S.; Leung, P. C. Life Sci. 2000, 67, 733-742.
(19) Mac´ıas, F.; Massanet, G.; Rodriguez-Luis, F.; Salva´, J . Magn. Reson.
Chem. 1989, 27, 653-658.
(20) Crystallographic data for the structures reported in this paper have
been deposited with the Cambridge Crystallograpic Data Centre.
Copies of the data can be obtained, free of charge, on application to
the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK [fax:
+44 (0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk].
(21) Altomare, A.; Cascarano, G.; Giacovazzi, C.; Guagliardi, A.; Burla,
M.; Polidori, G.; Camalli, M. J . Appl. Crystallogr. 1994, 27, 435.
(22) Sheldrick, G. SHELXTL97: Program for Refinement of Crystal
Structures; University of Go¨ttingen: Germany, 1997.
(23) Becerril, J .; Duke, M.; Nadihalli, U.; Matsumoto, H.; Duke, S. Phys.
Plant. 1992, 86, 6-16.
Ack n ow led gm en t. This investigation was supported by
grants from DGAPA-UNAM (IN 207300), CONACyT (Conve-
nio 27978N), and PAEP (Nos. 108333 and 108321). Thanks
are due to Isabel Cha´vez, Luis Velasco-Ibarra and J avier
Pe´rez-Flores (Instituto de Qu´ımica UNAM), Oscar Ya´n˜ez,
Marisela Gutie´rrez, and Georgina Duarte (Facultad de Quı´m-
ica UNAM) for recording NMR, MS, UV, and IR spectra. The
technical assistance of Laura Acevedo, Isabel Rivero, Gustavo
Morales, Myrna Mendoza, and Gilberto Ma´rquez is also
aknowledged. N.V. acknowledges the fellowship awared by
CONACyT and DGEP to carry out graduate studies.
(24) Hiscox, J .; Israelstam, G. Can. J . Bot. 1979, 57, 1332-1334.
NP010448T