Structure-activity relationships (SAR) studies of benzoxazinones, their degradation products and analogues. Phytotoxicity on standard target species (STS)

Article abstract of DOI:10.1021/jf0484071

Benzoxazinones 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) and 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) have been considered key compounds for understanding allelopathic phenomena in Gramineae crop plants such as corn (Zea mays L.), wheat (Triticum aestivum L.), and rye (Secale cereale L.). The degradation processes in the environment observed for these compounds, in which soil microbes are directly involved, could affect potential allelopathic activity of these plants. We present in this work a complete structure-activity relationships study based on the phytotoxic effects observed for DIMBOA, DIBOA, and their main degradation products, in addition to several synthetic analogues of them. Their effects were evaluated on standard target species (STS), which include Triticum aestivum L. (wheat) and Allium cepa L. (onion) as monocots and Lepidium sativum L. (cress), Lactuca sativa L. (lettuce), and Lycopersicon esculentum Will, (tomato) as dicots. This permitted us to elucidate their ecological role and to propose new herbicide models based on their structures. The best phytotoxicity results were shown by the degradation chemical 2-aminophenoxazin-3-one (APO) and several 2-deoxy derivatives of natural benzoxazinones, including 4-acetoxy-(2H)-1,4-benzoxazin- 3(4H)-one (ABOA), 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), and 4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIMBOA). They showed high inhibitory activity over almost all species growth. The fact that APO is a degradation product from DIBOA with high phytotoxicity and stability makes it possible to assign an important ecological role regarding plant defense mechanisms. 2-Deoxy derivatives of natural benzoxazinones display a wide range of activities that allow proposing them as new leads for natural herbicide models with a 1,4-benzoxazine skeleton.

Full text of DOI:10.1021/jf0484071

538 J. Agric. Food Chem. 2005, 53, 538−548
Structure−Activity Relationships (SAR) Studies of
Benzoxazinones, Their Degradation Products and Analogues.
Phytotoxicity on Standard Target Species (STS)
FRANCISCO A. MAC´ıAS,* DAVID MAR´ıN, ALBERTO OLIVEROS-BASTIDAS,†
DIEGO CASTELLANO, ANA M. SIMONET, AND JOSEÄ M. G. MOLINILLO
Grupo de Alelopat´ıa, Departamento de Qu´ımica Orga´nica, Facultad de Ciencias,
Universidad de Ca´diz. C/Repu´blica Saharaui, s/n, 11510 Puerto Real, Ca´diz, Spain
Benzoxazinones 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) and 2,4-dihy-
droxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) have been considered key compounds for understand-
ing allelopathic phenomena in Gramineae crop plants such as corn (Zea mays L.), wheat (Triticum
aestivum L.), and rye (Secale cereale L.). The degradation processes in the environment observed
for these compounds, in which soil microbes are directly involved, could affect potential allelopathic
activity of these plants. We present in this work a complete structure-activity relationships study
based on the phytotoxic effects observed for DIMBOA, DIBOA, and their main degradation products,
in addition to several synthetic analogues of them. Their effects were evaluated on standard target
species (STS), which include Triticum aestivum L. (wheat) and Allium cepa L. (onion) as monocots
and Lepidium sativum L. (cress), Lactuca sativa L. (lettuce), and Lycopersicon esculentum Will.
(tomato) as dicots. This permitted us to elucidate their ecological role and to propose new herbicide
models based on their structures. The best phytotoxicity results were shown by the degradation
chemical 2-aminophenoxazin-3-one (APO) and several 2-deoxy derivatives of natural benzoxazinones,
including 4-acetoxy-(2H)-1,4-benzoxazin-3(4H)-one (ABOA), 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-
one (D-DIBOA), and 4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIMBOA). They showed
high inhibitory activity over almost all species growth. The fact that APO is a degradation product
from DIBOA with high phytotoxicity and stability makes it possible to assign an important ecological
role regarding plant defense mechanisms. 2-Deoxy derivatives of natural benzoxazinones display a
wide range of activities that allow proposing them as new leads for natural herbicide models with a
1,4-benzoxazine skeleton.
KEYWORDS: Allelochemicals, DIMBOA, DIBOA, soil degradation products, wheat, phytotoxicity, SAR,
STS.
INTRODUCTION
which DIBOA is preserved inside the plant prior to its release
(7). Benzoxazolin-2-one (BOA) and 6-methoxybenzoxazolin-
2-one (MBOA) (Table 2, A) are the first chemicals in the
DIBOA and DIMBOA degradation series, respectively (8-10).
There are interesting precedents regarding their bioactivity on
different systems. Lactam 2-hydroxy-(2H)-1,4-benzoxazin-
3(4H)-one (HBOA) (Table 1, B) has been proposed as a
biosynthetic precursor of DIBOA (11, 12). 2-Hydroxy-7-
methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HMBOA) (Table 1,
B) has been detected as a degradation product for DIMBOA in
wheat crop soil (9). Both compounds have been isolated from
crop soils of plants with high prodcution of benzoxazinones.
Benzoxazinones containing the hydroxamic acid moiety
have attracted attention of phytochemistry researchers since the
first isolation of 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one
(DIBOA) (1) in 1959 and 2,4-dihydroxy-7-methoxy-(2H)-1,4-
benzoxazin-3(4H)-one (DIMBOA) in 1962 (2) (Table 1, A).
Interesting bioactivity was observed for both compounds, some
of their degradation products, and also some synthetic analogues,
being antimicrobial (3), antifeedant, insecticide (4), and phy-
totoxic behaviors (5, 6) widely described. The DIBOA natural
glycoside (2-O-â-D-glucopyranosyl-4-hydroxy-(2H)-1,4-benz-
oxazin-3(4H)-one (DIBOA-Glc) (Table 1, A) is the form in
Antifeedant and antiaphid capabilities for DIMBOA and
DIBOA, together with their related benzoxazolinones (Table
2, A) benzoxazinoid lactams (Table 1, B), and synthetic
analogues (2-deoxybenzoxazinones, Table 1, C), were evaluated
by Escobar et al. (4) on aphid Sitobion aVenae. A complete
* Author to whom correspondence should be addressed. Phone:
+34-956.016.370. Fax: +34-956.016.193. E-mail: famacias@uca.es.
† Permanent address: Laboratorio de Qu´ımica Ecolo´gica, Departamento
de Qu´ımica, Facultad de Ciencias, Universidad de Los Andes, Nu´cleo
Universitario Pedro Rinco´n Gutie´rrez, La Hechicera, Me´rida 5101-A,
Venezuela.
10.1021/jf0484071 CCC: $30.25
© 2005 American Chemical Society
Published on Web 01/19/2005

SAR Studies of Benzoxazinones
J. Agric. Food Chem., Vol. 53, No. 3, 2005 539
electron-donor group at position C-7 (as it occurs in DIMBOA)
increased the antifeedant index and the toxicity, and the presence
of a hydroxyl group at positions C-2 and C-4 increased or
decreased bioactivities depending on the aromatic substitution.
Degradation processes from benzoxazinones to benzoxazolino-
nes resulted in a partial detoxification.
Table 1. Natural Allelochemicals, Degradation Products/Biosynthetic
Precursors, and Synthetic Analogs with Benzoxazinone Skeleton
Employed for SAR Studya
Several authors evaluated phytotoxic activity of DIMBOA,
DIBOA, and some related compounds (5, 6). Bravo and Lazo
(13) provided interesting results regarding antialgal and anti-
fungal activity by means of experiments with Chlorella xanthella
and Candida albicans, respectively.
The alga C. xanthella has been proposed as a model for
phytotoxicity evaluation. DIBOA showed more inhibitory
activity than DIMBOA. The effect of 2-deoxy derivatives was
different depending on the aromatic substitution since D-
DIMBOA (Table 1, C) was more active than DIMBOA and
D-DIBOA (Table 1, C) was less active than DIBOA. Lactam
2-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA) (Table 1,
B) and the synthetic analogue 4-acetoxy-2H-1,4-benzoxazin-
3(4H)-one (ABOA) (Table 1, C) showed very low phytotox-
icities. The inhibition experiments on the fungus C. albicans
allowed the authors to conclude that the 7-methoxy group
increases fungistatic behavior and the presence of a hydroxyl
moiety at C-2 also increases antifungal activity. The absence
of this hydroxyl group blocks the opening of the heterocycle
by means of hemiacetal hydrolysis on degradation processes
(13) so it is possible to connect bioactivity with the keto form
of the heterocyclic hemiacetal. Nevertheless, all the activity
seemed to be controlled by the hydroxamic acid moiety. The
authors did not provide comparisons with commercial herbicides
or fungicides. Herein, we present a complete phytotoxicity pro-
file of several synthetic derivatives with the lack of a hydroxyl
at C-2 and the comparison of their bioactivity levels with those
shown by natural allelochemicals DIMBOA and DIBOA.
Antimicrobial activity of DIMBOA and MBOA was evalu-
ated and compared on Agrobacterium tumefaciens (14).
DIMBOA showed higher toxicity, being the population devel-
opment of this organism totally inhibited at 5 × 10-4 M (100%
inhibition of colony diameter). Nevertheless, it was described
as a bacteriostatic compound instead of a bactericide since it
affected expression of A. tumefaciens virulency genes.
In general terms, natural 2,4-dihydroxybenzoxazin-3-ones
(DIMBOA, DIBOA) (Table 1, A) are the most active com-
pounds. Their corresponding benzoxazolinones (MBOA, BOA)
(Table 2, A) are much less active, and 2-deoxy analogues of
DIMBOA and DIBOA (Table 1, C) have different behaviors
depending on the different species assayed. Lactams (Table 1,
B) are nonactive compounds.
In addition to this bioactivity research, the low stability of
DIMBOA, DIBOA, and their related benzoxazolinones in
several conditions, such as biotransformation by fungi (15), and
degradation in crop soil (16, 17) and in aqueous solution (8-
10), has been investigated. We have recently developed a
complete degradation study of these allelochemicals in wheat
crop soil in which we observed and characterized the conversion
dynamics of these compounds into several molecules carried
out by soil microbial population (9, 10). 2-Aminophenoxazin-
3-one (APO) and 2-amino-7-methoxyphenoxazin-3-one (AMPO)
(Table 2, B) are the final products for the DIBOA and DIMBOA
degradation route found by us in wheat crop soil. Several authors
reported their presence in other systems in which these degrada-
tions take place (16, 17). Their N-acetyl derivatives, 2-aceta-
midophenoxazin-3-one (AAPO) and 2-acetamido-7-methoxy-
phenoxazin-3-one (AAMPO) (Table 2, B), have been proposed
as detoxification compounds produced by nonpathogenic
a Functionalization, systematic name, and acronym are shown for each
compound.
structure-activity relationship (SAR) study was made. Com-
pounds with a 1,4-benzoxazin-3-one skeleton (six-membered
heterocyclic ring, Table 1) were more active as antifeedants
than benzoxazolinones (five-membered heterocyclic rings, Table
2, A). Hydroxamic acids (hydroxyl group on the N-4 position)
were much more active than their corresponding lactams (lack
of hydroxyl group at N-4). The absence of a hydroxyl group at
position C-2 (Table 1, C) strongly decreased the effect. A
mortality test made on these organisms indicated DIMBOA and
DIBOA to be the most toxic compounds. In general terms, an

540 J. Agric. Food Chem., Vol. 53, No. 3, 2005
Mac´ıas et al.
Table 2. Base Structures, Functionalizations, Systematic Names, and Acronyms for the Degradation Products Employed for SAR Study
organisms associated to Gramineae (15, 18). APO has been
already described as a potent phytotoxic agent for Echinochloa
crus-galli (barnyardgrass) (16). This compound inhibited ger-
mination of this weed (50% inhibition at 5 × 10-4 M) more
than its precursor 2-benzoxazolinone (BOA, 50% inhibition at
0.7 × 10-3 M). We present in this work a complete phytotox-
icity profile for APO, its activity correlated with some analogue
aminophenoxazines found in such degradation processes.
2-Hydroxyphenylmalonamic acids (Table 2, C) have been
described as biotransformation products from benzoxazolinones,
produced by endophytic fungi asymptomatically associated to
several cereal plants (15, 18, 19). N-[2-Hydroxyphenyl]-
malonamic acid (HPMA) and N-[2-hydroxy-7-methoxyphenyl]-
malonamic acid (HMPMA) have been tested over fungal
colonies, and its influence over Lepidium satiVum L. (cress) root
length has been recorded, although comparison with commercial
fungicides or herbicides was not made (18). Their activities have
been compared with those for their precursors BOA and MBOA
(Table 2, A). The lack of phytotoxicity observed for these
malonamic acids allowed some researchers (18) to propose them
as benzoxazolinone detoxification products. Herein, we present
a complete phytotoxic bioactivity profile with three parameters
(germination, root, and shoot lengths) and a wider variety of
plant species.
analogues were included in order to locate the required
functionalization for phytotoxicity enhancement, with the
purpose of new herbicide models development. Thus, all
possible combinations regarding the presence or absence of
hydroxyl groups at positions C-2 and N-4 and of a methoxy
group at C-7 were tested (2-deoxy-4-hydroxy, 2-hydroxy-4-
deoxy, 2,4-dihydroxy, and 2,4-dideoxy) for both series (7-
methoxy- and 7-H-benzoxazinones). 4-N-Acetoxy derivatives
were also introduced to test the influence of a potential leaving
group over N-4 (Table 1, C). A commercial herbicide is
included as internal standard in order to control bioassay
accuracy and to compare bioactivity levels with a real model.
Our objective in this study was to evaluate a complete
phytotoxicity profile of these compounds considering effects
on standard target species (STS) germination and growth (20).
The results were correlated with compound structures. Degrada-
tion products (Table 2) (benzoxazolinones, aminophenoxazines,
hydroxyphenylmalonamic acids) were included in order to
evaluate their phytotoxic effect and to compare it with the
chemicals from which they were transformed. This would allow
us to establish their roles in Gramineae chemical defense. The
relation between phytotoxic effect and soil persistence of
degradation products investigated by us allowed the drawing
of interesting conclusions about the ecological role of DIMBOA,
DIBOA, and their main derivatives. All of these data will allow
us to obtain an accurate scope of the allelopathic phenomena
associated to benzoxazinoids and their derivatives present in
cultivars and to select which chemical structures would be useful
to develop new herbicide models.
Although many bioactivity data have been presented in the
literature for benzoxazinones and their degradation derivatives,
the development of natural herbicide models based on their
structures needs a complete study in which all compounds
involved in plant defense and detoxification routes could be
compared. This would also allow us to elucidate the different
ecological roles of the degradation products. Then, a complete
SAR study was designed on the basis of their phytotoxic
bioactivities. Several nonnatural (2H)-1,4-benzoxazin-3(4H)-one
MATERIALS AND METHODS
General Methods. The purity of the assayed compounds was
determined by 1NMR and HPLC analyses and was found to be >98%.

SAR Studies of Benzoxazinones
J. Agric. Food Chem., Vol. 53, No. 3, 2005 541
Scheme 1. Summary of Reactions and Conditions for the Synthesis of 2-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA) from 2-Nitrophenol (I)
1H and 13C NMR spectra were recorded using MeOH-d4 as solvent in
a Varian INOVA spectrometer at 399.99 and 100.577 MHz, respec-
tively. Chemical shifts are given in ppm with respect to residual CH3OH
or CD3OD signals (δ = 3.30 and 49.0, respectively). For HPLC analysis,
an HPLC PDA detector (diode array UV-vis system), a column
Phenomenex SYNERGI 4 µm Fusion RP-80 (250 mm × 460 mm),
and a Varian 1200L quadrupole MS/MS detector were used.
Scheme
2.
2-Hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one
(HMBOA) Was Obtained from DIMBOA after Reduction with Samarium
Diiodide
Isolation and Synthesis of Natural Allelochemicals, Degradation
Products, and Their Derivatives. The studied chemicals belong to
three different groups representing natural benzoxazinones, their
degradation products, and several nonnatural analogues:
Hydroxyphenylmalonamic Acids (Table 2, C). They have been
synthesized in our laboratory (Scheme 3) by employing a modification
from a previously reported procedure (18). Although this reported
method leads directly to both compounds, we wanted to modify it in
the search with the main objective of controlling protection and
deprotection of both phenolic and ammine moiety, so that we could
access to modified 2-aminophenol with selectivity. We also wanted to
avoid aminophenol dimerization, which would lead to aminophenox-
azines, as commented below. Thus, we got access to tert-butyldim-
ethylsilyloxyanilines (both 5-methoxy and 5-H), and after catalytic
hydrogenation, amidation and basic hydrolysis, both malonamic acids
were obtained in high yield (68%).
Natural Benzoxazinones (Table 1, A). DIBOA and DIMBOA were
obtained from natural sources by means of previously reported isolation
procedures (21, 22) modified by us. The DIBOA natural glycoside (2-
O-â-D-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DI-
BOA-Glc) was isolated from natural sources. Its isolation protocol,
adapted from the literature, has been already described by us regarding
its degradation study in wheat crop soil (10).
Biosynthetic Precursors (Table 1, B) and Degradation Products
(Table 2). They have been selected according to the precedents
mentioned above, belonging to four different structural types:
Aminophenoxazin-3-ones (Table 2, B). APO and AAPO have been
synthesized in large scale by using previously reported procedures (16).
2-Amino-7-methoxyphenoxazin-3-one (AMPO) and 2-acetamido-7-
methoxyphenoxazin-3-one (AAMPO) were obtained in our laboratory
by novel synthesis methods (Scheme 4). In the search for 5-methoxy-
2-aminophenol, we proceeded with the reduction of the corresponding
nitro derivative 5-methoxy-2-nitrophenol, employing conditions similar
to those shown for reductive cyclization mentioned above which are
compatible with that reduction. Otherwise, 70% yield of the dimer
AMPO was obtained. Further amine acylation yielded AAMPO. The
investigation of the Pd catalyst role in the dimerization process is one
of our current efforts regarding aminophenoxazines chemistry.
Nonnatural 1,4-Benzoxazin-3-one Analogues (Table 1, C). Six
synthetic analogues with a 1,4-benzoxazin-3-one skeleton have been
included in the study with the main purpose of studying the influence
of hydroxyl groups at positions C-2 and N-4 of benzoxazinones with
hydroxamic acid moiety and benzoxazinoid lactams: D-DIBOA,
4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3-(4H)-one (D-DIMBOA),
(2H)-1,4-benzoxazin-3-(4H)-one (D-HBOA) and 7-methoxy-(2H)-1,4-
benzoxazin-3(4H)-one (D-HMBOA). ABOA and 4-acetoxy-7-methoxy-
(2H)-1,4-benzoxazin-3(4H)-one (AMBOA) have been included to study
the influence of an N-OH blockage in the bioactivity. The lack of
hydroxyl group at position C-2 is common for all these derivatives
due to its presence in natural benzoxazinones being directly associated
to the proposed degradation processes mechanisms (24). Thus, these
compounds are stable models for the 1,4-benzoxazin skeleton. They
have been obtained by synthetic methods adapted from the literature
as commented above. The changing of the reductive agent-starting
material ratio afforded either hydroxamic acids or lactams at N-4 at
the reductive cyclization step (Scheme 1, Step b) (1:1 and 1:2,
respectively).
Benzoxazolin-2-ones (Table 2, A). Both of them are commercial
compounds. They were purchased from Fluka Chemika and Lancaster
Synthesis, respectively. They were used as received.
Benzoxazinoid Lactams (Table 1, B). They have been obtained at
our laboratory by means of novel synthesis procedures in amounts
enough to elucidate their phytotoxicity profiles. The synthetic route
employed for HBOA is described in Scheme 1. The key to this method
is an acyl [3.3] sigmatropic rearrangement from 4-acetoxy-(2H)-1,4-
benzoxazin-3(4H)-one (ABOA, IV), which allowed functionalization
at position C-2 (Step d). This reaction was previously described by
Hashimoto et al. (23). Changing benzene for toluene as solvent allowed
a higher reflux temperature, and reaction yield increased from the
original 30% to 70%. The resulting acetyl ester at C-2 was selectively
cleaved, searching for the preservation of the lactam moiety, by using
magnesium methoxide in methanol at room temperature (Step e, 90%
yield). This afforded the desired HBOA after five reaction steps with
55% yield. 4-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA, III)
was obtained by adapting the method described by Atkinson et al.
(24), in which THF in Step a was substituted with DMF in the search
for a higher alkoxide solubility. Reductive cyclization (Step b) was
also modified by changing the sodium borohydride proportion, so
that the hydroxamic acid moiety was preferently obtained instead of
the lactam. A double proportion of reductive agent provided benzox-
azinoid lactams in a selective manner. This procedure is general for
all the 2-deoxy derivatives indicated below. DIMBOA analogues were
obtained by changing starting material 2-nitrophenol by 5-methoxy-
2-nitrophenol.
Nevertheless, this procedure was not valid for HMBOA (Table 1,
B) synthesis since acyl sigmatropic rearrangement was not successful.
Thus, a hemisynthesis from natural DIMBOA was optimized with just
one reaction step (Scheme 2). The N-OH moiety of natural DIMBOA
was reduced to N-H by employing samarium diiodide, a reagent
described for selective reduction of oximes, hydroxylamines, and
hydroxamic acids (25), which has never been employed in this kind of
compounds.
Additional Compounds EValuated. To characterize its bioactivity and
to discuss the phytotoxicity of DIBOA degradation route chemicals,
2-aminophenol (Table 2, D) (purchased from Sigma-Aldrich, used as
received) was evaluated. 2-Amino-7-hydroxyphenoxazin-3-one (AHPO)
(Table 2, B) was included in the study of the activity-lipophilicity

542 J. Agric. Food Chem., Vol. 53, No. 3, 2005
Mac´ıas et al.
Scheme 3. Summary of Reactions and Conditions for the Synthesis of Hydroxyphenylmalonamic Acids HPMA and HMPMA
Scheme 4. Summary of Reactions and Conditions for the Synthesis of 7-Methoxyphenoxazines from 5-Methoxy-2-nitrophenol
relationships for aminophenoxazines described below. It was obtained
from AMPO by demethylation with hydrobromic acid at 100 °C (99%
yield), and its phytotoxicity was evaluated.
the compounds in study. Control samples (buffered aqueous solutions
with DMSO and without any tested compound) were used for all the
plant species assayed.
Bioassay Data Acquisition. Evaluated parameters (germination rate,
root length, and shoot length) were recorded by using a Fitomed system
(26) that allowed automatic data acquisition and statistical analysis by
its associated software.
Statistical Analysis. Data were statistically analyzed using Welch’s
test, with significance fixed at 0.01 and 0.05. They are presented as
percentage differences from control. Zero represents control, positive
values represent stimulation of the studied parameter, and negative
values represent inhibition (20, 26).
Once the germination and growth data are acquired, cluster analysis
was used to group compounds with similar phytotoxicity behaviors and
associate them with their molecular structure. Complete linkage was
used as amalgamation rule and the distance measurement was based
on squared euclidean distances (27), given by this equation:
Phytotoxic Activity Bioassay. Target Plants. Selection of target
plants is based on an optimization process made by us in the search
for a standard phytotoxicity evaluation bioassay (20). After this process,
several STS were proposed, including monocots Triticum aestiVum L.
(wheat) and Allium cepa L. (onion) and dicots Lycopersicon esculentum
Will. (tomato), Lepidium satiVum L. (cress) and Lactuca satiVa L.
(lettuce), which were assayed for this study.
Methodology. Bioassays used Petri dishes (90 mm diameter) with
one sheet of Whatman No.1 filter paper as support. Germination and
growth were conducted in aqueous solutions at controlled pH by using
10-2 M 2-[N-morpholino]ethanesulfonic acid (MES) and NaOH 1 M
(pH ) 6.0). Solutions (0.2, 0.1, 0.02, 0.01, and 0.002 M) of the
compounds to be assayed were prepared in DMSO and then diluted
with buffer (5 µL DMSO/mL buffer) to reach the test concentrations
for each compound (10-3, 5 × 10-4, 10-4, 5 × 10-5, and 10-5 M).
This procedure facilitated the solubility of the assayed compounds. The
number of seeds in each Petri dish depended on the seed size. Twenty
five seeds were used for tomato, lettuce, cress, and onion and 10 seeds
were used for wheat. Five milliliters of treatment, control, or internal
reference solution were added to each Petri dish. Four replicates were
used for tomato, cress, onion, and lettuce (100 seeds); 10 replicates
(100 seeds) were used for wheat.
After adding seeds and aqueous solutions, Petri dishes were sealed
with Parafilm to ensure closed-system models. Seeds were further
incubated at 25 °C in a Memmert ICE 700 controlled-environment
growth chamber in the absence of light. Bioassays took 4 days for cress,
5 days for lettuce, tomato, and wheat, and 7 days for onion. After
growth, plants were frozen at -10 °C for 24 h to avoid subsequent
growth during the measurement process. This helped the handling of
the plants and allowed a more accurate measurement of root and shoot
lengths.
d(x,y) )
(xi - yi)2
∑
i
Where d(x,y) is the squared euclidean distance (i-dimensional), i
represents the number of variables, and x and y the observed values.
The cluster was obtained by using Statistica v. 5.0 software. The
lack of consistent effects over germination for almost all assayed species
forced us to make cluster analysis on the basis of growth paramenters
(root length and shoot length). Results for all STS, except lettuce, which
did not show good correspondence between concentration and phyto-
toxic effect in some cases, were included in order to acquire an overall
view of the phytotoxicity and its relation with chemical structure.
EC50 values were obtained after adjusting phytotoxicity data to
concentration (logarithmic scale), to a sigmoidal dose-response curve,
defined by the equation:
The commercial herbicide Logran, a combination of N-(1,1-
dimethylethyl)-N′-ethyl-6-(methylthio)-1,3,5-triazine-2,4-diamine (Ter-
butryn, 59.4%) and 2-(2-chloroethoxy)-N-[[(4-methoxy-6-methyl-1,3,5-
triazin-2-yl)amino]carbonyl]benzenesulfonamide (Triasulfuron, 0.6%)
was used as internal reference, according to a comparison study
Ymax - Ymin
1 + 10log EC50-X
Y ) Ymin
+
Where X indicates the logarithm of the concentration, Y indicates the
response (phytotoxicity), and Ymax and Ymin are the maximum and
minimum values of the response, respectively. Goodness of fit is
previously reported (20). It was used at the same concentrations (10-3
,
5 × 10-4, 10-4, 5 × 10-5, and 10-5 M) and in the same conditions as

SAR Studies of Benzoxazinones
J. Agric. Food Chem., Vol. 53, No. 3, 2005 543
Figure 1. Phytotoxicity bioassay results (root length, % from control) for monocot species (Allium cepa L. and Triticum aestivum L.). If it is not indicated,
P > 0.05 for Welch’s test. (a) Values significantly differ with P < 0.01. (b) Values significantly differ with 0.01 < P < 0.05.
described by determination coefficient (r2). The adjustment and the r2
were obtained by using GraphPad Prism software v. 4.00.
Lipophilicity Calculations. Lipophillicity, expressed as log P (water/
n-octanol partition coefficient) value, was obtained by computational
degradation products, is not involved in intraspecific competition
phenomena for wheat. DIBOA-Glc and DIBOA moderately
affected wheat. Although these compounds and APO inhibited
wheat growth, the concentrations assayed here are significantly
methods according to the Ghose, Prichett, and Crippen methodology
higher than the natural ones according to several estimation
(28). This algorithm is implemented in Hyperchem v. 7.0 software.
previously discussed by us (9). Their synthetic analogues did
not affect wheat germination or growth, especially at the lowest
concentrations, so the chemicals belonging to this series could
RESULTS AND DISCUSSION
be good candidates for the development of new herbicide models
for wheat weed control. A similar structure-activity relation-
ships study for common wheat weeds (i.e., Lolium rigidum L.
and AVena fatua L.) is needed to confirm this hypothesis, and
it is currently taking place in our laboratory.
Phytotoxic Bioactivity Profiles. Effects of the assayed
compounds on monocots (root length) are shown in Figure 1.
The same parameter for cress and tomato is shown in Figure
2.The most affected parameter was root length for all the active
compounds, followed by shoot length. Effects over Lactuca
satiVa L. were diverse, but a good correspondence between
concentration and effect was not observed. Thus, lettuce growth
results were not included in the cluster analysis.
All chemicals showed inhibitory profiles, except AMPO and
its acetate AAMPO (Table 2, B) in cress and lettuce and BOA
and MBOA (Table 2, A) for wheat, although these effects are
not very consistent. Natural allelochemicals and their synthetic
analogues were the most active compound groups. The rest of
the analyzed chemicals, which are present in natural benzox-
azinone degradation routes, had moderate or null activity, except
APO (Table 2, B), which showed the highest inhibitory effects
of the degradation products.
The presence of wheat in the bioassay is especially valuable
since this would allow for discovering autotoxicity phenomenon
associated to these wheat allelochemicals and their potential
utility to weed management for wheat and analogous crops. All
assayed species were highly affected by the most active
chemicals, except wheat, which was moderately inhibited. This
selective behavior, specially observed for DIMBOA allows us
to conclude that this natural allelochemical, together with its
Benzoxazinones. DIBOA and its glycoside showed similar
inhibition profiles. Allium cepa was the most affected plant
according to inhibition recorded at high and at low doses (70%
and 91% inhibition for root length at 10-3 M, respectively).
These compounds maintained phytotoxicity at 5 × 10-4 M for
this plant (60% and 73% inhibition for root length at for DIBOA
and DIBOA-Glc respectively). DIBOA phytotoxic activity is
higher over roots than over shoots, and there is lower activity
over monocotyledonous that even disappear when the target
species is wheat. This fact clearly showed a lack of autotoxicity
for this compound. Regarding DIMBOA bioactivity, significant
inhibition levels were recorded for root lengths in all assayed
species, except wheat and lettuce (A. cepa: 68%; L. esculen-
tum: 86%; L. satiVum: 74% at 5 × 10-4 M.)
Benzoxazinoid Synthetic Analogues. The effects shown by
these compounds were highly inhibitory in the cases of L.
satiVum, L. esculentum, and A. cepa. DIBOA-related compounds
(D-DIBOA, D-HBOA, and especially ABOA) (Table 1, C)
showed the highest inhibition levels, and the effects were
significantly preserved at 10-4 M for D-DIBOA and ABOA

544 J. Agric. Food Chem., Vol. 53, No. 3, 2005
Mac´ıas et al.
Figure 2. Phytotoxicity bioassay results (root length, % from control) for dicot species (L. sativum L. and L. esculentum Will.). If it is not indicated, P >
0.05 for Welch’s test. (a) Values significantly differ with P < 0.01. (b) Values significantly differ with 0.01 < P < 0.05.
(52%, 68%-A. cepa, root length). Almost complete inhibition
effects were recorded at higher concentrations (D-DIBOA, 90%;
D-HBOA, 86%; ABOA, 89%, A. cepa, root length, 10-3 M).
The N-acetyl derivative of D-DIBOA (ABOA) showed the most
important effect at low concentrations, as it can be observed
for A. cepa and L. satiVum root growth inhibition from 10-3 to
10-4 M. This product also presented interesting shoot inhibition
for L. esculentum, L. satiVa, A. cepa, and L. satiVum. D-HBOA
presented null effects at highly diluted treatments.
Degradation Products. The bioactivity observed for natural
benzoxazinones was not preserved for their degradation prod-
ucts. Benzoxazolinones BOA and MBOA were slightly inhibi-
tory at the highest concentration toward lettuce (root growth,
39%, 20%), and significant inhibition values were also observed
for onion (51%, 64%), tomato (70%, 68%), and cress (51%,
49%) roots at 10-3 M. All these effects were lost at any lower
concentrations. As in the case of DIBOA and DIMBOA, their
phytotoxic activity is higher over roots than over shoots, in
which the inhibitory effect over monocotyledonous species was
not shown. This fact agrees with other studies carried out in
lettuce about the mode of action of BOA (29). It is possible
that this compound causes an integrity disruption at a radicular
cell membrane system. When the target species is wheat, there
are even promoting effects.
crop soil degradation experiments carried out with DIMBOA
(9), and its phytotoxicity profile allows this compound to be
proposed as a detoxification metabolite. AAPO and AAMPO
(Table 2, B) showed some effect, but they were not significant
in comparison to the rest of the bioassay. This is in good
agreement with their detoxification role proposed in the literature
(31).
In contrast to these results, aminophenoxazine APO showed
strong inhibition profiles for root and shoot lengths of all assayed
species except wheat in which it hade moderate effect over root
length and lettuce in which germination rate was the most
affected parameter (76% at 10-3 M and 78% at 10-4 M) and
growth was moderately inhibited (∼50% at 10-3 M). Effects
over tomato (EC50 ) 153 µM, r2 ) 0.95), onion (EC50 ) 72.9
µM, r2 ) 0.99), and cress (EC50 ) 38.2 µM, r2 ) 0.99) roots
are especially significant. There was also high inhibition in shoot
length for these three species.
Structure-Activity Relationships. Cluster analysis for
growth results in STS for all compounds is shown in Figure 3.
Activities can be divided in two main groups (G1 for high and
moderate activities and G2 for low effects). G1 is divided into
two subgroups (G1A for the highest effects and G1B for
moderate inhibitions).
The most active group (G1A) is formed by the commercial
herbicide Logran, aminophenoxazin APO (Table 2, B), three
2-deoxy derivatives (ABOA, D-DIBOA, and D-DIMBOA)
(Table 1, C), and natural benzoxazinones DIBOA and
DIMBOA (Table 1, A). Moderate activity levels were recorded
for acetamidophenoxazin AAPO (Table 2, B) benzoxazolinones
(Table 2, A), DIBOA-Glc (Table 1, A), 2-deoxy lactams (D-
HBOA and D-HMBOA), and AMBOA (Table 1, C). The lower
effects were observed for malonamic acids HPMA and HMPMA
Benzoxazinoid lactams (HBOA, HMBOA) (Table 1, B) and
malonamic acids (HPMA, HMPMA) (Table 2, C) were not
significantly active in any case. HBOA has been proposed as
an intermediate in DIBOA biosynthesis, and its lack of
phytotoxic effect agrees with the idea that the 4-hydroxy group
of benzoxazinoids is crucial for the biological activity. HMBOA
is not a precursor for DIMBOA synthesis, as it would be
expected by its structure (30). It has been detected in wheat

SAR Studies of Benzoxazinones
J. Agric. Food Chem., Vol. 53, No. 3, 2005 545
Figure 3. Cluster analysis for STS growth inhibition (effects on root length and shoot length); (a) benzoxazinones; (b) benzoxazolinones; (c)
aminophenoxazines; and (d) malonamic acids.
(Table 2, C), lactams HBOA and HMBOA (Table 1, B), and
7-methoxyphenoxazines AMPO and AAMPO (Table 2, B).
Benzoxazines. The synthetic benzoxazines (Table 1, C) have
been selected to study the influence of the different functional
group combinations at C-7, C-2, and N-4. The 7-methoxyben-
zoxazines can be structurally correlated with DIMBOA and
HMBOA, while the lack of functional group at C-7 corresponds
to the DIBOA and HBOA series. Both of these groups have
compounds with the 2-hydroxy moiety (D-HMBOA and D-
HBOA, respectively), 4-hydroxy (D-DIMBOA and D-DIBOA),
and 2-acetoxy (AMBOA and ABOA). Their activities can be
correlated with the ones observed for the natural products
DIMBOA and DIBOA, together with their benzoxazolinones
MBOA and BOA (Table 2, A).
DIMBOA and DIBOA have low activities, especially in
wheat, lettuce, and cress. There is no evidence about the
degradation of 2-deoxy derivatives of benzoxazinones in any
conditions, while DIMBOA and DIBOA are degraded to their
benzoxazolinones. Their first degradation products, BOA and
MBOA, have activities much lower than them. 2-Deoxy
derivatives are stable through all the assay time, while DIMBOA
and DIBOA are degraded to their benzoxazolinones (31).
D-DIMBOA and D-DIBOA have very similar effects, and
they could be candidates for further development of herbicide
models based on the 1,4-benzoxazine skeleton. Different
functional groups at N-4 have to be assayed for bioactivity
enhancement, as ABOA results suggest. The observed phyto-
toxicity values lead to structural modifications at DIBOA
derivatives in the search for the most inhibitory compounds.
This fact will lead us to a more detailed study of 2-deoxy
derivatives and their properties and biological effects. The lack
of a hydroxyl group at position 2 enhances bioactivity. It can
be also observed by comparing 2-hydroxy lactams (HBOA and
HMBOA, with low or null activities) and their 2-deoxy
analogues (D-HBOA and D-HMBOA, with moderate inhibition
levels).
stabilized electrophilic intermediate (23), but the low phyto-
toxicity results obtained for the N-4-acetoxy derivative of
D-DIMBOA (AMBOA) suggests the opposite statement. This
modification does not enhance phytotoxicity at 7-methoxyben-
zoxazinones.
Aminophenoxazines. 2-Aminophenoxazin-3-one (APO) is
the only aminophenoxazin belonging to G1A group (Figure 3).
Its acetate (AAPO) has moderate activity (G1B) and 7-meth-
oxyphenoxazines AMPO and AAMPO are the least active
compounds of the whole assay.
Previous works about the occurrence of aminophenoxazin
skeleton (Table 2, B) in the degradation processes of natural
benzoxazinones suggest acetamidophenoxazines to be detoxi-
fication products of the amines APO and AMPO (31). This
hypothesis can be accepted in the case of APO and AAPO due
to the lack of activity observed for the last one. Phytotoxicity
for AMPO is much lower than expected in comparison to its
nonmethoxylated analogue APO. Taking into account the
similarity of APO and AMPO structures, their big differences
in activity could be due to other phenomena, like aqueous and
lipid solubility and their consequences regarding chemicals’
diffusion through the cell membrane.
According to Lipinskii (32) and Tice (33) models for
bioactivity in pharmaceuticals and agrochemicals, the solubility
in lipids or aqueous media is a very important parameter in the
search for bioactive compounds. These solubilities can be
quantified by using log P (water/n-octanol partition coefficient).
Previous studies performed by us regarding bioactivity of
sesquiterpene lactones allowed us to establish correlations
between log P and EC50 for several bioactivity parameters. Thus,
log P was theoretically calculated for all assayed aminophe-
noxazines (Table 2, B) by computational methods (28). To add
another analogous compound, phytotoxicity of 7-hydroxy-2-
aminophenoxazin-3-one (AHPO) was evaluated for L. esculen-
tum. A correlation between phytotoxicity values (root length
of Lycopersicon esculentum) and log P can be observed in
Figure 4. When log P > 0.6, a significant activity value can
be observed. For lower log P, effects are constrained between
+20 and -20%, which constitutes a drastic phytotoxicity loss.
In addition to this, we have observed precipitation for APO
solutions at concentrations higher than 5 × 10-4 M. This would
The high bioactivity levels shown by the N-acetoxy derivative
of D-DIBOA (ABOA) could lead future research in 4-hydroxy-
1,4-benzoxazin structure modification. Some authors have
suggested the presence of this acetyl leaving group at the N-4
position to enhance toxicity of these 2-deoxy benzoxazinones,
acting as leaving group and provoking the formation of a highly

546 J. Agric. Food Chem., Vol. 53, No. 3, 2005
Mac´ıas et al.
Figure 5. Phytotoxicity (Allium cepa L., root length) and persistence in
soil (half-life) for DIBOA degradation series compounds in wheat crop
soil.
Conclusions. Regarding new herbicide model development,
benzoxazinones constitute the most interesting group since they
are afforded by high-yield and easy-to-scale synthetic methods.
In addition to this, their molecular structure and functionalization
permits different modifications at the heterocycle and the
aromatic ring, so a large number of candidates could be
developed and tested in further research about this matter.
The bioactivity profiles shown by benzoxazinones suggest
2-deoxy derivatives of natural allelochemicals DIBOA and
DIMBOA (D-DIMBOA and D-DIBOA) (Table 1, C) to be the
best leads for new herbicides with this structural base. D-DIBOA
matches all the conditions that increase phytotoxicity on the
species assayed: lack of hydroxyl group at C-2, hydroxyl at
N-4, and absence of a methoxy group in the aromatic ring. The
fact that acetylation of the hydroxyl group at position N-4
increases phytotoxicity of this compound’s analogues, points
to an interesting direction regarding further chemical modifica-
tions of this structure with the purpose of phytotoxicity
enhancement. The N-OH moiety can constitute the base of a
more accurate adequation of this benzoxazinones to the Lipinskii
(32) and Tice (33) rules mentioned above. The introduction of
longer side chains via esterification or etherification could
increase molecular weight, as well as lipophillicity. The lower
phytotoxicity levels shown by 2,4-dihydroxy compounds (DIM-
BOA and DIBOA) in some cases could be due to degradation
phenomena leading to less active compounds such as BOA or
MBOA, which are much more stable. A degradation study for
these compounds in the buffer employed in these bioassays will
allow us to elucidate the role of degradation processes in DIBOA
and DIMBOA bioactivity evaluation.
Figure 4. Phytotoxicity (Lycopersicon esculentum Will., root length) (A)
and lipophilicity (log P) (B) for aminophenoxazines.
explain the similarity observed for phytotoxic activities at the
two higher concentrations for almost all tested bioactivity
parameters.
Ecological Role of Benzoxazinones and Degradation
Products. DIBOA-Glc, DIBOA, BOA, APO, and AAPO,
together with 2-aminophenol (APH) constitute a complete
degradation series carried out by soil microbes, as well as by
other organisms (31). DIBOA-Glc is produced by the plant and
liberated as the aglucon DIBOA, which participates in the
different degradation processes (7, 9, 10). Thus, it is interesting
to compare bioactivity values for the complete series since all
these compounds are together in soil while the degradation
process takes place. This comparison would lead us to describe
the role that soil microbes could play in the chemical defense
strategies employed by plants with high production of benzox-
azinones. The half-lives of compounds were obtained by means
of the wheat crop soil degradation study mentioned above (10).
The phytotoxicity values, expressed as root growth inhibition
for Allium cepa and the half-life values observed for each
compound (except for DIBOA-Glc, which is not released to
soil, and AAPO, which was not detected in our degradation
experiments) are shown in Figure 5. The high persistence in
soil shown by APO, much higher than its precursors, in addition
to its high phytotoxicity, leads us to assign a critical role
regarding chemicals defense in DIBOA producer plants, other
than DIBOA or BOA, that have been proposed as the main
responsibles for several plant-plant interaction phenomena (6,
34, 35). The occurrence of degradation in buffered aqueous
solution (8, 10) also enforces a careful revision of the bioassay
methodologies employed in the phytotoxicity evaluation of 2,4-
dihydroxybenzoxazinones and benzoxazolinones since the ac-
tivities observed could be due to degradation products instead
of the original materials dosed.
Aminophenoxazin APO was the most active degradation
compound, with high phytotoxicity levels and also high
maintenance of the effect at low concentrations. The lack of
phytotoxic effect observed for its structurally related compounds,
such as AMPO and AHPO, could be related to their lipophilia.
The effects observed for acetamidophenoxazines AAPO and
AAMPO confirm them as detoxification derivatives. Their
bioactivity values are in good accordance with the correlation

SAR Studies of Benzoxazinones
J. Agric. Food Chem., Vol. 53, No. 3, 2005 547
(6) Wolf, R. B.; Spencer, G. F.; Plattner, R. D. Benzoxazolinone,
2,4-dihydroxy-1,4-benzoxazin-3-one, and its glucoside from
Acanthus mollis seeds inhibit velvetleaf germination and growth.
J. Nat. Prod. 1985, 48, 59-63.
(7) Cambier, V.; Hance, T.; De Hoffmann, E. Noninjured maize
contains several 1,4-benzoxazin-3-one related compounds but
only as glucoconjugates. Phytochem. Anal. 1999, 10, 119-126.
(8) Woodward, M. D.; Corcuera, L. J.; Helgeson, J. P.; Upper, C.
D. Decomposition of 2,4-dihydroxy-7-methoxy-2H-1,4-benzox-
azin-3(4H)-one in aqueous solutions. Plant Physiol. 1978, 61,
796-802.
(9) Mac´ıas, F. A.; Oliveros-Bastidas, A.; Mar´ın, D.; Castellano, D.;
Simonet, A. M.; Molinillo, J. M. G. Degradation Studies on
Benzoxazinoids. Soil Degradation Dynamics of 2,4-Dihydroxy-
7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) and Its
Degradation Products, Phytotoxic Allelochemicals from Gramine-
ae. J. Agric. Food Chem. 2004, 52, 6402-6413.
(10) Mac´ıas, F. A.; Oliveros-Bastidas, A.; Mar´ın, D.; Castellano, D.;
Simonet, A. M.; Molinillo, J. M. G. Degradation studies on
benzoxazinoids. Soil degradation dynamics of (2R)-2-O-beta-
D-glucopyranosyl-2,4-dihydroxy-(2H)-1,4-Benzoxazin-3(4H)-
one (DIBOA-Glc) and its degradation products, phytotoxic
allelochemicals from Gramineae. J. Agric. Food Chem. 2005,
53, in press.
(11) Peng, S.; Chilton, W. S. Biosynthesis of DIMBOA in maize using
deuterium oxide as a tracer. Phytochemistry 1994, 37, 167-171.
(12) Kumar, P.; Moreland, D. E.; Chilton, W. S. 2H-1,4-benzoxazin-
3(4H)-one, an intermediate in the biosynthesis of cyclic hydrox-
amic acids in maize. Phytochemistry 1994, 36, 893-898.
(13) Bravo, R.; Lazo, W. Antialgal and antifungal activity of natural
hydroxamic acids and related compounds. J. Agric. Food Chem.
1996, 44, 1569-1571.
(14) Sahi, S. V.; Chilton, M.; Chilton, W. S. Corn metabolites affect
growth and virulence of Agrobacterium tumefaciens. Proc. Natl.
Acad. Sci. U.S.A. 1990, 87, 3879-3883.
(15) Zikmundova, M.; Drandarov, K.; Bigler, L.; Hesse, M.; Werner
C. Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4-
benzoxazin-3-one by endophytic fungi isolated from Aphelandra
tetragona. Appl. EnViron. Microb. 2002, 68, 4863-4870.
(16) Gagliardo, R. W.; Chilton, W. S. Soil transformation of 2(3H)-
benzoxazolone of rye into phytotoxic 2-amino-3H-phenoxazin-
3-one. J. Chem. Ecol. 1992, 18, 1683-1691.
with lipophilia expressed above. The high persistence of this
compound in wheat crop soil recorded by us (10) makes this
compound a weak candidate for new herbicide models develop-
ment at least at this research stage. Further research will be
needed to discover its utility in pest management. Taking into
consideration reports from the literature (31) describing deg-
radation for aminophenoxazines, which proceeds with detoxi-
fication, as phytotoxicity for AAPO and AAMPO data show,
further research is needed to determine the final fate of
aminophenoxazines in soil and other environments. Antifungal
activity shown by APO (31) could affect its own degradation
dynamics in soil, at least at the higher dosed employed in its
degradation experiments (10).
Nevertheless, APO showed high phytotoxicity and also higher
persistence in soil that its precursors BOA and DIBOA. These
facts allow us to conclude that some of the allelopathic behaviors
observed in plants that produce DIBOA could be due to its
degradation compound rather than the compound originally
released by plant. The low phytotoxicity of AMPO points to
the opposite direction regarding the DIMBOA series, so a more
accurate study about its interaction with soil biological popula-
tion and inorganic materials will be needed to discover its
ecological role. This fact, in addition to the higher persistence
in soil shown by DIBOA degradation series compounds (10),
allows us to conclude that DIBOA producing species take more
advantage of allelopathic defense strategies based on benzox-
azinones than DIMBOA producing ones.
The many different bioactivities shown by degradation
products of DIBOA and DIMBOA have to be taken into account
in the research of their biological activities and mode of action
since the effects observed could be related to the presence of
such derivatives in stock solutions and standards employed for
these evaluations, especially in the longer time experiments.
Our current efforts are directed toward the evaluation of these
compounds over common wheat weeds and other target organ-
isms.
ACKNOWLEDGMENT
(17) Kumar, P.; Gagliardo, R. W.; Chilton, W. S. Soil transformation
of wheat and corn metabolites MBOA and DIM2BOA into
aminophenoxazinones. J. Chem. Ecol. 1993, 19, 2453-2461.
(18) Friebe, A.; Vilich, V.; Hennig, L.; Kluge, M.; Sicker, D.
Detoxification of benzoxazolinone allelochemicals from wheat
by Gaeuannomyces graminis var. tritici, G. graminis var.
graminis, G. graminis var. avenae, and Fusarium culmorum.
Appl. EnViron. Microb. 1998, 64, 2386-2391.
(19) Yue, Q.; Bacon, C. W.; Richardson, M. D. Biotransformation
of 2-benzoxazolinone and 6-methoxy-benzoxazolinone by Fusar-
ium moniliforme. Phytochemistry 1998, 48, 451-454.
(20) Macias, F. A.; Castellano, D.; Molinillo, J. M. G. Search for a
standard phytotoxic bioassay for allelochemicals. Selection of
standard target species. J. Agric. Food. Chem. 2000, 48, 2512-
2521.
This work was financially supported by the program ‘Quality
of life and management of living resources (1998 to 2002)’ of
the European Union, FATEALLCHEM Contract No. QLRT-
2000-01967. Fellowships from Universidad de los Andes-
Venezuela (A. O. B.), European Comission (European Union),
and Junta de Andaluc´ıa-Spain (D. M.) are also gratefully
acknowledged.
LITERATURE CITED
(1) Virtanen, A. I.; Hietala, P. K. The structures of the precursors
of benzoxazolinone in rye plants. II. Suom. Kemistil. B 1959,
32B, 252.
(21) Larsen, E.; Christensen, L. P. Simple method for large scale
isolation of the cyclic arylhydroxamic acid DIMBOA from maize
(Zea mays L.). J. Agric. Food Chem. 2000, 48, 2556-2558.
(22) Barnes, J. P.; Putnam, A. R. Role of benzoxazinones in
allelopathy by rye (Secale cereale L.). J. Chem. Ecol. 1987, 13,
889-906.
(23) Hashimoto, Y.; Ishizaki, T.; Seudo, K.; Okamoto, T. Rearrange-
ment of 4-acetoxy-2H-1,4-benzoxazin-3(4H)-one. Chem. Pharm.
Bull. 1983, 31, 3891-3896.
(24) Atkinson, J.; Morand, P.; Arnason, J. T.; Niemeyer, H. M.; Bravo,
H. R. Analogues of the cyclic hydroxamic acid 2,4-dihydroxy-
7-methoxy-2H-1,4-benzoxazin-3-one (DIMBOA): decomposi-
tion to benzoxazolinones and reaction with â-mercaptoethanol.
J. Org. Chem. 1991, 56, 1788-1800.
(2) Hamilton, R. H.; Bandurski, R. S.; Reusch, W. H. Isolation and
characterization of a cyclic hydroxamate from Zea mays. Cereal
Chem. 1962, 39, 107-113.
(3) Honkanen, E.; Virtanen, A. I. Synthesis of some 1,4-benzoxazine
derivatives and their antimicrobial activity. Acta Chem. Scand.
1960, 14, 1214-1217.
(4) Escobar, C. A.; Sicker, D.; Niemeyer, H. M. Evaluation of
DIMBOA analogues as antifeedants and antibiotics towards the
aphid Sitobion aVenae in artificial diets. J. Chem. Ecol. 1999,
25, 1543-1554.
(5) Schulz, M.; Friebe, A.; Kueck, P.; Seipel, M.; Schnabl, H.
Allelopathic effects of living quack grass (Agropyron repens L.).
Identification of inhibitory allelochemicals exuded from rhizome
borne roots. Angew. Bot. 1994, 68, 195-200.

548 J. Agric. Food Chem., Vol. 53, No. 3, 2005
Mac´ıas et al.
(25) Keck, G. E.; Wager, T. T.; McHardy, S. F. Reductive cleavage
of N-O bonds in hydroxylamines and hydroxamic acid deriva-
tives using samarium diiodide. Tetrahedron 1999, 55, 11755-
11772.
(26) Castellano, D.; Mac´ıas, F. A.; Castellano, M.; Cambronero, R.
FITOMED (Automated system for the measurement of Variable
lengths). Patent No. P9901565.
(27) Mac´ıas, F. A.; Velasco, R. F.; Vin˜olo, V. M. I.; Castellano, D.;
Galindo, J. C. G.; Molinillo, J. M. G. Cluster analysis. A valuable
tool for allelopathic SAR studies? In Allelopathy: from molecules
to ecosystems; Reigosa, M. J., Pedrol, N., Eds.; Science Publish-
ers, Inc.: Enfield, NH, 2002.
(28) Ghose, A. K.; Pritchett, A.; Crippen, G. M. Atomic physico-
chemical parameters for three-dimensional structure directed
quantitative structure-activity relationships III: modeling hy-
drophobic interactions. J. Comput. Chem. 1988, 9, 80-90.
(29) Sa´nchez-Moreiras, A. M.; Coba de la Pen˜a, T.; Mart´ınez, A.;
Gonza´lez, L.; Pellisier, F.; Reigosa, M. J. Mode of action of the
hydroxamic acid BOA and other related compounds. In Allel-
opathy; Mac´ıas, F. A., Ed.; CRC Press LLC: Boca Raton, FL,
2004; pp 239-252.
(31) Fomsgaard. I. S.; Mortensen, A. G.; Carlsen, S. C. K. Microbial
transformation products of benzoxazolinone and benzoxazinone
allelochemicals-a review. Chemosphere 2004, 54, 1025-1038.
(32) Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J.
Experimental and computational approaches to estimate solubility
and permeability in drug discovery and development settings.
AdV. Drug DeliVery ReV. 2001, 46, 3-26.
(33) Tice, C. M. Selecting the right compounds for screening: Does
Lipinski’s rule of 5 for pharmaceuticals apply to agrochemicals?
Pest Manage. Sci. 2001, 57, 3-16.
(34) Barnes, J. P.; Putnam, A. R.; Burke, B. A. Allelopathy activity
of rye (Secale cereale L.). In The Science of Allelopathy; Putnam,
A. R., Tang, C. S., Eds.; Wiley-Interscience: New York, 1986;
pp 271-286.
(35) Sicker, D.; Frey, M.; Schulz, M.; Gierl, A. Role of natural
benzoxazinones in survival strategies of plants. In International
reView of cytologysA surVey of cell biology; Jeong, K. W., Ed.;
Academic Press: San Diego, CA, 2000; Vol. 198, pp 319-346.
Received for review September 22, 2004. Revised manuscript received
November 19, 2004. Accepted November 22, 2004.
(30) Glawischnig, E.; Gru¨n, S.; Frey, M.; Gierl, A. Cytochrome P450
monooxygenases of DIBOA biosynthesis: specificity and con-
servation among grasses. Phytochemistry 1999, 50, 925-930.
JF0484071