Isolation and synthesis of allelochemicals from gramineae: Benzoxazinones and related compounds

Article abstract of DOI:10.1021/jf050896x

Compounds with a (2H)-1,4-benzoxazin-3(4H)-one skeleton have attracted the attention of phytochemistry researchers since 2,4-dihydroxy-(2H)-1,4-benzoxazin- 3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) were isolated from plants belonging to the Poaceae family. These compounds exhibit interesting biological properties, such as phytotoxic, antimicrobial, antifeedant, antifungal, and insecticidal properties. These chemicals, in addition to a wide variety of related compounds involved in their metabolism, detoxification mechanisms, and degradation on crop soils and other systems, have high interest and in some cases potential agronomic utility. This paper presents a complete review of the methods employed for their synthetic obtention in addition to some of the authors' own contributions to their chemistry. The degradation and phytotoxicity experiments carried out in ongoing research into the potential agronomic utility of these compounds required large amounts of them, which were obtained from natural sources. This paper presents a modified methodology to access DIMBOA from Zea mays cv. Apache and to obtain 2-O-β-D-glucopyranosyl-2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and DIBOA from Secale cereale L. New synthetic methodologies were employed for the obtention of the lactams 2-hydroxy-(2H)-1,4-benzoxazin-3(4H)- one and 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one and the malonamic acids N-(2-hydroxyphenyl)malonamic acid and N-(2-hydroxy-7-methoxyphenyl)- malonamic acid. The aminophenoxazines 2-amino-7-methoxyphenoxazin-3-one and 2-acetamido-7-methoxyphenoxazin-3-one have been synthesized in the authors' laboratory by novel procedures. All of the methodologies employed allowed the desired compounds to be obtained in high yield and in an easy-to-scale manner.

Full text of DOI:10.1021/jf050896x

J. Agric. Food Chem. 2006, 54, 991−1000 991
Isolation and Synthesis of Allelochemicals from Gramineae:
Benzoxazinones and Related Compounds
FRANCISCO A. MACIÄAS,* DAVID MARIÄN, ALBERTO OLIVEROS-BASTIDAS,†
DAVID CHINCHILLA, 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
Compounds with a (2H)-1,4-benzoxazin-3(4H)-one skeleton have attracted the attention of phyto-
chemistry researchers since 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-
7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) were isolated from plants belonging to the
Poaceae family. These compounds exhibit interesting biological properties, such as phytotoxic,
antimicrobial, antifeedant, antifungal, and insecticidal properties. These chemicals, in addition to a
wide variety of related compounds involved in their metabolism, detoxification mechanisms, and
degradation on crop soils and other systems, have high interest and in some cases potential agronomic
utility. This paper presents a complete review of the methods employed for their synthetic obtention
in addition to some of the authors’ own contributions to their chemistry. The degradation and
phytotoxicity experiments carried out in ongoing research into the potential agronomic utility of these
compounds required large amounts of them, which were obtained from natural sources. This paper
presents a modified methodology to access DIMBOA from Zea mays cv. Apache and to obtain 2-O-
â-D-glucopyranosyl-2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and DIBOA from
Secale cereale L. New synthetic methodologies were employed for the obtention of the lactams
2-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one and 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one
and the malonamic acids N-(2-hydroxyphenyl)malonamic acid and N-(2-hydroxy-7-methoxyphenyl)-
malonamic acid. The aminophenoxazines 2-amino-7-methoxyphenoxazin-3-one and 2-acetamido-
7-methoxyphenoxazin-3-one have been synthesized in the authors’ laboratory by novel procedures.
All of the methodologies employed allowed the desired compounds to be obtained in high yield and
in an easy-to-scale manner.
KEYWORDS: Allelochemicals; DIMBOA; DIBOA; soil degradation products; wheat; isolation; synthesis;
preparation
INTRODUCTION
have new modes of action, high biodegradability, and also low
environmental impact (3).
The isolation, structural elucidation, and synthesis of natural
products constitute the main origin of new molecules with
biological activity. Biological effects of natural products are
very diverse. Pharmacological properties and the possibility to
control the development of natural or cultivated ecosystems have
acquired special relevancy in recent years (1).
Benzohydroxamic acids containing a 1,4-benzoxazin moiety
have attracted the attention of phytochemistry researchers since
the first isolation of 2,4-dihydroxy-7-methoxy-(2H)-1,4-ben-
zoxazin-3(4H)-one (DIMBOA) (4) and 2,4-dihydroxy-(2H)-1,4-
benzoxazin-3(4H)-one (DIBOA) (5) (Table 1A). These com-
pounds are present in the seedlings of several Poaceae as 2-O-
â-D-glucosides (6-8). The main hydroxamic acid in maize is
2-O-â-D-glucopyranosyl-4-hydroxy-7-methoxy-(2H)-1,4-ben-
zoxazin-3(4H)-one (DIMBOA-Glc, Table 1A) with lesser
amounts of 2-O-â-D-glucopyranosyl-4-hydroxy-(2H)-1,4-ben-
zoxazin-3(4H)-one (DIBOA-Glc, Table 1) and 2-O-â-D-glu-
copyranosyloxy-4-hydroxy-7,8-dimethoxy-(2H)-1,4-benzoxazin-
3(4H)-one (DIM2BOA-Glc, Table 1) (9). DIMBOA-Glc is also
the major cyclic hydroxamic acid in wheat, whereas DIBOA-
Glc is found in rye (10).
During the past 30 years, large efforts have been dedicated
to the discovery of new allelochemicals (natural plant toxins)
with potential application in weed management (2). The biocides
developed from allelochemicals have important advantages with
respect to traditional herbicides, fungicides, or insecticides: they
* Author to whom correspondence should be addressed (telephone +34-
956.016.370; fax +34-956.016.295; 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/jf050896x CCC: $33.50
© 2006 American Chemical Society
Published on Web 01/24/2006

992 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Table 1. Benzoxazinone Allelochemical Structures
Mac´ıas et al.
There are interesting works that deal with the biological
activities of benzohydroxamic acids (11-14) together with some
of their degradation derivatives (15, 16) in different environ-
ments and also synthetic analogues (17) investigated to discover
structure-activity relationships.
There are a wide variety of metabolites related to 1,4-
benzoxazin-3-ones, which have also attracted interest in phyto-
chemical and allelopathic studies. The benzoxazinone lactams
were first described by Niemeyer et al. (18). The most interesting
compounds with this base structure are 2-hydroxy-(2H)-1,4-
benzoxazin-3(4H)-one (HBOA) and 2-hydroxy-7-methoxy-(2H)-
1,4-benzoxazin-3(4H)-one (HMBOA) (Table 1A). They have
been proposed as biosynthetic precursors as well as degradation
products from benzoxazinones (19-21). The degradability of
benzoxazinones in different environments (21-26) moved the
attention to their degradation products, some of them being more
stable than the original plant metabolites.
(HMPMA) (Table 1D) have been proposed as fungal detoxi-
fication metabolites from benzoxazolinones BOA and MBOA,
respectively (29-31). Their detoxification role has been con-
firmed (17).
Natural Sources of DIMBOA, DIBOA, and Their Gluco-
sides. Most of the methods for benzoxazinone glycosides
isolation employed HPLC procedures (6, 7, 32-38), which have
analytical value being unsuitable for large-scale isolation,
according to Larsen and Christensen (39). They developed a
convenient high-scale purification procedure for DIMBOA-Glc
and DIMBOA from maize. We made some modifications (21,
23) to this process to obtain DIMBOA from maize, and novel
extraction methods were developed to produce DIBOA-Glc and
DIBOA from rye. The results of these procedures are com-
mented on below. The sources of the aglycones DIMBOA and
DIBOA are similar to those of their glucoside precursors (9,
10, 23, 33, 40-44).
The degradation products 2-benzoxazolinone (BOA) and
6-methoxy-2-benzoxazolinone (MBOA) (Table 1B) are pro-
duced by means of spontaneous degradation in aqueous solutions
(21-23, 27) as well as biological processes (21, 23, 24). There
are interesting works about their bioactivity (11, 14-17) and
mode of action (28). Benzoxazolinones, when degraded to the
corresponding 2-aminophenols, followed by further dimeriza-
tion, yield aminophenoxazines (Table 1C). They have been
found to occur in different crop soils (21, 23, 25, 26) as well as
by means of microbial transformations (29).
Naturally Occurring Benzoxazolinones, Aminophenox-
azines, and Malonamic Acids. BOA and MBOA (Table 1B)
have been isolated from plants of agronomic (6, 45-47) and
nonagronomic interest (37, 48).
Aminophenoxazines 2-aminophenoxazin-3-one (APO), 2-
amino-7-methoxyphenoxazin-3-one (AMPO), and their aceta-
mide derivatives (Table 1C) have been described as microbial
degradation products of the benzoxazolinones BOA and MBOA
(30, 49). From an agronomic point of view, the production of
these metabolites in crop soils by root colonizing bacteria (21,
23, 25, 26) has attracted attention to the elucidation of their
phytotoxicity and ecological role (17).
The malonamic acids N-(2-hydroxyphenyl)malonamic acid
(HPMA) and N-(2-hydroxy-4-methoxyphenyl)malonamic acid

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 993
Scheme 1. Overview of Benzoxazinone Synthetic Methods Based on Oxidation of 1,4-Benzoxazine Heterocycle in C-2
Scheme 2. Overview of Benzoxazinone Synthetic Methods Based on Cyclization of Functionalized Side Chains
The malonamic acids HPMA and HMPMA (Table 1D) have
been described as detoxification products produced by both
pathogenic (31) and nonpathogenic fungi (50) associated with
crop plants. Zikmundova´ et al. isolated HPMA from Aphelandra
tetragona (29).
proposed different methodologies for this purpose. HBOA and
HMBOA (Table 1A) were obtained in our laboratory with novel
methods (17) according to this strategy.
The need for large amounts of the benzoxazinone glycosides
also forced some researchers to develop interesting methods for
their synthetic obtention (58, 59).
Synthesis of Degradation Derivatives from Benzoxazin-
ones. Some efforts have been made in the benzoxazolinone
(Table 1B) derivatives synthetic obtention in the search for
antimicrobial agents (60-63).
The synthesis of a wide variety of compounds with an
aminophenoxazine skeleton has been extensively developed
more for their pharmaceutical interest (64-66) than for their
agronomical use (24). All of the synthetic methodologies
employed have the dimerization of two phenol units to access
the tricyclic structure in common (67-69), mainly dealing with
the synthesis of 2-aminophenoxazin-3-one (APO, Table 1C).
Taking into consideration the structural analogy of this
compound to 2-amino-7-methoxyphenoxazin-3-one (AMPO)
(Table 1C), there have not been a large number of efforts toward
its synthesis. In fact, it has been obtained just as a byproduct in
the search for organometallic reagents (69, 70).
Synthesis of Compounds with a (2H)-1,4-Benzoxazin-
3(4H)-one Skeleton. The synthetic methods employed to access
(2H)-1,4-benzoxazin-3(4H)-ones start with a functionalized
phenol, to which a suitable side chain is added (51). The first
successful method for the synthetic obtention of benzoxazinones
(52) employed several potassium 2-nitrophenoxides as starting
materials (Scheme 1, Jernow method). As an alternative, the
patent also suggested using a reductive cyclization method
developed by Coutts (53) (Scheme 1, Coutts method). The
reductive conditions are generated through sodium borohydride
and palladium over charcoal, suspended in aqueous dioxane.
This convenient method has become general for the obtention
of these compounds.
The need for a lactol deprotection step was avoided by three
alternative methods, which have the oxidation of the heterocycle
after its formation in common. Matlin et al. (54) (Scheme 1,
Matlin method), Sicker and Hartenstein (55) (Scheme 1, Sicker
method), Sicker (56) (Scheme 2, Sicker method), and again
Hartenstein and Sicker (57) (Scheme 2, Hartenstein method)
The detoxification metabolites HPMA and HMPMA (Table
1D) were synthesized by direct acylation of aminophenols (31).

994 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Mac´ıas et al.
Scheme 3. Summary of Reactions and Conditions Employed To Obtain 2-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA) and
N-(2-Hydroxyphenyl)malonamic Acid (HPMA)
To obtain DIBOA (Table 1A), the ethyl acetate extracts discarded
in the isolation of DIBOA-Glc were combined, dried on anhydrous
Na2SO4, concentrated in vacuo, and purified by column chromatography
(normal phase) by using chloroform/n-hexane increasing polarity
solutions (0-30% CHCl3). Fractions containing DIBOA were combined
and dried by distillation at reduced pressure. Recrystallization from
ethyl acetate/hexane afforded pure DIBOA.
Synthesis of Benzoxazinones and Their Degradation Products.
Synthesis of 2-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA). Reac-
tions and conditions are summarized in Scheme 3. Analytical data for
the synthetic intermediates matched exactly with those previously
reported by Atkinson et al. (27) (2-5) and Hashimoto et al. (71)
(6).
Ethyl 2-(2′-Nitrophenoxy)acetate (2). One gram of 2-nitrophenol (1)
(purchased from Sigma Aldrich Co., used as received) was dissolved
in a solution of 0.1 M KOH in absolute ethanol. After 1 h, the solvent
was removed at reduced pressure. The resulting alkoxide was redis-
solved in N,N-DMF (50 mL), and 1.2 mol equiv of ethyl 2-bromoacetate
was added. The reaction mixture was stirred under argon for 24 h. After
this time, 50 mL of ethyl acetate was added, and the resulting organic
solutions were washed with five portions of distilled water. The organic
layers were combined and dried over anhydrous sodium sulfate, and
the solvent was distilled at reduced pressure. Reaction crude was
chromatographed (CC, ethyl acetate/hexane, 20:80) to obtain ethyl 2-(2′-
nitrophenoxy)acetate (2) in quantitative yield.
Obtention of 4-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (3) and
(2H)-1,4-Benzoxazin-3(4H)-one (4). One hundred milligrams of Pd/C
(10%) was suspended in an aqueous solution of 1,4-dioxane (1:1) (50
mL). After this, sodium borohydride (650 mg) was added and the
solution was vigorously stirred. To this stirred suspension was added
dropwise a solution of 5 in 1,4-dioxane (0.5 g/mL). The reaction
evolution was controlled by TLC. Once the reaction was completed,
the suspension was filtered through Celite to remove the catalyst, and
the filtrate was treated with 10% HCl until pH 2 was reached. This
solution was further extracted with ethyl acetate (three times). The
organic layers were combined and dried over anhydrous sodium sulfate,
and the solvent was distilled at reduced pressure. The obtained residue
was chromatographied (CC, ethyl acetate/hexane, 30%) to obtain
4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (3) in 70% yield. When a
double proportion of sodium borohydride and Pd/C was used, compound
4 was obtained in the same yield.
The low stability showed by these chemicals in a wide variety
of conditions forced us to look for the alternative method
described below.
MATERIALS AND METHODS
General Methods. The purity of the isolated and synthetic com-
pounds was determined by 1H NMR and HPLC analyses and was found
to be >98%. 1H and 13C NMR spectra were recorded using MeOH-d4,
CDCl3, and D2O as solvents in a Varian INOVA spectrometer at 399.99
and 100.577 MHz, respectively. The resonance of residual methanol
for 1H was set to δ 3.30, that of residual chloroform to δ 7.25, and that
of residual water to δ 4.73. The solvent peak for 13C was set to δ 49.00
(methanol) or δ 77.00 (chloroform) and used as internal reference. When
D2O is used, 10 µL of MeOH-d4 is added for referencing 13C NMR
spectra. An HPLC-PDA detector (diode array UV-vis system),
equipped with a Phenomenex SYNERGI 4 micron Fusion RP-80 (250
× 460 mm) column, was used for acquiring UV-vis spectra. Mass
spectra were recorded by means of a Varian 1200L quadrupole MS/
MS detector. FTIR spectra were obtained by means of a Spectrum BX
Perkin-Elmer FTIR system. Frequency values are given in cm-1
.
Polarimetry data were acquired on a Perkin-Elmer model 341 pola-
rimeter. R values are given in degrees.
Isolation of Benzoxazinones DIMBOA, DIBOA-Glc, and DIBOA.
DIMBOA (Table 1A) was isolated from maize (Z. mays L. cv. Apache)
seedlings according to the method reported by Larsen and Christensen
(39), in which several modifications were included. Seven-day-old
maize shoots were frozen and then crushed and homogenized in water
(1 kg of plant/L of water). The resulting mixture was filtered and the
remaining solid discarded. After this, the liquid phase was allowed to
stand for 1 h at room temperature to allow enzymes to degrade the
DIMBOA glucoside. Then, 100 g of Amberlite XAD-7 (purchased from
Sigma Aldrich Co., used as received) was added and stirred for 1 h at
room temperature. Then, the mixture was filtered through cheesecloth,
the Amberlite washed with water, and the aqueous phase discarded.
The Amberlite was further washed with acetone (2 × 250 mL), yielding
an aqueous acetone solution, from which acetone was selectively
evaporated at reduced pressure. The remaining aqueous phase was
extracted with ethyl acetate (four times). The last extraction was aided
by using an ultrasound bath. The organic layers obtained were
combined, dried over anhydrous sodium sulfate, filtered, and distilled
in vacuo. The remaining solid was recrystallized from hexane (-20
°C, 12 h) to afford pure DIMBOA.
DIBOA-Glc (Table 1A) was isolated from 7-day-old rye seedlings
by means of the following procedure: the frozen shoots (1 kg) were
crushed and homogenized in an acetone/methanol mixture (1:1, 1 L).
The obtained mixture was filtered, and the filtrate was then extracted
with n-hexane and ethyl acetate. These organic phases were discarded,
and the aqueous phase was re-extracted with n-butanol. The n-butanol
layers were combined, filtered, and concentrated under reduced pressure.
The obtained residue was chromatographed (MPLC, RP C-18, H2O/
methanol 7:3, 1% AcOH) to obtain pure DIBOA-Glc.
4-Acetoxy-(2H)-1,4-benzoxazin-3(4H)-one (5). Two hundred and fifty
milligrams of 4 was dissolved in dry pyridine (10 mL), and 2 mL of
acetic anhydride was added with stirring under argon. After 12 h, ethyl
acetate (10 mL) was added to the reaction mixture, and the organic
solution was washed with five portions of aqueous HCl (10%). The
organic layers were combined and dried over anhydrous sodium sulfate,
and the solvent was distilled at reduced pressure. No further purification
was needed to isolate 4-acetoxy-(2H)-1,4-benzoxazin-3(4H)-one (5) in
quantitative yield.
2-Acetoxy-(2H)-1,4-benzoxazin-3(4H)-one (6). Compound 5 (100
mg) was dissolved in dry toluene (10 mL) and heated (95 °C, 24 h).

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 995
was filtered to obtain an orangish residue, which was washed with cold
water to yield AAMPO.
Scheme 4. Summary of Reactions and Conditions Employed To
Obtain
2-Amino-7-methoxyphenoxazin-3-one
(AMPO)
and
Synthesis of N-(2-Hydroxyphenyl)malonamic acid (HPMA).
Reactions and conditions employed for the synthesis of this compound
are summarized in Scheme 3. The structure of the synthetic intermedi-
2-Acetamido-7-methoxyphenoxazin-3-one (AAMPO)
1
ates was confirmed by H NMR.
O-tert-Butyldimethylsilyl-2-nitrophenol (7). tert-Butyldimethylsilyl
chloride (1.2 mol equiv) was added to a stirred solution of 500 mg of
2-nitrophenol (1) (purchased from Sigma Aldrich Co., used as received)
in dry pyridine (20 mL). The flask was set to argon atmosphere and
stirred. After 24 h, ethyl acetate (50 mL) was added to the reaction
mixture, and the obtained solution was washed with three portions (50
mL) of aqueous HCl (10%). The organic layers were combined, dried
over anhydrous sodium sulfate, filtered, and concentrated at reduced
pressure. The obtained residue was chromatographed (10% ethyl acetate/
hexane) to obtain compound 7 in quantitative yield.
O-tert-Butyldimethylsilyl-2-aminophenol (8). Two hundred and fifty
milligrams of compound 7 was dissolved in ethyl acetate (20 mL). To
this solution was added 15 mg of Pd/C (10%), and the mixture was
vigorously stirred under 1 atm of H2. Once the reaction was completed
(8 h), the catalyst was removed by filtration through Celite, and the
remaining solution was concentrated under reduced pressure to obtain
8 in quantitative yield and without further purification.
N-(2′-tert-Butyldimethylsilyloxyphenyl)malonamic Acid Ethyl Ester
(9). One hundred milligrams of compound 8 was dissolved in dry
toluene and treated with 1.2 mol equiv of ethyl 3-bromo-3-oxopropi-
onate. After 24 h, the solvent was removed by distillation at reduced
pressure, and the remaining residue was chromatographed (CC, ethyl
acetate/hexane 60%) to obtain compound 9 with 70% yield.
N-(2-Hydroxyphenyl)malonamic Acid (HPMA). One hundred mil-
ligrams of compound 9 was dissolved in methanol/water (30:70, 10
mL) and heated at 60 °C for 6 h. After this, methanol was removed by
distillation at reduced pressure, and the remaining aqueous solution
was acidified to pH 4 by means of the addition of aqueous HCl (10%).
This solution was extracted with three portions of ethyl acetate (60
mL), and after the combination, drying, and evaporation of the solvent
in the organic layers, the residue was chromatographed (CC) with ethyl
acetate/hexane mixtures of increasing polarity to obtain HMPA in
quantitative yield.
After this, the solvent was evaporated in vacuo, and the residue was
chromatographed (CC, ethyl acetate/hexane mixtures of increasing
polarity) to obtain compound 6 in 70% yield.
2-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA). One hundred
milligrams of 6 was dissolved in methanol and stirred under argon
atmosphere. After this, 360 µL of a magnesium methoxide solution in
dry methanol (7.4% w/w) was added through a syringe. Once the
reaction was completed (45 min), the solution was treated with diluted
HCl (5%) to reach pH 4. After this, ethyl acetate was added and the
resulting solution was washed with brine (one time) and distilled water
(two times). The organic layers were combined, dried, filtered, and
concentrated at reduced pressure. The obtained solid residue was
chomatographied to obtain HBOA in quantitative yield.
Synthesis of 2-Hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-
one (HMBOA). One hundred and fifty milligrams of 2,4-dihydroxy-
7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA (Table 1A),
isolated in our laboratory (see above for details), was suspended in
dry THF (20 mL). This solution was vigorously stirred under argon
atmosphere. After this, 1.2 mol equiv of samarium diiodide (0.1 M in
dry THF, 8 mL) was added. Once the reaction was completed, a solution
of sodium thiosulfate (1%, 20 mL) was added, and the resulting solution
was extracted with ethyl acetate (three times). The organic layers were
combined, dried, filtered, and concentrated at reduced pressure to obtain
a brown oil, which was chromatographed (CC, ethyl acetate/hexane,
40:60) to obtain HMBOA (Table 1A) in 70% yield.
Synthesis of Degradation Products 2-Amino-7-methoxyphenox-
azin-3-one (AMPO) and 2-Acetamido-7-methoxyphenoxazin-3-one
(AAMPO). Synthetic methodology for aminophenoxazines obtention
is summarized in Scheme 4.
2-Amino-7-methoxyphenoxazin-3-one (AMPO, Table 1C). Sodium
borohydride (185 mg) and 5-methoxy-2-nitrophenol (12) (250 mg) were
added to a stirred suspension of 10% Pd/C (18 mg) in aqueous dioxane
(1:1, 15 mL). After 48 h, the reaction mixture was filtered, and the
solid obtained was washed with cold water and dried at 80 °C to yield
pure AMPO.
The analogues ethyl 7-methoxy-2-nitrophenylacetate (11), 4-hydroxy-
7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (12), 7-methoxy-(2H)-1,4-
benzoxazin-3(4H)-one (13), 4-acetoxy-7-methoxy-(2H)-1,4-benzoxazin-
3(4H)-one (14), and the malonamic acid HMPMA (Scheme 5) were
obtained according to methods analogous to those for compounds 2,
3, 4, 5, and HPMA (Scheme 3), respectively.
RESULTS AND DISCUSSION
2-Acetamido-7-methoxyphenoxazin-3-one (AAMPO, Table 1C). Fifty
milliliters of acetic anhydride was added to a solution of AMPO in
glacial acetic acid (100 mL, 0.05 g/mL), and the resulting suspension
was stirred for 48 h at room temperature. After this time, the suspension
Isolation of Benzoxazinones DIBOA, DIBOA-Glc, and
DIMBOA. The novel procedures for the preparative isolation
of DIBOA-Glc and DIBOA were successful. They afforded the
Scheme 5. Summary of Reactions and Conditions Employed To Obtain DIMBOA Analogues 2-Hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one
(12), 7-Methoxy-(2H)-1,4-benzoxazin-3(4H)-one (13), 2-Acetoxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (14), and
N-(2-Hydroxy-4-methoxyphenyl)malonamic Acid (HMPMA)

996 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Mac´ıas et al.
desired compounds at the required scales and permitted their
bioactivity evaluation (17) and the description of their degrada-
tion behavior in wheat crop soil (23). Recent works that include
DIBOA preparative isolation (74) deal with lower DIBOA yields
of ∼0.04% w/w from fresh plants (not reported by the authors,
estimated by us), by means of more complicated methodologies
in which three preparative TLC methods are used. In addition
to this, the authors used oven-dried plant tissue. The temperature
employed (60 °C) could accelerate spontaneous degradation of
DIBOA.
reported by Atkinson et al. (27). Compounds 5 and 6 had the
same spectral data as those previously reported by Hashimoto
et al. (71).
Atkinson et al. (27) prepared a very wide variety of
benzoxazinones analogues, which included methoxy, dimethoxy,
halo, and cyanide derivatives of 2,4-dihydroxybenzoxazinones,
lactams, and some 2-deoxy derivatives for the evaluation of their
reactivity and decomposition kinetics. The interest in those
2-deoxy compounds has been recently discussed by us (17),
and the methodology employed by Atkinson et al. was modified
with the purpose of yield optimization in the 2-deoxy derivatives
obtention. The nucleophilic substitution used potassium nitro-
phenoxides solved in N,N-DMF instead of acetone or THF.
Under these conditions, the reaction proceeds with excellent
yield at room temperature. This modification increases the yield
for 2-deoxy compounds from the original 40 to 70%.
The starting material for this reaction, 4-acetoxy-(2H)-1,4-
benzoxazin-3(4H)-one (Schemes 3 and 5), was obtained by the
usual pyridine/Ac2O acetylation procedure, which gave quantita-
tive yield, instead of the DCM/Ac2O/Na2CO3 system employed
by the authors (40% yield). The magnesium methoxide proce-
dure for acyl group cleavage was simple and yielded the desired
HBOA with mild conditions. HBOA was obtained in a 48%
overall yield after five reaction steps.
Analytical data for 2-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one
(HBOA, Table 1A): λmax (nm) 208.5, 247.6; FTIR (cm-1) 3182,
3026, 1696, 1500, 1016; MS (ESI), m/z found for C8H7NO3
166 (100%, [M + 1]+); 1H NMR (MeOH-d4, 400 MHz) δ 5.53
[s, 1H (H-2)], 6.95 [m, 4H (H-5, H-6, H-7, H-8)]; 13C NMR
(MeOH-d4, 100 MHz) δ 92.0 (C-2), 116.8 (C-8), 118.7 (C-5),
123.7 (C-7), 124.9 (C-6), 127.4 (C-10), 142.3 (C-9), 165.2 (C-
3).
Synthesis of 2-Hydroxy-7-methoxy-(2H)-1,4-benzoxazin-
3(4H)-one (HMBOA). Samarium diiodide has been described
as a useful reagent for the cleavage of the N-O bond, and it
has been used for the reduction of hydroxylamines, oximes, and
hydroxamic acids (73). This reagent was employed to access
the benzoxazinone lactam moiety for the first time and allowed
us the direct obtention of HMBOA (Table 1A) from readily
available DIMBOA (Table 1A) in an easy way.
This methodology can be general in the obtention of lactams
from benzohydroxamic acids, facilitating the synthetic access
to those compounds. HMBOA was obtained in 70% yield.
Analytical data for 2-hydroxy-7-methoxy-(2H)-1,4-benz-
oxazin-3(4H)-one (HMBOA, Table 1A): λmax (nm) 204.7, 256.3;
FTIR (cm-1) 3181, 1686, 1513, 1157, 1030; MS (ESI), m/z
found for C9H9NO4 196 (100%, [M + 1]+); 1H NMR (MeOH-
d4, 400 MHz) δ 3.74 [s, 3H (-OCH3)], 5.56 [d, 1H, J ) 6.0
Hz (H-2)], 6.59 [d, 1H, J ) 2.0 Hz (H-8)], 6.59 [dd, 1H, J )
9.0, 2.0 Hz (H-6)], 6.84 [d, 1H, J ) 6.0 Hz (-OH)], 6.92 [d,
1H, J ) 9.0 Hz (H-5)], 9.58 [s, 1H (-NH)]; 13C NMR (MeOH-
d4, 100 MHz) δ 55.8 (-OCH3), 92.0 (C-2), 104.5 (C-8), 108.7
(C-6), 116.7 (C-5), 121.2 (C-10), 157.0 (C-9), 162.7 (C-7), 170.9
(C-3).
Synthesis of Aminophenoxazines. 2-Amino-7-methoxy-
phenoxazin-3-one (AMPO, Table 1C) was obtained in a single
reaction step in 70% yield. The described method is a very
simple way of obtaining this chemical at a large scale and
avoiding the inconveniences associated with the use of orga-
nometallics.
2,4-Dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA, Table
1A): λmax (nm) 253, 281; FTIR (cm-1) 3300, 3234, 1678, 1495,
1037; MS (ESI), m/z found for C8H7NO4 182 (100%, [M +
1]+); 1H NMR (MeOH-d4, 400 MHz) δ 5.73 [s, 1H (H-2)], 7.05
[m, 3H (H-6, H-7, H-8)], 7.36 [d, 1H, J ) 7.3 Hz (H-5)]; 13C
NMR (MeOH-d4, 100 MHz) δ 93.5 (C-2), 114.3 (C-5), 118.3
(C-8), 123.7 (C-7), 125.5 (C-6), 129.3 (C-10), 142.3 (C-9), 159.9
(C-3); yield, 0.1% w/w from the fresh plant.
2R-2-O-â-D-Glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-
3(4H)-one (DIBOA-Glc, Table 1A): λmax (nm) 255, 278; FTIR
(cm-1) 3200, 1739, 1263, 1077, 1030; [R]30 69°; MS (ESI),
1
m/z found for C13H16NO9 366 (100%, [M + Na]+); H NMR
(D2O, 400 MHz) δ 3.10 [dd, 1H, J ) 9.3 Hz, 8.0 (H-2′)], 3.21
[dd, 1H, J ) 9.3, 9.0 Hz (H-4′)], 3.34 [ddd, 1H, J ) 9.5, 5.4,
2.2 Hz (H-5′)], 3.37 [dd, 1H, J ) 9.3, 9.0 Hz (H-3′)], 3.56 [dd,
1H, J ) 12.2, 5.4 Hz (H-6′b)], 3.74 [dd, 1H, J ) 12.2, 2.2 Hz
(H-6′a)], 4.71 [d, 1H, J ) 7.8 Hz (H-1′)], 5.84 [s, 1H (H-2)],
7.02 [dd, 1H, J ) 7.6, 1.7 Hz (H-8)], 7.07 [m, 2H (H-6, H-7)],
7.37 [br d, 1H, J ) 7.6 Hz (H-5)]; 13C NMR (MeOH-d4, 100
MHz) δ 60.9 (C-6′), 69.5 (C-4′), 73.1 (C-2′), 75.8 (C-5′), 76.7
(C-3′), 97.2 (C-2), 102.3 (C-1′), 114.1 (C-5), 118.0 (C-8), 124.3
(C-7), 126.1 (C-6), 118.3 (C-8), 127.3 (C-10), 140.5 (C-9), 129.3
(C-10), 157.7 (C-3); yield, 0.0088% w/w from the fresh plant.
The DIMBOA yield we have obtained is similar to that
obtained by Larsen et al. (0.136 ( 0.12% w/w from fresh plants)
(39). The additional ethyl acetate extraction provoked higher
yields than the unmodified Larsen protocol when applied in our
rye variety and culture conditions.
2,4-Dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one
(DIMBOA, Table 1A): λmax (nm) 262; FTIR (cm-1) 3273, 1681,
1505, 1442, 1025; MS (ESI), m/z found for C9H9NO5 212
1
(100%, [M + 1]+); H NMR (MeOH-d4, 400 MHz) δ 3.76 [s,
3H (-OCH3)], 5.67 [s, 1H (H-2)], 6.62 [d, 1H, J ) 2.6 Hz
(H-8)], 6.67 [dd, 1H, J ) 8.8, 2.6 Hz (H-6)], 7.26 [d, 1H, J )
8.8 Hz (H-5)]; 13C NMR (MeOH-d4, 100 MHz) δ 56.1
(-OCH3), 94.0 (C-2), 104.7 (C-8), 108.9 (C-6), 115.2 (C-5),
123.3 (C-10), 143.6 (C-9), 158.7 (C-7), 159.4 (C-3); yield,
0.12% w/w from the fresh plant.
Synthesis of 2-Hydroxy-(2H)-1,4-benzoxazin-3(4H)-one
(HBOA). Among the different possibilities of obtaining benz-
oxazinones with the hydroxyl moiety at C-2 mentioned above,
and after several tests, we decided to take advantage of the [3.3]-
acyl sigmatropic rearrangement described by Hashimoto et al.
(71) (Scheme 3), which was employed to obtain some non-
natural derivatives useful for bioactivity and chemical reactivity
studies. The obtained acyl group (5) was further rearranged to
position C-2 by heating in dry toluene (85 °C, 24 h). The authors
employed benzene, which is highly toxic and has a lower boiling
point (80 °C). The higher temperature increased the reaction
yield in a significant manner (from 30 to 70%). After this, a
transesterification with magnesium methoxide in methanol at
room temperature yielded 99% of HBOA. This method was
chosen for its simplicity, selectivity, and mild conditions (72).
Analytical data for 2-4 matched exactly those previously
The easy access to AMPO allowed the obtention of its acetate
derivative 2-acetamido-7-methoxyphenoxazin-3-one (AAMPO,
Scheme 4) in high yield and in an easy-to-scale manner. The
low solubility of AMPO in the common solvents used for

Fate and Effects of Allelochemicals
J. Agric. Food Chem., Vol. 54, No. 4, 2006 997
1
acylation forced us to use acetic acid, in which AAMPO
precipitated once the reaction was completed. Overall yield from
5-methoxy-2-nitrophenol was 56%. It is interesting to note the
high reactivity difference between the amino group of ami-
nophenoxazines APO and AAMPO (Table 1C). Acetylation of
APO (Table 1C) can be made in good yields by means of the
usual pyridine procedure (25), yielding 2-acetamidophenoxazin-
3-one (AAPO, Table 1C), whereas base-catalyzed acylation
did not proceed successfully with AMPO.
- CO2 - 1]+); H NMR (MeOH-d4, 400 MHz) δ 3.85 [s, 2H
(H-2)], 6.90 [ddd, 1H, J ) 8.0, 7.0, 1.0 Hz (H-4′)], 6.98 [dd,
1H, J ) 8.0, 1.0 Hz (H-6′)], 7.06 [ddd, 1H, J ) 8.0, 7.0, 1.0
Hz (H-5′)], 8.08 [dd, 1H, J ) 8.0, 1.0 Hz (H-3′)]; 13C NMR
(MeOH-d4, 100 MHz) δ 55.4 (C-2), 110.8 (C-6′), 120.4 (C-4′),
121.3 (C-5′), 124.9 (C-3′), 127.2 (C-1′), 149.8 (C-2′), 165.6 (C-
3), 170.6 (C-1).
Synthesis of N-(2-Hydroxy-4-methoxyphenyl)malonamic
Acid (HMPMA, Table 1D). Reactions and conditions employed
are summarized in Scheme 5. The same synthetic methodology
as the employed for HMPA obtention was used for the
preparation of its analogue, HMPMA, from 5-methoxy-2-
nitrophenol (purchased from Sigma Aldrich Co., used as
received) (10). The structures of the synthetic intermediates were
2-Amino-7-methoxyphenoxazin-3-one (AMPO, Table 1C):
λ
max (nm) 235.0; FTIR (cm-1) 3450, 3299, 1577, 847; MS (ESI),
m/z found for C13H10N2O3 243 (100%, [M + 1]+); H NMR
(MeOH-d4, 400 MHz) δ 3.85 [s, 3H (-OCH3)], 6.33 [s, 1H
(H-4)], 6.35 [s, 1H (H-1)], 7.01 [dd, 1H, J ) 7.0, 2.7 Hz (H-
8)], 7.11 [d, 1H, J ) 2.7 Hz (H-6)], 7.64 [d, 1H, J ) 9.0 Hz
(H-9); 13C NMR (MeOH-d4, 100 MHz) δ 56.1 (-OCH3), 98.8
(C-1), 100.1 (C-6), 103.3 (C-4), 113.5 (C-8), 128.4 (C-9a), 129.0
(C-9), 143.3 (C-5a), 145.7 (C-2), 146.6 (C-10a), 148.6 (C-4a),
160.0 (C-7), 179.9 (C-2).
1
1
confirmed by H NMR.
O-tert-Butyldimethylsilyl-5-methoxy-2-nitrophenol (15): 1H
NMR (CDCl3, 400 MHz) δ 7.90 [1H, d, J ) 9.0 Hz (H-3)],
6.52 [1H, dd, J ) 9.0, 2.6 Hz (H-4)], 6.40 [1H, d, J ) 2.6 Hz
(H-6)], 3.82 [s, 3H (CH3O)], 1.00 [s, 9H (H-8)], 0.24 [s, 6H
(H-7)].
2-Acetamido-7-methoxyphenoxazin-3-one (AAMPO, Table
1C): λmax (nm) 235.4; FTIR (cm-1) 3298, 2924, 1526, 1257,
805; MS (ESI), m/z found for C15H12N2O4 285 (100%, [M +
1
O-tert-Butyldimethylsilyl-2-amino-5-methoxyphenol (16): H
NMR (CDCl3, 400 MHz) δ 6.63 [1H, d, J ) 8.3 Hz (H-3)],
6.39 [1H, dd, J ) 8.3, 2.6 Hz (H-4)], 6.36 [1H, d, J ) 2.6 Hz
(H-6)], 3.70 [s, 3H (CH3O)], 1.02 [s, 9H (H-8)], 0.25 [s, 6H
(H-7)].
N-(2′-tert-Butyldimethylsilyloxy-4′-methoxyphenyl)malon-
amic acid ethyl ester (17): 1H NMR (CDCl3, 400 MHz) δ 8.07
[1H, d, J ) 8.9 Hz (H-6′)], 6.39 [2H, m, (H-3′, H-5′)], 4.15
[2H, q, J ) 7.2 Hz (H-9′)], 3.66 [3H, s, (CH3O)], 3.37 [2H, s,
(H-2)], 1.20 [3H, t, J ) 7.2 Hz (H-10′)], 1.16 [9H, s, (H-8′)],
0.23 [s, 6H (H-7′)].
N-(2-Hydroxy-7-methoxyphenyl)malonamic acid (HMPMA,
Table 1D): λmax (nm) 208.1, 249.8, 287.4; FTIR (cm-1) 3215,
3081, 1698, 1433, 1202; MS (ESI), m/z found for C10H11NO5
226 (100%, [M + 1]+); 1H NMR (MeOH-d4, 400 MHz) δ 3.30
[s, 2H (H-2)], 3.52 [s, 3H (-OCH3)], 6.19 [dd, 1H, J ) 2.0,
9.0 Hz (H-5′)], 6.25 [d, 1H, J ) 2.0 Hz (H-3′)], 7.38 [d, 1H, J
) 9.0 Hz (H-6′)]; 13C NMR (MeOH-d4, 100 MHz) δ 41.9 (C-
2), 54.8 (-OCH3), 102.0 (C-3′), 104.6 (C-5′), 119.0 (C-1′),
123.9 (C-6′), 150.1 (C-2′), 158.5 (C-4′), 166.3 (C-3), 170.8 (C-
1).
The synthetic procedures described above yielded HMPA and
HMPMA in a 68% overall yield after four reactions. After two
reaction steps, Friebe et al. reported yields of 66% for HPMA
and 45% for HMPMA (31). In addition to the yield increase at
HMPMA obtention, the methods described here avoided the
undesired dimerizations and provided useful intermediates for
further research on benzoxazinone bioactivity and modes of
action.
1
1]+); H NMR (CDCl3, 400 MHz) δ 2.20 [s, 3H (-OCCH3)],
δ 3.87 [s, 3H (-OCH3)], 6.38 [s, 1H (H-4)], 6.83 [d, 1H, J )
3.0 Hz (H-6)], 6.94, [dd, 1H, J ) 9.0, 3.0 Hz (H-8)], 7.72 [d,
1H, J ) 9.0 Hz (H-9)], 8.36 [s, 1H (H-1)]; 13C NMR (CDCl3,
100 MHz) δ 25.3 (-OCCH3), 56.1 (-OCH3), 91.5 (C-4), 99.8
(C-6), 103.8 (C-8), 114.7 (C-9), 126.9 (C-9a), 131.2 (C-1), 140.5
(C-5a), 144.4 (C-4a), 147.3 (C-2), 149.5 (C-10a), 159.4 (C-7),
179.3 (C-2).
Synthesis of N-(2-Hydroxyphenyl)malonamic Acid (HPMA,
Table 1D). To avoid the inconvenience of the coexistence of
amino and hydroxyl moieties (dimerization of nitrophenols to
give aminophenoxazines), a procedure in which aromatic
nitrogen could be functionalized with the hydroxyl moiety
protected was optimized. 2-Nitrophenol was protected by
generating its tert-butyldimethylsilyl ether. After this, the nitro
group was reduced to amino by catalytic hydrogenation. This
2-amino-tert-butyldimethylsilyloxybenzene has potential utility
in the obtention of heterocycle ring-opened derivatives of
benzoxazinones, which have been proposed as metabolic
intermediates of natural benzoxazinones with an important role
in their biological activity (27). Once the amino group was
obtained, the side chain was introduced by amidation with ethyl
3-chloro-3-oxopropionate. The final alkaline hydrolysis afforded
the final desired product by cleaving the silyl ether and the ethyl
ester simultaneously. The structures of the synthetic intermedi-
1
ates were confirmed by H NMR.
O-tert-Butyldimethylsilyl-2-nitrophenol (7): 1H NMR (CDCl3,
400 MHz) δ 7.77 [1H, dd, J ) 1.6, 8.1 Hz (H-3)], 7.41 [m, 2H
(H-4, H-5)], 6.99 [m, 1H (H-6)], 0.99 [s, 9H (H-8)], 0.24 [s,
6H (H-7)].
Synthesis of DIMBOA Analogues Ethyl 5-Methoxy-2-
nitrophenylacetate (11), 4-Hydroxy-7-methoxy-(2H)-1,4-ben-
zoxazin-3(4H)-one (12), 7-Methoxy-(2H)-1,4-benzoxazin-
3(4H)-one (13), and 4-Acetoxy-7-methoxy-(2H)-1,4-benzoxazin-
3(4H)-one (14). To obtain these compounds, the same
methodology as the one employed for the obtention of DIBOA
analogues 2, 3, 4 and 5, respectively, was used (see above).
Analytical data for 11-13 matched exactly those previously
reported by Atkinson et al. (27). Compound 14 had the same
spectral data as those previously reported by Hashimoto et al.
(71).
O-tert-Butyldimethylsilyl-2-aminophenol (8): 1H NMR (CDCl3,
400 MHz) δ 7.78 [1H, dd, J ) 1.6, 8.0 Hz (H-6)], 7.42 [1H,
ddd, J ) 1.6, 8.0, 8.0 Hz (H-4)], 6.99 [m, 2H (H-3, H-5)], 0.99
[s, 9H (H-8)], 0.25 [s, 6H (H-7)].
N-(2′-tert-Butyldimethylsilyloxyphenyl)malonamic acid ethyl
1
ester (9): H NMR (CDCl3, 400 MHz) δ 8.28 [m, 1H (H-6′)],
6.90 [m, 2H (H-4′, H-5′)], 6.84 [m, 1H (H-3′)], 4.19 [2H, q, J
) 7.7 Hz (H-9′)], 3.43 [2H, s, (H-2)], 1.21 [3H, t, J ) 7.7 Hz
(H-10′)], 0.99 [9H, s, (H-8′)], 0.27 [s, 6H (H-7′)].
Reactions and conditions are summarized in Scheme 5. The
starting material employed was 5-methoxy-2-nitrophenol (10)
(purchased from Sigma Aldrich Co., used as received). The
synthesis of these compounds was made to obtain HMBOA in
N-(2-Hydroxyphenyl)malonamic acid (HPMA, Table 1D):
λ
max (nm) ) 208.9, 243.5, 283.7; FTIR (cm-1) 3292, 2924, 1723,
1542, 1254; MS (ESI), m/z found for C9H9NO4 150 (67%, [M

998 J. Agric. Food Chem., Vol. 54, No. 4, 2006
Mac´ıas et al.
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rearrangement was not successful when applied on 7-methoxy-
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Efficient isolation and synthetic methodologies exist for the
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on their chemical and biological properties and will be useful
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ACKNOWLEDGMENT
We acknowledge the collaboration of Dr. Rau´l F. Velasco
(Universidad de Ca´diz) for the optimization of benzoxazinone
synthetic methods.
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Received for review April 19, 2005. Revised manuscript received May
31, 2005. Accepted December 6, 2005. 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 QLRT-2000-01967. Fellowships from the Universidad de los
Andes-Venezuela (A.O.B.) and from the European Commission
(European Union) and Junta de Andaluc´ıa-Spain (D.M.) are gratefully
acknowledged.
JF050896X