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Structure-activity relationships of benzoxazinoids on Echinochloa crus galli (L.) P./Beauv.

Francisco A. Macías, Nuria Chinchilla, Rosa M. Varela, David Marín and José M.G. Molinillo

Grupo de Alelopatía, Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Cádiz. Avda. República Saharaui, s/n, 11510 Puerto Real (Cádiz), Spain. *


Echinochloa crus-galli (L.) P./Beauv. (barnyardgrass) is a widely distributed weed. It constitutes a serious weed problem in 42 countries and has been found in at least 27 more. It is the world's main weed of rice affecting besides to 36 crops worldwide. Several cases of resistance to herbicides with different modes of action have been achieved for this plant. In our ongoing studies regarding the application of benzoxazinones and their degradation products in soil for weed control, a complete structure-activity relationships study was made by using E. crus-galli as target plant. Compounds used in this study include natural allelochemicals 2-O-β-D-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-Glc), 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), together with some degradation derivatives found in wheat crop soil, and some synthetic analogues. The most active compounds were the degradation product 2-aminophenol (APH) and the synthetic analogue 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA). Their activities confirm the direction proposed in a previous SAR study, which establishes D-DIBOA to be the best lead for natural herbicide models development with benzoxazinone structure.

Media summary

Compounds belonging to the structural types benzoxazinone, benzoxazolinone, aminophenoxazine, and malonamic acid were tested barnyardgrass. Highest activities were displayed by benzoxazinone D-DIBOA and aminophenoxazine APO.

Key Words

Benzoxazinones, barnyardgrass, ABOA, D-DIBOA, phytotoxicity, SAR.


Barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.) is a plant originally from Europe and India. It constitutes a serious weed problem in 42 countries and has been found in at least 27 more (Holm et al. 1979). Barnyardgrass has been also detected in 36 crops worldwide. It is particularly problematic for rice crops all over the world. It is also a main weed in many other crops like cotton, corn, and potatoes, for example, in the United States (Holm et al. 1977).

The success of barnyardgrass is attributed to prolific seeding, seed dormancy, ability to grow rapidly, flowering in a wide range of photoperiods, and relative resistance to herbicides (Carey et al. 1995; De Prado et al. 2004).

Benzoxazinones containing the hydroxamic acid moiety acquired high relevancy on phytochemistry research after the isolation of 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA) (Virtanen and Hietala 1960), and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA) (Walroos and Virtanen 1959) (Figure 1). The necessity of obtaining new herbicides with new and multiple modes of action that could prevent resistant phenomena lead us to study the influence of benzoxazinones and related compounds on the growth of barnyardgrass (Figure 1).

In addition to this bioactivity research, the low stability of DIMBOA and DIBOA, and the moderate degradability of their related benzoxazolinones in several conditions, like biotransformation by fungi, and degradation in crop soil and in aqueous solution has been investigated (Fomsgaard et al. 2004). After the characterization of conversion dynamics of these compounds in model wheat crop soils, 2-aminophenoxazin-3-one (APO) and 2-amino-7-methoxyphenoxazin-3-one (AMPO) were the final products for DIBOA and DIMBOA degradation routes found by us (Macías et al. 2004, 2005a). Their N-acetyl derivatives, 2-acetamidophenoxazin-3-one (AAPO) and 2-acetamido-7-methoxyphenoxazin-3-one (AAMPO) have been proposed as detoxification compounds produced by non-pathogenic organisms

associated to Gramineae (Fomsgaard et al. 2004) (Figure 1). We recently reported a complete structure-activity relationships study dealing with 21 chemicals, including natural benzoxazinones, a wide variety

Figure 1. Allelochemicals, synthetic analogues and degradation compounds evaluated.

of synthetic analogues of them, and degradation products belonging to four different structural types (Macías et al. 2005b). The bioactivity profiles shown by benzoxazinones suggested 2-deoxy derivatives of natural allelochemicals DIBOA and DIMBOA (D-DIMBOA and D-DIBOA) (Figure 1) to be the best leads for new herbicides with this structural base.

The main objective of this work was to evaluate phytotoxicity of benzoxazinones and related compounds on barnyardgrass, in the search for new compounds able to be applied as part of strategies directed to weed control on barnyardgrass affected crops. Statistical treatment of the acquired data would allow discovering structure-activity relationships useful in further development of highly phytotoxic and compounds based on natural product structures.


Natural Benzoxazinones

DIBOA and DIMBOA were obtained from natural sources by means of previously reported isolation procedures (Larsen and Christensen 2000) modified by us. The DIBOA natural glycoside (DIBOA-Glc) was isolated from natural sources. Its isolation protocol, adapted from literature, has been already described by us regarding its degradation study in wheat crop soil (Macías et al. 2005a).

Synthetic Benzoxazinones

They were obtained in our laboratory by means of previously reported methods (Macías et al. 2005c).

Degradation Products

They have been selected according to the precedents mentioned above, belonging to four different structural types:

  • Benzoxazolinones: 2-Benzoxazolinone (BOA) and 6-methoxy-2-benzoxazolinone (MBOA) (Figure 1) are commercial compounds. They were purchased from Fluka Chemika and Lancaster Synthesis respectively. They were used as received.
  • Aminophenoxazin-3-ones: They were obtained in our laboratory by previously reported synthesis procedures (Macías et al. 2005c).
  • Additional Compounds Evaluated: In order to characterize its bioactivity and to discuss the phytotoxicity of DIBOA degradation route chemicals on barnyardgrass, 2-aminophenol (purchased from Sigma-Aldrich Co., used as received) (Figure 1) was evaluated.

Phytotoxicity Bioassays

Bioassays used Petri dishes (90 mm. diameter) with one sheet of Whatman No.1 filter paper as sustrate. Germination and growth were conducted in aqueous solutions at controlled pH by using 10-2 M 2-[N-morpholino]ethanesulfonic acid (MES) and addition of solution of NaOH 1 M to reach 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 (1, 0.5, 0.1, 0.05, and 0.01 mM).

Barnyardgrass seeds were purchased from Herbiseed Co. (Twyford, England). They were used as received. The number of seeds in each Petri dish was 25, and 5 mL of treatment, control or internal reference solution were added to each Petri dish. Four replicates were used (100 seeds).

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. The bioassay took 5 days. After growth, plants were frozen at -10 ºC 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.

The commercial herbicide Logran®, a combination of N-(1,1-dimethylethyl)-N-ethyl-6-(methylthio)-1,3,5-triazine-2,4-diamine (Terbutryn, 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 previously reported (Macías et al. 2000). It was used at the same concentrations (1, 0.5, 0.1, 0.05, and 0.01 mM), and in the same conditions as the compounds in study. Buffered aqueous solutions with DMSO and without any tested compound were used as control samples. Once acquired, the phytotoxicity data are statistically treated and compared by means of a cluster analysis (Macías et al. 2002).

Result and Discussion

Cluster analysis for growth parameters is shown in Figure 2. Compounds can be classified according to their phytotoxic effects in two main groups: G1 for high or moderate effects and G2 for low or null activities. G1 is divided in two subgroups: G1A for the most active compounds and G1B for those which showed moderate effects.

Figure 2. Cluster analysis (effects on growth) for allelochemicals, degradation products and synthetic analogues. a) benzoxazinones; b) benzoxazolinones; c) aminophenoxazines.

All compounds assayed with benzoxazinone skeleton belong to G1 group, except D-HMBOA. Regarding the behavior of natural allelochemicals, all of them had moderate activity levels, preserved just at the highest concentrations. In fact, all these compounds belong to G1B group. Thus, the benzoxazinone skeleton was the most active structure assayed. The synthetic benzoxazinone D-DIBOA was the most active compound of all the bioassay, with phytotoxicity profiles very similar to the commercial herbicide Logran®, which had IC50 values of 261 μM (r2=0.95) and 108 μM (r2=0.96) for root and shoot lengths, respectively. As it occurred in our previous phytotoxicity study, its 7-methoxy analogue D-DIMBOA showed weaker phytotoxicity. The same occurs with the assayed benzoxazinone lactams, showing D-HBOA higher inhibition than D-HMBOA. The influence of the 7-methoxy moiety seems to be the opposite analyzing phytotoxicity of natural allelochemicals, since DIMBOA effects are stronger than DIBOA and DIBOA-Glc ones. In our previous study, which included the aminophenoxazines assayed here, we offered a possible explanation of the lack of phytotoxic effect observed for AAPO, AMPO and AAMPO. In this case, the same behaviour is observed, so that these phytotoxicity values can correlated with aqueous solubility and lipophillicity, in the context of Hansch (Hansch and Leo 1995) models for bioactivity in pharmaceuticals and agrochemicals. Lipophillicity, calculated as logarithmic water/n-octanol partition coefficient (LogP) has a value close to 0.8 for APO, whereas AMPO, AAMPO and AAPO roughly reach 0.6, which gives much lower phytotoxic effects. The fact that DIBOA-Glc, DIBOA, BOA, APO and AAPO constitute a complete degradation series in wheat crop soil models (Macías et al. 2005a), as well as in other systems (Fomsgaard et al. 2004), allow to relate phytotoxicity of these compounds with their persistence in soil, in order to describe the influence of the complete series in the development of barnyardgrass.Taking into account the phytotoxicities observed, the most active compound (APH) has the shortest half-life of the whole series, while the most persistent compound (APO), had just moderate effects.


Regarding to the phytotoxicity of degradation products, the only one that shows moderate effects was APO. If natural compounds and their synthetic analogues are compared, it can be concluded that the DIMBOA or DIBOA detoxification capacity is not effective when barnyardgrass is exposed to synthetic analogues like D-DIBOA. Then, the lower effects caused by natural benzoxazinones are debt to two factors: degradation in the bioassay conditions and potential detoxification capacity. Our current efforts are directed towards the evaluation of the phytotoxic potential of this compound at different barnyardgrass and rice growth stages, in order to discover its utility for pest management in rice.


This research was supported by the Ministry of Science and Technology, Spain, Project No. AGL2001-3556 (AGR) and by the Commission of the European Communities, contract No. QLK5-CT-2001-01967. Fellowships from the Ministry of Education and Science (N. Ch.) and from the European Commission and the Regional Gobernment of Andalusia (D. M.) are also gratefully acknowledged.


Carey VF, Duke SO, Hoagland RE and Talbert RE (1995). Resistance mechanism of propanil-resistant barnyardgrass. I. Absorption, translocation, and site of action studies. Pesticide Biochemistry and Physiology 52, 182-189.

De Prado R and Franco AR (2004). Cross-resistance and herbicide metabolism in grass weeds in Europe: Biochemical and physiological aspects. Weed Science 52, 441-447.

Fomsgaard IS, Mortensen AG and Carlsen SCK (2004). Microbial transformation products of benzoxazolinone and benzoxazinone allelochemicals-a review. Chemosphere 54, 1025-1038.

Hansch C and Leo A (1995). In ‘Exploiting QSAR. Fundamentals and applications in chemistry and biology’ (American Chemical Society:USA).

Holm LG, Pancho JV, Herberger JP and Plucknett DL (1977). In ‘The world’s worst weeds’ (University Press of Hawaii, Honolulu).

Holm LG, Pancho JV, Herberger JP and Plucknett DL (1979). In ‘A geographical atlas of world weeds’ (John Wiley and Sons, New York).

Larsen E and Christensen LP (2000). Simple method for large scale isolation of the cyclic arylhydroxamic acid DIMBOA from maize (Zea mays L.). Journal of Agricultural and Food Chemistry 48, 2556-2558.

Macías FA, Castellano D and Molinillo JMG (2000). Search for a standard phytotoxic bioassay for allelochemicals. Selection of standard target species. Journal of Agricultural and Food Chemistry 48, 2512-2521.

Macías FA, Velasco RF, Viñolo VMI, Castellano D, Galindo JCG and Molinillo JMG (2002). Cluster analysis. A valuable tool for allelopathic SAR studies? In ‘Allelopathy: from molecules to ecosystems’ (Eds MJ Reigosa and N Pedrol) pp. 305-316 (Science Publishers Incorporated:Enfield, USA).

Macías FA, Oliveros-Bastidas A, Marín D, Castellano D, Simonet AM and Molinillo JMG (2004). 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 Gramineae. Journal of Agricultural and Food Chemistry 52, 6402-6413.

Macías FA, Oliveros-Bastidas A, Marín D, Castellano D, Simonet AM and Molinillo JMG (2005a). Degradation studies on benzoxazinoids. Soil degradation dynamics of (2R)-2-O-β -D-Glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-Glc) and its degradation products, Phytotoxic allelochemicals from Gramineae. Journal of Agricultural and Food Chemistry 53, 554-561.

Macías FA, Marín D, Oliveros-Bastidas A, Castellano D, Simonet AM and Molinillo JMG (2005b). Structure-activity Relationships (SAR) studies of benzoxazinones, their degradation products and analogues. Phytotoxicity on Standard Target Species (STS). Journal of Agricultural and Food Chemistry 53, 538-548.

Macías FA, Marín D, Oliveros-Bastidas A, Castellano D, Simonet AM and Molinillo JMG (2005c). Isolation and synthesis of allelochemicals from Gramineae: Benzoxazinones and related compounds. Journal of Agricultural and Food Chemistry 53 (In press).

Virtanen AI and Hietala PK (1960). Precursors of benzoxazolinone in rye plants. Acta Chemica Scandinavica 14, 499-501.

Wahlroos Ö and Virtanen I (1959). The precursors of 6-methoxy-benzoxazolinone in maize and wheat plants, their isolation and some of their properties. Acta Chemica Scandinavica 13, 1906-1908.

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