Imazethapyr targets the enzyme acetolactate synthase (ALS) (also known as acetohydroxyacid synthase, AHAS) in plants. ALS catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine. These amino acids are essential for protein synthesis and plant growth. By binding to the active site of ALS and inhibiting its catalytic activity, Imazethapyr depletes branched-chain amino acid pools, leading to the cessation of cell division, plant growth arrest, and eventual plant death. Imazethapyr is selective because it has a higher affinity for the ALS enzyme in susceptible weeds compared to the enzyme in tolerant crops, though some crops have been genetically engineered for resistance. Imazethapyr does not target any mammalian enzymes (mammals do not synthesize branched-chain amino acids and lack ALS).

Imazethapyr is a herbicide that is part of the imidazolinones family, which is widely used in many cropping systems to increase crop yields and shield crops from weeds and annual grasses in soybean and peanut cultivation. Imazethapyr would influence the transcription of genes linked to photosynthesis, suppress the plant's antioxidant system, and have an impact on the synthesis of chlorophyll[2].

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In vitro, Imazethapyr is a potent inhibitor of plant acetolactate synthase (ALS) activity. In a standard cell-free enzyme assay, ALS is extracted from plant tissues (e.g., corn or pea seedlings) or recombinant ALS is expressed in E. coli. The enzyme is incubated with pyruvate (substrate), thiamine pyrophosphate (cofactor), and FAD in the presence of Imazethapyr (1 nM to 100 uM). After incubation at 37degC for 60 minutes, the reaction is stopped with sulfuric acid, and the product (acetolactate) is decarboxylated to acetoin, which is quantified by colorimetry (creatinine/alpha-naphthol assay). Imazethapyr inhibits ALS with an IC50 typically in the low nanomolar to micromolar range, depending on the plant species. For example, in ALS from sensitive weeds such as Amaranthus retroflexus, the IC50 is 10-50 nM, while in tolerant crops like soybeans, the IC50 is higher. The compound does not inhibit any mammalian enzyme, as the branched-chain amino acid synthesis pathway is absent in humans and animals. In plant cells, Imazethapyr reduces the levels of free valine, leucine, and isoleucine, leading to growth arrest.

In vivo, Imazethapyr is used as a post-emergence herbicide in crops. After application (typically as a spray or granule), the compound is absorbed through the leaves, stems, and roots of weeds and is translocated to the growing points (meristems), where it inhibits ALS activity. Symptoms appear 5-14 days after application and include chlorosis (yellowing), stunting, necrosis, and eventual plant death. The compound is selective in tolerant crops such as soybeans, which metabolize the herbicide more rapidly than sensitive weeds. In fields, Imazethapyr provides residual control, remaining active in the soil for several weeks to months, depending on environmental conditions (pH, moisture, temperature). Imazethapyr is not intended for use in animals or humans; it has no therapeutic activity in mammals. However, it can be used as a research tool in laboratory animals (e.g., rats) to study the effects of branched-chain amino acid depletion on physiology, though this is uncommon.

A non-cellular (cell-free) protocol for evaluating the inhibitory activity of Imazethapyr on acetolactate synthase (ALS) involves the extraction of ALS from plant tissue. Plant leaves (e.g., corn seedlings, 5-7 days old) are homogenized in liquid nitrogen and suspended in extraction buffer (100 mM K2HPO4, pH 7.5, 10 mM sodium pyruvate, 5 mM MgCl2, 0.5 mM thiamine pyrophosphate (TPP), 10 uM FAD, 10% glycerol, and 1 mM DTT). The homogenate is filtered, and the supernatant is clarified by centrifugation at 10,000 g for 15 minutes. ALS is precipitated with 50% ammonium sulfate and dialyzed. The enzyme reaction is performed in microcentrifuge tubes: 50 uL of enzyme extract, 25 uL of reaction buffer (200 mM K2HPO4, pH 7.5, 20 mM sodium pyruvate, 10 mM MgCl2, 1 mM TPP, 20 uM FAD, 2 mM DTT), and increasing concentrations of Imazethapyr (0-100 uM) in a total volume of 100 uL. The reaction is incubated at 37degC for 60 minutes. The reaction is stopped by adding 50 uL of 6% H2SO4, followed by incubation at 60degC for 15 minutes to decarboxylate acetolactate to acetoin. The acetoin is quantified by adding 100 uL of 0.5% creatine in 60% NaOH and 100 uL of 5% alpha-naphthol in 60% NaOH, followed by incubation at 60degC for 15 minutes. The mixture is cooled, and the absorbance is read at 525 nm. The amount of acetoin is calculated from a standard curve. The percentage of inhibition is plotted against the concentration of Imazethapyr, and the IC50 is determined.

Since Imazethapyr is an herbicide targeting plants, an in vitro cellular protocol typically uses plant cell cultures rather than animal cells. For example, a protocol using Nicotiana tabacum (tobacco) Bright Yellow-2 (BY-2) cells is described. BY-2 cells are maintained in Murashige and Skoog (MS) medium containing 3% sucrose, 0.2 ug/mL 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 uM KH2PO4 at 25degC in the dark on a rotary shaker at 130 rpm. Cells are subcultured weekly. For the assay, 1 mL of 3-day-old BY-2 cell suspension is transferred to each well of a 24-well plate. Imazethapyr (dissolved in DMSO or ethanol) is added to the culture medium at final concentrations ranging from 0.1 nM to 100 uM. A solvent control (0.1% DMSO or ethanol) is included. Cells are incubated for 2-7 days at 25degC in the dark. Cell growth is assessed by measuring the packed cell volume (PCV) after centrifugation, or by determining dry weight. Cell viability is measured using fluorescein diacetate (FDA) staining (viable cells take up FDA and fluoresce green) or by the reduction of triphenyltetrazolium chloride (TTC) to formazan. The concentration inhibiting growth by 50% (IC50) is calculated. For mechanistic studies, cells can be harvested after 24-48 hours and analyzed for branched-chain amino acid (valine, leucine, isoleucine) levels by HPLC.

An in vivo animal protocol is not standard for Imazethapyr since it is an herbicide, but a plant model protocol is provided for evaluating herbicidal activity in a greenhouse setting. Seeds of a target weed (e.g., Amaranthus retroflexus, redroot pigweed) and a tolerant crop (e.g., Glycine max, soybean) are sown separately in 10 cm diameter pots containing a standard potting mix. The pots are placed in a greenhouse under natural light (or supplemented with 600 W high-pressure sodium lamps, 14-hour photoperiod) at 25/20degC day/night temperature with 60-70% relative humidity. Plants are thinned to 5-10 plants per pot after emergence. Imazethapyr is formulated as a soluble concentrate (e.g., as the ammonium salt). At the 2-4 leaf stage (approximately 10-14 days after sowing), Imazethapyr is applied at various concentrations (0, 1, 5, 10, 25, 50, 100, and 200 g active ingredient per hectare) using a laboratory sprayer calibrated to deliver 200 L/ha. The spray nozzle is set 50 cm above the plant canopy. Each treatment is applied to 4 replicate pots per species. After application, the plants are returned to the greenhouse. Visual assessments of phytotoxicity (percentage of necrosis, chlorosis, and growth reduction) are made at 7, 14, and 21 days after treatment (DAT) on a scale of 0 (no effect) to 100 (complete death). At 21 DAT, the shoots are cut at the soil level, and fresh weight is measured. The fresh weight reduction (%) is calculated relative to the untreated control, and the GR50 (dose causing 50% growth reduction) is calculated.

Absorption, Distribution and Excretion
In rats, 92% of the drug is excreted in the urine and 5% in the feces within 24 hours after oral administration. After 48 hours, residual levels in blood, liver, kidneys, muscle, and adipose tissue are below 0.01 ppm. Metabolism/Metabolites In a rat metabolism study, almost 100% of the radiolabeled test substance/imidazoline/ was recovered from excretion within 96 hours (89-95% in urine, 6-11% in feces). Over 95% of the oral dose is excreted within the first 31 hours. The main residues in urine and feces are the parent compound. Approximately 2% of the oral dose is metabolized and excreted as CL 288511 (a 1-hydroxyethyl derivative of imidazoline). The majority of the administered substance is excreted in the urine as the unmodified parent compound (>97%), while the excretion of CL 288511 is minimal. In the high-dose group, the unmodified parent compound was the major component in the feces of both male and female animals, especially within 12 hours or less after administration. CL 288511 was the major metabolite. Additionally, a high level of an unknown metabolite was detected. In the low-dose group, six components were detected in the feces: the parent compound, CL 288511, the aforementioned unknown metabolite, and three unknown metabolites at lower levels. Metabolism was rapid in non-sensitive plants (50% loss within 1.6 days in soybeans).

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Pharmacokinetic (PK) data for Imazethapyr are primarily available from agricultural and environmental toxicology studies. After oral administration in rats, Imazethapyr is rapidly absorbed and reaches peak plasma concentrations (Cmax) within 1-2 hours (Tmax). The elimination half-life (t½) in plasma is approximately 2-5 hours. The compound is extensively metabolized, primarily by hydroxylation of the pyridine ring, and excreted in the urine (50-70%) and feces (20-30%). Imazethapyr has low systemic toxicity in mammals and is not accumulated in tissues. In plants, Imazethapyr is absorbed by roots and leaves and is translocated via the xylem and phloem to the growing points. It is metabolized in tolerant crops (e.g., soybeans) by cytochrome P450 enzymes to inactive hydroxylated metabolites, which are rapidly conjugated to glucose and sequestered. In sensitive weeds, metabolism is slow, and the compound accumulates at the growing points. Soil degradation occurs via microbial metabolism, with a half-life in soil of 30-60 days, depending on temperature, pH, and moisture.

Toxicity Data
LC50 (Rat) = 3,270 mg/m³ Non-human Toxicity Values LD50 (Rats, Oral) >5000 mg/kg LD50 (Rabbit, Dermal) >2000 mg/kg LC50 (Rats, Inhalation) 3.27 mg/L / Duration Unspecified /

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Imazethapyr is an herbicide and, as such, has a well-characterized toxicological profile in mammals and the environment. In acute toxicity studies in rats, the oral LD50 is >5000 mg/kg, indicating low acute toxicity (US EPA toxicity category III/IV). The dermal LD50 is >2000 mg/kg, and the inhalation LC50 is >5.4 mg/L. Imazethapyr is not a skin or eye irritant and is not a skin sensitizer. In subchronic studies (90-day feeding in rats), the no-observed-adverse-effect level (NOAEL) is approximately 1000 mg/kg/day, with adverse effects only at high doses (reduced body weight gain, increased liver weight). Imazethapyr is not genotoxic (negative in Ames test, chromosome aberration test, and micronucleus test) and is not carcinogenic in long-term studies. It is not a reproductive or developmental toxicant at doses up to 1000 mg/kg/day. The compound is highly toxic to aquatic plants (algae) but has low to moderate toxicity to fish, daphnia, and birds. It is for agricultural use only and is not intended for human consumption or therapeutic use. Standard safety precautions should be followed when handling the technical compound.

[1]. Luca Carena, et al. Modelling the photochemistry of imazethapyr in rice paddy water. Sci Total Environ. 2018 Dec 10;644:1391-1398.
[2]. Yanqiang Zhou, et al. Preparation of Imazethapyr Surface Molecularly Imprinted Polymers for Its Selective Recognition of Imazethapyr in Soil Samples. J Anal Methods Chem. 2018 Sep 30;2018:7535417.

Imazethapyr is an aromatic carboxylic acid belonging to the pyridine class of compounds. Mechanism of Action After being absorbed by plant roots and leaves, IZR is transported to the meristematic tissue region, where it inhibits the biosynthesis of valine, leucine, and isoleucine, thereby preventing cell division. IZR, imidazolinone (IMZC), imidazolinone (IMZT), imidazolinone (IMZX), and imidazoquinoline (IMZQ)... all belong to the imidazolinone (IMI) class of herbicides. Their mechanism of action is the inhibition of acetylhydroxy acid synthase (AHAS), an enzyme involved in the biosynthesis of leucine, isoleucine, and valine.

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Imazethapyr is an imidazolinone herbicide that inhibits acetolactate synthase (ALS), a key enzyme in branched-chain amino acid biosynthesis. It is used for pre- and post-emergence control of broadleaf weeds and grasses in crops such as soybeans, peanuts, and dry beans. The compound provides residual activity, giving extended weed control. It is selective due to differential metabolism in tolerant crops. Imazethapyr has a molecular formula of C15H19N3O3 and a molecular weight of 289.33. It is a white to off-white solid with a melting point of 140-145degC. The compound is slightly soluble in water (approximately 1.4 g/L at 25degC, pH 7) and soluble in organic solvents. As of 2026, Imazethapyr is approved for agricultural use in many countries but is not approved for human or animal therapeutic applications. It is for research use and agricultural purposes only.

C15H19N3O3

289.33

289.143

81335-77-5

101917-66-2 (ammonium-salt)

54740

White to off-white crystalline solid
Off-white to tan solid

1.28 g/cm3

446.8ºC at 760 mmHg

173 °C

224ºC

1.397

2

5

4

21

475

0

O=C(C1C(C2NC(C(C)C)(C)C(=O)N=2)=NC=C(CC)C=1)O

XVOKUMIPKHGGTN-UHFFFAOYSA-N

InChI=1S/C15H19N3O3/c1-5-9-6-10(13(19)20)11(16-7-9)12-17-14(21)15(4,18-12)8(2)3/h6-8H,5H2,1-4H3,(H,19,20)(H,17,18,21)

5-ethyl-2-(4-methyl-5-oxo-4-propan-2-yl-1H-imidazol-2-yl)pyridine-3-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 83.33 mg/mL (288.01 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.19 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.19 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.19 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)