| Targets |
Protoporphyrinogen oxidase (PPO, also known as Protox), a key enzyme in the chlorophyll and heme biosynthesis pathway. PPO catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. Bifenox binds competitively to the active site of PPO, inhibiting its activity. This inhibition results in the accumulation of protoporphyrinogen IX, which leaks out of the chloroplast and is non-enzymatically oxidized to protoporphyrin IX, leading to light-dependent lipid peroxidation and cell membrane disruption.
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|---|---|
| ln Vitro |
Bifenox is a potent inhibitor of plant PPO. In cell-free assays, the concentration required for 50% inhibition (IC50) of PPO activity is typically in the low nanomolar range (e.g., 10-50 nM). The compound does not exhibit significant inhibitory activity against mammalian PPO at the same concentrations due to differences in the active site structure. It shows no activity against other enzymes involved in tetrapyrrole biosynthesis.
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| ln Vivo |
In vivo, bifenox is absorbed through the leaves and roots of target weeds. After application, it translocates primarily in the apoplast (via the xylem). The herbicidal effect is light-dependent and manifests as rapid desiccation, necrosis, and bleaching of the treated foliage. In crops with metabolic tolerance (e.g., soybeans, corn), bifenox is rapidly detoxified by O-demethylation and conjugation with glutathione, whereas susceptible weeds metabolize it slowly. The half-life in plants is approximately 1-3 days.
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| Enzyme Assay |
The PPO enzyme inhibition assay is performed using purified PPO from a plant source (e.g., corn or pea). The reaction mixture (1 mL) contains 50 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 0.1% Tween 20, 10 uM protoporphyrinogen IX (freshly prepared by chemical reduction of protoporphyrin IX with sodium amalgam), and varying concentrations of bifenox (0.1-1000 nM). The reaction is initiated by adding PPO (5-10 ug protein). The increase in fluorescence due to the oxidation of protoporphyrinogen IX to protoporphyrin IX (Ex=405 nm, Em=630 nm) is measured continuously for 10 min at 25degC. The IC50 is calculated.
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| Cell Assay |
Not applicable for mammalian cells, as bifenox is a herbicide. For plant cell cultures, a sensitive plant line (e.g., Lemna minor or duckweed) is treated with bifenox (0.1-100 uM) for 24-48 h under continuous light (100 umol/m2/s). Chlorophyll content is extracted with 80% acetone and quantified spectrophotometrically at 648 and 664 nm. The EC50 for chlorophyll loss is calculated. Alternatively, electrolyte leakage (membrane damage) is measured using a conductivity meter.
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| Animal Protocol |
In a greenhouse efficacy study, weed species (e.g., Amaranthus retroflexus, Echinochloa crus-galli) are grown in pots to the 2-4 leaf stage. Bifenox is applied as a post-emergence spray at rates of 0.5-2 kg active ingredient per hectare. Crop species (e.g., soybean, corn) are also treated to assess selectivity. Visual injury ratings (0-100% scale) are recorded at 7, 14, and 21 days after treatment. Fresh and dry shoot weights are measured at study termination. The ED90 (dose for 90% weed control) is determined.
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| ADME/Pharmacokinetics |
Absorption, Distribution and Excretion
Biphenylnorone is absorbed and eliminated relatively rapidly in vivo; 5-(2,4-dichlorophenyl)-2-nitrobenzoic acid is the main urinary metabolite, and biphenylnorone was not detected. Biphenylnorone and 5-(2,4-dichlorophenyl)-o-aminobenzoic acid were detected in feces. …Technical-grade biphenylnorone (98% purity) was suspended in 1% carboxymethyl cellulose solution and orally administered at a dose of 2 g/kg to six equal numbers of young adult goats to study its oral pharmacokinetics. Blood samples were collected from each goat at different time intervals, and the concentration of biphenylnorone in the blood was determined by gas chromatography. Biphenylnorone was detected in the blood at 2 hours after administration (1.32 ± 0.07 μg/ml), reaching a peak at 12 hours (6.76 ± 0.17 μg/ml), then gradually decreasing in concentration, reaching a minimum at 24 hours. The pharmacokinetic behavior of biphenylnorone conforms to a two-compartment open model. The volume area of distribution and the blood clearance rate were 2.73±0.28 L/kg and 0.88±0.07 L/kg/hour, respectively, indicating that it was widely distributed in the body and had a short duration of action. Biological Half-Life Bifenox technical grade drug with a purity of 98% was suspended in a 1% carboxymethyl cellulose solution and administered orally at a dose of 2 g/kg to six young adult goats (half male and half female) for oral pharmacokinetic studies. The half-life was 2.04 ± 0.04 hours. In mammals, bifenox is rapidly absorbed from the gastrointestinal tract after oral administration, but it is also rapidly metabolized and excreted. The elimination half-life in rats is approximately 5-10 hours. The primary route of metabolism is O-demethylation to the corresponding phenol, followed by glucuronidation or sulfation. The metabolites are excreted in urine and feces. Bifenox has low oral bioavailability due to extensive first-pass metabolism. In plants, the half-life is longer (1-3 days). |
| Toxicity/Toxicokinetics |
Non-Human Toxicity Values
Oral LD50 in rats >5000 mg/kg Oral LD50 in mice 4556 mg/kg Dermal LD50 in rabbits >2000 mg/kg Oral LD50 in rats >6400 mg/kg Toxicity Data LC50 (rat) > 910 mg/m3 Bifenox has low acute toxicity to mammals. The acute oral LD50 in rats is >5,000 mg/kg, indicating very low acute toxicity. It is not a skin or eye irritant and is not a dermal sensitizer. In subchronic and chronic feeding studies, the no-observed-adverse-effect-level (NOAEL) in rats is 10-30 mg/kg/day. Bifenox is not classified as a carcinogen, mutagen, or reproductive toxicant by the EPA or IARC. However, it is highly toxic to aquatic plants and algae (EC50 < 1 mg/L). |
| References | |
| Additional Infomation |
Bifenox is a type of nitrobenzoic acid.
Bifenox is a registered herbicide for agricultural use in many countries, but it is not a pharmaceutical. It has no FDA approval or clinical trial status for human use. It belongs to the diphenyl ether class of PPO-inhibiting herbicides, which includes other compounds such as acifluorfen, fomesafen, and oxyfluorfen. Bifenox is used in integrated weed management programs, typically at application rates of 0.5-2 kg/ha. It is formulated as an emulsifiable concentrate (EC) or a flowable concentrate (SC). |
| Exact Mass |
340.986
|
|---|---|
| CAS # |
42576-02-3
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| PubChem CID |
39230
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| Appearance |
Solid
Yellow tan crystals
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| Density |
1.5±0.1 g/cm3
|
| Boiling Point |
421.0±45.0 °C at 760 mmHg
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| Melting Point |
85 °C
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| Flash Point |
208.4±28.7 °C
|
| Vapour Pressure |
0.0±1.0 mmHg at 25°C
|
| Index of Refraction |
1.608
|
| LogP |
4.52
|
| Hydrogen Bond Donor Count |
0
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
22
|
| Complexity |
417
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
SUSRORUBZHMPCO-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C14H9Cl2NO5/c1-21-14(18)10-7-9(3-4-12(10)17(19)20)22-13-5-2-8(15)6-11(13)16/h2-7H,1H3
|
| Chemical Name |
methyl 5-(2,4-dichlorophenoxy)-2-nitrobenzoate
|
| Synonyms |
Modown; MC-4379
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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