Insect GABA-gated chloride channel (RDL GABA receptor) non-competitive antagonist. [1]
Desmethyl-broflanilide (active metabolite): IC50 = 1.3 nM against wild-type Spodoptera litura RDL GABA receptor homomer expressed in cells (membrane potential assay). [1]
The primary target of broflanilide is the insect γ-aminobutyric acid (GABA)-gated chloride channel, specifically the resistant-to-dieldrin (RDL) GABA receptor. Broflanilide itself acts as a prodrug and is metabolized in insects to the active metabolite desmethyl-broflanilide (DMBF). DMBF acts as a potent and selective non-competitive antagonist of the insect RDL GABA receptor, binding to a novel site (M1-M3 intersubunit cavity) and irreversibly blocking the chloride channel. This inhibits chloride influx, leading to hyperexcitation, convulsions, paralysis, and death in insects. Unlike conventional GABA antagonists like fipronil and dieldrin, broflanilide binds to a distinct site, showing no cross-resistance with these insecticides.

Desmethyl-broflanilide, a strong and specific antagonist of insect anti-dieldrin (RDL) GABA receptors, is produced when the pesticide broflanilide metabolizes. Its IC50 value for inhibiting Spodoptera litura RDL GABAR is 1.3 nM. Desmethyl-broflanilide has relatively mild inhibitory effects on GlyR α1-A288Gβ and GlyR α1-A288G. mild (IC50s, 0.706, 0.149 μM) [1]. It also has poor action against human GABAA α1β2η2 receptor, mammalian GABAA α1β3η2 receptor, and human glycine receptor (GlyR) α1β.

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A membrane potential assay showed that the active metabolite, desmethyl-broflanilide, exhibited high inhibitory potency against the wild-type Spodoptera litura RDL GABA receptor subunit homomer, with an IC50 value of 1.3 nM. This potency was significantly higher than that of conventional non-competitive antagonists (NCAs) such as fipronil (IC50 = 105 nM), EBOB, picrotoxin, lindane, dieldrin, and α-endosulfan. [1]
Two-electrode voltage-clamp (TEVC) recordings using Xenopus oocytes expressing wild-type S. litura RDL GABA receptor showed that the antagonist activity of desmethyl-broflanilide was 1000 times higher than that of the parent compound broflanilide. [1]
In membrane potential assays using Drosophila S2 cells, a linear correlation (R² = 0.94) was observed between the larvicidal activity (LD50) of various meta-diamides against S. litura and their inhibitory potency against the S. litura RDL GABA receptor homomer, supporting RDL GABA receptor as the target. [1]
Mutation studies in Drosophila RDL GABA receptor revealed that the G336M mutation (equivalent to G319M in S. litura) abolished the inhibitory activity of desmethyl-broflanilide but had little effect on NCAs like fipronil. In contrast, mutations at the A2' position (e.g., A2'S, A2'N, A2'G), which confer resistance to cyclodienes and reduce fipronil activity, had no effect on the activity of desmethyl-broflanilide. [1]
Desmethyl-broflanilide showed high selectivity for insect RDL GABA receptors. Its IC50 against human GABA_A receptor (α1β2γ2 and α1β3γ2) and human glycine receptor (α1 and α1β) was >3 µM, compared to 1.3 nM for the insect receptor. An A288G mutation in the human glycine receptor α1 subunit (equivalent to G336 in Drosophila) dramatically increased sensitivity to desmethyl-broflanilide (IC50 decreased from >3 µM to 149 nM for α1 and 706 nM for α1β). [1]
The binding site of desmethyl-broflanilide overlaps with that of macrocyclic lactones (e.g., ivermectin, milbemectin), as evidenced by complete inhibition of [³H]BPB 1 (a desmethyl-broflanilide analog) binding by these compounds. However, the effects of mutations (e.g., I277F, L281C, V340Q/N, A2'S/N) on the activity of macrocyclic lactones (changing them from agonists to antagonists) differed from their effects on desmethyl-broflanilide (modest reduction or no change). [1]
Homology modeling and docking studies suggested that desmethyl-broflanilide binds to an intersubunit cavity (M1-M3 pocket) near G336 in the closed conformation of the Drosophila RDL GABA receptor, distinct from the pore-binding site of fipronil. [1]
Broflanilide (and BPB 3, an N-methyl analog) did not inhibit the binding of [³H]BPB 1 to housefly RDL GABA receptor membranes, while desmethyl-broflanilide (and BPB 1) completely inhibited it, indicating that N-methyl meta-diamides require demethylation to become active. [1]

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In vitro studies demonstrate that broflanilide is metabolized to the active metabolite desmethyl-broflanilide. In membrane potential assays using cells expressing wild-type Spodoptera litura RDL GABA receptors, desmethyl-broflanilide exhibited potent inhibitory activity with an IC₅₀ value of 1.3 nM, significantly more potent than conventional non-competitive antagonists like fipronil (IC₅₀=105 nM). Two-electrode voltage clamp recordings in Xenopus oocytes expressing the receptor showed that desmethyl-broflanilide was approximately 1000 times more active than the parent compound broflanilide. This active metabolite showed very weak inhibition of mammalian GABA_A receptors (α1β2γ2 and α1β3γ2) and human glycine receptor α1β (IC₅₀ >3 μM), but its activity was enhanced against glycine receptor α1 subunit containing the A288G mutation (IC₅₀=149 nM). Furthermore, cytotoxicity studies on human SH-SY5Y neuroblastoma cells showed that the commercial broflanilide formulation, mainly due to the active ingredient BFL, causes mitochondrial damage (collapse of membrane potential and opening of permeability transition pore), DNA damage (single-strand breaks, double-strand breaks, and oxidative damage), and induces mitochondria-mediated apoptosis.

Broflanilide and desmethyl-broflanilide caused the same excitatory symptoms (convulsions and paralysis) in Spodoptera litura larvae. [1]
There was no difference in the larvicidal potency between broflanilide and desmethyl-broflanilide. [1]
BPB 3 (an N-methyl meta-diamide analog) showed the same level of insecticidal activity against both dieldrin-susceptible and dieldrin-resistant houseflies (carrying the A2'S mutation). [1]

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Broflanilide demonstrates potent insecticidal activity against a wide range of agricultural and public health pests. A strong linear correlation (R²=0.94) was observed between its inhibitory potency against the Spodoptera litura RDL GABA receptor (IC₅₀=1.3 nM) and its larvicidal activity, confirming RDL GABA receptor as the functional target for its insecticidal action. In indoor residual spraying (IRS) applications, the broflanilide-based formulation VECTRON™ T500 (100 mg ai/m²) showed extended lethal effects against both laboratory-susceptible and field populations of Anopheles gambiae mosquitoes, maintaining over 80% mortality against wild populations for up to six months post-spraying on cement and mud surfaces, with induced mortality rates of 83.2-95.0%. The compound exhibits low knockdown but effectively exposes mosquitoes to lethal doses. For agricultural pest control, broflanilide is highly effective against chewing pests including Lepidoptera (diamondback moth, fall armyworm), Coleoptera (striped flea beetle), and Thysanoptera, and remains effective against pests resistant to fipronil. After ten generations of continuous exposure to broflanilide, the Spodoptera frugiperda population developed only 2.71-fold resistance, indicating low metabolic resistance risk, though with a significant fitness cost (relative fitness of only 0.35).

In vitro enzyme/receptor binding assays are primarily performed using radioligand binding techniques and functional assays. 1. Radioligand Binding Assay: Membranes prepared from cells or housefly heads expressing RDL GABA receptors are incubated with tritiated ligands such as [³H]BPB 1 (a desmethyl-broflanilide analog) and varying concentrations of test compounds. Bound and free radioligands are separated by filtration, and bound radioactivity is measured by scintillation counting. 2. Membrane Potential Assay: Drosophila S2 cells or other cell lines expressing RDL GABA receptors are stimulated with GABA at EC₅₀ concentration in the presence of test compounds. Changes in membrane potential are detected using fluorescent or potentiometric dyes, and concentration-response curves are generated to calculate IC₅₀ values. 3. Two-Electrode Voltage Clamp (TEVC): Xenopus laevis oocytes injected with cRNA encoding RDL GABA receptor subunits are voltage-clamped, and GABA-induced chloride currents are recorded to evaluate the antagonistic activity of compounds.

Membrane Potential Assay: The inhibitory activities of compounds against RDL GABA receptors were determined using a membrane potential assay. Drosophila S2 cells or other suitable cell lines expressing wild-type or mutant RDL GABA receptor subunit homomers were used. Changes in membrane potential upon application of GABA (at an EC50 concentration) in the presence or absence of test compounds were measured, typically using fluorescent or potentiometric dyes. Concentration-response curves were generated to calculate IC50 values. This assay was used to compare the potency of desmethyl-broflanilide with NCAs and to assess the effects of various mutations. [1]
Two-Electrode Voltage Clamp (TEVC): Xenopus laevis oocytes were injected with cRNA encoding wild-type or mutant RDL GABA receptor subunits. After expression, oocytes were voltage-clamped, and chloride currents elicited by GABA application were recorded. The inhibitory effects of test compounds (like broflanilide and desmethyl-broflanilide) on these GABA-induced currents were measured to determine antagonist potency. [1]
Radioligand Binding Assay: Membranes prepared from housefly heads or cells expressing RDL GABA receptors were used. The binding of tritiated ligands such as [³H]EBOB, [³H]BPB 1 (a desmethyl-broflanilide analog), or [³H]avermectin to the receptor was measured in the presence or absence of competing compounds (e.g., broflanilide, desmethyl-broflanilide, fipronil, dieldrin). This assay helped characterize the binding site and interactions between different classes of insecticides. [1]

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In vitro cell-based assays are conducted using both insect and mammalian cell lines. 1. Insect Cell Assays: Drosophila S2 cells expressing wild-type or mutant RDL GABA receptors are used in membrane potential assays. Cells are stimulated with GABA at EC₅₀ concentration, and changes in membrane potential in the presence of varying test compound concentrations are detected using fluorescent dyes, generating concentration-response curves to calculate IC₅₀ values. 2. Mammalian Cytotoxicity Assays: Human SH-SY5Y neuroblastoma cells are treated with broflanilide formulation. Cell viability is assessed by MTT assay, apoptosis rate by Annexin V-FITC/PI double staining flow cytometry, mitochondrial membrane potential collapse by JC-1 staining, ROS levels by DCFH-DA fluorescent staining, DNA damage (single- and double-strand breaks) by single-cell gel electrophoresis (comet assay), and apoptosis-related protein expression by Western blot.

In vivo animal studies primarily involve insect bioassays and WHO cone bioassays for public health applications. 1. Agricultural Pest Bioassays: Using the diet-overlay method, broflanilide is serially diluted and incorporated into artificial diet for feeding newly-hatched or 3rd instar Spodoptera frugiperda larvae for multiple generations (e.g., 10 generations). Parameters including mortality, body weight, pupation rate, emergence rate, and fecundity are recorded. LC₅₀ values, resistance ratios, and relative fitness are calculated. 2. Public Health Mosquito Bioassays (Indoor Residual Spraying): Following the standard WHO cone bioassay method, VECTRON™ T500 formulation is sprayed on cement and mud panels at a rate of 100 mg ai/m². Monthly testing involves placing laboratory-reared susceptible or field-collected Anopheles gambiae mosquitoes into cones attached to treated panels for 30 minutes. One-hour knockdown is recorded, and mosquitoes are transferred to holding tubes for 24-hour mortality assessment. Residual efficacy is monitored for up to six months.

Studies have shown that Broflanilide is metabolized in vivo to the active form desmethylBroflanilide (N-demethylation). This hypothesis is supported by the following facts: 1) the two compounds have the same larvicidal effect and potency; 2) the in vitro antagonistic activity of desmethylBroflanilide is 1000 times higher than that of Broflanilide; 3) N-methyl analogs (such as BPB 3) have insecticidal activity, but unlike desmethyl analogs, they do not bind to the RDL GABA receptor in vitro. [1]

Broflanilide exhibits high selective safety for mammals but poses certain toxicity risks to aquatic organisms. Regarding receptor selectivity, the active metabolite desmethyl-broflanilide shows approximately 2300-fold higher potency against insect RDL GABA receptors (IC₅₀=1.3 nM) compared to mammalian GABA_A receptors (IC₅₀ >3 μM), demonstrating excellent selectivity. Cytotoxicity studies on human SH-SY5Y neuroblastoma cells show that broflanilide formulation can cause mitochondrial damage (collapse of membrane potential and opening of permeability transition pore), DNA damage (single-strand breaks, double-strand breaks, and oxidative damage), and induce mitochondria-mediated apoptosis. Environmental toxicology studies indicate that prolonged exposure to low concentrations of broflanilide causes chronic toxicity in zebrafish, posing a potential risk to aquatic ecosystems. In agricultural applications, broflanilide presents high toxicity risk to non-target arthropods including Apis mellifera (honeybee) and Trichogramma ostriniae (a parasitoid wasp).

[1]. Broflanilide: A meta-diamide insecticide with a novel mode of action. Bioorg Med Chem. 2016 Feb 1;24(3):372-7.

Broflanilide is a benzamide formed by the condensation of the carboxyl group of 3-[benzoyl(methyl)amino]-2-fluorobenzoic acid with the amino group of 2-bromo-4-(1,1,1,2,3,3,3-heptafluoroprop-2-yl)-6-(trifluoromethyl)aniline. It is an insecticide with high killing activity against the larvae of the beet armyworm and is also effective against pests resistant to cyclodienes and fipronil. It can be used as an agrochemical, GABA antagonist, and insecticide. It belongs to the benzamide class, monofluorobenzene class, organofluorine insecticides, bromobenzene class, and (trifluoromethyl)benzene class. Bromofluoroamide is a m-diamide insecticide discovered by Mitsui Chemicals & Agriculture Co., Ltd. [1]
Its main mechanism of action is as a non-competitive antagonist (NCA) of insect GABA-gated chloride channels (RDL GABA receptors), but its site of action is different from that of classic NCAs such as cyclodienes, lindane, and fipronil. [1]
The binding site of its active metabolite, desmethylbromofluoroamide, is located at or near the G336 residue of the M3 transmembrane region of the Drosophila RDL GABA receptor (intersubunit cavity). [1]
This novel site of action is considered to be the key to its effective resistance against pests resistant to cyclodienes and fipronil due to mutations at the A2' site in the M2 region of the RDL GABA receptor. [1]
DesmethylBroflanilide has a much higher selectivity for insect GABA and glycine receptors than that for mammals, which gives it good toxicological properties. The glycine residue corresponding to the G336 site of the mammalian receptor is the key factor determining this selectivity. [1]
Although their binding sites overlap with those of macrolides (e.g., ivermectin), their mechanisms of action differ (antagonist vs. allosteric agonist), and receptor mutations have different effects on their activity. [1]

C25H14BRF11N2O2

663.277304172516

662.006

C, 45.27; H, 2.13; Br, 12.05; F, 31.51; N, 4.22; O, 4.82

1207727-04-5

53341374

White to off-white solid powder

1.6±0.1 g/cm3

467.9±45.0 °C at 760 mmHg

236.8±28.7 °C

0.0±1.2 mmHg at 25°C

1.538

5.3

1

13

5

41

922

0

BrC1=CC(=CC(C(F)(F)F)=C1NC(C1C=CC=C(C=1F)N(C)C(C1C=CC=CC=1)=O)=O)C(C(F)(F)F)(C(F)(F)F)F

QSLZKWPYTWEWHC-UHFFFAOYSA-N

InChI=1S/C25H14BrF11N2O2/c1-39(21(41)12-6-3-2-4-7-12)17-9-5-8-14(18(17)27)20(40)38-19-15(23(29,30)31)10-13(11-16(19)26)22(28,24(32,33)34)25(35,36)37/h2-11H,1H3,(H,38,40)

3-[benzoyl(methyl)amino]-N-[2-bromo-4-(1,1,1,2,3,3,3-heptafluoropropan-2-yl)-6-(trifluoromethyl)phenyl]-2-fluorobenzamide

broflanilide; 1207727-04-5; 5M24RC688M; CHEBI:131598; DTXSID50894815;

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 : ~250 mg/mL (~376.91 mM)

Solubility in Formulation 1: 2.08 mg/mL (3.14 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (3.14 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.)