URAT1 (urate transporter 1); non-competitive inhibitor of XO/xanthine oxidase; GLUT9; CYP2C9
The primary therapeutic target of Benzbromarone is Human Uric Acid Transporter 1 (hURAT1, SLC22A12). By non-competitively inhibiting URAT1 on the apical membrane of renal proximal tubular cells, it blocks the reabsorption of uric acid, thereby promoting its excretion. Additionally, Benzbromarone is a potent inhibitor of Cytochrome P450 2C9 (CYP2C9) and also inhibits CYP2C19 and AKR1C family members. It also shows activating/agonistic effects on Peroxisome Proliferator-Activated Receptors (PPARα and PPARγ).
Benzbromarone targets CYP2C9 (Ki = 19.3 ± 7.5 nM, competitive inhibition) [1]
Benzbromarone affects mitochondrial targets: inhibits electron transport chain complexes, uncouples oxidative phosphorylation, and inhibits mitochondrial β-oxidation (IC50 for β-oxidation inhibition ~2 μmol/L) [2]

In vitro activity: Benzbromarone (20 μM) decreases mitochondrial membrane potential by 81% in isolated rat hepatocytes. Benzbromarone decreases state 3 oxidation and respiratory control ratios for L-glutamate with IC50 < 1 μM in isolated rat liver mitochondria. Benzbromarone (50 μM) uncouples oxidative phosphorylation and increases oxygen consumption by hepatocytes starting at 10 μM in isolated rat hepatocytes. Benzbromarone also inhibits the formation of acid-soluble β-oxidation products in a dose-dependent manner with IC50 of 2 μM. Benzbromarone (100 μM) inhibits the electron transport chain and are uncouplers of oxidative phosphorylation in isolated rat liver mitochondria. Benzbromarone (1 μM) leads to concentration-dependent increasion of ROS production in HepG2 cells. Benzbromarone (100 μM) leads to a significant increase in mitochondrial size of isolated rat liver mitochondria. Benzbromarone is associated with leakage of cytochrome c into the cytoplasm of HepG2 cells. Benzbromarone (100 μM) results in the proportion of apoptotic cells of 11% in rat hepatocytes. Benzbromarone significantly reduces the oxypurinol uptake at a concentration as low as 10 nM and completely blocks it at 1 μM. Benzbromarone (1 μM) uptakes the typical substrate of OCTN1 (tetraethylammonium) and OCTN2 (carnitine) in the HEK293 cells expressed with human OCTN1 by 96.7% and 111% of control, respectively. Benzbromarone completely inhibits urate uptake at 50 μM in URAT1-expressing oocytes, with IC50 of less than 0.1 μM. Benzbromarone activates through sequential hydroxylation of the benzofuran ring to a catechol, which can then be further oxidized to a reactive quinone intermediate capable of adducting protein. [1,2]

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In vitro studies demonstrate that Benzbromarone is a highly potent inhibitor of hURAT1, with an IC₅₀ value of 26 nM. Its major Phase I metabolite, 6-hydroxy-benzbromarone, retains inhibitory activity with an IC₅₀ of 189 nM. Regarding metabolic enzymes, Benzbromarone potently inhibits CYP2C9 with an IC₅₀ of 0.016 μM and CYP2C19 with an IC₅₀ of 18.2 μM. It also exhibits direct scavenging activity against superoxide radicals independent of its uric acid-lowering effects.

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In vitro, Benzbromarone is a potent CYP2C9 inhibitor with a Ki of 19.3 ± 7.5 nM, determined using (S)-warfarin as substrate and measuring 7-OH-warfarin formation in reconstituted enzyme systems. The pKa of Benzbromarone is 4.5 ± 0.6, and its log P is 6.6 ± 0.9, with log D7.0 of 4.4. Non-halogenated analogs have higher pKa and lower affinity, supporting the role of an anionic group for binding. [1]
In isolated rat hepatocytes, Benzbromarone (20 μmol/L) decreased mitochondrial membrane potential by 81% relative to control. In isolated rat liver mitochondria, Benzbromarone decreased state 3 oxidation and respiratory control ratio (RCR) for L-glutamate and succinate in a concentration-dependent manner. The concentration causing 50% decrease in RCR was <1 μmol/L for both substrates. At 0.1 μmol/L, RCR for L-glutamate was 6.3 ± 1.9 vs. control 6.5 ± 1.0; at 1 μmol/L, RCR was 2.0 ± 0.3; at 10 μmol/L, RCR was 1.0 ± 0.0. Benzbromarone also uncoupled oxidative phosphorylation in isolated hepatocytes, increasing oxygen consumption in the presence of oligomycin at concentrations starting from 10 μmol/L. Benzbromarone inhibited mitochondrial β-oxidation of palmitate with an IC50 of approximately 2 μmol/L; at 50 μmol/L, activity was 0.26 ± 0.06 nmol/min/mg vs. control 0.61 ± 0.08. Ketogenesis was not affected. Benzbromarone increased reactive oxygen species (ROS) production in HepG2 cells starting at 0.1 μmol/L. At 100 μmol/L, it induced mitochondrial swelling (permeability transition) as measured by forward scatter in flow cytometry. Benzbromarone at 100 μmol/L caused release of cytochrome c from mitochondria into cytoplasm in HepG2 cells, as shown by immunocytochemistry. In isolated rat hepatocytes, Benzbromarone (100 μmol/L) induced apoptosis (11% apoptotic nuclei by Hoechst 33342 staining) and necrosis (only 11% viable cells by Annexin V/PI staining). Cellular ATP content dropped to 18 ± 5% of control at 100 μmol/L. The antioxidant ascorbate (250 μmol/L) reduced but did not completely prevent apoptosis induced by Benzbromarone. [2]

The in vivo urate lowering effects of JNS4 were evaluated with Benzbromarone (BM) as positive control. Potassium oxonate and hypoxanthine were used for inducing hyperuricemia in KM mice, lesinurad was used as another positive drug. As shown in Fig. 7, the serum urate levels of model group (950.4 μM) were significantly increased (P

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In vivo, Benzbromarone effectively lowers serum uric acid levels in animal models. In rat models of angiotensin II-induced hypertension, oral administration of Benzbromarone significantly reduced oxidative stress markers (such as advanced oxidation protein products) in the kidneys, indicating direct antioxidant effects. In a rat model of pulmonary arterial hypertension, chronic treatment with Benzbromarone (targeting TMEM16A) reversed vascular remodeling. In quails, oral administration showed a Cmax of 44.01 μg/mL and an AUC of 220.94 h·μg/mL.

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Methodology for CYP Inhibition (Microsomal Assay) : The metabolic activation of Benzbromarone is studied using human liver microsomes. To identify the enzymes responsible for its bioactivation, specific chemical inhibitors (e.g., Ketoconazole for CYP3A4) are used. Incubations are carried out in the presence of an NADPH-generating system at 37°C. The samples are then analyzed via HPLC-MS/MS to detect reactive metabolites, such as epoxide-derived N-acetylcysteine (NAC) conjugates.

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CYP2C9 Inhibition Assay (Ki determination): Reconstituted human CYP2C9 (20 pmol) was incubated with (S)-warfarin at concentrations between 2.0 and 40 μM, three concentrations of inhibitor (below, at, or above the Ki), 1000 U catalase, in 1 mL of 50 mM KPi buffer (pH 7.4) at 37°C. The reaction was initiated by adding 1 μmol NADPH, quenched after 30-40 min with 0.6 mL acetone. d5-7-OH-warfarin (0.1 nmol) was added as internal standard. Metabolites were extracted and derivatized, then analyzed by GC/MS. Data were fit to a competitive inhibition model using nonlinear regression. The Km for (S)-warfarin was 4.2 ± 2.6 μM, Vmax was 22.6 ± 0.9 pmol/min/pmol CYP2C9. [1]
pKa Measurement: 10 or 100 nmol of Benzbromarone was added into 1 mL of 0.1 M KPi buffer at pH values between 2.0 and 12.0. UV-Vis absorbance was measured at λmax between 355-360 nm (phenolate form). Absorbance vs. pH was plotted and fitted to a sigmoidal function using GraphPad Prism. [1]
Mitochondrial Membrane Potential Assay in Isolated Hepatocytes: Freshly isolated rat hepatocytes were labeled with 40 nM [3H]-tetraphenylphosphonium bromide, then seeded in 96-well plates and incubated with test compounds for 1 hour at 37°C. Cells were harvested on a unifier GF/B, mixed with scintillation cocktail, and counted for 3H-radioactivity. Results were expressed as percentage of control (1% DMSO). [2]
Mitochondrial Oxygen Consumption Assay: Oxygen uptake was monitored polarographically using a 1-mL chamber with a Clark-type oxygen electrode at 30°C. Substrates used were 20 mmol/L L-glutamate or succinate. Test compounds were dissolved in DMSO and added at indicated concentrations. State 3, state 4, and respiratory control ratio (RCR) were determined. For uncoupling assay, isolated rat hepatocytes (1×10^6 cells) were treated with oligomycin (5 μg/mL) to inhibit ATPase, then test compounds were added, and oxygen consumption was measured. [2]
Mitochondrial β-Oxidation Assay: Formation of 14C-acid-soluble β-oxidation products from [1-14C]palmitic acid by isolated rat liver mitochondria was measured in the presence of test compounds. Incubations were carried out as described, and supernatants were analyzed for radioactivity. [2]
Ketogenesis Assay: Ketone body formation was measured using freeze-thawed mitochondria with an acetyl-CoA generating system. Acetoacetate in supernatants was determined by an enzyme-catalyzed reaction measuring NADH changes. [2]
Acyl-CoA Dehydrogenase and β-Ketothiolase Assays: Freeze-thawed mitochondria were treated with 5% cholic acid to disrupt membranes, then diluted 100-fold. Acyl-CoA dehydrogenase activity was determined spectrophotometrically; β-ketothiolase activity was measured similarly. [2]
Reactive Oxygen Species (ROS) Assay: Confluent HepG2 cells in 96-well plates were incubated with 5 mmol/L 2,7-dichlorofluorescin diacetate in DMEM without serum. After incubation, medium was replaced with PBS, and cellular fluorescence (λex=485 nm, λem=520 nm) was measured at room temperature using a microtiter plate reader. [2]
Mitochondrial Swelling Assay: Rat liver mitochondria (1 mg protein) were mixed with 200 μL MSM buffer, and test compounds were added. Swelling was measured by flow cytometry (FACScalibur) as mean forward scatter. CaCl2 (1 mmol/L) was used as positive control, and cyclosporin A (2 μmol/L) as inhibitor. [2]
Cytochrome c Release Immunocytochemistry: HepG2 cells grown on poly-D-lysine-coated chamber slides were treated with test compounds for 8 hours. Cytochrome c was visualized using an anti-cytochrome c antibody followed by an anti-sheep IgG antibody conjugated with Cy3. [2]

Methodology (PPAR Transactivation Assay) : NIH/3T3 cells are seeded in 24-well plates. After 20 hours, they are transfected with plasmids encoding the ligand-binding domain of PPAR (α or γ) fused to a GAL4 DNA-binding domain, along with a luciferase reporter. Following transfection, cells are treated with Benzbromarone (0.001–100 μM) or control vehicle (0.1% DMSO) for 48 hours. Cells are then lysed, and luciferase activity is measured using a luminometer to quantify PPAR activation.

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Hepatocyte Viability and Apoptosis/Necrosis Assay: Freshly isolated rat hepatocytes were cultured on poly-D-lysine-coated slides and treated with test compounds for 8 hours. For Hoechst 33342 nuclear staining, cells were incubated with 50 μmol/L Hoechst 33342 for 30 min at room temperature and visualized by fluorescence microscopy. Apoptotic cells were identified by chromatin condensation and/or fragmentation. For Annexin V/PI staining, cells were stained with Annexin V-Alexa Fluor 488 and propidium iodide (1.5 μg/L final) for 15 min at room temperature, then analyzed by flow cytometry. Viable cells (unstained), early apoptotic (Annexin V only), and late apoptotic/necrotic (double-stained) populations were quantified. [2]
Cellular ATP Content Assay: Freshly isolated rat hepatocytes (200,000 cells/well) in 12-well plates were treated with test compounds for 8 hours. Cells were washed with PBS, suspended in 1 mL water, snap-frozen, and stored at -80°C. ATP was extracted by boiling water, and concentration was determined using a luciferin-luciferase reagent kit, comparing against an ATP standard curve. [2]

4 mg/kg of BM; PO
Hyperuricemia model in KM mice

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Methodology (Rat Pharmacokinetics) : Male Sprague-Dawley rats are typically used. To determine absolute bioavailability, animals are divided into two groups: an intravenous (IV) group and an oral (PO) group. For the PO group, a suspension of Benzbromarone is administered via gavage. Blood samples are collected at various time points (e.g., 0, 0.5, 1, 2, 4, 6, 8, and 24 hours post-dose). Plasma concentrations are measured by LC-MS/MS, and parameters such as Tmax, Cmax, and AUC are calculated using non-compartmental analysis.

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In animal models, Benzbromarone is rapidly absorbed with an oral Tmax of approximately 1.56 hours in quails. It exhibits moderate to good oral bioavailability (F~0.58 in rats). The elimination half-life (T1/2) is relatively short; in quails, the elimination half-life (K10_HL) is approximately 2.06 hours. It is extensively metabolized in the liver, primarily via hydroxylation (CYP2C9) to 6-hydroxy-benzbromarone, and also undergoes further bioactivation to reactive epoxide intermediates.

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Hepatotoxicity
The incidence of liver dysfunction during benzbromarone treatment has been reported to be very low, with only 0.1% of patients experiencing abnormalities in clinical trials. Furthermore, benzbromarone was widely used for many years until reports of hepatotoxicity emerged in the late 1980s. Since then, there have been reports of acute liver injury and acute liver failure during benzbromarone treatment. Liver injury typically appears 1 to 6 months after treatment, manifesting as jaundice and fatigue, usually accompanied by elevated hepatocyte enzymes. Immune allergic symptoms (rash, fever) are uncommon. Some case reports show low levels of autoantibodies, and liver histology reveals chronic active hepatitis, especially if benzbromarone is not discontinued promptly. Symptoms usually subside within 1 to 3 months after discontinuation. Liver injury caused by benzbromarone is similar to that reported with benzalkonium chloride (a structure-related drug formerly used to treat peripheral vascular disease but also withdrawn from the market due to hepatotoxicity issues). Probability Score: B (Very likely, but rare, to cause clinically significant liver injury).

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The primary toxicity concern for Benzbromarone is hepatotoxicity (liver injury) . It was withdrawn from the European market in 2003 due to reports of serious liver damage, including fatalities. The mechanism is linked to the formation of reactive metabolites (epoxides and quinone intermediates) by CYP3A4 and CYP2C9, which can covalently bind to proteins. In animal studies, it induces hepatic hypertrophy and peroxisome proliferation (via PPARα activation). Accidental ingestion is toxic; GHS classification indicates Acute Toxicity Category 3 (H301: Toxic if swallowed).

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Hepatic toxicity: Benzbromarone has been associated with hepatic injury in patients. In vitro, it is a mitochondrial toxin: it decreases mitochondrial membrane potential (81% decrease at 20 μmol/L in rat hepatocytes), inhibits mitochondrial respiration (RCR decreased by 50% at <1 μmol/L), uncouples oxidative phosphorylation, inhibits β-oxidation (IC50 ~2 μmol/L), induces ROS production (starting at 0.1 μmol/L in HepG2 cells), triggers mitochondrial permeability transition (swelling at 100 μmol/L), causes cytochrome c release, and induces both apoptosis and necrosis in isolated rat hepatocytes (11% viable cells at 100 μmol/L). Cellular ATP content drops to 18% of control at 100 μmol/L. The toxicity is structure-dependent; the benzofuran core alone is not toxic; side chains at the furan ring are necessary. [2]
Drug-drug interaction: Co-administration of Benzbromarone with the anticoagulant (S)-warfarin increases bleeding due to enantioselective metabolic inhibition of warfarin clearance by CYP2C9. [1]

[1]. Drug Metab Dispos.2003 Jul;31(7):967-71;
[2]. Hepatology.2005 Apr;41(4):925-35.

Benzbromarone is a 1-benzofuran with ethyl and 3,5-dibromo-4-hydroxybenzoyl groups substituted at the C-2 and C-3 positions, respectively. It is a CYP2C9 inhibitor used to treat gout. It is also a uricosuric drug. Benzbromarone belongs to the 1-benzofuran class of compounds and is an aromatic ketone. Its structure is similar to 2,6-dibromophenol. Benzbromarone has been used in basic science research and clinical trials for the treatment of heart failure, hyperuricemia, chronic kidney disease, renal dysfunction, gout, and asymptomatic hyperuricemia. Benzbromarone is a non-purine xanthine oxidase inhibitor used to treat gout, but it has never been approved for use in the United States due to reports of acute liver injury and death. It is a uricosuric drug that works by increasing uric acid clearance. It is used to treat gout.

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Background: Benzbromarone (CAS# 3562-84-3) is a uricosuric agent that reduces proximal tubular reabsorption of uric acid. Its structure consists of a benzofuran ring with a 2-ethyl group, a 3-(3,5-dibromo-4-hydroxybenzoyl) group. It is structurally similar to amiodarone and benzarone. [1][2]
Mechanism of CYP2C9 inhibition: Benzbromarone binds to CYP2C9 with high affinity. The proposed binding mode involves an anionic interaction (the phenolate ion at physiological pH) with a cationic site (likely Arg105 or Arg108) in the active site, hydrophobic interactions, and positioning of the benzofuran ring near the heme for metabolism at position 6. [1]
Metabolism: The primary metabolite of Benzbromarone occurs at position 6 of the benzofuran heterocycle. [1]
Clinical relevance: Hepatic toxicity cases have been reported with Benzbromarone and its analog benzarone, sometimes with fatal outcome. The toxicity is attributed to mitochondrial dysfunction. [2]

C17H12BR2O3

424.08

421.915

C, 48.15; H, 2.85; Br, 37.68; O, 11.32

3562-84-3

Benzbromarone-d5

2333

White to off-white solid powder

1.7±0.1 g/cm3

514.1±50.0 °C at 760 mmHg

161 - 163ºC

264.7±30.1 °C

0.0±1.4 mmHg at 25°C

1.673

6.64

1

3

3

22

405

0

O=C(C1=CC(Br)=C(O)C(Br)=C1)C2=C(CC)OC3=CC=CC=C23

WHQCHUCQKNIQEC-UHFFFAOYSA-N

InChI=1S/C17H12Br2O3/c1-2-13-15(10-5-3-4-6-14(10)22-13)16(20)9-7-11(18)17(21)12(19)8-9/h3-8,21H,2H2,1H3

(3,5-dibromo-4-hydroxyphenyl)(2-ethylbenzofuran-3-yl)methanone

L2214;MJ10061; L 2214; MJ-10061; L-2214; L2214-Labaz; Narcaricin; Normurat; Benzbromarone; MJ 10061

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.90 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 (4.90 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (4.90 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.)