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| Targets |
FXR agonist 3 targets the farnesoid X receptor (FXR, NR1H4), a nuclear receptor that is highly expressed in the liver and intestine. FXR is activated by bile acids and regulates the expression of genes involved in bile acid homeostasis, lipid metabolism (lipogenesis and fatty acid oxidation), glucose metabolism, and inflammation. FXR activation reduces hepatic steatosis by decreasing de novo lipogenesis and increasing fatty acid oxidation. FXR also has anti-fibrotic effects by inhibiting hepatic stellate cell activation and reducing the expression of fibrogenic genes such as COL1A1, TGF-beta1, alpha-SMA, and TIMP1.
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| ln Vitro |
FXR agonist 3 (compound 3a) (5 μM; 24 h) shows anti-fibrogenic activity, decreases multiple fibrogenic biomarkers level in LX-2 cells in a dose-dependent manner[1]. FXR agonist 3 shows cytotoxic concentration against LX2 cells with an CC50 value of 70.36 μM[1]. Metabolic stability of FXR agonist 3 in human, rat and mouse liver microsomes[1] Species T1/2 (h) CLInt (mic) (μg/min/mg) CLInt (liver) ( μg/min/mg) Remaining Ratio (%) (T=60 min) Human 53.3 26.0 23.4 44.1 Rat 7.4 187.8 338.0 0.4 Mouse 7.4 187.9 744.1 39.0
In vitro, FXR agonist 3 (compound 3a) shows anti-fibrogenic activity, decreasing multiple fibrogenic biomarkers in LX-2 cells (human hepatic stellate cell line) in a dose-dependent manner at 5 microM for 24 hours. It inhibits the expression of COL1A1 (IC50 = 8.19 micromol/L), TGF-beta1, alpha-SMA, and TIMP1. The compound shows a cytotoxic concentration (CC50) against LX-2 cells of 70.36 microM. Metabolic stability studies show half-lives of 53.3 hours (human liver microsomes), 7.4 hours (rat), and 7.4 hours (mouse), with remaining ratios of 44.1% (human), 0.4% (rat), and 39.0% (mouse) after 60 minutes. No specific EC50 for FXR activation is reported. |
| ln Vivo |
FXR agonist 3 (compound 3a) (200 mg/kg; po; daily for 4 weeks) significantly attenuates the degree of liver fibrosis in choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD)-induced NASH mice model[1]. FXR agonist 3 (200 mg/kg; po; daily for 4 weeks) also exerts liver-protective and anti-fibrosis activities in bile duct ligation (BDL)-induced fibrosis rat model[1].
In vivo, FXR agonist 3 (200 mg/kg; oral; daily for 4 weeks) significantly attenuates the degree of liver fibrosis in a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD)-induced NASH mouse model. It also exerts liver-protective and anti-fibrotic effects in a bile duct ligation (BDL)-induced fibrosis rat model. The compound activates FXR, leading to reduced expression of COL1A1, TGF-beta1, alpha-SMA, and TIMP1, reduced liver steatosis and inflammation, and improved liver fibrosis levels. No other in vivo data (e.g., effects on serum bile acids, liver enzymes, or lipids) are reported. |
| Enzyme Assay |
The binding of FXR agonist 3 to FXR is measured by a standard in vitro FXR reporter gene assay (luciferase reporter assay). HEK293 or COS-7 cells are co-transfected with a plasmid encoding the human FXR ligand-binding domain (LBD) fused to a Gal4 DNA-binding domain, and a Gal4-responsive luciferase reporter. Cells are treated with FXR agonist 3 at graded concentrations (0.01-100 microM) for 24 hours. Luciferase activity is measured, and the EC50 for FXR activation is calculated. Alternatively, a competitive binding assay using [3H]-chenodeoxycholic acid (CDCA) and purified FXR protein can be performed. No specific EC50 is reported in the search results. The compound‘s inhibition of COL1A1 (IC50 = 8.19 micromol/L) is measured in LX-2 cells by qPCR or by measuring COL1A1 protein levels via Western blot.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: LX-2 Tested Concentrations: 0, 2.5, 5, 7.5, and 10 μM Incubation Duration: 24 hours; with or without 2 ng/mL TGF-β1 for another 24 hr Experimental Results: Decreased COL1A1, TGF-β1, α-SMA, and TIMP1 protein expressions in a dose-dependent manner. For in vitro cellular assays, LX-2 human hepatic stellate cells are seeded in 6- or 96-well plates. Cells are treated with FXR agonist 3 (0, 2.5, 5, 7.5, and 10 microM) for 24 hours, with or without 2 ng/mL TGF-beta1 for an additional 24 hours. Cell viability is assessed by MTT or CellTiter-Glo assays to determine the CC50 (70.36 microM). Protein expression of COL1A1, TGF-beta1, alpha-SMA, and TIMP1 is assessed by Western blot or ELISA. mRNA expression is measured by qPCR. For FXR activation assays, HepG2 cells transfected with an FXR response element (FXRE)-luciferase reporter are treated with FXR agonist 3, and luciferase activity is measured. |
| Animal Protocol |
Animal/Disease Models:C57BL/6 N mice fed CDAHFD diet for 16 weeks[1]
Doses: 200 mg/kg Route of Administration: Oral gavage; daily for 4 weeks after CDAHFD-induced Experimental Results: Decreased expression of IL-1β and IL-6 in livers, indicating the liver-protective effect of 3a in CDAHFD mice may partially through inhibiting inflammasome activation. Lowered the serum levels of biochemical markers of ALT, AST, ALP, LDH, LDL and TBiL significantly, while raised HDL and GLU levels. For in vivo evaluation of anti-fibrotic activity, CDAHFD-induced NASH mouse model: male C57BL/6 mice (6-8 weeks old) are fed a CDAHFD diet for 4-6 weeks to induce NASH. FXR agonist 3 is administered orally (200 mg/kg, once daily) for 4 weeks. At the study endpoint, blood is collected for measurement of ALT, AST, and total bilirubin. Livers are harvested for histological analysis: H&E staining (for steatosis and inflammation), Sirius Red staining (for collagen deposition/fibrosis), and immunohistochemistry for alpha-SMA and TGF-beta1. Hepatic triglyceride and cholesterol levels are quantified. Fibrosis is scored using the NASH CRN system or the Ishak score. In the BDL-induced fibrosis rat model, Sprague-Dawley rats undergo bile duct ligation for 2-3 weeks, followed by oral administration of FXR agonist 3 (200 mg/kg, daily). The same endpoints are measured. |
| ADME/Pharmacokinetics |
FXR agonist 3 (C2₈H2₈BrNO4, MW = 522.43, CAS 3037847-72-3) is a solid powder. For storage, the powder should be kept at -20degC for up to 3 years, sealed, and protected from light. For in vitro use, stock solutions in DMSO (250 mg/mL, 478.53 mM) can be stored at -80degC for up to 6 months or at -20degC for 1 month. For in vivo oral administration, it can be formulated in 0.5% methylcellulose/0.1% Tween-80, 10% DMSO / 90% corn oil, or other suitable vehicles. Purity is typically ≥98% (HPLC). No detailed PK parameters (Cmax, Tmax, half-life, AUC) are reported, but metabolic stability data (half-life in liver microsomes) suggests moderate to low stability.
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| Toxicity/Toxicokinetics |
No specific toxicity data for FXR agonist 3 are reported beyond the CC50 value of 70.36 microM in LX-2 cells. As a research-grade FXR agonist, it is not intended for human or veterinary use. Standard laboratory safety precautions for handling chemicals should be followed, including the use of gloves, lab coat, and safety goggles. FXR agonists as a class can cause side effects such as pruritus (itching) and elevation of LDL-cholesterol. No LD50 or formal toxicology studies are available for this specific compound.
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| References | |
| Additional Infomation |
FXR agonist 3 is a research-grade, non-steroidal, synthetic FXR agonist with anti-NASH and anti-fibrotic activity. FXR (farnesoid X receptor) is a bile acid-activated nuclear receptor that plays a critical role in bile acid, lipid, and glucose homeostasis. FXR activation has been shown to reduce hepatic steatosis (fatty liver), inflammation, and fibrosis in preclinical models of NASH. Several FXR agonists (e.g., obeticholic acid, OCA) have been investigated in clinical trials for NASH and primary biliary cholangitis (PBC). However, OCA was not approved for NASH due to safety concerns. FXR agonist 3 is a research tool for studying the role of FXR in NASH and liver fibrosis. The compound is for research use only and has not entered clinical trials or received regulatory approval. It was reported in the literature (Zhang N, et al. Eur J Med Chem, 2023).
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| Molecular Formula |
C28H28BRNO4
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| Molecular Weight |
522.43
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| Appearance |
Solid powder
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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)
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| Solubility (In Vitro) |
DMSO : ≥ 250 mg/mL (478.53 mM)
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9141 mL | 9.5707 mL | 19.1413 mL | |
| 5 mM | 0.3828 mL | 1.9141 mL | 3.8283 mL | |
| 10 mM | 0.1914 mL | 0.9571 mL | 1.9141 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
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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