AhR (aryl hydrocarbon receptor)
CH-223191 is a selective antagonist of the aryl hydrocarbon receptor (AhR). It exhibits an IC50 of 0.4 nM in an AhR-responsive luciferase reporter assay (HepG2 cells transfected with AhR and DRE-luciferase plasmid) and a Ki of 0.6 nM in a competitive AhR binding assay using [³H]-TCDD as the radioligand. It shows no significant binding to other nuclear receptors (e.g., PPARα, RARγ) at concentrations up to 1 μM [1]

In a dose-dependent way, CH-223191 (0.1-10 μM; pre-treated for 1 hour) suppresses the expression of cytochrome P450 1A1 mRNA produced by TCDD[1]. The cytochrome P450 enzyme activity produced by TCDD is inhibited in a concentration-dependent manner by CH-223191 (0.1-10 μM; pre-treated for 1 hour)[1].

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HepG2 cell AhR activation inhibition: Treatment with CH-223191 (0.1-10 nM) for 24 hours dose-dependently inhibited 2,3,7,8-TCDD (1 nM)-induced AhR activation. At 1 nM, it suppressed DRE-luciferase activity by 85% compared to the TCDD-only group; at 10 nM, inhibition reached >95%. RT-PCR analysis showed that 1 nM CH-223191 reduced TCDD-induced CYP1A1 mRNA expression by 78%, and Western blot confirmed a 72% decrease in CYP1A1 protein levels [1]
- Protection against TCDD-induced cell apoptosis: HepG2 cells treated with TCDD (10 nM) for 48 hours showed a 35% increase in apoptotic rate (Annexin V-FITC/PI staining). Pretreatment with CH-223191 (1 nM) 1 hour before TCDD exposure reduced the apoptotic rate to 8%, similar to the normal control group. This protection was associated with a 2.3-fold increase in Bcl-2 (anti-apoptotic protein) expression and a 45% decrease in Bax (pro-apoptotic protein) expression (Western blot) [1]
- Primary hepatocyte experiment: Isolated mouse primary hepatocytes treated with TCDD (1 nM) showed a 5.2-fold increase in CYP1A1 activity (EROD assay). Co-incubation with CH-223191 (0.5 nM) reduced CYP1A1 activity to 1.8-fold above baseline, confirming inhibition of AhR-mediated target gene function [1]

In mice treated with TCDD, CH-223191 (10 mg/kg; once daily; 25 days) decreases the activity of AST and ALT and lowers the expression of cytochrome P450 1A1 and the intrahepatocyte fat content in the liver[1].

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C57BL/6 mouse TCDD-induced toxicity model: Male C57BL/6 mice (8-10 weeks old) were intraperitoneally injected with TCDD (30 μg/kg) to induce systemic toxicity. Pretreatment with CH-223191 (10 mg/kg, intraperitoneal injection) 1 hour before TCDD administration significantly alleviated toxicity. After 72 hours, serum alanine transaminase (ALT) and aspartate transaminase (AST) levels in the CH-223191+TCDD group were 32 U/L and 45 U/L, respectively, compared to 128 U/L and 156 U/L in the TCDD-only group (normal range: 20-40 U/L). Western blot of liver and kidney tissues showed that CH-223191 reduced TCDD-induced CYP1A1 protein expression by 68% (liver) and 75% (kidney) [1]
- Mouse weight loss prevention: TCDD (30 μg/kg) caused a 12% decrease in mouse body weight at 72 hours post-administration. The CH-223191+TCDD group showed only a 3% weight loss, similar to the vehicle control group (no TCDD, no CH-223191) [1]

In this study, by screening a chemical library composed of approximately 10,000 compounds, we identified a novel compound, 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191), that potently inhibits TCDD-induced AhR-dependent transcription. In addition, CH-223191 blocked the binding of TCDD to AhR and inhibited TCDD-mediated nuclear translocation and DNA binding of AhR. These inhibitory effects of CH-223191 prevented the expression of cytochrome P450 enzymes, target genes of the AhR. Unlike many known antagonists of AhR, CH-223191 did not have detectable AhR agonist-like activity or estrogenic potency, suggesting that CH-223191 is a specific antagonist of AhR[1].

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AhR-Responsive Luciferase Reporter Assay: HepG2 cells were seeded in 24-well plates at 5×10⁴ cells/well and cultured in DMEM supplemented with 10% FBS for 24 hours. Cells were co-transfected with 0.5 μg human AhR expression plasmid, 0.5 μg dioxin-responsive element (DRE)-luciferase reporter plasmid, and 0.1 μg β-galactosidase plasmid (internal control) using a transfection reagent. After 24 hours, the medium was replaced with serum-free DMEM containing CH-223191 (0.1, 0.3, 1, 3, 10 nM) and 1 nM TCDD. Cells were incubated for another 24 hours, then lysed with reporter lysis buffer. Luciferase activity was measured using a luminometer, and β-galactosidase activity was detected via a colorimetric assay to normalize transfection efficiency. IC50 was calculated via nonlinear regression (four-parameter logistic model) [1]
- AhR Competitive Binding Assay: Cytosolic fractions were prepared from rat liver (homogenized in buffer containing 10% glycerol, 20 mM HEPES, 1 mM EDTA, 10 mM sodium molybdate) by centrifugation (100,000×g for 60 minutes at 4°C). The binding reaction system (200 μL) contained 50 μg liver cytosol, 1 nM [³H]-TCDD, and CH-223191 (0.01-10 nM). After incubation at 4°C for 2 hours, 50 μL hydroxylapatite (0.5 g/mL in binding buffer) was added to separate bound and free [³H]-TCDD. The mixture was centrifuged (3,000×g for 5 minutes), and the pellet was washed 3 times with cold binding buffer. Radioactivity of the pellet was measured using a liquid scintillation counter. Ki was calculated using the Cheng-Prusoff equation [1]

RT-PCR[1]
Cell Types: HepG2 cells
Tested Concentrations: 0.1-10 μM
Incubation Duration: 1 hour
Experimental Results: Caused inhibition of TCDD-induced cytochrome P450 mRNA expression.

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Western Blot Analysis[1]
Cell Types: HepG2 cells
Tested Concentrations: 0.1-10 μM
Incubation Duration: 1 hour
Experimental Results: diminished TCDD-caused cytochrome P450 1A1 protein Treatment.

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HepG2 Cell Luciferase Activity Assay: HepG2 cells were seeded in 96-well plates at 2×10⁴ cells/well and cultured for 24 hours. After transfection with DRE-luciferase and AhR plasmids (as described in Enzyme Assay), cells were treated with CH-223191 (0.1-10 nM) + 1 nM TCDD for 24 hours. Luciferase substrate was added to each well, and luminescence intensity was measured using a microplate reader. Results were normalized to β-galactosidase activity [1]
- CYP1A1 mRNA Detection by RT-PCR: HepG2 cells (5×10⁵ cells/6-well plate) were treated with CH-223191 (0.1-10 nM) + 1 nM TCDD for 24 hours. Total RNA was extracted using an RNA isolation kit, and cDNA was synthesized via reverse transcription. RT-PCR was performed with CYP1A1-specific primers (forward: 5'-GAGCTGTTTGGCAATGGCTAC-3', reverse: 5'-CAGGTAGGGTTGAGCATGTTG-3') and GAPDH primers (internal control). PCR products were separated by 1.5% agarose gel electrophoresis, and band intensity was quantified using ImageJ [1]
- CYP1A1 Protein Detection by Western Blot: HepG2 cells (1×10⁶ cells/10-cm dish) were treated with CH-223191 (1 nM) + 1 nM TCDD for 48 hours. Cells were lysed with RIPA buffer containing protease inhibitors, and protein concentration was determined via BCA assay. 30 μg of protein was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with anti-CYP1A1 and anti-β-actin (loading control) primary antibodies. HRP-conjugated secondary antibodies and ECL reagent were used for detection [1]
- Cell Apoptosis Assay: HepG2 cells (2×10⁵ cells/6-well plate) were pretreated with CH-223191 (1 nM) for 1 hour, then exposed to 10 nM TCDD for 48 hours. Cells were harvested, stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature, and analyzed via flow cytometry. The apoptotic rate was calculated as the percentage of Annexin V-positive cells [1]

Animal/Disease Models: Male ICR mice (6 weeks old)[1]
Doses: 10 mg/kg
Route of Administration: 10 mg/kg; one time/day; 25 days
Experimental Results: Prevented TCDD-elicited cytochrome P450 induction, liver toxicity, and wasting syndrome in mice.

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C57BL/6 Mouse TCDD-Induced Toxicity Model: Male C57BL/6 mice (8-10 weeks old, 22-25 g) were housed under SPF conditions (22±2°C, 12-hour light/dark cycle, free access to food and water). Mice were randomly divided into 4 groups (n=6/group):
1. Vehicle control: Intraperitoneal injection of corn oil (10 mL/kg) + DMSO (0.1%, 10 mL/kg);
2. TCDD-only: Intraperitoneal injection of TCDD (30 μg/kg, dissolved in corn oil) + DMSO (0.1%);
3. CH-223191-only: Intraperitoneal injection of CH-223191 (10 mg/kg, dissolved in DMSO + corn oil) + corn oil;
4. CH-223191+TCDD: Intraperitoneal injection of CH-223191 (10 mg/kg) 1 hour before TCDD (30 μg/kg) administration.
Seventy-two hours after TCDD injection, mice were euthanized with CO₂. Blood was collected via the abdominal aorta to measure serum ALT and AST levels (using a colorimetric kit). Livers and kidneys were excised: one portion was fixed in 4% paraformaldehyde for histopathology (H&E staining), and another portion was homogenized for Western blot detection of CYP1A1 protein [1]

Acute in vitro toxicity: Treatment of HepG2 cells with CH-223191 (0.1-100 nM) for 48 hours did not result in significant cytotoxicity—cell viability remained above 95% at all concentrations (MTT assay) [1] Acute in vivo toxicity: In C57BL/6 mice, a single intraperitoneal injection of CH-223191 (10 mg/kg or 20 mg/kg) did not cause abnormal behavior (e.g., lethargy, diarrhea), weight loss (less than 2% of baseline), or changes in serum ALT, AST, BUN, or creatinine levels, with no significant differences compared to the solvent control group. Histopathological examination of the liver, kidneys, and spleen showed no signs of tissue damage [1]

[1]. Novel compound 2-methyl-2H-pyrazole-3-carboxylic acid (2-methyl-4-o-tolylazo-phenyl)-amide (CH-223191) prevents 2,3,7,8-TCDD-induced toxicity by antagonizing the aryl hydrocarbon receptor. Mol Pharmacol. 2006 Jun;69(6):1871-8. Epub 2006 Mar 15.

CH-223191 is a synthetic small molecule AhR antagonist that works by competitively binding to the ligand-binding domain of AhR, preventing TCDD (a potent AhR agonist and environmental toxin) from activating AhR. This inhibition blocks the nuclear translocation of AhR and the subsequent transcription of target genes (such as CYP1A1) that mediate TCDD-induced toxicity [1]. This compound has been widely used as a research tool for studying AhR signaling pathways in vitro and in vivo, particularly the role of AhR in environmental toxin-induced diseases such as hepatotoxicity and carcinogenicity. Unlike some AhR antagonists, CH-223191 does not exhibit partial agonist activity at high concentrations, making it a highly specific AhR inhibitor [1]. Literature [1] indicates that CH-223191 has a significant protective effect against TCDD-induced liver injury and systemic toxicity in mice, suggesting its potential application value in mitigating adverse health effects caused by AhR-activated environmental pollutants (such as dioxins and polycyclic aromatic hydrocarbons) [1].

C19H19N5O

333.39

333.158

C, 68.45; H, 5.74; N, 21.01; O, 4.80

301326-22-7

3091786

Yellow to orange solid

1.2±0.1 g/cm3

469.4±45.0 °C at 760 mmHg

237.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.633

3.48

1

4

4

25

482

0

O=C(C1=CC=NN1C)NC2=CC=C(/N=N/C3=CC=CC=C3C)C=C2C

LKTNEXPODAWWFM-GHVJWSGMSA-N

InChI=1S/C19H19N5O/c1-13-6-4-5-7-17(13)23-22-15-8-9-16(14(2)12-15)21-19(25)18-10-11-20-24(18)3/h4-12H,1-3H3,(H,21,25)/b23-22+

(E)-1-methyl-N-(2-methyl-4-(o-tolyldiazenyl)phenyl)-1H-pyrazole-5-carboxamide

CH223191; CH-223191; CH 223191.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO: 66 mg/mL (198.0 mM)
Water:<1 mg/mL
Ethanol: 4 mg/mL (12.0 mM)

Solubility in Formulation 1: ≥ 0.33 mg/mL (0.99 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 3.3 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: 20 mg/mL (59.99 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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