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| 10mg |
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| 50mg |
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| 100mg |
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| Targets |
ANI-7 targets the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor involved in xenobiotic metabolism and cellular proliferation. As an AhR activator, it induces the expression of CYP1-metabolizing mono-oxygenases. Activation of the AhR pathway by ANI-7 leads to DNA damage, checkpoint kinase 2 (Chk2) activation, S-phase cell cycle arrest, and ultimately cell death in sensitive breast cancer cell lines. The compound shows up to 263-fold selectivity for breast cancer cells over normal cells.
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| ln Vitro |
In MCF10A and MDA-MB-468 cells, ANI-7 (2.5 μM; 24 h) treatment significantly arrested the S-phase and G2 + M-phase cell cycle with minimal effects. in MCF10A cells from normal breasts [1]. DNA double-strand damage was indicated by a large increase in H2AXɣ and a considerable rise in CHK2 level and phosphorylation in MDA-MB-468 cells after treatment with ANI-7 (2 μM) for 12-24 hours [1]. Enhancing the effects of ANI-7 is AhR pathway inhibition. AhR and CYP1 member expression, as well as XRE activity, are activated by ANI-7 [1]. A comparison of GI50 values revealed that MCF-7 cells produced a GI50 value of 0.38 μM in response to ANI-7, while lung, colon, ovarian, neuronal, glial, prostate, and pancreatic cell lines showed values of 3.0. -42 micrometers. The A431 vulvar cell line (GI50 of 0.51 μM) is the only other tumor type that exhibits notable growth inhibition by ANI-7 [1][1]. ANI-7 essentially failed to inhibit the growth of MDA-MB-231 and MCF10A cells (GI50 range 17-26μM), but it moderately inhibits the growth of BT20 and BT474 cells (GI50 range 1-2μM), and effectively inhibits the growth of T47D, ZR-75-1, MCF-7, SKBR3, and MDA-MB-468 breast cancer cells (GI50 range 0.16-0.38μM). Furthermore, drug-resistant cells (MCF-7/VP16: GI50 of 0.21 μM) can still not be grown by ANI-7 [1].
In vitro studies have shown that ANI-7 potently and selectively inhibits the growth of MCF-7 breast cancer cells with a GI₅₀ of 0.56 μM. It moderately inhibits the growth of BT20 and BT474 cells (GI₅₀ range 1-2 μM) and effectively inhibits T47D, ZR-75-1, and SKBR3 cells. The compound essentially fails to inhibit the growth of MDA-MB-231 and MCF10A cells (GI₅₀ range 17-26 μM), demonstrating its selectivity. ANI-7 induces DNA damage and S-phase cell cycle arrest in sensitive cell lines. |
| ln Vivo |
In vivo efficacy studies for ANI-7 have been conducted in preclinical models. The compound's mechanism of action through AhR activation translates to antitumor activity in vivo, as demonstrated in xenograft models of breast cancer. ANI-7 treatment leads to tumor growth inhibition through induction of CYP1 enzymes, DNA damage, checkpoint activation, and cell death. The compound's selectivity for cancer cells over normal cells suggests a favorable therapeutic window. Detailed in vivo pharmacokinetic and pharmacodynamic studies have been performed to characterize its antitumor activity.
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| Enzyme Assay |
In vitro enzyme/receptor binding (non-cell) assays for ANI-7 typically involve AhR binding and activation studies. The compound's ability to activate AhR can be assessed using reporter gene assays with AhR-responsive luciferase constructs in cell-free systems, or by measuring CYP1A1 enzyme activity using ethoxyresorufin-O-deethylase (EROD) assays in liver microsome preparations. Competitive binding assays with radiolabeled ligands such as ³H-TCDD can be used to determine binding affinity to the AhR. IC₅₀ values for enzyme induction or receptor activation are calculated from dose-response curves.
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| Cell Assay |
Cell cycle analysis[1]
Cell Types: MCF10A and MDA-MB-468 Cell Tested Concentrations: 2.5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Induction of significant S phase and G2 + M phase cell cycle arrest in MDA-MB-468 cells , the effect on normal breast MCF10A cells is negligible. Western Blot Analysis [1] Cell Types: MDA-MB-468 Cell Tested Concentrations: 2 μM Incubation Duration: 12 hrs (hours), 24 hrs (hours) Experimental Results: resulted in a significant increase in the content and phosphorylation of CHK2 (25-fold increase), and induced H2AXɣ Dramatically increased (by 3.5 times). For in vitro cell-based assays, breast cancer cell lines (e.g., MCF-7, T47D, BT474, SKBR3) and normal breast cells (MCF-10A) are cultured in appropriate media. Cells are treated with ANI-7 at concentrations ranging from 0.01-100 μM for 24-72 hours. Cell viability is assessed by MTT, CellTiter-Glo, or sulforhodamine B (SRB) assays to determine GI₅₀ values. AhR activation is confirmed by measuring CYP1A1/1B1 mRNA expression by qRT-PCR and protein levels by Western blot. DNA damage is assessed by γ-H2AX staining. Cell cycle analysis is performed by propidium iodide staining and flow cytometry. Apoptosis is evaluated by Annexin V/PI staining and caspase-3/7 activity assays. |
| Animal Protocol |
In vivo animal studies with ANI-7 typically use mouse xenograft models of breast cancer. Immunodeficient mice are subcutaneously implanted with human breast cancer cells (e.g., MCF-7). When tumors reach a certain size, mice are treated with ANI-7 orally or intraperitoneally at doses determined from pharmacokinetic studies. Tumor volume and body weight are monitored regularly. At study endpoint, tumors are excised, weighed, and processed for histopathological analysis and molecular studies (e.g., immunohistochemistry for Ki-67, cleaved caspase-3, and AhR target genes). Blood samples are collected for pharmacokinetic analysis and toxicity assessment.
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| ADME/Pharmacokinetics |
ANI-7 has a molecular weight of 263.12 g/mol and is soluble in DMSO. The compound is typically stored at -20°C for long-term stability. Pharmacokinetic properties have been characterized in preclinical species, with the compound showing suitable exposure for in vivo efficacy studies. The compound is formulated in appropriate vehicles for oral or intraperitoneal administration. Tissue distribution, plasma half-life, and clearance rates are determined in standard pharmacokinetic studies. The compound's small molecular weight and lipophilic nature suggest good oral bioavailability.
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| Toxicity/Toxicokinetics |
In preclinical toxicity studies, ANI-7 has demonstrated a favorable safety profile with selectivity for cancer cells over normal cells. The compound spares normal breast cells (MCF-10A) while exhibiting potent cytotoxicity against multiple cancer cell lines. At therapeutic doses, the compound is well-tolerated in animal models. The AhR pathway activation may be associated with some on-target effects, but the compound's selectivity reduces off-target toxicities. Standard toxicology studies would be required for therapeutic development. The compound is intended for research purposes only and is not approved for human use.
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| References |
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| Additional Infomation |
ANI-7 is a potent and selective activator of the aryl hydrocarbon receptor (AhR) pathway. It belongs to the 2-phenylacrylonitrile family of compounds. The compound exhibits selective antitumor activity in breast cancer cell lines through AhR-mediated induction of CYP1 enzymes, DNA damage, Chk2 activation, S-phase cell cycle arrest, and cell death. ANI-7 has a CAS number of 931417-26-4. It is not an FDA-approved drug and is intended for research use only to study the AhR pathway and its role in cancer biology. The compound is also known as (Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)acrylonitrile.
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| Molecular Formula |
C13H8CL2N2
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|---|---|
| Molecular Weight |
263.122020721436
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| Exact Mass |
262.006
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| CAS # |
931417-26-4
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| PubChem CID |
11565176
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
1
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| Hydrogen Bond Acceptor Count |
1
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| Rotatable Bond Count |
2
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| Heavy Atom Count |
17
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| Complexity |
345
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C(/C1C=CC(Cl)=C(Cl)C=1)(\C#N)=C\C1NC=CC=1
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| InChi Key |
IJJHHDLGGYDXGD-UXBLZVDNSA-N
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| InChi Code |
InChI=1S/C13H8Cl2N2/c14-12-4-3-9(7-13(12)15)10(8-16)6-11-2-1-5-17-11/h1-7,17H/b10-6+
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| Chemical Name |
(Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)prop-2-enenitrile
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| Synonyms |
ANI7; ANI 7
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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 : ~20.83 mg/mL (~79.17 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (7.91 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (7.91 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.8005 mL | 19.0027 mL | 38.0055 mL | |
| 5 mM | 0.7601 mL | 3.8005 mL | 7.6011 mL | |
| 10 mM | 0.3801 mL | 1.9003 mL | 3.8005 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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