ANI-7 is an activator of the AhR pathway. ANI-7 suppresses the growth of a variety of cancer/tumor cells and selectively suppresses the growth of MCF-7 breast cancer/tumor cells with a GI50 of 0.56 μM. ANI-7 induces CYP1 metabolic monooxygenase through activation of the AhR pathway and induces DNA damage, checkpoint kinase (Chk2) activation, S-phase cell cycle arrest, and cell death in sensitive breast cancer/tumor cell lines.

Physicochemical Properties

Molecular Formula	C13H8CL2N2
Molecular Weight	263.122020721436
Exact Mass	262.006
CAS #	931417-26-4
PubChem CID	11565176
Appearance	Light yellow to yellow solid powder
LogP	3.8
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	1
Rotatable Bond Count	2
Heavy Atom Count	17
Complexity	345
Defined Atom Stereocenter Count	0
SMILES	C(/C1C=CC(Cl)=C(Cl)C=1)(\C#N)=C\C1NC=CC=1
InChi Key	IJJHHDLGGYDXGD-UXBLZVDNSA-N
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+
Chemical Name	(Z)-2-(3,4-dichlorophenyl)-3-(1H-pyrrol-2-yl)prop-2-enenitrile
Synonyms	ANI7; ANI 7
HS Tariff Code	2934.99.9001
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)

Biological Activity

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].
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).
References	[1]. (Z)-2-(3,4-Dichlorophenyl)-3-(1H-Pyrrol-2-yl)Acrylonitrile Exhibits Selective Antitumor Activity in Breast Cancer Cell Lines via the Aryl Hydrocarbon Receptor Pathway. Mol Pharmacol. 2018 Feb;93(2):168-177. [2]. Dichlorophenylacrylonitriles as AhR Ligands That Display Selective Breast Cancer Cytotoxicity in vitro. ChemMedChem. 2018 Jul 18;13(14):1447-1458. [3]. Library synthesis and cytotoxicity of a family of 2-phenylacrylonitriles and discovery of an estrogen dependent breast cancer lead compound.  Medicinal Chemistry Communication. January 20112. (1):31-37.

Solubility Data

Solubility (In Vitro)

DMSO : ~20.83 mg/mL (~79.17 mM)

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.