Apoptosis Activator 2 is a novel and potent small molecule apoptosis activator with an IC50 value of about 4 μM. Apoptosis Activator 2 is a cell permeable substance that encourages apoptosis by triggering caspases in a way that depends on cytochrome c and Apaf-1. When there is 0.15 mM cytochrome c, 20 μM apoptosis activator 2 increases the fraction of Apaf-1 in the apoptosome. It was discovered that Apaf-1 oligomerization was induced by Apoptosis Activator 2. Apoptosis Activator 2 prominently causes DNA fragmentation, PARP cleavage, and caspase-3 activation through the induction of apoptosis.

Physicochemical Properties

Molecular Formula	C15H9CL2NO2
Molecular Weight	306.14
Exact Mass	305.001
Elemental Analysis	C, 58.85; H, 2.96; Cl, 23.16; N, 4.58; O, 10.45
CAS #	79183-19-0
Related CAS #	79183-19-0
PubChem CID	1901244
Appearance	Solid powder
Density	1.5±0.1 g/cm3
Boiling Point	470.6±55.0 °C at 760 mmHg
Flash Point	238.4±31.5 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.671
LogP	3.42
Hydrogen Bond Donor Count	0
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	2
Heavy Atom Count	20
Complexity	415
Defined Atom Stereocenter Count	0
SMILES	O=C1N(CC2=CC=C(Cl)C(Cl)=C2)C3=C(C=CC=C3)C1=O
InChi Key	KGRJPLRFGLMQMV-UHFFFAOYSA-N
InChi Code	InChI=1S/C15H9Cl2NO2/c16-11-6-5-9(7-12(11)17)8-18-13-4-2-1-3-10(13)14(19)15(18)20/h1-7H,8H2
Chemical Name	1-[(3,4-dichlorophenyl)methyl]indole-2,3-dione
Synonyms	Apoptosis Activator 2; AAII; N-(3,4-dichlorobenzyl) Isatin. MDK-83190; MDK 83190; MDK83190
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

Targets	Caspase-3
ln Vitro	Apoptosis Activator 2 (20 μM) at the reduced cyto c concentration increases the fraction of Apaf-1 in the apoptosome by 1.5-fold to 33%. At the decreased level of cytochrome c and caspase-3 activation, Apoptosis Activator 2 causes a 4-fold increase in the extent of caspase-3 activation. With an IC50 of 4 μM, Apoptosis Activator 2 kills cells by strongly inducing caspase-3 activation, PARP cleavage, and DNA fragmentation. With an IC50 of 50 μM, 43 μM, 4 μM, 6 μM, 9 μM, 20 μM, 44 μM and 35 μM. , Apoptosis Activator 2 induces apoptosis in PBL, HUVEC, Jurkat, Molt-4, CCRF-CEM, BT-549, MDA-MB-468, and NCI-H23. The majority of tumor cell lines tested exhibit a cytostatic response to apoptosis activator 2, which inhibits cell growth by 50–100% at 10 M. In 40 of the 48 tumor cell lines examined, Apoptosis Activator 2 inhibits cell growth by 50–100% at 10 μM, having a cytostatic effect on the majority of the tumor cell lines examined. [1] Through the induction of apoptosome formation, Apoptosis Activator 2 causes cell death. The survival rates of Ventral midbrain cultures for Apoptosis Activator 2 (-8.1 ± 6.0%) are not significantly influenced by En1 expression levels. Use of the other three reagents has no appreciable impact on the survival rate for Apoptosis Activator 2 (-10.7 ± 4.7%). [2] The Tunel assay and apoptotic DNA ladder are used to determine whether or not apoptosis activator 2 (10 μM) induces apoptosis in AGS cells. Anti TROP2 conjugated liposomes induce apoptosis more effectively when apoptosis activator 2 (10 μM) is added. [3] In neuronal cultures, zVAD (50 μM) or cyclohexamide (10 μg/mL) significantly reduce the toxicity of Apoptosis Activator 2. Numerous neurones with pyknotic nuclei suggestive of cell death involving apoptosis are produced in response to apoptosis activator 2 (3 μM). In neuronal cultures, DHT (10 nM) or E2 (10 nM) significantly reduce the toxicity of Apoptosis Activator 2. [4]
ln Vivo	N/A
Enzyme Assay	According to previously published reports, HeLa cell cytoplasmic extracts are created. Apoptosis Activator 2 is diluted in DMSO to a final concentration of 1 mM, with a final DMSO concentration of 1% vol/vol, and then distributed into 96-well microtiter plates. 250 μg of total protein from cytoplasmic extracts in HEB buffer (50 mM Hepes, pH 7.4/50 mM KCl/5 mM EGTA/2 mM MgCl) are added to each well along with 150 μL of the DEVD-AFC (Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) substrate. Fluorescence is measured in a LJL Biosystems plate reader at 10-minute intervals while the plates are incubated at 37 °C.
Cell Assay	An experimenter blinded to condition counts all viable cells using a manual mechanical counter within the defined field of a microscope reticle grid. The vital dye calcein acetoxymethyl ester and the morphological criterion of a smooth, spherical soma are both used to determine whether a cell is viable. Per culture well, counts of viable cells are performed in four non-overlapping fields, with three separate wells for each condition. For vehicle-treated control conditions, there were 100–200 viable cell counts per well. At least three different culture preparations are used for each experiment. One-way ANOVA is used to statistically analyze the raw cell count data, and the Fisher LSD test is used to compare between groups (significance is denoted by P < 0.05). Cell viability is represented graphically as a proportion of alive cells in the vehicle-treated control condition.
Animal Protocol	N/A N/A
References	[1]. Proc Natl Acad Sci U S A . 2003 Jun 24;100(13):7533-8. [2]. Neural Dev . 2009 Mar 16:4:11. [3]. N Am J Med Sci . 2012 Nov;4(11):582-5. [4]. J Neuroendocrinol . 2010 Sep;22(9):1013-22.
Additional Infomation	1-[(3,4-dichlorophenyl)methyl]indole-2,3-dione is a member of indoles.

Solubility Data

Solubility (In Vitro)

DMSO: ~61 mg/mL (~199.3 mM) Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.17 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 25.0 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.2665 mL	16.3324 mL	32.6648 mL
	5 mM	0.6533 mL	3.2665 mL	6.5330 mL
	10 mM	0.3266 mL	1.6332 mL	3.2665 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.