A-196 (A196) is a potent, selective and first-in-class inhibitor of SUV420H1 and SUV420H2 (IC50= 25 and 144 nM) with anticancer activity. It inhibits the di- and trimethylation of H4K20me in multiple cell lines. The in vitro activity of A-196 includes inhibition of SUV420H1 with IC50 of 25 nM and SUV420H2 with IC50 of 144 nM for methylation of H4K20me and greater than 100-fold selectivity over other histone methyltransferases and non-epigenetic targets. A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

Physicochemical Properties

Molecular Formula	C18H16CL2N4
Molecular Weight	359.25244140625
Exact Mass	358.075
CAS #	1982372-88-2
Related CAS #	1982372-88-2
PubChem CID	117072548
Appearance	Light brown to brown solid powder
LogP	4.5
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	24
Complexity	412
Defined Atom Stereocenter Count	0
SMILES	ClC1C(=CC2=C(C3C=CN=CC=3)N=NC(=C2C=1)NC1CCCC1)Cl
InChi Key	ABGOSOMRWSYAOB-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H16Cl2N4/c19-15-9-13-14(10-16(15)20)18(22-12-3-1-2-4-12)24-23-17(13)11-5-7-21-8-6-11/h5-10,12H,1-4H2,(H,22,24)
Chemical Name	6,7-dichloro-N-cyclopentyl-4-pyridin-4-ylphthalazin-1-amine
Synonyms	A-196; A 196; A196
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	After 48 hours of treatment with A-196 (0-5 μM; U2OS cells), H4K20me1 (EC50 value of 735 nM) increases, but H4K20me2 and H4K20me3 (EC50 values of 262 and 370 nM, respectively) decrease (see reference 1). In cells from various tissue types, A-196 (10 μM; 72 hours; wild-type, Suv4-20h double knockout, and inhibitor-treated mouse embryonic fibroblasts) inhibits the SUV4-20 enzyme without showing any signs of toxicity [1].
ln Vivo
Cell Assay	Western Blot Analysis[1] Cell Types: U2OS cells Tested Concentrations: 0 μM, 0.075 μM, 0.15 μM, 0.3 μM, 0.6 μM, 1.25 μM, 2.5 μM, 5 μM Incubation Duration: 48 hrs (hours) Experimental Results: Increased in H4K20me1 and diminished in both H4K20me2 and H4K20me3.
Animal Protocol
References	[1]. The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity. Nat Chem Biol. 2017 Mar;13(3):317-324.

Solubility Data

Solubility (In Vitro)

DMSO:10 mM Water:N/A Ethanol:N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 1 mg/mL (2.78 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 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: 1 mg/mL (2.78 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Solubility in Formulation 3: ≥ 1 mg/mL (2.78 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 10.0 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	2.7836 mL	13.9179 mL	27.8358 mL
	5 mM	0.5567 mL	2.7836 mL	5.5672 mL
	10 mM	0.2784 mL	1.3918 mL	2.7836 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.