A-366 (A366) is a novel, potent and peptide-competitive inhibitor of the histone methyltransferase G9a with anticancer activity. It inhibits G9a with an IC50 3 nM and > 100-fold selectivity over other methyltransferases and other non-epigenetic targets. A-366 has been shown to inhibit H3K9 methylation in cells with an IC50 of 100 nM and exhibits minimal cellular toxicity compared with previous quinazoline-based probes. A-366 selectively inhibits G9a and the closely related GLP (EHMT1), but not other histone methyltransferases. A-366 has significantly less cytotoxic effects on the growth of tumor cell lines compared to other known G9a/GLP small molecule inhibitors despite equivalent cellular activity on methylation of H3K9me2.

Physicochemical Properties

Molecular Formula	C19H27N3O2
Molecular Weight	329.4366
Exact Mass	329.21
CAS #	1527503-11-2
PubChem CID	76285486
Appearance	Light yellow to yellow solid powder
LogP	3.057
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	6
Heavy Atom Count	24
Complexity	471
Defined Atom Stereocenter Count	0
InChi Key	BKCDJTRMYWSXMC-UHFFFAOYSA-N
InChi Code	InChI=1S/C19H27N3O2/c1-23-16-12-14-15(21-18(20)19(14)6-4-7-19)13-17(16)24-11-5-10-22-8-2-3-9-22/h12-13H,2-11H2,1H3,(H2,20,21)
Chemical Name	5'-methoxy-6'-(3-(pyrrolidin-1-yl)propoxy)spiro[cyclobutane-1,3'-indol]-2'-amine
Synonyms	A 366A-366 A366
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	A-366 (0.01-10 μM; 14 days) impacts the viability of MV4;11 cells and causes differentiation [4]. In PC-3 prostate cancer cells, A-366 (0.3-3 μM; 72 hours) decreases overall H3K9me2 levels in a concentration- and time-dependent manner, with a cellular EC50 of roughly 300 nM. A-366 (0.01-10 μM; 4 days; HL-60 cells) decreased differentiation and concomitant proliferation in a dose-dependent manner. A-366-treated HL-60 cells' DNA content analysis revealed an increase of G1 phase cells, which is consistent with cell arrest [4].
ln Vivo	Growth suppression occurs when MV4;11 xenografts are treated with A-366 (30 mg/kg; osmotic minipump; once daily for 14 days) [4].
Cell Assay	Cell Viability Assay [4] Cell Types: MV4; 11 cells Tested Concentrations: 0.01-10 μM Incubation Duration: 14 days Experimental Results: Resulted in inhibition of proliferation and diminished viability, corresponding to the dose response observed by CD11b staining.
Animal Protocol	Animal/Disease Models: 6-8 weeks old SCID beige female mice (MV4; 11 xenografts) [4] Doses: 30 mg/kg Route of Administration: via mini-osmotic pump; one time/day for 14 days Experimental Results: In In the model, A-366 treatment produced a modest 45% tumor growth inhibition.
References	[1]. Epigenetics meets GPCR: inhibition of histone H3 methyltransferase (G9a) and histamine H3 receptor for Prader-Willi Syndrome. Sci Rep. 2020;10(1):13558. Published 2020 Aug 11. [2]. Identification of a small-molecule ligand of the epigenetic reader protein Spindlin1 via a versatile screening platform. Nucleic Acids Res. 2016;44(9):e88. [3]. Discovery and development of potent and selective inhibitors of histone methyltransferase g9a. ACS Med Chem Lett. 2014;5(2):205-209. Published 2014 Jan 2. [4]. The Histone Methyltransferase Inhibitor A-366 Uncovers a Role for G9a/GLP in the Epigenetics of Leukemia. PLoS One. 2015;10(7):e0131716. Published 2015 Jul 6.

Solubility Data

Solubility (In Vitro)

DMSO : ~50 mg/mL (~151.77 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.59 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 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (7.59 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.0355 mL	15.1773 mL	30.3545 mL
	5 mM	0.6071 mL	3.0355 mL	6.0709 mL
	10 mM	0.3035 mL	1.5177 mL	3.0355 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.