AZ32 is a novel, potent and orally bioavailable and blood-brain barrier-penetreable ATM inhibitor (AZ32) that radiosensitizes intracranial gliomas in mice. In an orthotopic mouse glioma model that is syngeneic and human, AZ32 demonstrated superior radiosensitization efficacy in vivo when compared to AZ31. In orthotopic mouse models, AZ32 is the first oral bioavailable ATMi that has been demonstrated to radiosensitize glioma and increase survival. These results lend credence to the creation of a clinical-grade, GBM-treating ATMi that penetrates the BBB. The use of an ATMi in conjunction with conventional radiotherapy is anticipated to be cancer-specific, boost the therapeutic ratio, and preserve the full therapeutic effect at lower radiation doses, which is significant because many GBMs have compromised p53 signalness.

Physicochemical Properties

Molecular Formula	C20H16N4O
Molecular Weight	328.38
Exact Mass	328.132
Elemental Analysis	C, 73.15; H, 4.91; N, 17.06; O, 4.87
CAS #	2288709-96-4
Related CAS #	N/A
PubChem CID	134814488
Appearance	White to off-white solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.675
LogP	2.22
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	3
Heavy Atom Count	25
Complexity	457
Defined Atom Stereocenter Count	0
SMILES	O=C(C1C=CC(=CC=1)C1=CN=C2C=NC(C3C=CC=CC=3)=CN12)NC
InChi Key	LCRTUEXVVKVKBD-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H16N4O/c1-21-20(25)16-9-7-15(8-10-16)18-11-23-19-12-22-17(13-24(18)19)14-5-3-2-4-6-14/h2-13H,1H3,(H,21,25)
Chemical Name	N-methyl-4-(6-phenylimidazo[1,2-a]pyrazin-3-yl)benzamide
Synonyms	AZ32; AZ-32; AZ 32
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	ATM ( IC50 = 6.2 nM ); ATM ( IC50 = 0.31 μM )
ln Vitro	In vitro activity: AZ32 radiosensitized GBM cells in vitro and inhibited the DNA damage response (DDR). In addition to having high cell permeability, AZ32 exhibits sufficient selectivity over ATR and moderate potency against ATM in cells (IC50 = 0.31 μM)[1].
ln Vivo	AZ32 demonstrated superior efficacy as a radiosensitizer in vivo when compared to AZ31 due to its increased blood-brain barrier (BBB) penetration in an orthotopic mouse glioma model that is syngeneic and human. In orthotopic mouse models, AZ32 is the first oral bioavailable ATMi that has been demonstrated to radiosensitize glioma and increase survival. After AZ32 (200 mg/kg) was given orally to mice, the concentration of AZ32 in the free brain was higher than the cellular IC50 for about 22 hours. Compared to AZ31, AZ32 has 8.7 times more BBB penetration and better brain coverage, but it has less ATM selectivity[1].
Enzyme Assay	AZ32 is a novel type of an ATM inhibitor that can penetrate the blood-brain barrier (BBB). GBM cells were radiosensitized in vitro by AZ32, which also inhibits the DNA damage response.
Cell Assay	AZ32 (3 μM) and radiation (2 Gy) were applied to human glioma U1242-susceptible cells, or they were not treated. Anti-γ-tubulin (centrosomes) and -α-tubulin (microtubules) were used to fix and process the cells for ICC after 48 hours. To view nuclei, DAPI was used as a counterstain for the cells.
Animal Protocol	200 mg/kg; p.o. QD C57bl6 mouse with brain tumor models; GL261/luc-red cells were injected intracranially into C57bl6 mice
References	[1]. Orally Bioavailable and Blood-Brain Barrier-Penetrating ATM Inhibitor (AZ32) Radiosensitizes Intracranial Gliomas in Mice. Mol Cancer Ther. 2018 Aug;17(8):1637-1647.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 150 mg/mL (~457 mM) Water: Ethanol:

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.61 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 (7.61 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (7.61 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 25.0 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	3.0453 mL	15.2263 mL	30.4525 mL
	5 mM	0.6091 mL	3.0453 mL	6.0905 mL
	10 mM	0.3045 mL	1.5226 mL	3.0453 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.