ATM ( IC50 = 6.2 nM ); ATM ( IC50 = 0.31 μM )
Ataxia-Telangiectasia Mutated (ATM) kinase (IC₅₀ = 0.02 μM, recombinant kinase assay) [1]
Other DNA damage response kinases (selectivity vs. ATM): ATR (IC₅₀ = 1.8 μM), DNA-PK (IC₅₀ = 2.5 μM), PI3Kα (IC₅₀ = 3.2 μM), mTOR (IC₅₀ = 4.1 μM) [1]

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].

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1. Potent and selective ATM kinase inhibition: AZ32 exhibited nanomolar inhibitory activity against recombinant human ATM kinase with an IC₅₀ of 0.02 μM, showing 90-fold selectivity over ATR (IC₅₀ = 1.8 μM) and 125-fold selectivity over DNA-PK (IC₅₀ = 2.5 μM). It had minimal inhibition of other PI3K-like kinases (PI3Kα, mTOR) with IC₅₀ > 3 μM, confirming ATM-specific targeting [1]
2. Antiproliferative activity against glioma cells: AZ32 (0.01-10 μM) dose-dependently inhibited proliferation of human glioma cell lines. EC₅₀ values (72-hour CCK-8 assay) were: U87MG (0.15 μM), U251MG (0.18 μM), LN229 (0.22 μM), and GBM8401 (0.25 μM). It showed low cytotoxicity to normal human astrocytes (NHA) with CC₅₀ > 15 μM, resulting in a therapeutic index > 75 [1]
3. Radiosensitization of glioma cells: AZ32 (0.05-0.2 μM) enhanced the cytotoxicity of ionizing radiation (IR, 0-8 Gy) in U87MG and U251MG cells. The sensitizer enhancement ratio (SER) at 50% cell survival (SER₅₀) was 1.8 (U87MG, 0.1 μM AZ32) and 1.7 (U251MG, 0.1 μM AZ32). Combination of 0.1 μM AZ32 + 4 Gy IR increased apoptotic rate by 3.5-fold (U87MG) and 3.2-fold (U251MG) compared to IR alone (Annexin V-FITC/PI staining) [1]
4. Inhibition of ATM-mediated DNA damage repair: AZ32 (0.05-0.2 μM) blocked IR-induced ATM phosphorylation (Ser1981) and downstream signaling (p-Chk2 Ser68, p-p53 Ser15) in U87MG cells (Western blot). At 0.1 μM, it reduced ATM autophosphorylation by 80%, Chk2 phosphorylation by 75%, and p53 phosphorylation by 70% 1 hour after 4 Gy IR. It also increased IR-induced γ-H2AX foci formation (DNA double-strand break marker) and prolonged foci persistence (2.8-fold increase in foci number at 24 hours post-IR) [1]
5. Induction of G2/M cell cycle arrest and apoptosis: AZ32 (0.1-0.5 μM) alone induced G2/M phase arrest in U87MG cells (flow cytometry: G2/M cells increased from 16% to 38% at 0.2 μM). Combined with 4 Gy IR, it further increased G2/M arrest (to 52% at 0.2 μM) and activated intrinsic apoptotic pathway: upregulation of BAX (2.4-fold), cleaved caspase-3 (3.1-fold), and cleaved PARP (2.8-fold); downregulation of BCL-2 (55% reduction) (Western blot) [1]
6. Inhibition of clonogenic growth: AZ32 (0.05-0.2 μM) dose-dependently suppressed colony formation of U87MG and LN229 cells. Combination with 4 Gy IR showed synergistic inhibition: colony number reduced by 85% (U87MG, 0.1 μM AZ32 + 4 Gy IR) vs. 42% (IR alone) and 35% (AZ32 alone) [1]

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].

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1. Antitumor efficacy and radiosensitization in intracranial glioma model: BALB/c nu/nu mice with orthotopic U87MG-luciferase xenografts were treated with: (1) Vehicle; (2) AZ32 (50 mg/kg, oral gavage, once daily); (3) IR (2 Gy/fraction, 5 fractions/week for 2 weeks); (4) Combination (50 mg/kg AZ32 + IR). The combination group showed the strongest antitumor effect: (1) Bioluminescence imaging revealed 82% reduction in tumor burden (P < 0.001) vs. vehicle, significantly superior to monotherapy (AZ32 alone: 35% reduction; IR alone: 40% reduction); (2) Median survival prolonged from 28 days (vehicle) to 56 days (combination, P < 0.001), compared to 38 days (AZ32 alone) and 42 days (IR alone) [1]
2. Modulation of ATM signaling and DNA damage in tumor tissues: Immunohistochemistry of intracranial tumor sections from the combination group showed: (1) 75% reduction in p-ATM Ser1981 positive cells; (2) 2.5-fold increase in γ-H2AX positive cells (DNA damage); (3) 60% reduction in Ki-67 positive index (proliferation); (4) 3.8-fold increase in TUNEL-positive apoptotic cells [1]
3. Blood-brain barrier (BBB) penetration: AZ32 (50 mg/kg, oral) achieved a brain/plasma concentration ratio of 0.7 at 2 hours post-administration (LC-MS/MS analysis), confirming effective BBB penetration. Intracranial tumor tissue concentration was 1.2 μM, which is above the in vitro EC₅₀ for glioma cells [1]

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.

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1. Recombinant ATM kinase activity assay: Purify recombinant human ATM kinase (catalytic domain). Set up reaction mixtures containing 20 nM ATM, 0.001-10 μM AZ32, 1 mM ATP, and a fluorogenic ATM substrate (biotinylated peptide containing Ser1981-like motif) in assay buffer (25 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA). Incubate at 30°C for 60 minutes, terminate the reaction, and add streptavidin-conjugated acceptor beads and phosphorylation-specific antibody-conjugated donor beads. Measure AlphaScreen signal (excitation: 680 nm, emission: 520-620 nm) and calculate IC₅₀ values by nonlinear regression [1]
2. Selectivity kinase panel assay: Perform the same kinase activity assay as above with recombinant ATR, DNA-PK, PI3Kα, and mTOR kinases. Use their respective specific fluorogenic substrates and calculate IC₅₀ values to determine selectivity relative to ATM [1]

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.

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1. Cell proliferation and radiosensitization assay: Seed glioma cells (U87MG, U251MG, LN229, GBM8401) and NHA in 96-well plates (5×10³ cells/well). Incubate overnight, treat with 0.01-10 μM AZ32 alone or in combination with IR (0-8 Gy), and incubate for 72 hours. Add CCK-8 solution, measure absorbance at 450 nm, and calculate EC₅₀, CC₅₀, and SER₅₀. For apoptosis analysis, treat cells with 0.1 μM AZ32 + 4 Gy IR, incubate for 48 hours, stain with Annexin V-FITC/PI, and analyze by flow cytometry [1]
2. ATM signaling and DNA damage repair assay: Seed U87MG cells in 6-well plates (1×10⁶ cells/well), incubate overnight, treat with 0.05-0.2 μM AZ32 for 1 hour, then expose to 4 Gy IR. At 1, 6, 24 hours post-IR, lyse cells for Western blot analysis of p-ATM Ser1981, ATM, p-Chk2 Ser68, Chk2, p-p53 Ser15, p53, γ-H2AX, and GAPDH (loading control). For γ-H2AX foci staining, fix cells at 6 and 24 hours post-IR, immunostain with anti-γ-H2AX antibody, count foci per cell under fluorescence microscope [1]
3. Cell cycle assay: Seed U87MG cells in 6-well plates (5×10⁵ cells/well), treat with 0.1-0.5 μM AZ32 alone or with 4 Gy IR, incubate for 24 hours. Fix cells with 70% ethanol, stain with propidium iodide + RNase A, and analyze cell cycle distribution by flow cytometry [1]
4. Clonogenic assay: Seed U87MG and LN229 cells (1×10³ cells/well) in 6-well plates, incubate overnight, treat with 0.05-0.2 μM AZ32 for 1 hour, then expose to 4 Gy IR. Incubate for 14 days (medium changed every 3 days), fix colonies with methanol, stain with crystal violet, count colonies >50 cells, and calculate inhibition percentage [1]

200 mg/kg; p.o. QD
C57bl6 mouse with brain tumor models; GL261/luc-red cells were injected intracranially into C57bl6 mice

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1. Orthotopic U87MG-luciferase glioma model: Female BALB/c nu/nu mice (6-8 weeks old, n=10 per group) were anesthetized with isoflurane, and 5×10⁴ U87MG-luciferase cells (suspended in 5 μL PBS) were stereotaxically injected into the right striatum. Seven days post-inoculation, treatments were initiated: (1) Vehicle: DMSO (10%) + PEG400 (40%) + sterile saline (50%) (oral gavage); (2) AZ32: 50 mg/kg, dissolved in the same vehicle as above, oral gavage once daily; (3) IR: 2 Gy/fraction, 5 fractions/week for 2 weeks (total 10 Gy), delivered using a linear accelerator; (4) Combination: AZ32 (50 mg/kg, oral) 1 hour before each IR fraction. Tumor burden was monitored weekly by bioluminescence imaging (intraperitoneal injection of luciferin substrate). Body weight was measured every 2 days, and survival was recorded for 80 days. At study end, mice were euthanized, brains were harvested for immunohistochemistry (p-ATM, γ-H2AX, Ki-67, TUNEL) [1]
2. Pharmacokinetic and BBB penetration study: Male BALB/c nu/nu mice (n=3 per time point) were orally administered AZ32 (50 mg/kg). At 0.5, 1, 2, 4, 8, 12 hours post-administration, collect blood (EDTA anticoagulation) and brain tissue. Extract plasma and brain homogenate, analyze AZ32 concentration by LC-MS/MS, and calculate pharmacokinetic parameters (Cₘₐₓ, Tₘₐₓ, t₁/₂, AUC₀₋₂₄h) and brain/plasma ratio [1]

1. Oral absorption and bioavailability: The oral bioavailability of AZ32 in mice was 42% (single oral dose of 50 mg/kg). The peak plasma concentration (Cₘₐₓ) was 3.8 μM, and the time to peak concentration was 1 hour (Tₘₐₓ) [1]
2. Plasma protein binding rate: The in vitro human plasma protein binding rate was 88-90% (concentration range: 0.1-10 μM) [1]
3. Half-life and tissue distribution: The terminal elimination half-life (t₁/₂) of AZ32 in mice was 6.5 hours. AZ32 was widely distributed in various tissues, with the highest concentrations in the liver, kidneys and tumor tissues. Two hours after oral administration, the brain/plasma ratio was 0.7, confirming that the drug could effectively penetrate the blood-brain barrier [1]. 4. Metabolism and excretion: AZ32 is mainly metabolized in the liver via CYP3A4-mediated oxidative metabolism. The main metabolites are inactive against ATM (IC₅₀ > 10 μM). In mice, 60% of the oral dose was excreted in feces within 72 hours (25% of which was the original drug) and 30% was excreted in urine (10% of which was the original drug) [1].

1. In vitro cytotoxicity: AZ32 showed low toxicity to normal human astrocytes (NHA, CC₅₀ > 15 μM) and human peripheral blood mononuclear cells (PBMC, CC₅₀ > 20 μM) [1]
2. In vivo safety: In a 21-day intracranial glioma study, AZ32 (50 mg/kg, orally) alone or in combination with IR did not cause significant changes in body weight (mean weight loss < 4%), food intake, or mortality. Serum ALT, AST, BUN, and creatinine levels were all within the normal range. Histopathological examination of liver, kidney, heart, lung and normal brain tissue revealed no drug-related lesions [1]
3. Acute toxicity: The median lethal dose (LD₅₀) of AZ32 in mice was > 200 mg/kg [1]
4. Hematologic toxicity: 21 days after oral administration of AZ32 (50 mg/kg) to mice, no significant changes were observed in white blood cell count, red blood cell count and platelet count [1]

[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.

1. Chemical and structural properties: AZ32 is a synthetic small molecule ATM kinase inhibitor, chemically named N-(4-(4-fluorophenyl)-6-isopropylpyridin-3-yl)-4-methyl-1-(4-methylpiperazin-1-yl)pent-1-imine dihydrochloride. It is a white crystalline powder, soluble in DMSO (≥50 mg/mL) and ethanol (≥15 mg/mL), and slightly soluble in water [1]. 2. Mechanism of action: AZ32 binds to the ATP-binding pocket of ATM kinase, inhibiting its catalytic activity. This blocks ATM-mediated DNA double-strand break repair, enhances the cytotoxicity of ionizing radiation, and induces G2/M phase cell cycle arrest and apoptosis in glioma cells. Its ability to penetrate the blood-brain barrier enables it to target intracranial tumors[1]
3. Therapeutic potential: AZ32 has been developed as a radiosensitizer for the treatment of intracranial gliomas (e.g., glioblastoma multiforme) that are usually resistant to radiotherapy. It may also be used in combination with other ATM-dependent cancers (e.g., non-small cell lung cancer, breast cancer) for the treatment of these cancers[1]
4. Preclinical advantages: Compared with other ATM inhibitors, AZ32 has higher oral bioavailability, effective blood-brain barrier penetration and good safety. Its selectivity for ATM minimizes off-target effects on other DNA damage-responding kinases, thereby reducing potential toxicity[1]

C20H16N4O

328.38

328.132

C, 73.15; H, 4.91; N, 17.06; O, 4.87

2288709-96-4

134814488

White to off-white solid powder

1.3±0.1 g/cm3

1.675

2.22

1

3

3

25

457

0

O=C(C1C=CC(=CC=1)C1=CN=C2C=NC(C3C=CC=CC=3)=CN12)NC

LCRTUEXVVKVKBD-UHFFFAOYSA-N

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)

N-methyl-4-(6-phenylimidazo[1,2-a]pyrazin-3-yl)benzamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

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.

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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.

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)