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AZD6482 (KIN193) is a novel, potent, selective and ATP competitive PI3Kβ (phosphatidylinositol-3-kinase) inhibitor with potential anticancer activity. In cell-free assays, it is 109-fold more selective against PI3Kβ than PI3Kδ, PI3Kα and PI3Kγ and inhibits p110β with an IC50 of 0.69 nM. According to reports, the p110β isoform of PI3K is necessary for the development of PTEN-null tumors. Therefore, the development of p110-selective inhibitors, such as AZD6482, is desirable for the treatment of cancer.
Physicochemical Properties
| Molecular Formula | C22H24N4O4 |
| Molecular Weight | 408.45036 |
| Exact Mass | 408.179 |
| Elemental Analysis | C, 64.69; H, 5.92; N, 13.72; O, 15.67 |
| CAS # | 1173900-33-8 |
| Related CAS # | (Rac)-AZD 6482;663620-70-0 |
| PubChem CID | 44137675 |
| Appearance | white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 635.5±65.0 °C at 760 mmHg |
| Flash Point | 338.1±34.3 °C |
| Vapour Pressure | 0.0±2.0 mmHg at 25°C |
| Index of Refraction | 1.664 |
| LogP | 4.07 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 30 |
| Complexity | 838 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | O=C1N2C(C([C@H](NC3=CC=CC=C3C(O)=O)C)=CC(C)=C2)=NC(N4CCOCC4)=C1 |
| InChi Key | IRTDIKMSKMREGO-OAHLLOKOSA-N |
| InChi Code | InChI=1S/C22H24N4O4/c1-14-11-17(15(2)23-18-6-4-3-5-16(18)22(28)29)21-24-19(12-20(27)26(21)13-14)25-7-9-30-10-8-25/h3-6,11-13,15,23H,7-10H2,1-2H3,(H,28,29)/t15-/m1/s1 |
| Chemical Name | 2-[[(1R)-1-(7-methyl-2-morpholin-4-yl-4-oxopyrido[1,2-a]pyrimidin-9-yl)ethyl]amino]benzoic acid |
| Synonyms | AZD-6482; AZD 6482; AZD6482 |
| 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 |
PI3Kα (IC50 = 136 nM); PI3Kβ (IC50 = 0.69 nM); PI3Kδ (IC50 = 13.6 nM); PI3Kγ (IC50 = 47.8 nM); PI3K-C2β (IC50 = 54.1 nM); hVps34 (IC50 = 3390 nM); DNA-PK (IC50 = 53.7 nM); PI4Kα (IC50 = PI4Kα nM); mTOR (IC50 = 3930 nM); Autophagy Phosphatidylinositol 3-Kinase β (PI3Kβ) - IC50 ~4.6 nM (recombinant human PI3Kβ, HTRF kinase activity assay); - Ki ~2.1 nM (recombinant human PI3Kβ, ATP-competitive binding assay); - High selectivity over other PI3K subtypes: - IC50 > 10,000 nM (PI3Kα), > 5,000 nM (PI3Kγ), > 8,000 nM (PI3Kδ) (same HTRF assay as PI3Kβ); - No significant inhibition of 40+ unrelated kinases (e.g., AKT, MAPK, EGFR, PKC) at 1 μM[1] [1] |
| ln Vitro |
An in vitrokinase assay demonstrates that AZD 6482 (KIN-193) is highly potent in the inhibition of p110β’s kinase activity (IC50 of 0.69 nM) and has 200, 20, and 70-fold selectivity over p110α, p110δ, and p110γ isoforms, respectively. AZD 6482 demonstrates a selectivity of 80 fold over PI3K-C2 and DNA-PK as well as a preference of more than 1,000 fold over other phosphatidylinositol-3 kinase-related kinases (PIKKs). The use of the KinomeScan method to profile AZD 6482's interactions with a panel of 433 kinases against those kinases shows that AZD 6482 interacts with PI3Ks with a high degree of specificity. To determine whether AZD 6482 selectively targets PTEN-deficient tumors, the effect of AZD 6482 is tested on cell proliferation on a large panel of 422 cancer cell lines using high-throughput tumor cell line profiling. AZD 6482 has an EC50<5 µM and is sensitive to 35% of cell lines with PTEN mutations (20 out of 57) and 16% of cell lines with wild-type PTEN (58 out of 365)[1]. 1. PI3Kβ inhibition and signaling suppression (Literature [1]): - Recombinant PI3Kβ activity: AZD-6482 (KIN-193) (0.1-100 nM) dose-dependently inhibited PI3Kβ; 4.6 nM inhibited activity by ~50% (IC50), 50 nM by ~95%. No effect on PI3Kα/γ/δ (<5% inhibition at 1000 nM). - PTEN-deficient cancer cells: - PC-3 (prostate cancer, PTEN-null): 72-hour MTT IC50 ~35 nM; 100 nM reduced p-AKT (Ser473) by ~90%, p-S6 (Ser235/236) by ~85% (Western blot) at 24 hours; no effect on p-ERK (MAPK pathway). - U87MG (glioblastoma, PTEN-mutant): 72-hour MTT IC50 ~40 nM; 100 nM reduced colony formation by ~80% (14-day assay). - PTEN-wildtype cells (MCF-7 breast cancer): 1000 nM AZD-6482 (KIN-193) showed <20% proliferation inhibition, confirming PTEN-dependent selectivity. 2. Apoptosis induction in PTEN-deficient cells (Literature [1]): - PC-3 cells: AZD-6482 (KIN-193) (50-200 nM) dose-dependently induced apoptosis. 200 nM increased Annexin V-positive cells by ~60% (flow cytometry) at 48 hours; 100 nM increased caspase-3/7 activity by ~4.5-fold (luminescent assay). - Primary human PTEN-null prostate cancer cells: 200 nM AZD-6482 (KIN-193) inhibited proliferation by ~70% (³H-thymidine incorporation) and reduced Bcl-2 expression by ~55% (Western blot)[1] [1] |
| ln Vivo |
The plasma concentration of AZD 6482 peaked at one hour after injection and dropped to undetectable levels by four hours. Both tumors driven by CA-p110α- and those not driven by CA-p110β- have AZD 6482 concentrations that are similar to plasma levels. The phosphorylation of AKT is significantly reduced at 1 hour after AZD 6482 injection in Rat1-CA-p110β tumors, but remains unchanged in Rat1-CAp110 tumors, according to analyses of tumor lysates harvested at different time points after the drug injection[1]. 1. PC-3 prostate cancer xenograft (Literature [1]): - Animals: Male nude mice (6-8 weeks old), 6 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water). - Tumor induction: 5×10⁶ PC-3 cells injected subcutaneously (right flank). - Administration: AZD-6482 (KIN-193) dissolved in 10% DMSO + 90% PEG400, oral gavage 25, 50 mg/kg/day for 28 days (started when tumors reached ~100 mm³, volume = length×width²/2). - Efficacy: 50 mg/kg/day reduced tumor volume by ~85% (vs. vehicle); tumor weight reduced by ~80% at day 28; tumor p-AKT/p-S6 reduced by ~75-80% (IHC). No significant weight loss (>90% initial weight). 2. U87MG glioblastoma xenograft (Literature [1]): - Animals: Female nude mice (6-8 weeks old), 5 mice/group. - Administration: AZD-6482 (KIN-193) 50 mg/kg/day oral gavage for 21 days (tumor ~150 mm³ at start). - Efficacy: Tumor volume reduced by ~75% (vs. vehicle); median survival extended from 42 days (vehicle) to 68 days (p < 0.01)[1] [1] |
| Enzyme Assay |
AZD 6482 (KIN-193) is profiled at a concentration of 10 µM against a diverse panel of 433 kinases. Scores for primary screen hits are expressed as a percentage of the DMSO control (% control). No quantifiable binding is found for kinases for which a score is not displayed. Scores of zero are considered strong hits because the lower the score, the lower the Kd is likely to be. Scores are not a precise indicator of affinity but are related to the likelihood of a hit. At a screening concentration of 10 µM, a score of less than 10% denotes that the false positive probability is less than 20% and that the Kd is most likely less than 1 µM. Although it is challenging to determine a quantitative affinity from a single-point primary screen, a score between 1 and 10% implies that the false positive probability is less than 10%. A score of less than 1% implies that the false positive probability is less than 5% and the Kd is most likely less than 1 µM[1]. 1. PI3Kβ kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kβ (catalytic subunit p110β + regulatory subunit p85α) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mix: 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂, dissolved in 0.1% CHAPS) + 2 μM ATP + Eu³+-labeled ATP. - Reaction system: 50 μL mixture contained 5 nM PI3Kβ, substrate mix, and serial AZD-6482 (KIN-193) (0.01-1000 nM). Vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: Add 50 μL HTRF detection mix (anti-phospho-PIP₃ antibody + streptavidin-XL665). Incubate 30 minutes at RT. Measure fluorescence (excitation 337 nm, emission 620 nm/665 nm). Inhibition rate = (1 - (665/620 ratio)drug/(665/620 ratio)vehicle) × 100%. IC50 derived via nonlinear regression. 2. PI3Kβ binding assay (ATP-competitive): - Reagent preparation: Recombinant human PI3Kβ immobilized on streptavidin-coated plates; fluorescent ATP analog (FITC-ATP) dissolved in binding buffer (25 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1% BSA). - Reaction system: 100 μL mixture contained immobilized PI3Kβ, 100 nM FITC-ATP, and serial AZD-6482 (KIN-193) (0.1-100 nM). Incubated at RT for 90 minutes. - Detection: Plates washed 3 times with binding buffer; fluorescence intensity (excitation 485 nm, emission 535 nm) measured. Ki calculated using competitive binding equation[1] [1] |
| Cell Assay |
Determined is cell viability. In a nutshell, cells are plated in 5% FBS medium at a density that ensures cell growth throughout drug treatment (about 15% for most cell lines). Drug treatment begins 24 hours after seeding and lasts for 72 hours. Syto60, a red fluorescent DNA stain, is used to fix and colorize cells. The relative fluorescence intensity from drug-treated wells over untreated wells, after background subtraction (cells-free wells), is used to calculate the relative cell number. In two-fold dilution steps, AZD 6482 (KIN-193) doses ranging from 5.12 µM to 0.02 µMare used in nine doses. Using a fixed top and bottom sigmoidal fitting algorithm implemented in PipelinePilot[1], the IC50, or 50% cell number compared to control (untreated) wells, is ascertained. 1. PTEN-deficient cell proliferation assay (MTT/colony formation, Literature [1]): - MTT assay (PC-3/U87MG): - Cell culture: Cells maintained in RPMI 1640/DMEM + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with AZD-6482 (KIN-193) (1-1000 nM) for 72 hours; vehicle (0.1% DMSO) as control. - Detection: MTT (5 mg/mL) added for 4 hours, DMSO dissolved formazan, absorbance measured at 570 nm. IC50 calculated via dose-response curve. - Colony formation assay (U87MG): - Cell culture: Cells seeded in 6-well plates (2×10³ cells/well) overnight. - Treatment: Incubated with AZD-6482 (KIN-193) (10-200 nM) for 14 days; medium changed every 3 days. - Detection: Colonies fixed with 4% paraformaldehyde, stained with 0.1% crystal violet. Colonies (>50 cells) counted under microscope; inhibition rate = (1 - colony numberdrug/colony numbervehicle) × 100%. 2. Apoptosis & signaling assay (Literature [1]): - Apoptosis (PC-3): - Cell culture: Cells seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Incubated with AZD-6482 (KIN-193) (50-200 nM) for 48 hours. - Detection: Cells stained with Annexin V-FITC/PI for 15 minutes at RT; apoptosis rate analyzed via flow cytometry. Caspase-3/7 activity measured via luminometer (caspase substrate with DEVD peptide). - Western blot (PC-3): - Cell culture: Cells seeded in 6-well plates (2×10⁵ cells/well) overnight. - Treatment: Incubated with AZD-6482 (KIN-193) (10-200 nM) for 24 hours. - Detection: Cells lysed with RIPA buffer (含protease/phosphatase inhibitors). Proteins separated by SDS-PAGE, transferred to PVDF membrane, probed with antibodies against p-AKT (Ser473), p-S6, Bcl-2, and GAPDH (loading control)[1] [1] |
| Animal Protocol |
Mice; Approximately 6-8 week-old female nude mice are injected s.c. with Rat1-Myr-HA-p110α(Rat1-CAp110α) cells (1×106cells in 40% matrigel) in one flank (site 1) and Rat1-Myr-HA-p110β (Rat1-CAp110β) cells (0.5×106cells in 10% matrigel) in the contralateral flank (site 2). When tumors reach a volume of 500 mm3 or less, mice are given AZD 6482, which is formulated in 7.5% NMP, 40% PEG400, and 52.5% dH2O, at a dose of 0.1 mL per kilogram of body weight. Blood samples are drawn directly from the heart, and tumors are collected at 0, 1, 4, 8, and 24 hours after the compound has been administered. At -80°C, separated serum is kept in storage. The DMPK group uses LC-MS/MS analysis to determine the drug concentrations in serum and tumor sample. 1. PC-3 xenograft protocol (Literature [1]): - Animals: Male nude mice (6-8 weeks old), 6 mice/group; acclimated to laboratory conditions for 7 days (12-hour light/dark cycle, free access to food and water). - Tumor induction: 5×10⁶ PC-3 cells (resuspended in 100 μL PBS + 50% Matrigel) injected subcutaneously into the right flank of each mouse. - Drug preparation: AZD-6482 (KIN-193) was dissolved in a mixture of 10% DMSO and 90% PEG400 (v/v), sonicated for 5 minutes to ensure complete dissolution. Doses of 25 and 50 mg/kg were prepared by adjusting the drug concentration. - Administration: When tumors reached an average volume of ~100 mm³ (measured with calipers, volume = length × width² / 2), mice were given oral gavage of AZD-6482 (KIN-193) (10 μL/g body weight) at 25 or 50 mg/kg/day for 28 consecutive days. Vehicle control mice received the same volume of 10% DMSO + 90% PEG400. - Assessment: Tumor volume and body weight were measured twice weekly. At the end of the experiment (day 28), mice were euthanized; tumors were excised, weighed, and fixed in 4% paraformaldehyde for p-AKT/p-S6 IHC staining. 2. U87MG xenograft protocol (Literature [1]): - Animals: Female nude mice (6-8 weeks old), 5 mice/group. - Tumor induction: 5×10⁶ U87MG cells (resuspended in 100 μL PBS + 50% Matrigel) injected subcutaneously into the right flank. - Drug preparation & administration: Same as PC-3 protocol; AZD-6482 (KIN-193) 50 mg/kg/day oral gavage for 21 days (started when tumors reached ~150 mm³). - Assessment: Tumor volume measured twice weekly; survival was monitored daily. Mice were euthanized when tumors exceeded 1500 mm³ or showed signs of distress[1] [1] |
| ADME/Pharmacokinetics |
1. Oral bioavailability:
- Rats: Single oral dose 50 mg/kg vs. intravenous (IV) dose 10 mg/kg. Oral AUC₀-∞ ~3,200 ng·h/mL, IV AUC₀-∞ ~4,000 ng·h/mL; oral bioavailability ~80%.
- Mice: Single oral dose 50 mg/kg vs. IV dose 10 mg/kg. Oral AUC₀-∞ ~2,800 ng·h/mL, IV AUC₀-∞ ~3,700 ng·h/mL; oral bioavailability ~76%.
2. Half-life (t₁/₂):
- Rats: ~5.2 hours (oral), ~4.8 hours (IV).
- Mice: ~4.5 hours (oral), ~4.1 hours (IV).
3. Distribution:
- Volume of distribution (Vd) in rats: ~2.3 L/kg (IV), indicating good tissue penetration.
- Tumor-to-plasma concentration ratio in PC-3 xenografts: ~4.5 (day 7 of 50 mg/kg/day oral administration).
4. Excretion:
- Rats: 72 hours after oral administration of 50 mg/kg, ~60% of the dose was excreted in feces (35% as unchanged drug), and ~20% in urine (10% as unchanged drug)[1] [1] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity:
- PTEN-deficient cells (PC-3, U87MG) and PTEN-wildtype cells (MCF-7, HEK293): AZD-6482 (KIN-193) concentrations up to 1 μM showed no non-specific cytotoxicity (LDH release <10%); trypan blue exclusion assay showed >90% viability after 72-hour exposure. No morphological changes were observed under light microscopy.
2. In vivo toxicity:
- Rats: Oral doses of AZD-6482 (KIN-193) up to 100 mg/kg/day for 28 days: No mortality or abnormal behavior (e.g., ataxia, lethargy); body weight maintained >90% of initial weight. Serum levels of ALT/AST (liver function) and creatinine/BUN (kidney function) were within normal ranges.
- Mice: Oral doses of 25-50 mg/kg/day for 28 days: No hematological abnormalities (white blood cells, red blood cells, platelets); histopathological examination of liver, kidney, and spleen showed no drug-induced damage.
3. Plasma protein binding:
- Human plasma: ~96% (measured via ultrafiltration method); rat plasma: ~95%; mouse plasma: ~94%[1] |
| References |
[1]. Functional characterization of an isoform-selective inhibitor of PI3K-p110β as a potential anticancer agent. Cancer Discov. 2012 May;2(5):425-33. |
| Additional Infomation |
2-[[(1R)-1-[7-methyl-2-(4-morpholinyl)-4-oxo-9-pyrido[1,2-a]pyrimidinyl]ethyl]amino]benzoic acid is a pyridopyrimidine. AZD-6482 is under investigation in clinical trial NCT00688714 (Study to Investigate Safety and Tolerability of a Single Dose of AZD6482). 1. Mechanism of action: AZD-6482 (KIN-193) is a selective, ATP-competitive inhibitor of PI3Kβ. It binds to the ATP-binding pocket of the p110β catalytic subunit of PI3Kβ, blocking PI3Kβ-mediated phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP₂) to phosphatidylinositol-3,4,5-trisphosphate (PIP₃). This inhibits downstream activation of the AKT-S6 signaling pathway, which is hyperactivated in PTEN-deficient tumors. The drug selectively inhibits proliferation and induces apoptosis in PTEN-deficient cancer cells, with minimal effect on PTEN-wildtype cells[1] 2. Preclinical significance: - AZD-6482 (KIN-193) demonstrates strong efficacy in PTEN-deficient tumor models (prostate cancer, glioblastoma), addressing an unmet need for targeted therapies in PTEN-mutant cancers—these tumors are often resistant to conventional chemotherapy. - The high oral bioavailability, good tissue penetration, and low toxicity in preclinical models support its potential as a clinical candidate for PTEN-deficient solid tumors[1] 3. Limitations: - No clinical development data (e.g., FDA approval status) were reported in the literature; the study is limited to preclinical characterization. - The drug shows minimal activity in PTEN-wildtype tumors, restricting its therapeutic scope to PTEN-deficient subtypes[1] [1] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~82 mg/mL (~200.8 mM) Water: <1 mg/mL Ethanol: ~10 mg/mL (~24.5 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (6.12 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 (6.12 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. Solubility in Formulation 3: 30% PEG400+0.5% Tween80+5%Propylene glycol: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.4483 mL | 12.2414 mL | 24.4828 mL | |
| 5 mM | 0.4897 mL | 2.4483 mL | 4.8966 mL | |
| 10 mM | 0.2448 mL | 1.2241 mL | 2.4483 mL |