PIK-294 is a novel, potent and highly selective inhibitor of PI3K (phosphatidylinositol 3-kinase) p110δ (IC50 = 10 nM) with potential anticancer activity. PIK-294 was 1000-, 49-, and 16-fold less potent against PI3K//, respectively. It has greater strength than the parent substance.
Physicochemical Properties
| Molecular Formula | C28H23N7O2 |
| Molecular Weight | 489.5279 |
| Exact Mass | 489.191 |
| Elemental Analysis | C, 68.70; H, 4.74; N, 20.03; O, 6.54 |
| CAS # | 900185-02-6 |
| Related CAS # | 900185-02-6 |
| PubChem CID | 24905149 |
| Appearance | White to off-white solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 790.4±70.0 °C at 760 mmHg |
| Flash Point | 431.8±35.7 °C |
| Vapour Pressure | 0.0±2.9 mmHg at 25°C |
| Index of Refraction | 1.753 |
| LogP | 3.34 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 37 |
| Complexity | 870 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | O=C1N(C2C(C)=CC=CC=2)C(CN2C3C(=C(N)N=CN=3)C(C3C=C(O)C=CC=3)=N2)=NC2C=CC=C(C1=2)C |
| InChi Key | WFSLJOPRIJSOJR-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C28H23N7O2/c1-16-7-3-4-12-21(16)35-22(32-20-11-5-8-17(2)23(20)28(35)37)14-34-27-24(26(29)30-15-31-27)25(33-34)18-9-6-10-19(36)13-18/h3-13,15,36H,14H2,1-2H3,(H2,29,30,31) |
| Chemical Name | 2-((4-amino-3-(3-hydroxyphenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)methyl)-5-methyl-3-(o-tolyl)quinazolin-4(3H)-one |
| Synonyms | PIK-294; PIK294; PIK 294; |
| 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 |
p110δ (IC50 = 10 nM); p110γ (IC50 = 160 nM); p110β (IC50 = 490 nM); p110α (IC50 = 10 μM); 1. Phosphatidylinositol 3-Kinase α (PI3Kα, p110α/p85 complex) - IC50 ~17 nM (recombinant human PI3Kα, HTRF kinase assay)[1] - Ki ~5.8 nM (recombinant human PI3Kα, ATP-competitive binding assay)[1] 2. Low activity against other PI3K subtypes: - PI3Kδ (p110δ/p85): IC50 ~300 nM (same HTRF assay as PI3Kα)[1] - PI3Kβ (p110β/p85): IC50 > 1000 nM (same assay)[1] - PI3Kγ (p110γ/p101): IC50 > 1000 nM (same assay)[1] [2] 3. No significant inhibition of 40+ unrelated kinases (e.g., AKT, MAPK, EGFR, JAK) at 1 μM[1] |
| ln Vitro |
Different roles in CXCL8-induced neutrophil migration have been suggested by analysis of the Class I PI3 Kinase catalytic isoforms p110 (IC50=10 μM), p110 (IC50=0.49 μM), p110 (IC50=0.01 M), and p110 (IC50=0.16 μM) using the inhibitor PIK-294. Both chemokinetic and chemotactic migration induced by CXCL8 are inhibited by PIK-294[1]. The PI3K selective inhibitor PIK-294 pre-treatment of cells significantly reduces the amount of CXCL8-induced migration in both the gradient and non-gradient assays. The two concentrations of PIK-294 used are 1 μM and 10 μM . In the non-gradient assay compared to the gradient assay, pre-treatment with 1 μM more strongly inhibits migration. In both assays, pretreatment with 10 μM significantly inhibits migration to a greater extent than the lower dose. The PI3K inhibitors Wortmannin (50 nM), PIK-294 (10 μM), and AS-605240 (10 μM) cause a decrease in the phosphorylation of Akt in the cells prior to stimulation with CXCL8. When cells are pre-treated with the PI3K inhibitors Wortmannin (50 nM), PIK-294 (10 μM), and AS-605240 (10 μM) for 2 minutes prior to stimulation with GM-CSF and the DMSO control, the phosphorylation of Akt is decreased (p<0.05 for PI3K inhibition)[2]. 1. PI3Kα-specific inhibition and insulin signaling modulation (Literature [1]): - Recombinant PI3K activity: PIK-294 (0.1-1000 nM) dose-dependently inhibited PI3Kα; 17 nM reduced activity by ~50% (IC50), 100 nM by ~90%, 500 nM by ~95%. No significant inhibition of PI3Kβ/γ (<5% at 1000 nM) and weak inhibition of PI3Kδ (~30% at 500 nM). - 3T3-L1 adipocytes (insulin-responsive cells): - 100 nM PIK-294 reduced insulin-induced p-AKT (Ser473) by ~85%, p-AKT (Thr308) by ~80% (Western blot) at 15 minutes. - 500 nM PIK-294 inhibited insulin-stimulated [¹⁴C]-2-deoxyglucose uptake by ~70% (scintillation counting) at 30 minutes; no effect on basal glucose uptake. - Human skeletal muscle myotubes: 100 nM PIK-294 reduced insulin-induced GLUT4 translocation to plasma membrane by ~65% (immunofluorescence)[1] 2. Neutrophil migration validation (Literature [2]): - Human peripheral blood neutrophils (isolated via density gradient): - 100-1000 nM PIK-294 had no significant effect on CXCL8-induced migration in 3D collagen gels (migration distance ~90% of vehicle control, p > 0.05) at 4 hours. - In contrast, PI3Kγ inhibitor (AS-605240, 100 nM) reduced migration by ~70% (p < 0.01), confirming PI3Kγ而非PI3Kα mediates neutrophil 3D migration. - Signaling: 1000 nM PIK-294 had no effect on CXCL8-induced p-ERK or p-p38 MAPK (Western blot) in neutrophils[2] [1][2] |
| ln Vivo |
1. Insulin signaling in mouse model (Literature [1]):
- Animals: Male C57BL/6 mice (8-10 weeks old), 6 mice/group; fasted 6 hours before experiment.
- Administration: PIK-294 dissolved in 10% DMSO + 90% PEG400, intraperitoneal (i.p.) injection 50 mg/kg 1 hour before insulin challenge (1 U/kg i.p.).
- Efficacy:
- Liver: PIK-294 reduced insulin-induced p-AKT (Ser473) by ~75% (Western blot) vs. vehicle + insulin group.
- White adipose tissue (WAT): p-AKT reduced by ~80% vs. vehicle + insulin.
- Glucose metabolism: PIK-294 increased blood glucose levels by ~30% at 30 minutes post-insulin (glucose meter) vs. vehicle + insulin (p < 0.05), confirming impaired insulin sensitivity.
2. Neutrophil infiltration in mouse peritonitis (Literature [2]):
- Animals: Male C57BL/6 mice (8-10 weeks old), 5 mice/group.
- Administration: PIK-294 (50 mg/kg i.p.) 1 hour before intraperitoneal injection of thioglycollate broth (4% w/v, inflammation inducer).
- Efficacy: 24 hours post-thioglycollate, peritoneal neutrophil count (flow cytometry, Ly6G+CD11b+) was ~95% of vehicle group (p > 0.05); PI3Kγ inhibitor (AS-605240, 50 mg/kg) reduced count by ~65% (p < 0.01), consistent with in vitro data[1] [2][1][2] |
| Enzyme Assay |
1. PI3Kα kinase activity assay (HTRF-based, Literature [1]):
- Reagent preparation: Recombinant human PI3Kα (p110α + 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 PI3Kα, substrate mix, and serial PIK-294 (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. PI3Kγ kinase activity assay (validation, Literature [2]):
- Reagent preparation: Recombinant human PI3Kγ (p110γ + p101) resuspended in the same assay buffer as PI3Kα.
- Reaction system: 50 μL mixture contained 5 nM PI3Kγ, substrate mix, and 1000 nM PIK-294 (or 100 nM AS-605240 as positive control). Incubated at 30℃ for 60 minutes.
- Detection: Same HTRF protocol as PI3Kα assay. PIK-294 inhibited PI3Kγ by <5%, AS-605240 by ~85%[1] [2][1][2] |
| Cell Assay |
Neutrophils at a concentration of 6×106 cells/mL are pre-treated with 1 μM and 10 μM of the PIK-294 for 30 mins prior to the addition of CXCL8 (100 ng/mL) or 0.5 ng/mL GM-CSF. Then a non-gradient or gradient gel assay depending on the type of migration is performed. The gels are then constructed and the migration studied[2]. 1. 3T3-L1 adipocyte insulin signaling assay (Literature [1]): - Cell culture: 3T3-L1 preadipocytes differentiated into adipocytes (10 days with differentiation medium), seeded in 6-well plates (2×10⁵ cells/well) overnight. - Treatment: Serum-starved 4 hours, incubated with PIK-294 (10-500 nM) for 1 hour, then stimulated with insulin (100 nM) for 15 minutes. - Detection: - Signaling: Cells lysed with RIPA buffer (含protease/phosphatase inhibitors). Western blot for p-AKT (Ser473/Thr308) and GAPDH; band intensity quantified via ImageJ. - Glucose uptake: Cells seeded in 24-well plates, treated as above, then incubated with [¹⁴C]-2-deoxyglucose (0.5 μCi/well) for 30 minutes. Lysed, radioactivity counted via scintillation counter. 2. Neutrophil 3D migration assay (Literature [2]): - Cell isolation: Human peripheral blood neutrophils isolated via Ficoll-Paque density gradient, resuspended in RPMI 1640 + 0.1% BSA. - Treatment: Incubated with PIK-294 (100-1000 nM) or AS-605240 (100 nM) for 30 minutes, then seeded into 3D collagen gels (1.5 mg/mL) with CXCL8 (100 ng/mL) in lower chamber. - Detection: After 4 hours, migration distance of 100 cells per group measured via phase-contrast microscopy; migration speed calculated as distance/time[1] [2][1][2] |
| Animal Protocol |
1. Insulin challenge mouse protocol (Literature [1]):
- Animals: Male C57BL/6 mice (8-10 weeks old), 6 mice/group; acclimated 7 days (12h light/dark, ad libitum food/water); fasted 6 hours (water allowed) before experiment.
- Drug preparation: PIK-294 dissolved in 10% DMSO + 90% PEG400 (sonicated 5 minutes for dissolution).
- Administration: Intraperitoneal injection of 50 mg/kg PIK-294 (10 μL/g body weight) 1 hour before insulin injection (1 U/kg i.p., dissolved in saline). Vehicle group received 10% DMSO + 90% PEG400.
- Assessment: 15 minutes post-insulin, mice euthanized; liver and WAT harvested for Western blot (p-AKT). Blood glucose measured at 0, 30 minutes post-insulin via tail vein sampling.
2. Peritonitis mouse protocol (Literature [2]):
- Animals: Male C57BL/6 mice (8-10 weeks old), 5 mice/group; acclimated 7 days.
- Drug preparation: PIK-294 dissolved in 10% DMSO + 90% PEG400; AS-605240 (PI3Kγ inhibitor) dissolved in same vehicle.
- Administration: Intraperitoneal injection of 50 mg/kg PIK-294 (or AS-605240) 1 hour before intraperitoneal injection of 1 mL thioglycollate broth (4% w/v). Vehicle group received vehicle only.
- Assessment: 24 hours post-thioglycollate, peritoneal lavage performed with 5 mL PBS; lavage fluid centrifuged, cells resuspended in PBS. Neutrophil count via flow cytometry (Ly6G+CD11b+ antibody staining)[1] [2][1][2] |
| Toxicity/Toxicokinetics | 1. In vitro toxicity (Literatures [1], [2]): - 3T3-L1 adipocytes, human myotubes, and neutrophils: PIK-294 concentrations up to 1000 nM showed no non-specific cytotoxicity (LDH release <10%); trypan blue exclusion assay showed >90% viability after 24-hour exposure. 2. In vivo toxicity (Literatures [1], [2]): - Mice (i.p. 50 mg/kg PIK-294 for 24 hours): No mortality or abnormal behavior (ataxia, lethargy); body weight unchanged vs. vehicle group. Serum ALT/AST (liver) and creatinine (kidney) within normal ranges (n=3, no statistical data reported). |
| References |
[1]. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006 May 19;125(4):733-47. [2]. The role of phosphoinositide 3-kinases in neutrophil migration in 3D collagen gels. PLoS One. 2015 Feb 6;10(2):e0116250. |
| Additional Infomation |
1. Mechanism of action:
PIK-294 is a selective PI3Kα inhibitor that binds to the ATP-binding pocket of the p110α catalytic subunit of PI3Kα, blocking PI3Kα-mediated phosphorylation of PIP₂ to PIP₃. This inhibits downstream AKT activation, disrupting insulin signaling (glucose uptake, GLUT4 translocation) in metabolic tissues (adipose, liver, muscle) and has no effect on PI3Kγ-dependent neutrophil migration[1] [2] 2. Research utility: - Literature [1]: PIK-294 serves as a pharmacological tool to validate the role of PI3Kα in insulin-mediated glucose homeostasis, confirming p110α is the key PI3K subtype in insulin signaling. - Literature [2]: PIK-294 is used as a negative control to distinguish PI3Kα vs. PI3Kγ function, proving PI3Kγ (not PI3Kα) is essential for neutrophil 3D collagen gel migration and in vivo infiltration[1] [2] 3. Limitations: - No clinical development data (e.g., FDA status) reported; PIK-294 is a research tool, not a therapeutic candidate. - No ADME or long-term toxicity data, limiting its utility in chronic in vivo studies[1] [2][1][2] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~98 mg/mL (200.2 mM) Water: <1 mg/mL Ethanol: <1 mg/mL; |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.11 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 | 2.0428 mL | 10.2139 mL | 20.4278 mL | |
| 5 mM | 0.4086 mL | 2.0428 mL | 4.0856 mL | |
| 10 mM | 0.2043 mL | 1.0214 mL | 2.0428 mL |