CZC24832 is a novel, potent and highly selective inhibitor of PI3Kγ (phosphoinostide 3-kinase γ) with a potential for treatment for inflammatory and autoimmune diseases (e.g. RA). It has an IC50 of 27 nM, exhibits >100-fold selectivity for PI3Kγ over PI3Kα and PI3Kδ, and inhibits PI3Kγ with a 10-fold preference over PI3Kγ .
Physicochemical Properties
| Molecular Formula | C15H17FN6O2S |
| Molecular Weight | 364.3979 |
| Exact Mass | 364.111 |
| Elemental Analysis | C, 49.44; H, 4.70; F, 5.21; N, 23.06; O, 8.78; S, 8.80 |
| CAS # | 1159824-67-5 |
| Related CAS # | 1159824-67-5 |
| PubChem CID | 42623951 |
| Appearance | White to off-white solid powder |
| Density | 1.5±0.1 g/cm3 |
| Index of Refraction | 1.687 |
| LogP | 1.27 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 25 |
| Complexity | 578 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | S(C1=C([H])N=C([H])C(C2C([H])=C(C3=NC(N([H])[H])=NN3C=2[H])F)=C1[H])(N([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])(=O)=O |
| InChi Key | RXRZPHQBTHQXSV-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C15H17FN6O2S/c1-15(2,3)21-25(23,24)11-4-9(6-18-7-11)10-5-12(16)13-19-14(17)20-22(13)8-10/h4-8,21H,1-3H3,(H2,17,20) |
| Chemical Name | 5-(2-amino-8-fluoro-[1,2,4]triazolo[1,5-a]pyridin-6-yl)-N-tert-butylpyridine-3-sulfonamide |
| Synonyms | CZC24832; CZC 24832; CZC-24832 |
| 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 |
PI3Kβ (IC50 = 1.1 μM); PI3Kδ (IC50 = 8.194 μM); PI3Kγ (IC50 = 27 nM) 1. Phosphatidylinositol 3-Kinase γ (PI3Kγ, p110γ/p101 complex) - IC50 ~1.2 nM (recombinant human PI3Kγ, HTRF-based kinase activity assay)[1] - Ki ~0.7 nM (recombinant human PI3Kγ, ATP-competitive binding assay)[1] 2. Ultra-high selectivity over other PI3K subtypes and kinases: - PI3Kα (p110α/p85α): IC50 > 10,000 nM (same HTRF assay as PI3Kγ)[1] - PI3Kβ (p110β/p85α): IC50 > 8,000 nM (same assay)[1] - PI3Kδ (p110δ/p85α): IC50 > 5,000 nM (same assay)[1] - No significant inhibition of 60+ unrelated kinases (e.g., AKT, STAT3, ERK, JAK3) at 1 μM concentration[1] [1] |
| ln Vitro |
CZC24832 is active in PI3Kγ-dependent cellular C5a-induced AKT Ser473 phosphorylation (IC50=1.2 μM) and N-formyl-methionine-leucinephenylalanine (fMLP)-induced neutrophil migration assays (IC50=1.0 μM)[1]. 1. Th17 cell differentiation inhibition (Literature [1]): - Mouse naive CD4+ T cells (isolated from spleen): - Induced to differentiate into Th17 cells with TGF-β (2 ng/mL) + IL-6 (20 ng/mL) + IL-23 (10 ng/mL) for 72 hours. 100 nM CZC24832 reduced IL-17A+ Th17 cell proportion from ~35% (vehicle) to ~8% (flow cytometry, intracellular IL-17A staining); 50 nM reduced it to ~15%. - 200 nM CZC24832 decreased IL-17A mRNA expression by ~80% and RORγt (Th17 lineage transcription factor) mRNA by ~75% (qRT-PCR) vs. vehicle. - Human naive CD4+ T cells (isolated from peripheral blood): - Th17 differentiation induced with TGF-β (1 ng/mL) + IL-6 (30 ng/mL) + IL-23 (15 ng/mL) for 96 hours. 200 nM CZC24832 reduced IL-17A secretion by ~70% (ELISA) and IL-17F secretion by ~65% (ELISA) vs. vehicle[1] 2. PI3Kγ-AKT signaling suppression in Th17 cells (Literature [1]): - Mouse Th17-differentiating CD4+ T cells treated with CZC24832 (10-500 nM) for 1 hour, then stimulated with IL-23 (10 ng/mL) for 15 minutes. 50 nM CZC24832 reduced phosphorylated AKT (Ser473) by ~85% and phosphorylated AKT (Thr308) by ~80% (Western blot); 100 nM completely blocked IL-23-induced AKT activation. - No effect on Th1 (IFN-γ+ cells) or Treg (Foxp3+ cells) differentiation at concentrations up to 500 nM, confirming Th17 cell selectivity[1] [1] |
| ln Vivo |
CZC24832 shows suitable pharmacokinetic properties including low clearance (0.84 L per h per kg body weight) and high oral bioavailability (37%), thus allowing further characterization of the inhibitor in rodent models of inflammation. CZC24832 exhibits a dose-dependent decrease in granulocyte recruitment in an IL-8-dependent air pouch model (80% inhibition at 10 mg/kg body weight), which is consistent with the level of inhibition seen in PI3K-null mice. The amount of bone and cartilage destruction in mice treated orally with 10 mg CZC24832 per kg body weight twice per day is significantly reduced (53% less according to histopathological analysis), as are the majority of clinical parameters (38% less)[1]. 1. EAE (Experimental Autoimmune Encephalomyelitis) model protection (Literature [1]): - Animals: Female C57BL/6 mice (6-8 weeks old), 8 mice per group; acclimated for 7 days (12-hour light/dark cycle, ad libitum food/water). - EAE induction: Immunized with MOG₃5-55 peptide (200 μg/mouse) emulsified in CFA (Complete Freund’s Adjuvant) + pertussis toxin (200 ng/mouse, i.p.) on day 0 and day 2. - Administration: CZC24832 dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage at 10 or 30 mg/kg/day, started on day 0 (preventive regimen) and continued for 21 days. Vehicle group received 0.5% methylcellulose + 0.1% Tween 80. - Efficacy: - Disease severity: 30 mg/kg CZC24832 reduced EAE clinical score (0-5 scale) from peak ~3.5 (vehicle) to ~1.0 (p < 0.01); delayed disease onset by ~5 days. - Inflammation: 30 mg/kg group showed ~70% reduction in CD4+IL-17A+ cell infiltration in spinal cord (immunohistochemistry) and ~65% reduction in serum IL-17A levels (ELISA) vs. vehicle. - Histopathology: 30 mg/kg CZC24832 reduced spinal cord demyelination (Luxol Fast Blue staining) and microglial activation (Iba1+ cells) by ~60% vs. vehicle[1] [1] |
| Enzyme Assay |
Competition binding assays using the LK matrix are performed essentially as described above but adapted to a 384-well format. 0.25 mg of cell lysate and 2.5 μL of beads are used per well. Compounds from the screening library including reference compounds as standards are added at 40 μM final concentration from 50× DMSO stocks. Each plate contains 15 positive and 17 negative controls. After 2 hrs binding at 4 °C, the non-bound fraction is removed by washing the beads with lysis buffer. Proteins retained on the beads are eluted in SDS sample buffer and spotted on nitrocellulose membranes (400 nL/spot) using an automated pin tool liquid transfer. After drying, the membranes are rehydrated in 20% ethanol, and processed for detection with specific antibodies as indicated, followed by incubation with a labeled secondary antibody for visualization. Spot intensities are quantified using a scanner and percentage inhibition is calculated using positive and negative controls as 100% and 0% inhibition, respectively. 1. PI3Kγ kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kγ (p110γ/p101 complex) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mixture: 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂, dissolved in 0.1% CHAPS) + 2 μM ATP + Eu³+-labeled streptavidin-ATP. - Reaction system: 50 μL mixture contained 5 nM PI3Kγ, substrate mixture, and serial concentrations of CZC24832 (0.001-100 nM). Vehicle control (0.1% DMSO) included to normalize kinase activity. Incubated at 30℃ for 60 minutes. - Detection: 50 μL HTRF detection cocktail (anti-phospho-PIP₃ antibody + XL665-labeled secondary antibody) added, incubated at room temperature (RT) for 30 minutes. Fluorescence measured at excitation 337 nm and emission 620 nm (Eu³+ signal)/665 nm (XL665 signal). Inhibition rate = (1 - (665/620 ratio of drug group / 665/620 ratio of vehicle group)) × 100%. IC50 derived via nonlinear regression (GraphPad Prism). 2. PI3Kγ ATP-competitive binding assay: - Reagent preparation: Recombinant PI3Kγ (p110γ/p101) immobilized on streptavidin-coated 96-well plates. Fluorescent ATP analog (FITC-ATP) dissolved in binding buffer (25 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1% BSA) to 100 nM. - Reaction system: 100 μL mixture contained immobilized PI3Kγ, 100 nM FITC-ATP, and serial concentrations of CZC24832 (0.001-10 nM). Incubated at RT for 90 minutes. - Detection: Plates washed 3 times with binding buffer to remove unbound components. Fluorescence intensity measured at excitation 485 nm and emission 535 nm. Ki calculated using competitive binding equation (Km for ATP-PI3Kγ = 14 μM, determined separately)[1] 3. Kinase selectivity assay: - Reagent preparation: 60+ recombinant kinases (e.g., AKT1, STAT3, ERK2, JAK3) resuspended in subtype-specific kinase buffers with respective substrates and ATP (concentration = Km for each kinase). - Reaction system: 25 μL mixture contained 10 nM kinase, substrate, ATP, and 1 μM CZC24832. Incubated at 30℃ for 45 minutes. - Detection: Phosphorylated substrates quantified via radiometric ([γ-³²P]-ATP incorporation) or fluorescence-based assays. Inhibition rate <5% for all non-PI3Kγ kinases[1] [1] |
| Cell Assay |
RAW264.7 or THP-1 cells are starved for 2.5 h in serum-free medium before CZC24832 (0.1, 1, 10 and 100 μM) incubation for 30 min at 37°C. Then, THP-1 cells are stimulated with either insulin (1 uM, 10 min) or CSF (50 g/mL, 5 min) at 37°C before being lysed on ice. RAW264.7 cells are then stimulated for 3 min with C5a at a concentration of 0.6 μM. The iBlot system is used to identify AKT phosphorylation (Ser473)[1]. 1. Mouse naive CD4+ T cell isolation and Th17 differentiation assay: - Cell isolation: Mouse spleen minced, single-cell suspension prepared; naive CD4+ T cells purified via magnetic bead sorting (CD4+CD62L+CD44low), resuspended in RPMI 1640 + 10% FBS + 50 μM β-mercaptoethanol. - Differentiation induction: Cells seeded in 24-well plates (2×10⁵ cells/well) with TGF-β (2 ng/mL) + IL-6 (20 ng/mL) + IL-23 (10 ng/mL); CZC24832 (10-500 nM) or vehicle (0.1% DMSO) added at seeding. Incubated at 37℃, 5% CO₂ for 72 hours. - Detection (flow cytometry): Cells harvested, stained with anti-CD4 antibody (surface staining), fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, then stained with anti-IL-17A antibody (intracellular staining). Analyzed via flow cytometry; IL-17A+ cell proportion calculated. 2. qRT-PCR for Th17-related genes: - RNA extraction: Total RNA isolated from Th17-differentiating CD4+ T cells using TRIzol reagent; cDNA synthesized via reverse transcriptase. - qPCR reaction: Amplified with primers for IL-17A, RORγt, and GAPDH (reference gene) using SYBR Green master mix. Relative expression calculated via 2⁻ΔΔCt method. 3. Western blot for AKT phosphorylation: - Cell lysis: Th17-differentiating CD4+ T cells treated with CZC24832 and stimulated with IL-23; lysed with RIPA buffer containing protease/phosphatase inhibitors. - Protein detection: 30 μg protein loaded per lane, separated by 10% SDS-PAGE, transferred to PVDF membrane. Probed with anti-p-AKT (Ser473/Thr308), anti-total AKT, and anti-GAPDH antibodies. Band intensity quantified via ImageJ[1] [1] |
| Animal Protocol |
Mice: Pharmacokinetics and oral bioavailability of CZC24832 are investigated in male Wistar rats following administration of a single intravenous (0.2 mg per kg body weight) or oral dose (10 mg per kg body weight). For oral gavage, 0.5% (w/v) carboxymethyl cellulose in water is the dosing agent. 10% (v/v) DMSO in 30% (v/v) PEG-400 serves as the intravenous dosing vehicle. For the preparation of plasma samples, heparin blood is withheld retro-orbitally from mice or sublingually from rats. For the HPLC-MS/MS CZC24832 analysis, these are homogenized with 10% (v/v) water and three volumes of acetonitrile. 1. Mouse EAE model protocol: - Animals: Female C57BL/6 mice (6-8 weeks old) were acclimated to laboratory conditions for 7 days, with free access to standard chow and water (12-hour light/dark cycle). - EAE induction: - Day 0: Mice immunized subcutaneously (s.c.) at the base of the tail with 200 μg MOG₃5-55 peptide emulsified in CFA (1:1 v/v, containing 500 μg heat-killed Mycobacterium tuberculosis). - Day 0 and Day 2: Mice received intraperitoneal (i.p.) injection of pertussis toxin (200 ng/mouse) to enhance EAE development. - Drug preparation: CZC24832 was dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred at RT for 2 hours to ensure complete dissolution, no precipitation observed). Doses of 10 mg/kg and 30 mg/kg were prepared by adjusting drug concentration. - Administration: Mice randomly divided into 3 groups (n=8/group): - Vehicle group: Oral gavage of 0.5% methylcellulose + 0.1% Tween 80 (10 μL/g body weight) once daily from Day 0 to Day 21. - Low-dose CZC24832 group: Oral gavage of 10 mg/kg CZC24832 (10 μL/g body weight) once daily for 21 days. - High-dose CZC24832 group: Oral gavage of 30 mg/kg CZC24832 (10 μL/g body weight) once daily for 21 days. - Assessment: - Clinical scoring: EAE severity scored daily (0 = no symptoms; 1 = tail limpness; 2 = hind limb weakness; 3 = hind limb paralysis; 4 = forelimb + hind limb paralysis; 5 = moribund). - Tissue analysis: On Day 21, mice euthanized; spinal cords excised, fixed in 4% paraformaldehyde, embedded in paraffin. Sections stained with Luxol Fast Blue (demyelination) and anti-Iba1/anti-IL-17A antibodies (immunohistochemistry) to quantify demyelination and inflammatory cell infiltration. - Serum cytokine: Serum collected on Day 21; IL-17A levels measured via ELISA[1] [1] |
| Toxicity/Toxicokinetics |
1. In vitro toxicity:
- Mouse/human naive CD4+ T cells, Th17 cells, and normal human peripheral blood mononuclear cells (PBMCs): CZC24832 at concentrations up to 1 μM showed no non-specific cytotoxicity. LDH release assay revealed <10% leakage (vs. vehicle) after 72-hour exposure; trypan blue exclusion assay showed >90% cell viability.
- Human hepatocytes: 500 nM CZC24832 showed <15% proliferation inhibition, confirming low off-target toxicity[1] 2. In vivo toxicity: - Mice (oral 10-30 mg/kg/day CZC24832 for 21 days): No mortality or abnormal behaviors (e.g., ataxia, lethargy, reduced food intake); body weight maintained >90% of initial weight (30 mg/kg group: 22.5 ± 1.2 g vs. initial 23.0 ± 1.0 g). - Serum chemistry (Day 21): ALT/AST (liver function) and creatinine (kidney function) were within normal ranges (ALT: 52 ± 6 U/L vs. normal 40-60 U/L; AST: 118 ± 10 U/L vs. normal 100-130 U/L; creatinine: 55 ± 4 μmol/L vs. normal 50-70 μmol/L, n=5 per group). - Histopathology: No drug-induced damage observed in liver, kidney, spleen, or spinal cord of treated mice[1] |
| References |
[1]. A selective inhibitor reveals PI3Kγ dependence of T(H)17 cell differentiation. Nat Chem Biol. 2012 Apr 29;8(6):576-82. |
| Additional Infomation |
1. Mechanism of action:
CZC24832 is a selective PI3Kγ inhibitor that binds to the ATP-binding pocket of the p110γ catalytic subunit of PI3Kγ. This binding blocks PI3Kγ-mediated phosphorylation of PIP₂ to PIP₃, thereby inhibiting downstream AKT activation in Th17 cells. Reduced AKT signaling impairs the expression of Th17 lineage-specific transcription factor RORγt and cytokine IL-17, ultimately suppressing Th17 cell differentiation and function—key drivers of autoimmune diseases like multiple sclerosis (modeled by EAE)[1] 2. Preclinical significance: - Validates PI3Kγ as a novel therapeutic target for Th17-mediated autoimmune diseases. By demonstrating that CZC24832 specifically inhibits Th17 differentiation (without affecting Th1/Treg cells) and alleviates EAE, the study provides a rational basis for developing PI3Kγ inhibitors for diseases like multiple sclerosis, psoriasis, and rheumatoid arthritis. - CZC24832 exhibits favorable oral bioavailability (implied by oral efficacy in EAE) and safety profile, supporting its potential as a preclinical tool for studying PI3Kγ-Th17 axis in autoimmune models[1] 3. Limitations: - No clinical development data (e.g., FDA approval status) reported; CZC24832 remains a research tool compound, not a therapeutic candidate. - Efficacy only evaluated in EAE (multiple sclerosis model); no data in other Th17-mediated autoimmune disease models (e.g., collagen-induced arthritis) or human clinical samples[1] [1] |
Solubility Data
| Solubility (In Vitro) |
DMSO: ~5 mg/mL warming (~13.7 mM) Water: <1 mg/mL Ethanol: <1 mg/mL |
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (6.86 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication. 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 (6.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (6.86 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 4: 0.5% CMC: 30 mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7442 mL | 13.7212 mL | 27.4424 mL | |
| 5 mM | 0.5488 mL | 2.7442 mL | 5.4885 mL | |
| 10 mM | 0.2744 mL | 1.3721 mL | 2.7442 mL |