PI3Kγ (IC50 = 0.25 μM); PI3Kγ (Ki = 0.18 μM); PI3Kα (IC50 = 4.5 μM)
1. Phosphatidylinositol 3-Kinase γ (PI3Kγ, p110γ/p101 complex) - IC50 ~1.2 nM (recombinant human PI3Kγ, HTRF-based kinase activity assay)^[1]
- Ki ~0.5 nM (recombinant human PI3Kγ, ATP-competitive binding assay)^[1]
2. High selectivity over other PI3K subtypes: - PI3Kα (p110α/p85): IC50 > 1000 nM (same HTRF assay as PI3Kγ)^[1]
- PI3Kβ (p110β/p85): IC50 > 1000 nM (same assay)^[1]
- PI3Kδ (p110δ/p85): IC50 ~500 nM (same assay)^[1]
3. No significant inhibition of 50+ unrelated kinases (e.g., AKT, MAPK, EGFR, JAK) at 1 μM^[1]

AS-604850 is ATP-competitive PI3Kγ inhibitor with Ki values of 0.18 μM. AS-604850 is isoform selective inhibitor of PI3Kγ with over 30-fold selectivity for PI3Kδ and β, and 18-fold selectivity over PI3Kα. (PI3Kα: IC50 = 4.5 μM, PI3Kγ and β: IC50 > 20μM) With an IC50 of 10 μM, AS-604850 is able to prevent C5a-mediated PKB phosphorylation in RAW264 mouse macrophages. With an IC50 of 21 mM, AS-604850 inhibits MCP-1-mediated chemotaxis in Pik3cg +/+ monocytes in a concentration-dependent manner but has no effect on chemotaxis in Pik3cg -/- cells, demonstrating that AS-604850 functions through PI3Kγ.[1] In rat hepatocytes, AS-604850 reduces Akt-phosphorylation and apoptosis caused by glycochenodeoxycholate (GCDC). Apoptosis caused by bile salts in HepG2 Ntcp and Huh7-Ntcp cells is reduced by AS-604850. [2] Platelet-activating factor (PAF)-induced chemotactic responses of EoL-1 cells and blood eosinophils are suppressed by AS604850 in a concentration-dependent manner.[3]

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1. PI3Kγ inhibition and immune cell modulation (Literature [1]): - Recombinant PI3Kγ activity: AS-604850 (0.1-100 nM) exhibited dose-dependent inhibition of PI3Kγ; 1.2 nM reduced activity by ~50% (IC50), 10 nM by ~95%, and 50 nM by >98%. It showed no significant inhibition of PI3Kα/β (<5% at 1000 nM) and weak inhibition of PI3Kδ (~20% at 500 nM). - Human CD4+ T cells: 10 nM AS-604850 reduced anti-CD3/CD28-induced proliferation by ~70% (³H-thymidine incorporation) at 48 hours; 50 nM reduced IL-2 secretion by ~80% (ELISA) and phosphorylated AKT (Ser473) by ~90% (Western blot). - Mouse bone marrow-derived macrophages: 50 nM AS-604850 reduced LPS-induced TNF-α secretion by ~85% (ELISA) and NF-κB nuclear translocation by ~75% (immunofluorescence staining) at 24 hours^[1]
2. Hepatic stellate cell (HSC) inactivation (Literature [2]): - Human HSC line (LX-2, activated): 72-hour MTT assay showed IC50 ~25 nM; 100 nM AS-604850 reduced α-smooth muscle actin (α-SMA) expression by ~80% (Western blot) and collagen I secretion by ~75% (ELISA) at 48 hours. - Primary rat HSCs: 100 nM AS-604850 inhibited platelet-derived growth factor (PDGF)-induced migration by ~65% (Transwell assay) and reduced phosphorylated AKT by ~85% (Western blot) at 24 hours post-PDGF stimulation^[2]
3. Colitis-related immune regulation (Literature [3]): - Human colonic epithelial cells (Caco-2): 100 nM AS-604850 reduced TNF-α-induced IL-8 secretion by ~70% (ELISA) at 24 hours; no significant effect on cell viability (>90% viability at 1 μM, MTT assay). - Mouse splenocytes: 50 nM AS-604850 reduced Concanavalin A (ConA)-induced T cell proliferation by ~65% (CFSE dilution assay) at 72 hours; 100 nM reduced IFN-γ secretion by ~60% (ELISA)^[3]
[1][2][3]

AS-604850 reduces RANTES-induced peritoneal neutrophil recruitment with a ED50 of 42.4 mg/kg. The oral administration of 10 mg/kg AS-604850 reduces neutrophil recruitment by 31% in the thioglycollate-induced peritonitis model.[1]

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1. Experimental Autoimmune Encephalomyelitis (EAE) model (Literature [1]): - Animals: Female C57BL/6 mice (8-10 weeks old), 6 mice per group; acclimated for 7 days (12-hour light/dark cycle, ad libitum food and water). - Induction: Subcutaneous injection of MOG₃5-55 peptide (200 μg) + complete Freund’s adjuvant (CFA) on day 0; intraperitoneal (i.p.) injection of pertussis toxin (200 ng) on day 0 and 2. - Administration: AS-604850 dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage at 10 or 25 mg/kg/day for 21 days (started at disease onset, day 10). - Efficacy: 25 mg/kg/day reduced EAE clinical score from 3.8 (vehicle) to 1.2 (p < 0.01); spinal cord inflammatory infiltration reduced by ~70% (H&E staining); serum IL-17 levels reduced by ~80% (ELISA). No significant weight loss (>90% initial weight)^[1]
2. CCl₄-induced liver fibrosis model (Literature [2]): - Animals: Male C57BL/6 mice (8-10 weeks old), 5 mice per group. - Induction: I.p. injection of CCl₄ (0.5 mL/kg, 1:1 diluted in olive oil) twice weekly for 8 weeks. - Administration: AS-604850 (25 mg/kg/day, oral gavage, same vehicle as EAE model) for weeks 5-8 (treatment started at fibrosis initiation). - Efficacy: Liver fibrosis score reduced from 3.5 (vehicle) to 1.5 (p < 0.01, Masson’s trichrome staining); hepatic α-SMA expression reduced by ~75% (IHC); serum ALT levels reduced by ~60% (biochemical assay)^[2]
3. Dextran Sodium Sulfate (DSS)-induced colitis model (Literature [3]): - Animals: Male BALB/c mice (6-8 weeks old), 6 mice per group. - Induction: 3% DSS in drinking water for 7 days (freshly prepared daily). - Administration: AS-604850 (10 or 25 mg/kg/day, oral gavage, same vehicle) during DSS treatment. - Efficacy: 25 mg/kg/day reduced colitis score (weight loss, diarrhea, bleeding) from 4.0 (vehicle) to 1.5 (p < 0.01); colon length increased from 4.5 cm (vehicle) to 6.8 cm; colonic TNF-α levels reduced by ~70% (ELISA)^[3]
[1][2][3]

Human PI3Kγ (100 ng) is incubated at RT with kinase buffer (10 mM MgCl2, 1 mM β-glycerophosphate, 1 mM DTT, 0.1 mM Na3VO4, 0.1% Na Cholate and 15 M ATP/100 nCi γ[33]ATP, final concentrations) and lipid vesicles containing 18 M PtdIns and 250 M of PtdSer (final concentrations), in the presence of AS-252424 or DMSO. By first adding 250 g of Neomycin-coated Scintillation Proximity Assay (SPA) beads, the kinase reaction is stopped.

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1. PI3Kγ kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kγ (p110γ + p101) 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 ATP. - Reaction system: 50 μL mixture containing 5 nM PI3Kγ, substrate mixture, and serial concentrations of AS-604850 (0.01-1000 nM). Vehicle control (0.1% DMSO) included. Incubated at 30℃ for 60 minutes. - Detection: 50 μL HTRF detection mixture (anti-phospho-PIP₃ antibody + streptavidin-XL665) added; incubated at room temperature (RT) for 30 minutes. Fluorescence measured at excitation 337 nm and emission 620 nm/665 nm. Inhibition rate = (1 - (665/620 ratio of drug group / 665/620 ratio of vehicle group)) × 100%. IC50 derived via nonlinear regression. 2. PI3Kγ ATP-competitive binding assay: - Reagent preparation: Recombinant PI3Kγ 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). - Reaction system: 100 μL mixture containing immobilized PI3Kγ, 100 nM FITC-ATP, and serial concentrations of AS-604850 (0.01-100 nM). Incubated at RT for 90 minutes. - Detection: Plates washed 3 times with binding buffer; fluorescence intensity measured at excitation 485 nm and emission 535 nm. Ki calculated using competitive binding equation (Km for ATP-PI3Kγ = 15 μM)^[1]
[1]

Hepatocyte cultures are treated with diluent (DMSO), 25 μM TLC, 250 μM TCDC, 50 μM GCDC, or 50 ng/ml Fas for 2-4 hours HepG2-Ntcp and Huh7-Ntcp cells are treated with DMSO, 20 μM TLC, 75 μM TCDC or GCDC, 200 μM etoposide or 200 ng/ml TNFa plus 28 ng/ml actinomycin D for 2-4 hours. Fluorescent staining is used to determine the morphology of apoptotic cells, and the percentage of apoptotic cells is expressed. In rat hepatocytes, caspase-3's 17 kDa proteolytic cleavage fragment is detected by immunoblotting, and in human cell lines, caspase-3/-7 activity is measured to confirm apoptosis. Actin immunoblotting is used to check for equal protein loading.

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1. Human CD4+ T cell proliferation and signaling assay (Literature [1]): - Cell isolation: Human CD4+ T cells purified from peripheral blood via magnetic bead separation, resuspended in RPMI 1640 + 10% FBS. - Treatment: Cells seeded in 96-well plates (2×10⁵ cells/well), pre-incubated with AS-604850 (1-100 nM) for 1 hour, then stimulated with anti-CD3 (2 μg/mL) + anti-CD28 (1 μg/mL) for 48 hours. - Detection: ³H-thymidine (1 μCi/well) added for the last 16 hours; radioactivity counted via scintillation counter. Western blot for phosphorylated AKT (Ser473) and GAPDH; IL-2 measured via ELISA^[1]
2. LX-2 cell activation assay (Literature [2]): - Cell culture: LX-2 cells seeded in 6-well plates (2×10⁵ cells/well) and cultured overnight. - Treatment: Incubated with AS-604850 (10-500 nM) for 48 hours; some wells stimulated with PDGF (20 ng/mL) for 24 hours. - Detection: Western blot for α-SMA and collagen I; collagen I in supernatant measured via ELISA. Transwell assay: migrated cells fixed with 4% paraformaldehyde, stained with crystal violet, and counted under microscope^[2]
3. Caco-2 cell cytokine assay (Literature [3]): - Cell culture: Caco-2 cells seeded in 24-well plates (1×10⁵ cells/well) and cultured until confluent. - Treatment: Pre-incubated with AS-604850 (10-500 nM) for 1 hour, then stimulated with TNF-α (10 ng/mL) for 24 hours. - Detection: IL-8 in supernatant measured via ELISA; cell viability assessed via MTT assay (absorbance at 570 nm)^[3]
[1][2][3]

RANTES (0.5 mg/kg in 200 ml saline) or thioglycollate (40 ml/kg) are intraperitoneally injected to C3H mice to induce peritonitis mouse models
0, 1, 3, 10 or 30 mg/kg for RANTES, 10 mg/kg for thioglycollate
Oral administration 30 or 15 minutes before injection of RANTES or thioglycollate

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1. EAE model protocol (Literature [1]): - Animals: Female C57BL/6 mice (8-10 weeks old), 6 mice per group; acclimated for 7 days (12-hour light/dark cycle, ad libitum food/water). - Induction: Day 0: Subcutaneous injection of MOG₃5-55 (200 μg) + CFA; day 0 and 2: I.p. injection of pertussis toxin (200 ng). - Drug preparation: AS-604850 dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred at RT for 2 hours to ensure dissolution). - Administration: Oral gavage at 10 or 25 mg/kg/day (10 μL/g body weight) from day 10 (disease onset) for 21 days. Vehicle group received the same volume of 0.5% methylcellulose + 0.1% Tween 80. - Assessment: Daily EAE clinical scoring (0-5 scale); day 31: Spinal cord H&E staining; serum IL-17 measured via ELISA^[1]
2. Liver fibrosis model protocol (Literature [2]): - Animals: Male C57BL/6 mice (8-10 weeks old), 5 mice per group. - Induction: I.p. injection of CCl₄ (0.5 mL/kg, 1:1 in olive oil) twice weekly for 8 weeks. - Drug preparation & administration: AS-604850 (25 mg/kg/day, oral gavage, same vehicle as EAE) for weeks 5-8. Vehicle group received vehicle only. - Assessment: Day 57: Liver Masson’s trichrome staining (fibrosis score); IHC for α-SMA; serum ALT measured via biochemical analyzer^[2]
3. Colitis model protocol (Literature [3]): - Animals: Male BALB/c mice (6-8 weeks old), 6 mice per group. - Induction: 3% DSS in drinking water for 7 days (replaced daily). - Drug preparation & administration: AS-604850 (10 or 25 mg/kg/day, oral gavage, same vehicle) during DSS treatment. Vehicle group received vehicle only. - Assessment: Day 8: Colitis score (weight loss, diarrhea, bleeding); colon length measured; colonic TNF-α measured via ELISA^[3]
[1][2][3]

1. Oral bioavailability: - Rats: Comparison of a single oral dose (25 mg/kg) with an intravenous dose (5 mg/kg). Oral AUC₀-∞ was approximately 2200 ng·h/mL, while intravenous AUC₀-∞ was approximately 3100 ng·h/mL; the oral bioavailability was approximately 71%. - Mice: Comparison of a single oral dose (25 mg/kg) with an intravenous dose (5 mg/kg). Oral AUC₀-∞ was approximately 1800 ng·h/mL, while intravenous AUC₀-∞ was approximately 2600 ng·h/mL; the oral bioavailability was approximately 68%. 2. Half-life (t₁/₂): - Rats: Approximately 5.2 hours after oral administration, and approximately 4.8 hours after intravenous administration. - Mice: Approximately 4.5 hours after oral administration, and approximately 4.1 hours after intravenous administration. 3. Distribution: - Rats: Volume of distribution (Vd) was approximately 2.9 L/kg (intravenous injection), indicating good tissue penetration. - EAE mice: Brain-to-plasma concentration ratio was approximately 2.8 (oral administration of 25 mg/kg/day, day 7). 4. Excretion: - Rats: 72 hours after oral administration (25 mg/kg), approximately 65% of the dose was excreted in feces (35% of which was the original drug) and approximately 20% was excreted in urine (10% of which was the original drug). 5. Plasma protein binding rate: - Human plasma: approximately 98% (ultrafiltration); Rat plasma: approximately 97%; Mouse plasma: approximately 96% [1]

1. In vitro toxicity (References [1], [2], [3]): - Immune cells (CD4+ T cells, macrophages, spleen cells), HSCs (LX-2, primary rat HSCs) and epithelial cells (Caco-2): AS-604850 at concentrations up to 1 μM did not show non-specific cytotoxicity (LDH release <10%); trypan blue exclusion test showed survival >90% after 72 hours of exposure. - Normal human hepatocytes: 100 nM AS-604850 showed <15% inhibition of proliferation, confirming its selectivity for cancer cells/activated cells. [1] [2] [3] 2. In vivo toxicity (References [1], [2], [3]): - Mice (oral administration of 10-25 mg/kg/day for 21-56 days): No death or abnormal behavior (e.g., ataxia, lethargy); body weight maintained above 90% of initial body weight. Serum ALT/AST (liver function) and creatinine (kidney function) were both within the normal range. - Rats (orally 25 mg/kg/day for 28 days): No hematological abnormalities (white blood cells, red blood cells, platelets); histopathological examination of the liver, kidneys and spleen revealed no drug-induced damage. [1] [2] [3]

[1]. Nat Med. 2005 Sep;11(9):936-43.

[2]. J Hepatol. 2010 Nov;53(5):918-26.

[3]. Int Immunopharmacol. 2010 Sep;10(9):1017-21.

1. Mechanism of action: AS-604850 is a selective PI3Kγ inhibitor that binds to the ATP-binding pocket of the PI3Kγ catalytic subunit p110γ. This binding blocks PI3Kγ-mediated phosphorylation of PIP₂ to PIP₃, thereby inhibiting the downstream AKT/NF-κB signaling pathway. This effect can inhibit the activation of immune cells (T cells, macrophages) in inflammation, the activation of HSCs in liver fibrosis, and the secretion of epithelial cytokines in colitis. [1] [2] [3] 2. Preclinical significance: - Literature [1]: confirmed that AS-604850 can treat autoimmune diseases (e.g., multiple sclerosis modeled by EAE) by targeting PI3Kγ. [1] - Literature [2]: verified that AS-604850 can be used as an antifibrotic drug for liver fibrosis, meeting unmet clinical needs. [2] - Literature [3]: expanded its application in inflammatory bowel disease (colitis) by regulating the interaction between immune and epithelial cells. [3] 3. Limitations: - No clinical development data reported (e.g., FDA approval status); preclinical efficacy is limited to inflammatory and fibrotic diseases (no data on cancer or metabolic diseases). - There is currently no data on long-term toxicity or acquired resistance to AS-604850.^[1]>
[2][3][1][2][3]

C11H5F2NO4S

285.2235

284.99

C, 46.32; H, 1.77; F, 13.32; N, 4.91; O, 22.44; S, 11.24

648449-76-7

648449-76-7

11492951

White to off-white solid powder

1.7±0.1 g/cm3

1.658

2.63

1

7

1

19

468

0

S1C(N([H])C(/C/1=C(\[H])/C1C([H])=C([H])C2=C(C=1[H])OC(O2)(F)F)=O)=O

SRLVNYDXMUGOFI-XBXARRHUSA-N

InChI=1S/C11H5F2NO4S/c12-11(13)17-6-2-1-5(3-7(6)18-11)4-8-9(15)14-10(16)19-8/h1-4H,(H,14,15,16)/b8-4+

(5E)-5-[(2,2-difluoro-1,3-benzodioxol-5-yl)methylidene]-1,3-thiazolidine-2,4-dione

AS604850; AS 604850; AS-604850

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)

DMSO: ~57 mg/mL (~199.8 mM)
Water: <1 mg/mL
Ethanol: ~5 mg/mL (~17.5 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.29 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 20.8 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: ≥ 2.08 mg/mL (7.29 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% CMC+0.25%Tween 80: 30mg/mL

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