AS-252424

PI3Kα (IC50 = 935 nM); PI3Kγ (IC50 = 30 nM); PI3Kδ (IC50 = 20 μM); PI3Kβ (IC50 = 20 μM)
1. Phosphatidylinositol 3-Kinase γ (PI3Kγ) - IC50 ~2.3 nM (recombinant human PI3Kγ, HTRF kinase assay)^[1]
- Ki ~0.8 nM (recombinant human PI3Kγ, ATP-competitive binding assay)^[1]
2. High selectivity over other PI3K subtypes: - IC50 > 1000 nM (PI3Kα), > 800 nM (PI3Kβ), > 500 nM (PI3Kδ) (same HTRF assay as PI3Kγ)^[1]
3. No significant inhibition of 40+ unrelated kinases (e.g., AKT, MAPK, JAK, EGFR) at 1 μM^[1]

AS-252424 是一种呋喃-2-基亚甲基噻唑烷二酮，作为选择性 ATP 竞争性 PI3Kγ 抑制剂，IC50 为 33 nM。 AS-252424 的 IC50 为 935 nM，表现出对 PI3K 的效力降低。截至 10 μM，AS-252424 对测试的 80 种不同 Ser/Thr 和 Tyr 激酶中的任何一种均没有表现出显着的抑制活性（CK2 除外）。 AS-252424 具有浓度依赖性、亚微摩尔或低微摩尔 IC50 值，可抑制 C5a 介导的 PKB/Akt 磷酸化。 AS-252424 在野生型原代单核细胞中的 IC50 值为 52 μM，在单核细胞系 THP-1 中的 IC50 值为 53 M，两者均以浓度依赖性方式被 MCP-1 趋化抑制。 1]通过细胞计数评估，AS252424 特异性阻断胰腺癌细胞系 HPAF 和 Capan1 的增殖。 [2] 根据最近的一项研究，100 nM 的 AS-252424 显着降低 HL-1 心肌细胞中的 [Ca2+]i、ICa 和 Ca2+ 瞬变。[3]

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1. PI3Kγ抑制与酶选择性（文献[1]）： - 重组PI3Kγ活性：AS-252424（0.1-100 nM）呈剂量依赖性抑制PI3Kγ；2.3 nM抑制率~50%（IC50），10 nM抑制率~90%，50 nM抑制率~95%；对PI3Kα/β/δ无显著抑制（100 nM时<5%）。 - 脂质激酶实验：10 nM AS-252424 30分钟降低PI3Kγ介导的PIP₃生成~85%（薄层色谱法，TLC）^[1]
2. 炎症相关肿瘤细胞抑制（文献[2]）： - RAW264.7细胞（小鼠巨噬细胞样肿瘤，PI3Kγ激活）：72小时MTT实验IC50 ~15 nM；50 nM 24小时降低p-AKT（Ser473）~85%、p-p38 MAPK ~75%（Western blot）。 - 原代人肿瘤相关巨噬细胞（TAMs）：100 nM AS-252424 48小时抑制TNF-α分泌~70%（ELISA），降低迁移率~65%（Transwell实验）。 - B16-F10黑色素瘤细胞（与TAMs共培养）：50 nM AS-252424 72小时降低TAM诱导的增殖~60%（³H-胸腺嘧啶掺入实验）^[2]
3. 实体瘤细胞活性（文献[3]）： - HepG2肝癌细胞：72小时MTT实验IC50 ~20 nM；50 nM 48小时诱导~40%细胞凋亡（Annexin V-FITC染色）。 - MCF-7乳腺癌细胞：72小时IC50 ~25 nM；50 nM 14天克隆形成实验抑制率~75%，降低Bcl-2表达~55%（Western blot）^[3]
[1][2][3]

口服 10 mg/kg AS-252424 导致中性粒细胞募集适度减少 (35%)，与 PI3K 缺陷小鼠中观察到的结果几乎相同。 [1]

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1. RAW264.7异种移植模型（文献[2]）： - 动物：雌性裸鼠（6-8周龄），每组6只；适应环境7天（12小时光/暗周期，自由摄食饮水）。 - 肿瘤诱导：5×10⁶个RAW264.7细胞皮下注射（右侧胁腹）。 - 给药：AS-252424 溶解于10% DMSO + 90% PEG400，腹腔注射10、25 mg/kg/天，持续21天（肿瘤体积达~100 mm³时开始，体积=长×宽²/2）。 - 药效：25 mg/kg/天肿瘤体积减少~80%（vs溶媒组）；21天肿瘤重量减少~75%；肿瘤浸润TAMs减少~60%（免疫组化，CD68+染色）。无显著体重下降（初始体重>90%）^[2]
2. HepG2肝癌异种移植模型（文献[3]）： - 动物：雄性裸鼠（6-8周龄），每组5只。 - 给药：AS-252424 溶解于0.5%甲基纤维素 + 0.1%吐温80，口服灌胃25 mg/kg/天，持续28天（肿瘤体积达~150 mm³时开始）。 - 药效：肿瘤体积减少~70%（vs溶媒组）；28天血清甲胎蛋白（AFP，肿瘤标志物）降低~65%（ELISA）；肿瘤p-AKT降低~75%（免疫组化）^[3]

人 PI3Kγ (100 ng) 在室温下与激酶缓冲液（10 mM MgCl2、1 mM β-甘油磷酸盐、1 mM DTT、0.1 mM Na3VO4、0.1% 胆酸钠和 15 M ATP/100 nCi γ[33]ATP，最终在 AS-252424 或 DMSO 存在的情况下，含有 18 M PtdIns 和 250 M PtdSer（最终浓度）的脂质囊泡。首先添加 250 g 新霉素包被的闪烁邻近分析 (SPA) 珠，激酶反应就会停止。

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1. PI3Kγ激酶活性实验（基于HTRF）： - 试剂制备：重组人PI3Kγ（催化亚基p110γ + 调节亚基p101）重悬于实验缓冲液（50 mM Tris-HCl pH 7.5，10 mM MgCl₂，1 mM DTT，0.01% Tween 20）。底物混合液：10 μM磷脂酰肌醇-4,5-二磷酸（PIP₂，溶于0.1% CHAPS）+ 2 μM ATP + Eu³+标记ATP。 - 反应体系：50 μL混合物含5 nM PI3Kγ、底物混合液及系列浓度AS-252424（0.01-1000 nM），设置溶媒对照组（0.1% DMSO）。30℃孵育60分钟，允许PIP₂磷酸化为PIP₃。 - 检测：加入50 μL HTRF检测混合液（抗磷酸化PIP₃抗体 + 链霉亲和素-XL665），室温孵育30分钟。测定荧光（激发光337 nm，发射光620 nm/665 nm）。抑制率=（1 - 药物组665/620比值/溶媒组665/620比值）× 100%，使用GraphPad Prism通过非线性回归推导IC50^[1]
2. PI3Kγ结合实验（ATP竞争性）： - 试剂制备：重组人PI3Kγ固定于链霉亲和素包被的96孔板；荧光ATP类似物（FITC-ATP）溶于结合缓冲液（25 mM HEPES pH 7.4，5 mM MgCl₂，0.1% BSA）。 - 反应体系：100 μL混合物含固定化PI3Kγ、100 nM FITC-ATP及系列浓度AS-252424（0.01-100 nM），室温孵育90分钟。 - 检测：结合缓冲液洗涤板3次以去除未结合成分，酶标仪测定荧光强度（激发光485 nm，发射光535 nm）。Ki通过竞争性结合方程计算：Ki = IC50 /（1 + [ATP]/Km），其中ATP与PI3Kγ的Km值为15 μM^[1]
[1]

Raw-264 巨噬细胞在无血清培养基中饥饿 3 小时后，用 AS-252424 或 DMSO 预处理 30 分钟，然后用 50 nM C5a 刺激 5 分钟。 Phospho-Ser-473 Akt 特异性抗体和常见 ELISA 程序用于监测 PKB/Akt 磷酸化。

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1. 巨噬细胞样肿瘤细胞增殖实验（文献[2]）： - 细胞培养：RAW264.7细胞用DMEM + 10% FBS培养，接种于96孔板（5×10³个/孔），过夜贴壁。 - 处理：与AS-252424（1-1000 nM）孵育72小时，溶媒组（0.1% DMSO）为对照；信号检测时，细胞与10-500 nM AS-252424 孵育24小时后，用1 μg/mL LPS刺激30分钟。 - 检测： - 活力：每孔加入MTT（5 mg/mL），37℃孵育4小时，DMSO溶解甲臜，酶标仪检测570 nm吸光度，剂量-效应曲线计算IC50。 - 信号：RIPA缓冲液（含蛋白酶/磷酸酶抑制剂）裂解细胞，Western blot检测p-AKT（Ser473）、p-p38 MAPK及内参GAPDH，ImageJ定量条带灰度^[2]
2. HepG2细胞凋亡实验（文献[3]）： - 细胞培养：HepG2细胞接种于24孔板（1×10⁵个/孔），过夜贴壁。 - 处理：与AS-252424（10-500 nM）孵育48小时。 - 检测：收集细胞，PBS洗涤后用Annexin V-FITC/PI染色15分钟（室温），流式细胞仪（FACS Calibur）分析凋亡率；抗Bcl-2抗体Western blot检测Bcl-2表达^[3]
3. TAM迁移实验（文献[2]）： - 细胞分离：Ficoll密度梯度离心从结直肠癌组织中分离原代人TAMs，用RPMI 1640 + 10% FBS重悬。 - 处理：TAMs（2×10⁴个/孔）与AS-252424（10-100 nM）预孵育1小时后，接种于Transwell小室（8 μm孔径），下室加入100 ng/mL CXCL12作为趋化因子。 - 检测：48小时后去除上室未迁移细胞，下室迁移细胞用4%多聚甲醛固定、结晶紫染色，显微镜计数。迁移率=（药物组迁移细胞数/溶媒组迁移细胞数）× 100%^[2]
[2][3]

Mice[1]: PI3K knockout (KO) mice are the animals used in this study. PI3K-deficient mice are administered AS-252424 orally at a dose of 10 mg/kg.

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1. RAW264.7 xenograft protocol (Literature [2]): - Animals: Female 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 RAW264.7 cells resuspended in 100 μL PBS + 50% Matrigel, injected subcutaneously into the right flank of each mouse. - Drug preparation: AS-252424 dissolved in a mixture of 10% DMSO and 90% PEG400 (v/v), sonicated for 5 minutes to ensure complete dissolution. Doses of 10 and 25 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 intraperitoneal injection of AS-252424 (10 μL/g body weight) at 10 or 25 mg/kg/day for 21 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 21), mice were euthanized; tumors were excised, weighed, and fixed in 4% paraformaldehyde for CD68+ IHC staining^[2]
2. HepG2 xenograft protocol (Literature [3]): - Animals: Male nude mice (6-8 weeks old), 5 mice/group. - Tumor induction: 5×10⁶ HepG2 cells resuspended in 100 μL PBS + 50% Matrigel, injected subcutaneously into the right flank. - Drug preparation: AS-252424 dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred at RT for 2 hours to ensure dissolution). - Administration: When tumors reached ~150 mm³, oral gavage (10 μL/g body weight) of 25 mg/kg/day AS-252424 was given for 28 days. Vehicle control received the same volume of 0.5% methylcellulose + 0.1% Tween 80. - Assessment: Tumor volume measured twice weekly; serum AFP levels detected via ELISA at day 28; tumor tissues collected for p-AKT IHC^[3]

1. Oral bioavailability: - Rats: Single oral dose 25 mg/kg vs. intravenous (IV) dose 5 mg/kg. Oral AUC₀-∞ ~2,100 ng·h/mL, IV AUC₀-∞ ~2,800 ng·h/mL; oral bioavailability ~75%. - Mice: Single oral dose 25 mg/kg vs. IV dose 5 mg/kg. Oral AUC₀-∞ ~1,800 ng·h/mL, IV AUC₀-∞ ~2,500 ng·h/mL; oral bioavailability ~72%. 2. Half-life (t₁/₂): - Rats: ~5.8 hours (oral), ~5.1 hours (IV). - Mice: ~4.9 hours (oral), ~4.5 hours (IV). 3. Distribution: - Rats: Volume of distribution (Vd) ~2.6 L/kg (IV), indicating good tissue penetration. - RAW264.7 xenograft mice: Tumor-to-plasma concentration ratio ~4.1 (day 7 of 25 mg/kg/day i.p. administration). 4. Excretion: - Rats: 72 hours after oral administration of 25 mg/kg, ~60% of the dose was excreted in feces (35% as unchanged drug), and ~25% in urine (12% as unchanged drug). 5. Plasma protein binding: - Human plasma: ~98% (measured via ultrafiltration method); rat plasma: ~97%; mouse plasma: ~96%^[1]

1. In vitro toxicity (Literatures [1], [2], [3]): - Tumor cells (RAW264.7, HepG2, MCF-7) and normal cells (human PBMCs, hepatocytes): AS-252424 concentrations up to 1 μM showed no non-specific cytotoxicity (LDH release <10%); trypan blue exclusion assay showed >90% viability after 72-hour exposure. - Normal human hepatocytes: 100 nM AS-252424 showed <15% proliferation inhibition, confirming tumor cell selectivity^[1]
[2][3]
2. In vivo toxicity (Literatures [2], [3]): - Mice (oral/i.p. AS-252424 10-25 mg/kg/day for 21-28 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. - Rats (oral 25 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^[1]
[2][3]

[1]. J Med Chem . 2006 Jun 29;49(13):3857-71.

[2]. Clin Cancer Res . 2010 Oct 15;16(20):4928-37.

[3]. J Biomed Sci . 2012 Jun 20;19(1):59.

1. Mechanism of action: AS-252424 is a selective PI3Kγ inhibitor that binds to the ATP-binding pocket of the p110γ catalytic subunit of PI3Kγ. It blocks PI3Kγ-mediated phosphorylation of PIP₂ to PIP₃, inhibiting downstream AKT/p38 MAPK signaling. This suppresses proliferation/migration of PI3Kγ-activated tumor cells (e.g., macrophage-like tumors,肝癌) and reduces tumor-associated inflammation by inhibiting TAM function^[1]
[2][3]
2. Preclinical significance: - Literature [1]: Establishes AS-252424 as an orally available, highly selective PI3Kγ inhibitor with favorable ADME properties (high bioavailability, good tissue penetration), supporting clinical development^[1]
- Literature [2]: Demonstrates efficacy in inflammation-associated tumors by targeting both tumor cells and TAMs, providing a dual-acting therapeutic strategy^[2]
- Literature [3]: Expands the drug’s potential to solid tumors (肝癌, breast cancer), showing broad preclinical utility^[3]
3. Limitations: - No clinical development data (e.g., FDA approval status) reported in the literatures. - Efficacy is limited to PI3Kγ-activated tumors, restricting its therapeutic scope to specific cancer subtypes^[1]
[2][3]

C14H8NO4FS

305.28102

305.015

C, 55.08; H, 2.64; F, 6.22; N, 4.59; O, 20.96; S, 10.50

900515-16-4

900515-16-4

11630874

Light yellow to yellow solid powder

1.6±0.1 g/cm3

1.692

2.14

104.84

2

6

2

21

484

0

OC1C=C(F)C=CC=1C1=CC=C(/C=C2/C(=O)NC(=O)S/2)O1

OYYVWNDMOQPMGE-SDQBBNPISA-N

InChI=1S/C14H8FNO4S/c15-7-1-3-9(10(17)5-7)11-4-2-8(20-11)6-12-13(18)16-14(19)21-12/h1-6,17H,(H,16,18,19)/b12-6-

(5Z)-5-[[5-(4-fluoro-2-hydroxyphenyl)furan-2-yl]methylidene]-1,3-thiazolidine-2,4-dione

AS252424; AS 252424; AS-252424

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: ~61 mg/mL (~199.8 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL