R406 free base 是一种具有口服活性的，ATP竞争性的Syk/FLT3抑制剂，Ki为 30 nM。它有效抑制 Syk 激酶活性，IC50为 41 nM，可减轻免疫复合物介导的炎症。它还抑制 Lyn 和 Lck，IC50分别为 63 nM和 37 nM。

R406 (free base) is a potent Syk inhibitor.

R406口服给药患免疫复合物导致炎症反应的小鼠,明显抑制皮肤反向被动Arthus反应,1 mg/kg和5 mg/kg R406处理,与对照组相比则抑制分别为72%和86%.用10 mg/kg R406处理用胶原抗体处理的小鼠,明显减少炎症和肿胀,将进行性关节炎降低至较低水平,且延迟K/BxN血清转移小鼠模型发病,降低临床关节炎达50%.

1 μM或4 μM R406处理DLBCL细胞系,诱导caspases 9和3激活,而不激活caspase 8,导致大部分细胞凋亡。R406在不同细胞中,选择性抑制Syk依赖的信号通路,EC50为33 nM到171 nM,比作用于Syk非依赖性通路效果好很多。R406抑制大型弥漫大B细胞淋巴瘤（DLBCL）细胞系的细胞增殖,EC50值范围为0.8 μM至8.1 μM。R406的预处理在B细胞受体（BCR）交联后完全阻断SYK525/526的磷酸化和R406敏感的DLBCL中SYK依赖性磷酸化BLNK。R406处理24和48小时后,有效降低MMP-9 mRNA水平,比对照组分别降低2.8和4.3倍,并降低RL细胞侵袭能力。

In-vitro Fluorescence Polarization Kinase Assays: R406 is serially diluted in DMSO and then diluted to 1% DMSO in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated BGG). ATP and substrate in kinase buffer are added at room temperature, resulting in a final DMSO concentration on 0.2%. The kinase reactions are performed in a final volume of 20 μL containing 5 μM HS1 peptide substrate and 4 μM ATP and started by addition of 0.125 ng of Syk in kinase buffer. The reaction is allowed to proceed for 40 minutes at room temperature. The reaction is stopped by the addition of 20 μL of PTK quench mix containing EDTA/anti-phosphotyrosine antibody (1X final)/fluorescent phosphopeptide tracer (0.5X final) diluted in FP Dilution Buffer. The plate is incubated for 30 minutes in the dark at room temperature and then read on a Polarion fluorescence polarization plate reader. Data are converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit. For IC50 determination, R406 is tested at eleven concentrations in duplicate and curve-fitting is performed by non-linear regression analysis using Prism GraphPad Software.

DLBCL cell lines are treated with serial dilutions of R406 (0.3, 0.6, 1.25, 2.5, or 5 μM) for 72 or 96 hours. Thereafter, cellular proliferation is determined by MTT assay, and cell apoptosis is assessed by using annexin V–FITC/propidium iodide (PI) staining. For the determination of caspase 9, 8, and 3, cells are lysed, size-fractionated by polyacrylamide gel electrophoresis (PAGE), and immunoblotted. (Only for Reference)

841290-80-0

C22H23FN6O5

470.461

R406 (free base)

H2O:<1 mgml
DMSO:20 mg/mL (42.5 mM)
Ethanol:<1 mgml
Powder: -20°C for 3 years
In solvent: -80°C for 2 years