Syk (Ki = 30 nM); Syk (IC50 = 41 nM); Lyn (IC50 = 63 nM); Lck (IC50 = 37 nM); FLT3

腺苷转运蛋白 (IC50=1.84 µM)、单胺转运蛋白 (IC50=2.74 µM) 和腺苷 A3 受体 (IC50=0.081 µM) 均被 R406 抑制[1]。 Huh7 肝细胞、A549 上皮细胞和 H1299 肺癌细胞系均受到 R406 的抑制，相应的 EC50 分别为 15.1、2.9 和 6.3 µM[1]。 R406 可防止肥大细胞的 Syk 底物 LAT 磷酸化和 B 细胞的 BLNK/SLP65 磷酸化[1]。

R406（5 和 10 mg/kg）可有效降低类风湿性关节炎 (RA) 的 K/BxN 和胶原抗体诱导关节炎 (CAIA) 模型的临床症状并改善 Arthus 反应。 R406 抑制 Fc 受体信号传导，从而减少免疫复合物 (IC) 介导的炎症[1]。

R406 在 DMSO 中连续稀释，在激酶缓冲液（20 mM HEPES，pH 7.4，5 mM MgCl2，2 mM MnCl2，1 mM DTT，0.1 mg/mL 乙酰化 BGG）中稀释，最后按体积稀释至 1% DMSO。在室温下将 ATP 和底物添加到激酶缓冲液中后，最终 DMSO 浓度为 0.2%。将 0.125 ng Syk 添加到激酶缓冲液中以启动激酶反应，该反应在最终体积为 20 mL 的条件下使用 5 mM HS1 肽底物和 4 mM ATP 进行。使反应在室温下继续四十分钟。添加在 FP 稀释缓冲液中稀释的 20 mL 含有 EDTA、抗磷酸酪氨酸抗体（1X 最终）和荧光磷酸肽示踪剂（0.5X 最终）的 PTK 淬灭混合物以终止反应。在室温下黑暗中孵育 30 分钟后，使用 Polarion 荧光偏振板读数器读取板。通过与酪氨酸激酶测定试剂盒中包含的磷酸肽竞争对手竞争，创建了一条校准曲线，用于将数据转换为存在的磷酸肽的量。使用非线性回归分析来拟合曲线并测试十一种不同浓度的R406以确定IC50.

蛋白质印迹分析[1]
细胞类型：培养的人类肥大细胞 (CHMC)
测试浓度： 0.016、0.08、0.4、2 µM
孵育持续时间： 40 分钟
实验结果： 根据目标蛋白的磷酸化判断，抑制所有其他测试激酶的效力比 Syk 低 5 至 100 倍。

Animal/Disease Models: Female balb/c (Bagg ALBino) mouse (6-8 weeks) with CAIA[1]
Doses: 5 and 10 mg/kg
Route of Administration: Administered orally, bid , for 14 days, starting 4 hrs (hours) after antibody challenge on day 0.
Experimental Results: decreased inflammation and swelling, and the arthritis progressed more slowly in animals treated than in vehicle controls.

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Animal/Disease Models: Female C57BL/6 mice with arthritis[1]
Doses: 10 mg/kg
Route of Administration: Administered orally one hour before serum injection; bid; for 13 days
Experimental Results: Delayed the onset and decreased the severity of clinical arthritis. Paw thickening and clinical arthritis were decreased by approximately 50%.

[1]. R406, an orally available spleen tyrosine kinase inhibitor blocks fc receptor signaling and reduces immune complex-mediated inflammation. J Pharmacol Exp Ther. 2006 Dec;319(3):998-1008.

[2]. A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes. J Pharmacol Exp Ther. 2006 May;317(2):571-8.

6-[[5-fluoro-2-(3,4,5-trimethoxyanilino)-4-pyrimidinyl]amino]-2,2-dimethyl-4H-pyrido[3,2-b][1,4]oxazin-3-one is a member of methoxybenzenes and a substituted aniline.

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Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.[1]

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Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like synoviocytes (FLS) using the novel Syk inhibitor N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406). Using immunohistochemistry, Syk was detected in RA synovial tissue (ST), primarily in the synovial intimal lining. Western blot analysis demonstrated significantly greater amounts of phospho-Syk expression in RA ST compared with osteoarthritis ST. The kinase was expressed and functionally activated by TNFalpha in FLS and was blocked by R406. Western blot analysis demonstrated that Syk inhibition by R406 markedly suppressed TNFalpha-induced c-Jun N-terminal kinase (JNK) phosphorylation in FLS, with a modest decrease in extracellular signal-regulated kinase phosphorylation. Surprisingly, p38 activation was not affected by R406. The Syk inhibitor also decreased TNFalpha-induced mitogen-activated protein kinase kinase (MKK) 4 phosphorylation but not MKK3 and MKK6 phosphorylation, which is consistent with its selective sparing of p38. The connection between Syk and JNK was confirmed by demonstrating decreased phospho-c-Jun protein expression and complete inhibition of JNK function in R406-treated cells. R406 also suppressed downstream actions of JNK, as determined by activator protein 1 binding, as well as matrix metalloproteinase 3 gene expression. These data demonstrate that Syk activation plays an essential role in TNFalpha-induced cytokine and matrix metalloproteinase production in RA FLS, especially by suppressing activation of the JNK pathway.[2]

C22H23FN6O5

470.45

470.171

C, 56.17; H, 4.93; F, 4.04; N, 17.86; O, 17.00

841290-80-0

R406;841290-81-1

11213558

White to light yellow solid powder

1.4±0.1 g/cm3

1.618

4.32

128.75

3

11

7

34

691

0

NHHQJBCNYHBUSI-UHFFFAOYSA-N

InChI=1S/C22H23FN6O5/c1-22(2)20(30)28-19-13(34-22)6-7-16(27-19)26-18-12(23)10-24-21(29-18)25-11-8-14(31-3)17(33-5)15(9-11)32-4/h6-10H,1-5H3,(H3,24,25,26,27,28,29,30)

6-((5-fluoro-2-((3,4,5-trimethoxyphenyl)amino)pyrimidin-4-yl)amino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one

R406 besylate; R406 benzenesulfonate; 841290-80-0; tamatinib; R-406; R406 (free base); R406 free base; 6-(5-fluoro-2-(3,4,5-trimethoxyphenylamino)pyrimidin-4-ylamino)-2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one; Tamatinib free base; 6-[[5-fluoro-2-(3,4,5-trimethoxyanilino)pyrimidin-4-yl]amino]-2,2-dimethyl-4H-pyrido[3,2-b][1,4]oxazin-3-one; Tamatinib; R 406, R406, R-406;

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)

配方 1 中的溶解度: 2.5 mg/mL (5.31 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.31 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。