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

体外活性：R406 是 Syk 的 ATP 竞争性抑制剂，Ki 值为 30 nM。 R406 选择性抑制 Syk 依赖性信号传导，EC50 值范围为 33 nM 至 171 nM，比不同细胞中的 Syk 独立途径更有效。 R406 可抑制大量弥漫性大 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 处理（1 μM 或 4 μM）诱导 caspase 9 和 3 激活，但不激活 caspase 8，导致大多数 DLBCL 细胞系显着凋亡。 R406 预处理可完全阻断 B 细胞受体 (BCR) 交联后 R406 敏感 DLBCL 中 SYK525/526 的磷酸化和 SYK 依赖性 BLNK 磷酸化。治疗 24 小时和 48 小时后，R406 有效降低 MMP-9 mRNA 水平，分别比对照低 2.8 倍和 4.3 倍，并降低 RL 细胞的侵袭能力。激酶测定：R406 在 DMSO 中连续稀释，然后在激酶缓冲液（20 mM HEPES、pH 7.4、5 mM MgCl2、2 mM MnCl2、1 mM DTT、0.1 mg/mL 乙酰化 BGG）中稀释至 1% DMSO。在室温下添加激酶缓冲液中的 ATP 和底物，最终 DMSO 浓度为 0.2%。激酶反应在含有 5 μM HS1 肽底物和 4 μM ATP 的最终体积 20 μL 中进行，并通过在激酶缓冲液中添加 0.125 ng Syk 开始。使反应在室温下进行40分钟。通过添加 20 μL PTK 淬灭混合物来终止反应，该混合物含有用 FP 稀释缓冲液稀释的 EDTA/抗磷酸酪氨酸抗体（1X 最终）/荧光磷酸肽示踪剂（0.5X 最终）。将板在室温下黑暗中孵育 30 分钟，然后在 Polarion 荧光偏振板读数器上读数。使用通过与酪氨酸激酶测定试剂盒中提供的磷酸肽竞争剂竞争生成的校准曲线将数据转换为存在的磷酸肽的量。对于 IC50 测定，R406 在 11 个浓度下进行重复测试，并使用 Prism GraphPad 软件通过非线性回归分析进行曲线拟合。细胞测定：DLBCL 细胞系用 R406（0.3、0.6、1.25、2.5 或 5 μM）系列稀释液处理 72 或 96 小时。此后，通过 MTT 测定测定细胞增殖，并使用膜联蛋白 V-FITC/碘化丙啶 (PI) 染色评估细胞凋亡。为了测定 caspase 9、8 和 3，细胞被裂解，通过聚丙烯酰胺凝胶电泳 (PAGE) 进行大小分级，并进行免疫印迹。

R406 在许多免疫疾病动物模型中显示出功效。与对照组相比，患有免疫复合物介导炎症的小鼠口服 R406 1 mg/kg 和 5 mg/kg 时，可显着抑制皮肤逆转被动阿蒂斯反应，分别约 72% 和 86%。 10 mg/kg 的 R406 治疗可显着减轻炎症和肿胀，将被动抗胶原抗体攻击小鼠的进行性关节炎降低至较低水平，并延迟 K/BxN 小鼠的发病时间并将爪子增厚和临床关节炎减少约 50%血清转移小鼠模型。

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IC5

R406 以系列稀释液（0.3、0.6、1.25、2.5 或 5 μM）应用于 DLBCL 细胞系，作用时间为 72 或 96 小时。之后，用MTT法测定细胞增殖，用膜联蛋白V-FITC/碘化丙啶（PI）染色评估细胞凋亡。将细胞裂解，使用聚丙烯酰胺凝胶电泳 (PAGE) 进行大小分级，并进行免疫印迹以确定 caspase 9、8 和 3 的存在。

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研究人员研究了R406对FcγRIIA介导的血小板活化的影响，R406是Syk的一种小分子抑制剂，被开发用于治疗自身免疫性疾病、过敏性疾病和B细胞相关的血液系统恶性肿瘤。为了进一步评估Syk抑制剂在HIT治疗中的潜在活性，还评估了R406对HIT抗体诱导的TF表达和单核细胞促凝活性的影响。
结果：我们发现R406是血小板信号传导和功能的强效抑制剂，由特异性抗体或HIT患者血清引发的FcγRIIA交联引发。Syk抑制可有效阻止FcγRIIA诱导的LAT磷酸化和磷酸肌醇3-激酶、Akt、磷脂酶Cγ2和p38 MAP激酶的激活。因此，R406强烈抑制FcγRIIA诱导的血小板聚集、颗粒分泌和微粒产生。此外，Syk抑制剂有效地损害了由HIT抗体引发的TF表达和人单核细胞的促凝活性。
结论：Syk抑制剂可能通过降低HIT抗体介导的血小板活化和单核细胞促凝活性，在治疗HIT方面具有治疗意义[3]。

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%.

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Formulated in DMSO and diluted in saline containing 35% TPGS, 60% PEG 400, and 5% propylene glycol; 10 mg/kg; Oral gavage

Female C57BL/6 mice challenged intravenously with 1% ovalbumin (OVA) in saline (10 mg/kg) containing 1% Evans blue dye, female Balb/c mice with the anticollagen antibody-induced arthritis, and female C57BL/6 mice with arthritis induced by intraperitoneal

[1]. J Pharmacol Exp Ther . 2006 Dec;319(3):998-1008.

[2]. Blood . 2009 Jul 30;114(5):1029-37.

[3]. J Thromb Haemost . 2011 Oct;9(10):2067-76.

[4]. Toxicol Appl Pharmacol . 2007 Jun 15;221(3):268-77.

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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Antigenic stimulation through the B-cell antigen receptor (BCR) is considered to promote the expansion of chronic lymphocytic leukemia (CLL) B cells. The spleen tyrosine kinase (Syk), a key component of BCR signaling, can be blocked by R406, a small-molecule Syk inhibitor, that displayed activity in CLL patients in a first clinical trial. In this study, we investigated the effects of BCR stimulation and R406 on CLL cell survival and migration. The prosurvival effects promoted by anti-IgM stimulation and nurselike cells were abrogated by R406. BCR triggering up-regulated adhesion molecules, and increased CLL cell migration toward the chemokines CXCL12 and CXCL13. BCR activation also enhanced CLL cell migration beneath marrow stromal cells. These responses were blocked by R406, which furthermore abrogated BCR-dependent secretion of T-cell chemokines (CCL3 and CCL4) by CLL cells. Finally, R406 inhibited constitutive and BCR-induced activation of Syk, extracellular signal-regulated kinases, and AKT, and blocked BCR-induced calcium mobilization. These findings suggest that BCR activation favors CLL cell homing, retention, and survival in tissue microenvironments. R406 effectively blocks these BCR-dependent responses in CLL cells, providing an explanation for the activity of R406 in patients with CLL.[2]

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Spleen tyrosine kinase (Syk) is a novel pharmaceutical target for treatment of allergic, autoimmune, and neoplastic disorders. Previous studies have indicated that Syk signaling plays critical roles in regulating the lymphohematopoietic system. These observations prompted us to investigate whether inhibition of Syk would promote immunotoxicity. In a series of studies, rats were treated orally with R406, at dose levels up to and including 100 mg/kg/day (or its prodrug R788 at dose levels up to and including 100 mg/kg/day, reduced to 50 mg/kg/day for females as MTD was exceeded), a potent Syk inhibitor, twice daily for 28 days. In addition to standard toxicological assessments, immunophenotyping by flow cytometric analysis, and a study of humoral immune response measuring anti-KLH IgM and IgG levels, were undertaken. Other immunotoxicity studies included three host resistance models in female Balb/c mice to further ascertain effects of R406 on innate and acquired immunity. Following R406 treatment, expected immunomodulating effects (e.g., decreased thymic and spleen weight, hypocellularity of bone marrow, and reduced lymphocyte counts, including T and B cells) were observed in the rat studies. These changes essentially resolved during a 14-day treatment-free recovery period. A KLH challenge in rats demonstrated no adverse effects on IgG or IgM response. R788/406, administered orally at dose levels up to and including 80 mg/kg/day for 28 days, did not affect bacterial or viral clearance in the Listeria, Streptococcal, or Influenza host resistance mouse models, respectively. This correlated with previous in vitro macrophage and neutrophil function assays (assessing migration, phagocytosis, oxidative burst and microbicidal activity), which revealed that R406 did not adversely affect macrophage or neutrophil function in innate immune responses. Collectively, these results demonstrate that R406 has minimal functional immunotoxicity notwithstanding its lymphocytopenic effect, suggesting that inhibition of Syk might not lead to unacceptable mechanism-based adverse effects. [3]

C28H29FN6O8S

628.63

628.175

C, 53.50; H, 4.65; F, 3.02; N, 13.37; O, 20.36; S, 5.10

841290-81-1

R406 free base;841290-80-0

11984591

Light yellow to khaki solid powder

5.227

198.22

4

14

8

44

874

0

O=C1C(C)(C)OC2C(=NC(NC3C(F)=CN=C(NC4C=C(OC)C(OC)=C(OC)C=4)N=3)=CC=2)N1.O=S(C1C=CC=CC=1)(O)=O

UXDRJPYSTZHIOE-UHFFFAOYSA-N

InChI=1S/C22H23FN6O5.C6H6O3S/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;7-10(8,9)6-4-2-1-3-5-6/h6-10H,1-5H3,(H3,24,25,26,27,28,29,30);1-5H,(H,7,8,9)

benzenesulfonic acid;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

R406 benzenesulfonate; R-406 benzenesulfonate; R-406 besylate; R406; R-406; R 406; R 406 besylate; R406 Benzenesulfonate; Tamatinib besylate; R406 besylate; 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 benzenesulfonate; 6-[[5-fluoro-2-[(3,4,5-trimethoxyphenyl)amino]-4-pyrimidinyl]amino]-2,2-dimethyl-2H-pyrido[3,2-b]-1,4-oxazin-3(4H)-one benzenesulfonate; 841290-81-1 (besylate); R406 besylate; R406 benzenesulfonate; Tamatinib

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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 (3.98 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), 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 (3.98 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% Propylene glycol: 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。