Syk (Ki = 7.5 nM); Syk (IC50 = 10 nM)

体外活性：BAY 61-3606 是一种高度选择性的 Syk 激酶抑制剂。其他选定的酪氨酸激酶，Lyn、Fyn、Src、Itk 和 Btk，浓度高达 4.7 μM 时不会被 BAY 61-3606 抑制。 BAY 61-3606 还被发现可以抑制 B 细胞受体 (BCR) 介导的信号传导。 Bay 61-3606 是 TRAIL 诱导的细胞凋亡的敏化剂。在 MCF-7 细胞中，Bay 61-3606 下调 Mcl-1 具有浓度和时间依赖性。在 MCF-7 和 T47D 细胞中，Bay 61-3606 会降低 Syk 的磷酸化。 Bay 61-3606 对 Mcl-1 的下调与乳腺癌细胞中的 Syk 无关。 Bay 61-3606 促进 MCF-7 细胞中 Mcl-1 蛋白的泛素/蛋白酶体依赖性降解。 Bay 61-3606 抑制 MCF-7 细胞中 CDK9、RNA 聚合酶 II 和 Mcl-1 表达的磷酸化。 Bay 61-3606 抑制 CDK9 激酶活性，体外 IC50 为 37 nM 激酶测定：BAY 61-3606 不仅抑制脱颗粒（IC50 值在 5 到 46 nM 之间），而且还抑制肥大细胞中的脂质介质和细胞因子合成。 BAY 61-3606 对从健康人类受试者获得的嗜碱性粒细胞非常有效 (IC50 = 10 nM)，并且对于从特应性（高血清 IgE）受试者获得的嗜碱性粒细胞似乎至少同样有效 (IC50 = 8.1 nM)。 BAY 61-3606 还可有效抑制嗜酸性粒细胞和单核细胞中 B 细胞受体的激活以及 IgG 信号传导 Fc 部分的受体。我们确定 BAY61-3606 是表达 K-RAS 突变形式的结直肠癌细胞的增殖抑制剂，但在表达野生型 K-RAS 的同基因细胞中则不然。除了在突变细胞中的抗增殖作用外，BAY61-3606 在野生型细胞中还表现出独特的生物学特性，因为它赋予对 RAF 抑制的敏感性。在这种情况下，BAY61-3606 通过抑制 MAP4K2 (GCK) 发挥作用，MAP4K2 (GCK) 通常会响应 RAF 的抑制而激活野生型细胞中的 NFκβ 信号传导。细胞测定：MCF-7细胞暴露于含有或不含Bay 61-3606 (2.5 μM)的TRAIL（指定浓度：0、12.5、25、37.5 ng/ml）中24小时；暴露于试剂 12 小时后，使用活性 Bak 抗体对 MCF-7 细胞进行免疫细胞化学分析；在暴露于 Bay 61-3606 (5 μM) 且有或没有 TRAIL (50 ng/ml) 的 MCF-7 细胞中测量 Caspase 活性 24 小时。

体内：大鼠口服 3 mg/kg BAY 61-3606 可显着抑制抗原诱导的被动皮肤过敏反应、支气管收缩和支气管水肿。此外，BAY 61-3606 减轻了大鼠中抗原诱导的气道炎症。

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脾酪氨酸激酶（Syk）酪氨酸激酶在多种炎症细胞中免疫球蛋白Fc部分受体和B细胞受体复合物信号传导中起重要作用；因此，Syk激酶抑制剂可能显示出作为平喘/过敏治疗药物的潜力。我们鉴定了2-[7-（3,4-二甲氧基苯基）-咪唑[1,2-c]嘧啶-5-氨基]-烟酰胺二盐酸（BAY 61-3606），一种有效的（Ki = 7.5 nM）选择性Syk激酶抑制剂。BAY 61-3606不仅能抑制肥大细胞的脱颗粒（IC50值在5 ~ 46 nM之间），还能抑制脂质介质和细胞因子的合成。BAY 61-3606对健康人的嗜碱性细胞非常有效（IC50 = 10 nM），对特应性（高血清IgE）患者的嗜碱性细胞也同样有效（IC50 = 8.1 nM）。在嗜酸性粒细胞和单核细胞中，B细胞受体的激活和IgG信号Fc部分的受体也被BAY 61-3606有效地抑制。大鼠口服BAY 61-3606 （3 mg/kg）可显著抑制抗原诱导的被动皮肤过敏反应、支气管收缩和支气管水肿。此外，BAY 61-3606还能减轻抗原诱导的大鼠气道炎症。基于BAY 61-3606的体内外抗炎作用，表明Syk可能在过敏反应的发病机制中起着非常关键的作用。[1]

在肥大细胞中，除了脱颗粒之外，BAY 61-3606 还能抑制脂质介质和细胞因子的合成（IC50 值范围为 5 至 46 nM）。在取自健康人类受试者的嗜碱性粒细胞中，BAY 61-3606 非常有效（IC50 = 10 nM），并且它似乎对取自特应性（高血清 IgE）受试者的嗜碱性粒细胞至少同样有效（IC50 = 8.1 nM）。 BAY 61-3606 还被发现能有效抑制 B 细胞受体激活以及嗜酸性粒细胞和单核细胞中 IgG 信号传导 Fc 部分的受体。在表达突变型 K-RAS 的结直肠癌细胞中，但在表达野生型 K-RAS 的同基因细胞中则不然，我们发现 BAY61-3606 抑制这些细胞的增殖。除了在突变模型中抑制细胞分裂的能力外，BAY61-3606 在野生型细胞中还表现出独特的生物学特性：它赋予 RAF 抑制的敏感性。在这种情况下，BAY61-3606 通过阻断 MAP4K2 (GCK) 发挥作用，在野生型细胞中，MAP4K2 通常会触发 NFκβ 信号传导以响应 RAF 抑制。

24 小时后，将 MCF-7 细胞暴露于含有或不含 Bay 61-3606 (2.5 μM) 的 TRAIL（指定浓度：0、12.5、25 和 37.5 ng/ml）。暴露后，细胞使用活性 Bak 抗体进行免疫细胞化学处理。将 MCF-7 细胞暴露于含有或不含 TRAIL (50 ng/ml) 的 Bay 61-3606 (5 μM) 24 小时后，测试 caspase 活性。

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细胞活力测定[1]
为了评估市售SYK抑制剂BAY61-3606、R406、GS-9973（托普替尼）和PRT062607单独使用以及BAY61-3606与紫杉醇、顺铂、阿霉素和替莫唑胺联合使用对SH-SY5Y和SK-N-BE(2)神经母细胞瘤细胞活力的影响，采用比色MTT（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四钠）测定法。细胞在全生长培养基中接种于96孔板。作为例外，用GS-9973和PRT062607处理的细胞在Opti-MEM中接种，以减少残留血清引起的细胞活力变化。24 h后，将细胞用Opti-MEM洗涤一次，然后单独与SYK抑制剂孵育24和48 h，或与BAY61-3606联合化疗药物孵育48和72 h。对照细胞以药物处理细胞中最高的浓度接受相应的药物载体。将BAY 61-3606和阿霉素溶于水，R406、GS-9973、PRT062607溶于DMSO，替莫唑胺溶于乙醇，顺铂溶于0.9%生理盐水。24、48或72 h后，每孔加入MTT溶液（5 mg MTT，每ml磷酸盐缓冲盐水10 μL），再孵育3 h。为促进甲酰胺晶体的溶解，每孔小心地取出70 μL溶液，加入100 μL含0.04 M HCl的异丙醇，充分混合。此外，在室温下将板置于轨道激振器上1小时。用CLARIOstar平板阅读器在590nm处测量吸光度。实验至少进行了三次，每次治疗至少有三次平行。细胞活力计算为处理细胞的平均OD值与对照细胞（100%活细胞）的比值。siRNA和SYK质粒的细胞活力测定在24孔板上进行。相应调整MTT溶液和酸性异丙醇的用量。

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细胞信号传导研究[1]
为了研究市售SYK抑制剂对MAPK和Akt介导的信号通路的影响，将SH-SY5Y细胞接种于全生长培养基的6孔板中。第二天，在Opti-MEM中洗涤细胞，并用BAY61-3606、R406、GS-9973（托替尼）、PRT062607或相应的对照（BAY 61-3606为水，R406、GS-9973和PRT062607为DMSO）处理4或24小时。孵育后，用PBS洗涤细胞，在含有Halt™蛋白酶和磷酸酶抑制剂鸡尾酒的RIPA裂解和提取缓冲液中收获细胞，并进行western blot分析。采用Fiji软件进行密度测定。磷酸化蛋白和总蛋白归一化到各自的GAPDH负载对照（pERK/GAPDH、ERK/GAPDH、pAkt/GAPDH、Akt/GAPDH）。使用归一化值计算pERK/ERK和pAkt/Akt的比值。将各车辆控制设为1，并计算比值。

Female BALB/c nude mice (5 weeks old) bearing MCF-7 tumor xenograft
50 mg/kg
TRAIL (10 mg/kg), Bay 61-3606 (50 mg/kg), or a combination of both (50 mg/kg) was intraperitoneally injected twice a week; TRAIL was administered two hours following the injection of Bay 61-3606 for a duration of two weeks.

[1]. The orally available spleen tyrosine kinase inhibitor 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]nicotinamide dihydrochloride (BAY 61-3606) blocks antigen-induced airway inflammation in rodents. J Pharmacol Exp Ther. 2003 Sep;306(3):1174-81.

[2]. SYK Inhibition Potentiates the Effect of Chemotherapeutic Drugs on Neuroblastoma Cells in Vitro. Cancers (Basel). 2019 Feb 10;11(2). pii: E202.

[3]. Quantitative phosphoproteomics revealed interplay between Syk and Lyn in the resistance to AMN107 in chronic myeloid leukemia cells. Blood. 2011 Aug 25;118(8):2211-21.

[4]. Bay 61-3606 Sensitizes TRAIL-Induced Apoptosis by Downregulating Mcl-1 in Breast Cancer Cells. PLoS One. 2015 Dec 31;10(12):e0146073.

2-[[7-(3,4-dimethoxyphenyl)-5-imidazo[1,2-c]pyrimidinyl]amino]-3-pyridinecarboxamide is a member of pyrimidines.
Neuroblastoma is a malignancy arising from the developing sympathetic nervous system and the most common and deadly cancer of infancy. New therapies are needed to improve the prognosis for high-risk patients and to reduce toxicity and late effects. Spleen tyrosine kinase (SYK) has previously been identified as a promising drug target in various inflammatory diseases and cancers but has so far not been extensively studied as a potential therapeutic target in neuroblastoma. In this study, we observed elevated SYK gene expression in neuroblastoma compared to neural crest and benign neurofibroma. While SYK protein was detected in the majority of examined neuroblastoma tissues it was less frequently observed in neuroblastoma cell lines. Depletion of SYK by siRNA and the use of small molecule SYK inhibitors significantly reduced the cell viability of neuroblastoma cell lines expressing SYK protein. Moreover, SYK inhibition decreased ERK1/2 and Akt phosphorylation. The SYK inhibitor BAY 613606 enhanced the effect of different chemotherapeutic drugs. Transient expression of a constitutive active SYK variant increased the viability of neuroblastoma cells independent of endogenous SYK levels. Collectively, our findings suggest that targeting SYK in combination with conventional chemotherapy should be further evaluated as a treatment option in neuroblastoma. [2]

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In this study, we have addressed how Lyn kinase signaling mediates nilotinib-resistance by quantitative phospho-proteomics using Stable Isotope Labeling with Amino acid in Cell culture. We have found an increased tyrosine phosphorylation of 2 additional tyrosine kinases in nilotinib-resistant cells: the spleen tyrosine kinase Syk and the UFO family receptor tyrosine kinase Axl. This increased tyrosine phosphorylation involved an interaction of these tyrosine kinases with Lyn. Inhibition of Syk by the inhibitors R406 or BAY 61-3606 or by RNA interference restored the capacity of nilotinib to inhibit cell proliferation. Conversely, coexpression of Lyn and Syk were required to fully induce resistance to nilotinib in drug-sensitive cells. Surprisingly, the knockdown of Syk also strongly decreased tyrosine phosphorylation of Lyn and Axl, thus uncovering interplay between Syk and Lyn. We have shown the involvement of the adaptor protein CDCP-1 in resistance to nilotinib. Interestingly, the expression of Axl and CDCP1 were found increased both in a nilotinib-resistant cell line and in nilotinib-resistant CML patients. We conclude that an oncogenic signaling mediated by Lyn and Syk can bypass the need of Bcr-Abl in CML cells. Thus, targeting these kinases may be of therapeutic value to override imatinib or nilotinib resistance in CML. [3]

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Breast cancer cells generally develop resistance to TNF-Related Apoptosis-Inducing Ligand (TRAIL) and, therefore, assistance from sensitizers is required. In our study, we have demonstrated that Spleen tyrosine kinase (Syk) inhibitor Bay 61-3606 was identified as a TRAIL sensitizer. Amplification of TRAIL-induced apoptosis by Bay 61-3606 was accompanied by the strong activation of Bak, caspases, and DNA fragmentation. In mechanism of action, Bay 61-3606 sensitized cells to TRAIL via two mechanisms regulating myeloid cell leukemia sequence-1 (Mcl-1). First, Bay 61-3606 triggered ubiquitin-dependent degradation of Mcl-1 by regulating Mcl-1 phosphorylation. Second, Bay 61-3606 downregulates Mcl-1 expression at the transcription level. In this context, Bay 61-3606 acted as an inhibitor of Cyclin-Dependent Kinase (CDK) 9 rather than Syk. In summary, Bay 61-3606 downregulates Mcl-1 expression in breast cancer cells and sensitizes cancer cells to TRAIL-mediated apoptosis. [4]

C20H18N6O3

390.4

390.144

C, 61.53; H, 4.65; N, 21.53; O, 12.29

732983-37-8

BAY 61-3606 dihydrochloride;648903-57-5; BAY 61-3606;732983-37-8; 1615197-10-8;

10200390

white solid powder

1.4±0.1 g/cm3

1.696

1.75

116.66

2

7

6

29

566

0

O=C(C1=CC=CN=C1NC2=NC(C3=CC=C(OC)C(OC)=C3)=CC4=NC=CN24)N

JWQOJVOKBAAAAR-UHFFFAOYSA-N

InChI=1S/C20H18N6O3/c1-28-15-6-5-12(10-16(15)29-2)14-11-17-22-8-9-26(17)20(24-14)25-19-13(18(21)27)4-3-7-23-19/h3-11H,1-2H3,(H2,21,27)(H,23,24,25)

2-[[7-(3,4-dimethoxyphenyl)imidazo[1,2-c]pyrimidin-5-yl]amino]pyridine-3-carboxamide

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)