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

The efficacy of Bay 61–3606 in combination with TRAIL was significantly (P <0.001) reduced in the volume of the xenografted tumor after 20 days of drug administration[2].
At 3 mg/kg, oral BAY 61-3606 administration to rats significantly reduces bronchoconstriction, bronchial edema, and antigen-induced passive cutaneous anaphylactic reaction. Moreover, rats' airway inflammation brought on by antigens is reduced by BAY 61-3606[1].

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Spleen tyrosine kinase (Syk) tyrosine kinase plays essential roles in receptors for Fc portion of immunoglobulins and B cell receptor complex signaling in various inflammatory cells; therefore, inhibitors of Syk kinase may show potential as antiasthmatic/allergic therapeutics. We identified 2-[7-(3,4-dimethoxyphenyl)-imidazo[1,2-c]pyrimidin-5-ylamino]-nicotinamide dihydrochloride (BAY 61-3606), a potent (Ki = 7.5 nM) and selective inhibitor of Syk kinase. BAY 61-3606 inhibited not only degranulation (IC50 values between 5 and 46 nM) but also lipid mediator and cytokine synthesis in mast cells. BAY 61-3606 was highly efficacious in basophils obtained from healthy human subjects (IC50 = 10 nM) and seems to be at least as potent in basophils obtained from atopic (high serum IgE) subjects (IC50 = 8.1 nM). B cell receptor activation and receptors for Fc portion of IgG signaling in eosinophils and monocytes were also potently suppressed by BAY 61-3606. Oral administration of BAY 61-3606 to rats significantly suppressed antigen-induced passive cutaneous anaphylactic reaction, bronchoconstriction, and bronchial edema at 3 mg/kg. Furthermore, BAY 61-3606 attenuated antigen-induced airway inflammation in rats. Based on these anti-inflammatory effects of BAY 61-3606 both in vitro and in vivo, it was demonstrated that Syk may play a very critical role in the pathogenesis of allergic reactions. [1]

In mast cells, BAY 61-3606 inhibited lipid mediator and cytokine synthesis in addition to degranulation (IC50 values ranging from 5 to 46 nM). In basophils taken from healthy human subjects, BAY 61-3606 was very effective (IC50 = 10 nM), and it appears to be at least as potent (IC50 = 8.1 nM) in basophils taken from atopic (high serum IgE) subjects. BAY 61-3606 was also found to potently suppress B cell receptor activation as well as receptors for the Fc portion of IgG signaling in eosinophils and monocytes. In colorectal cancer cells expressing mutant forms of K-RAS, but not in isogenic cells expressing wild-type K-RAS, we found that BAY61-3606 inhibits the proliferation of these cells. Beyond its ability to inhibit cell division in mutant models, BAY61-3606 demonstrated a unique biological characteristic in wild-type cells: it bestowed susceptibility to RAF inhibition. In this instance, BAY61-3606 worked by blocking MAP4K2 (GCK), which in wild-type cells typically triggers NFκβ signaling in reaction to RAF inhibition.

After a 24-hour period, MCF-7 cells are exposed to TRAIL (specified concentrations: 0, 12.5, 25, and 37.5 ng/ml) with or without Bay 61-3606 (2.5 μM). Following this exposure, the cells undergo immunocytochemistry using an active Bak antibody. MCF-7 cells exposed to Bay 61-3606 (5 μM) with or without TRAIL (50 ng/ml) for a 24-hour period are tested for caspase activity.

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Cell Viability Assay [1]
To assess the effect of the commercially available SYK inhibitors BAY61-3606, R406, GS-9973 (Entospletinib) and PRT062607 alone as well as BAY61-3606 in combination with paclitaxel, cisplatin, doxorubicin and temozolomide on the cell viability of SH-SY5Y and SK-N-BE(2) neuroblastoma cells the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazodium bromide)-assay was used. The cells were seeded in 96-well plates in full growth media. As an exception, cells treated with GS-9973 and PRT062607 were seeded in Opti-MEM to reduce cell viability variations attributed to residual serum. After 24 h, the cells were washed once with Opti-MEM before incubation with SYK inhibitors alone for 24 and 48 h or a combination of chemotherapeutic drugs with BAY61-3606 for 48 and 72 h. Control cells received the corresponding drug vehicle at the highest concentration present in the drug-treated cells. BAY 61-3606 and doxorubicin were dissolved in water, R406, GS-9973, PRT062607, and temozolomide in DMSO, paclitaxel in ethanol, and cisplatin in 0.9% saline. After 24, 48 or 72 h the MTT solution (10 μL of 5 mg MTT, per ml phosphate buffered saline) was added to each well and incubated for additional 3 h. To facilitate formazan crystal solubilizing, 70 μL of the solution were carefully removed from each well, 100 μL isopropanol containing 0.04 M HCl were added and mixed thoroughly. In addition, the plates were placed on an orbital shaker for 1 h at room temperature. The absorbance was measured with a CLARIOstar plate reader at 590 nm. The experiment was performed at least three times with at least three parallels per treatment. The cell viability was calculated as the ratio of the mean OD of treated cells over vehicle treated control cells (100% living cells). The cell viability assay for the siRNA and SYK plasmid studies were performed in 24-well plates. The amounts of MTT solution and acidic isopropanol were adjusted correspondingly.

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Cell Signaling Study [1]
To investigate the effect of commercially available SYK inhibitors on MAPK- and Akt- mediated signaling SH-SY5Y cells were seeded in 6-well plates in full growth medium. The next day, the cells were washed in Opti-MEM and treated with BAY 61-3606, R406, GS-9973 (entospletinib), PRT062607 or corresponding vehicle controls (water for BAY61-3606 and DMSO for R406, GS-9973, and PRT062607) for 4 or 24 h. Following incubation, the cells were washed with PBS and harvested in RIPA Lysis and Extraction Buffer containing Halt™ Protease and Phosphatase Inhibitor Cocktail and analyzed by western blot. Densitometry was performed using Fiji software. Phosphorylated and total protein were normalized to their respective GAPDH loading control (pERK/GAPDH, ERK/GAPDH, pAkt/GAPDH, Akt/GAPDH). Ratios of pERK/ERK and pAkt/Akt were calculated using the normalized values. The respective vehicle control was set as 1 and the ratios were calculated.

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

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)