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

Bay 61-3606 (50 mg/kg; administered twice a week for two weeks by intraperitoneal injection) alone produces more effective reductions than TNF-related apoptosis-inducing ligand (TRAIL; 10 mg/kg) alone in MCF-7 tumor xenograft-bearing BALB/c nude mice. The volume of the xenografted tumor is greatly reduced when Bay 61-3606 is used in conjunction with TRAIL[4].

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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.

Neuroblastoma is a malignant tumor originating from the developing sympathetic nervous system and is the most common and deadliest cancer in infants and young children. New treatments are urgently needed to improve the prognosis of high-risk patients and reduce toxicity and long-term effects. Spleen tyrosine kinase (SYK) has previously been identified as a potential drug target for various inflammatory diseases and cancers, but its application as a potential therapeutic target for neuroblastoma is currently insufficient. This study observed increased SYK gene expression in neuroblastoma compared to neural crest and benign neurofibromas. While SYK protein was detected in most neuroblastoma tissues examined, it was less detected in neuroblastoma cell lines. Knockdown of SYK using siRNA and the use of small-molecule SYK inhibitors significantly reduced cell viability in neuroblastoma cell lines expressing SYK protein. Furthermore, SYK inhibition also reduced phosphorylation levels of ERK1/2 and Akt. The SYK inhibitor BAY 613606 enhanced the efficacy of various chemotherapeutic agents. Transient expression of constitutively active SYK variants improved the survival of neuroblastoma cells, and this effect was independent of endogenous SYK levels. In summary, our findings suggest that the possibility of targeting SYK in combination with conventional chemotherapy as a treatment option for neuroblastoma should be further evaluated. [2]

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In this study, we used quantitative phosphorylation proteomics with amino acid stable isotope labeling in cell culture to investigate how the Lyn kinase signaling pathway mediates nilotinib resistance. We found that in nilotinib-resistant cells, the tyrosine phosphorylation levels of two other tyrosine kinases were increased: spleen tyrosine kinase Syk and UFO family receptor tyrosine kinase Axl. This increase in tyrosine phosphorylation involves the interaction of these tyrosine kinases with Lyn. Inhibition of Syk with the inhibitors R406 or BAY 61-3606, or inhibition of Syk by RNA interference, restored the ability of nilotinib to inhibit cell proliferation. In contrast, in drug-sensitive cells, co-expression of Lyn and Syk was necessary to fully induce nilotinib resistance. Surprisingly, knockdown of Syk also significantly reduced the tyrosine phosphorylation levels of Lyn and Axl, revealing an interaction between Syk and Lyn. We have confirmed that the adaptor protein CDCP-1 is involved in the development of nilotinib resistance. Interestingly, the expression of both Axl and CDCP-1 was elevated in nilotinib-resistant cell lines and in patients with nilotinib-resistant chronic myeloid leukemia (CML). We conclude that oncogenic signaling pathways mediated by Lyn and Syk can bypass the need for Bcr-Abl in CML cells. Therefore, targeting these kinases may have therapeutic value in overcoming imatinib or nilotinib resistance in CML. [3]

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Breast cancer cells often develop resistance to tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), thus requiring the assistance of sensitizers. In this study, we demonstrated that the spleen tyrosine kinase (Syk) inhibitor Bay 61-3606 is a TRAIL sensitizer. Bay 61-3606 enhances TRAIL-induced apoptosis while also strongly activating Bak, caspase, and fragmenting DNA. In terms of mechanism of action, Bay 61-3606 enhances the sensitivity of cells to TRAIL through two mechanisms that regulate myeloid leukemia sequence-1 (Mcl-1). First, Bay 61-3606 triggers ubiquitin-dependent degradation of Mcl-1 by regulating its phosphorylation. Second, Bay 61-3606 downregulates Mcl-1 expression at the transcriptional level. In this context, Bay 61-3606 inhibits cyclin-dependent kinase (CDK) 9, not Syk. In conclusion, Bay 61-3606 downregulates Mcl-1 expression in breast cancer cells and makes cancer cells more sensitive to TRAIL-mediated apoptosis. [4]

C20H18N6O3.2HCL

463.32

462.097

648903-57-5

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

11784504

Off-white to yellow solid

5.028

4

7

6

31

566

0

O=C(N)C1C(NC2N3C=CN=C3C=C(C3C=C(OC)C(OC)=CC=3)N=2)=NC=CC=1.[H]Cl.[H]Cl

SPMFEULFGGPQLN-UHFFFAOYSA-N

InChI=1S/C20H18N6O3.2ClH/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*1H

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 0.71 mg/mL (1.53 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 0.71 mg/mL (1.53 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 7.1 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)