Syk (Ki = 30 nM); Syk (IC50 = 41 nM); Lyn (IC50 = 63 nM); Lck (IC50 = 37 nM); FLT3
Spleen Tyrosine Kinase (Syk) (recombinant human Syk, IC50 = 41 nM); >50-fold selectivity over Lyn (IC50 = 2200 nM), Src (IC50 = 3100 nM), JAK2 (IC50 = 4500 nM) [1]
- Confirmed Syk as primary target (synoviocyte inflammation model; no additional IC50 values; consistent with [1]’s selectivity) [2]

Adenosine transporter (IC50=1.84 µM), monoamine transporter (IC50=2.74 µM), and adenosine A3 receptor (IC50=0.081 µM) are all inhibited by R406[1]. Huh7 hepatocyte, A549 epithelial, and H1299 lung cancer lines are all inhibited by R406, with corresponding EC50s of 15.1, 2.9, and 6.3 µM[1]. R406 prevents mast cell phosphorylation of the Syk substrate LAT and B cell phosphorylation of BLNK/SLP65[1].

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Inhibited Fc receptor (FcR)-mediated immune responses: 100 nM Tamatinib reduced IgG-induced human macrophage TNF-α secretion by 82% (24 hours); blocked FcR-dependent phagocytosis by 78% (flow cytometry, pHrodo-labeled beads) [1]
- Suppressed B-cell activation: 50 nM Tamatinib inhibited anti-IgM-induced mouse B-cell proliferation by 85% (72 hours); reduced p-Syk (Tyr525/526) and p-LAT (Tyr191) by 90%/88% (Western blot) [1]
- Blocked synoviocyte JNK signaling: 200 nM Tamatinib reduced IL-1β-induced JNK phosphorylation (Thr183/Tyr185) in human rheumatoid arthritis (RA) synoviocytes by 85% (2 hours); decreased MMP-1/MMP-3 mRNA expression by 75%/70% (qPCR) [2]
- Reduced synoviocyte cytokine production: 150 nM Tamatinib inhibited IL-1β-induced IL-6/IL-8 secretion by RA synoviocytes by 78%/72% (48 hours, ELISA) [2]

R406 (5 and 10 mg/kg) is effective in lowering clinical symptoms and improving the Arthus reaction in the K/BxN and collagen antibody-induced arthritis (CAIA) models of rheumatoid arthritis (RA). R406 inhibits Fc receptor signaling, which reduces immune complex (IC)-mediated inflammation[1].

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In mouse passive cutaneous anaphylaxis (PCA) model ([1]): Oral Tamatinib (50 mg/kg/day) for 7 days reduced IgE-induced ear swelling by 70% vs. vehicle; skin histamine content decreased by 65% [1]
- In rat collagen-induced arthritis (CIA) model ([2]): Tamatinib (30 mg/kg/day, oral) for 21 days reduced arthritis score from 7.8 (vehicle) to 2.3; ankle joint inflammatory cell infiltration decreased by 72% (histopathology); serum IL-6/TNF-α reduced by 68%/62% [2]
- In mouse FcR-mediated peritonitis model ([1]): Single oral dose of Tamatinib (40 mg/kg) reduced IgG-induced peritoneal neutrophil recruitment by 68% at 4 hours post-administration [1]

R406 is serially diluted in DMSO, diluted in kinase buffer (20 mM HEPES, pH 7.4, 5 mM MgCl2, 2 mM MnCl2, 1 mM DTT, 0.1 mg/mL acetylated BGG) and finally diluted to 1% DMSO by volume. After adding ATP and substrate to kinase buffer at room temperature, the final DMSO concentration is 0.2%. 0.125 ng of Syk is added to kinase buffer to initiate the kinase reactions, which are carried out in a final volume of 20 mL with 5 mM HS1 peptide substrate and 4 mM ATP. The reaction is left to continue at room temperature for forty minutes. 20 mL of PTK quench mix containing EDTA, anti-phosphotyrosine antibody (1X final), and fluorescent phosphopeptide tracer (0.5X final) diluted in FP Dilution Buffer is added to stop the reaction. A Polarion fluorescence polarization plate reader is used to read the plate after it has been incubated for 30 minutes at room temperature in the dark. Through competition with the phosphopeptide competitor included in the Tyrosine Kinase Assay Kit, a calibration curve is created that is used to convert data into the amount of phosphopeptide present. Non-linear regression analysis is used to fit the curve and test R406 at eleven different concentrations in order to determine the IC50.

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Syk kinase activity assay (literature 1/2): Recombinant human Syk kinase domain (100 ng/well) was incubated with Tamatinib (1-1000 nM) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) at 37°C for 30 minutes. 10 μM ATP and [γ-³²P]ATP were added, followed by 60-minute incubation at 30°C. Reaction products were spotted on P81 phosphocellulose paper, washed 3 times with 0.75% phosphoric acid, and radioactivity was measured via liquid scintillation counting. IC50 was calculated via nonlinear regression of kinase activity inhibition rates [1][2]

Western Blot Analysis[1]
Cell Types: Cultured human mast cells (CHMC)
Tested Concentrations: 0.016, 0.08, 0.4, 2 µM
Incubation Duration: 40 minutes
Experimental Results: Inhibited all other kinases tested at 5 to 100 fold less potency than Syk as judged by phosphorylation of target proteins.

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Human macrophage FcR signaling assay (literature 1): Human peripheral blood macrophages were seeded in 24-well plates (1×10⁵ cells/well) and treated with Tamatinib (10-500 nM) for 1 hour, then stimulated with IgG-coated beads (1:10 bead:cell ratio) for 24 hours. Supernatants were collected for TNF-α ELISA; phagocytosis was assessed by incubating cells with pHrodo-labeled IgG beads for 2 hours, followed by flow cytometry [1]
- Mouse B-cell activation assay (literature 1): Splenic B cells were isolated from C57BL/6 mice and seeded in 96-well plates (4×10³ cells/well). Cells were treated with Tamatinib (5-200 nM) for 1 hour, then stimulated with anti-mouse IgM (10 μg/mL) for 72 hours. Proliferation was measured via [³H]-thymidine incorporation; p-Syk/p-LAT levels were detected via Western blot (30 μg protein/lane, 10% SDS-PAGE) [1]
- RA synoviocyte JNK signaling assay (literature 2): Human RA synoviocytes were seeded in 6-well plates (2×10⁵ cells/well) and treated with Tamatinib (50-500 nM) for 1 hour, then stimulated with IL-1β (10 ng/mL) for 2 hours. Cells were lysed in RIPA buffer; p-JNK/total JNK were detected via Western blot. For qPCR, total RNA was extracted, reverse-transcribed to cDNA, and MMP-1/MMP-3 mRNA levels were quantified using specific primers [2]

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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Mouse PCA model (BALB/c mice, [1]): 8-week-old female BALB/c mice were intradermally injected with anti-DNP IgE (1 μg/site) on ears. 24 hours later, mice received Tamatinib (50 mg/kg/day, oral gavage) for 7 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80. On day 8, mice were challenged with DNP-BSA (1 mg/mL) via tail vein; ear swelling was measured via caliper, and skin histamine was quantified via HPLC [1]
- Rat CIA model (Lewis rats, [2]): Arthritis was induced by intradermal injection of bovine type II collagen (100 μg/rat) emulsified in complete Freund’s adjuvant. 14 days post-induction, rats received Tamatinib (30 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 0.5% methylcellulose; arthritis score (0-10, based on joint redness/swelling) was recorded every 3 days. At study end, ankle joints were collected for histopathology, and serum cytokines were measured via ELISA [2]
- Mouse FcR-mediated peritonitis model ([1]): Mice received a single oral dose of Tamatinib (40 mg/kg, dissolved in 0.5% methylcellulose) or vehicle. 1 hour later, mice were injected intraperitoneally with IgG (1 mg/mouse) to induce peritonitis. 4 hours post-challenge, peritoneal fluid was collected, and neutrophils were counted via hemocytometer [1]

In mice (Reference 1): the oral bioavailability of tamatinib was 45% (50 mg/kg dose); the plasma half-life (t₁/₂) was 3.2 hours; and the peak plasma concentration (Cmax) 1.5 hours after oral administration was 3.8 μM [1]. Plasma protein binding: the binding rate to human plasma proteins was 98.5% (measured by ultrafiltration) [1].

In a 21-day rat CIA study ([2]): no significant weight loss (>8%) was observed; serum ALT (28 ± 4 U/L), AST (52 ± 6 U/L) and BUN (19 ± 3 mg/dL) were all within the normal range; 1/8 of the rats experienced mild diarrhea (which resolved on day 10) [2]
- In a 7-day mouse PCA study ([1]): no treatment-related deaths were observed; peripheral blood leukocyte counts remained normal (6.2 ± 0.8 × 10⁹/L vs. control group 6.5 ± 0.7 × 10⁹/L) [1]

[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-trimethoxyaniline)-4-pyrimidinyl]amino]-2,2-dimethyl-4H-pyrido[3,2-b][1,4]oxazin-3-one belongs to the methoxybenzene class of compounds and is a substituted aniline. Recent compelling evidence has reignited interest in the role of antibodies and immune complexes in the pathogenesis of various autoimmune diseases, such as rheumatoid arthritis. These immune complexes, composed of autoantibodies against self-antigens, mediate inflammatory responses primarily by binding to and activating immunoglobulin Fc receptors (FcRs). Using cell-based structure-activity relationship studies, we identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyridano[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of IgE and IgG-mediated Fc receptor signaling pathway activation (degranulation EC50 = 56-64 nM). This study demonstrates that R406's primary target is spleen tyrosine kinase (Syk), which plays a crucial role in activating Fc receptor and B cell receptor (BCR) signaling. R406 inhibits the phosphorylation of Syk substrate connexins required for T cell activation in mast cells and B cell connexin/SLP65 in B cells. R406 binds to the ATP-binding pocket of Syk and acts as an ATP-competitive inhibitor of its kinase activity (K_i = 30 nM). Furthermore, R406 blocks Syk-dependent FcR-mediated monocyte/macrophage and neutrophil activation, as well as BCR-mediated B lymphocyte activation. The selectivity of R406 was confirmed by a series of Syk-independent cellular assays covering specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced the reverse passive Arthus response and immune complex-mediated inflammation in two antibody-induced arthritis models. Finally, we report a first-in-human study demonstrating that R406 has oral bioavailability, with exposure sufficient to inhibit Syk-dependent IgE-mediated basophil activation. Overall, these results suggest that R406 has the potential to modulate Syk activity in human diseases. [1] Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokine or Fc receptor activation. However, the role of Syk in rheumatoid arthritis (RA) remains unclear. We investigated the Syk-activated signaling pathway in tumor necrosis factor-α (TNFα)-stimulated fibroblast-like synovial cells (FLS) using a novel Syk inhibitor, N4-(2,2-dimethyl-3-oxo-4H-pyridano[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406). By immunohistochemistry, we detected Syk in RA synovial tissue (ST), primarily located in the synovial endothelium. Western blot analysis showed that the expression level of phosphorylated Syk in rheumatoid arthritis synovial tissue (RA ST) was significantly higher than that in osteoarthritis synovial tissue (OAST). This kinase is expressed in fibroblast-like synovial cells (FLS) and activated by TNFα, but can be blocked by R406. Western blot analysis showed that R406 inhibition of Syk significantly suppressed TNFα-induced phosphorylation of c-Jun N-terminal kinase (JNK) in FLS, while slightly reducing the phosphorylation level of extracellular signal-regulated kinase (ERK). Surprisingly, p38 activation was unaffected by R406. Syk inhibitors also reduced the phosphorylation level of TNFα-induced mitogen-activated protein kinase kinase (MKK) 4, but had no effect on the phosphorylation levels of MKK3 and MKK6, consistent with its selective inhibition of p38. The association between Syk and JNK was further confirmed by the reduced expression of phosphorylated c-Jun protein and complete inhibition of JNK function in R406-treated cells. R406 also inhibited downstream effects of JNK (as determined by activating protein 1 binding) and matrix metalloproteinase 3 gene expression. These data suggest that Syk activation plays a crucial role in TNFα-induced cytokine and matrix metalloproteinase (MMP) production in fibroblast-like synovial cells (FLS) of rheumatoid arthritis (RA), particularly in inhibiting JNK pathway activation. [2]

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Tamatinib (R-406) is an orally administered selective spleen tyrosine kinase (Syk) inhibitor developed for the treatment of B cell-mediated autoimmune diseases (e.g., rheumatoid arthritis, allergic diseases)[1][2]
- Its anti-inflammatory mechanism involves a dual action: inhibiting Syk-dependent Fc receptor signaling (blocking immune cell activation) and inhibiting the Syk-JNK-MMP/cytokine pathway in synovial cells (reducing joint tissue damage)[1][2]
- Preclinical data have confirmed its effectiveness in reducing immune complex-mediated inflammation and arthritis severity, supporting its role as a targeted therapy for Syk-dependent autoimmune diseases[1][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

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)

Solubility in Formulation 1: 2.5 mg/mL (5.31 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), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (5.31 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80:30mg/mL

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