Tie2 (IC50 = 250 nM)

Tie2 kinase inhibitors have a moderately strong inhibitory effect on Tie2 tyrosine kinase. With an IC50 of 232 nM in HEL cells, the Tie2 kinase inhibitor likewise exhibits moderate cellular activity. Moreover, Tie2 kinase inhibitor exhibits >10-fold selectivity over VEGFR2, VEGFR3, and PDGFR1β and a remarkable selectivity for Tie2 over p38 (IC50=50 μM).[1]

Tie2 kinase inhibitors at doses of 25 and 50 mg/kg (i.p., b.i.d.) reduce angiogenesis in a Matrigel mouse model of angiogenesis by 41% and 70%, respectively. Treatment with a Tie2 kinase inhibitor slows tumor growth in a dose-dependent manner in a MOPC-315 plasmacytoma xenograft model.[1]

Turned on an incubator shaker and adjusted temperature to 30 °C. To the Flashplate, 20 μL of 3× kinase buffer (final 20 mM Tris-HCl, pH 7, 100 mM NaCl, 12 mM MgCl2, 1 mM DTT) was added per well. except for background, 20 μL of protein were added to each well. Additional Tie2 Kinase Inhibitor, usually 1–2 μL in DMSO stocks. For each well, 20 μL of a 1:1 v/v combination of cold ATP and gamma 33p-ATP was added. Film made of translucent polyester was used to cover. five washes and two hours of incubation at 30 °C in a shaker. IC50 values were computed using standard methods after the plate was read on TopCount or another counting device.

Immunoblot Analysis [2]
HUVEC, SVR, and MS1-VEGF cells were grown to approximately 70% confluence. Cells were washed twice in cold PBS and protein isolated in cold NP-40 lysis buffer (50 mM HEPES (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na3VO4, 20 mM beta-glycerophosphate, 10 µg/ml of leupeptin and aprotinin). Proteins were quantified using a standard Bradford absorbance assay. Proteins were separated on 4% to 20% gradient SDS-PAGE gels and transferred to polyvinylidene fluoride membrane by electrophoretic transfer. Membranes were subsequently blocked in 5% nonfat milk or bovine serum albumin in 1 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20. Primary antibodies for Western blot detection of VEGFR2 (1:1000) or Tie2 were applied overnight at 4°C. Secondary antibody for detection was either goat antirabbit immunoglobulin G (IgG) HRP (Tie2, 1:4000; Santa Cruz Biotechnology, Inc) or goat antimouse IgG-HRP (VEGFR-2, 1:4000). Proteins were detected through enhanced chemiluminescence detection system.

---

Cell Survival Assays [2]
SVR and MS1-VEGF cells were plated at 1500 cells per well in 96-well plates and allowed to adhere overnight. The medium was aspirated and replaced with fresh medium containing varying concentrations of sunitinib, Tie2 kinase inhibitor, or DMSO vehicle control. The final concentration of DMSO was 0.1%. Cells were allowed to grow for 72 hours, and cell survival was quantified using the WST-1 cell proliferation reagent according to the manufacturer's instructions. Cell survival assays examining combined treatment were similarly performed at a fixed Tie2 kinase inhibitor-to-sunitinib molar ratio of 25:1 (to reflect relative drug potencies) at total drug concentrations of 26 to 7800 nM.

MOPC-315 plasmacytoma xenograft model.
≤50 mg
Administered via i.p.

---

In Vivo Studies [2]
Athymic nude mice (6- to 8-week-old females) were obtained from Harlan Laboratories, Inc (Indianapolis, IN). The care and treatment of experimental animals were in accordance with institutional guidelines. SVR and MS1-VEGF cells (2 x 106 cells) were implanted subcutaneously. For histopathologic confirmation of the angiosarcoma phenotype, SVR tumors were excised at days 3, 6, 9, and 12 after injection, and MS1-VEGF tumors were excised at days 5, 10, 15, and 20 after injection. For tumor growth delay studies, treatment was started when the tumor volume reached 200 mm3 (SVR) or when palpable (MS1-VEGF). Animals were randomized into four treatment groups. The control group and the Tie2 kinase inhibitor alone group were treated daily with 100 µl of vehicle (0.5% carboxymethylcellulose, 0.4% Tween 80, 1.8% NaCl, and 0.9% benzyl alcohol in distilled water, pH 6.0) by oral gavage. The sunitinib-alone and combination treatment groups were treated daily with 100 µl of vehicle containing sunitinib (60 mg/kg for SVR tumors, 30 mg/kg for MS1-VEGF tumors) by oral gavage. The control and sunitinib-alone groups were treated twice weekly with 100 µl of vehicle (5% ethanol, 5% Cremophor, and 90% distilled water) through intraperitoneal injection. The Tie2 kinase inhibitor-alone and combination treatment groups were injected twice weekly with 100 µl of vehicle containing Tie2 kinase inhibitor (50 mg/kg). Tumor volume was determined by direct caliper measurement and calculated by the following formula: volume = 0.5 x (large diameter) x (small diameter)2. For necrosis, apoptosis, and proliferation studies in vivo, SVR tumor-bearing animals (n = 4–6 per group) were randomized and treated as above for 11 days. On day 12, animals were killed, and tumors were harvested for histologic and immunohistochemical analyses. Percent tumor necrosis was estimated by a pathologist (D.B.) blinded to treatment using hematoxylin and eosin-stained whole tumor sections. The number of cleaved caspase 3 and PCNA-positive cells per high-power field (n = 5/tumor) was quantified by an observer blinded to treatment.

[1]. Pyridinylimidazole inhibitors of Tie2 kinase. Bioorg Med Chem Lett. 2007 Sep 1;17(17):4756-60. Epub 2007 Jun 27.

[2]. Efficacy of Tie2 receptor antagonism in angiosarcoma. Neoplasia. 2012 Feb;14(2):131-40.

4-[4-(6-methoxy-2-naphthalenyl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine is a member of imidazoles.

---

This communication details the evolution of the screening lead SB-203580, a known CSBP/p38 kinase inhibitor, into a potent and selective Tie2 tyrosine kinase inhibitor. The optimized compound 5 showed efficacy in an in vivo model of angiogenesis and a MOPC-315 plasmacytoma xenograft model. [1]

---

Angiosarcomas are malignant endothelial cell tumors with few effective systemic treatments. Despite a unique endothelial origin, molecular candidates for targeted therapeutic intervention have been elusive. In this study, we explored the tunica internal endothelial cell kinase 2 (Tie2) receptor as a potential therapeutic target in angiosarcoma. Human angiosarcomas from diverse sites were shown to be universally immunoreactive for Tie2. Tie2 and vascular endothelial growth factor receptor (VEGFR) antagonists inhibited SVR and MS1-VEGF angiosarcoma cell survival in vitro. In the high-grade SVR cell line, Tie2 and VEGF antagonists inhibited cell survival synergistically, whereas effects were largely additive in the low-grade MS1-VEGF cell line. Xenograft modeling using these cell lines closely recapitulated the human disease. In vivo, Tie2 and VEGFR inhibition resulted in significant angiosarcoma growth delay. The combination proved more effective than either agent alone. Tie2 inhibition seemed to elicit tumor growth delay through increased tumor cell apoptosis, whereas VEGFR inhibition reduced tumor growth by lowering tumor cell proliferation. These data identify Tie2 antagonism as a potential novel, targeted therapy for angiosarcomas and provide a foundation for further investigation of Tie2 inhibition, alone and in combinations, in the management of this disease. [2]

C26H21N3O2S

439.53

439.135

C, 71.05; H, 4.82; N, 9.56; O, 7.28; S, 7.29

948557-43-5

23625762

Light yellow to yellow solid powder

1.4±0.1 g/cm3

699.8±55.0 °C at 760 mmHg

377.0±31.5 °C

0.0±2.1 mmHg at 25°C

1.746

5.31

1

5

5

32

635

0

O=S(C)C1C=CC(C2NC(C3C=C4C(C=C(C=C4)OC)=CC=3)=C(C3C=CN=CC=3)N=2)=CC=1

SINQIEAULQKUPD-UHFFFAOYSA-N

InChI=1S/C26H21N3O2S/c1-31-22-8-5-19-15-21(4-3-20(19)16-22)25-24(17-11-13-27-14-12-17)28-26(29-25)18-6-9-23(10-7-18)32(2)30/h3-16H,1-2H3,(H,28,29)

4-[4-(6-methoxynaphthalen-2-yl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine

Tie2-inhibitor-5; Tie2IN5; 948557-43-5; Tie2 kinase inhibitor; Tie2 kinase-IN-1; Tie2 kinase inhibitor 1; Tie2-IN-5; 4-(4-(6-methoxynaphthalen-2-yl)-2-(4-(methylsulfinyl)phenyl)-1H-imidazol-5-yl)pyridine; CHEMBL237352; 4-[4-(6-methoxynaphthalen-2-yl)-2-(4-methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine; Tie2-IN-5; Tie2 kinase inhibitor 5; Tie2 inhibitor 5; Compound 5; Tie2 IN 5

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: ≥ 1.79 mg/mL (4.07 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 17.9 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.

---

Solubility in Formulation 2: ≥ 1.79 mg/mL (4.07 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 17.9 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.

---

View More

Solubility in Formulation 3: 2% Cremophor EL, 2% N,N-dimethylacetamide: 30 mg/mL

---

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