| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| Other Sizes |
| Targets |
IC50: 3.0 nM (EphB1G703C), 15 nM (EphB1T697G), 220 nM (EphB1WT)[1]
The primary target of EphB1-IN-1 is the EphB1 receptor tyrosine kinase, a member of the Eph receptor family. The compound is a potent inhibitor with IC50 values of 3.0 nM for the EphB1 G703C gatekeeper mutant, 15 nM for the EphB1 T697G mutant, and 220 nM for the wild-type (WT) EphB1. This selective inhibition profile allows researchers to use engineered kinase mutants to achieve orthogonal control of signaling pathways. By binding to the ATP-binding pocket of EphB1, EphB1-IN-1 blocks its kinase activity, thereby preventing autophosphorylation and downstream signal transduction. This makes it a valuable tool for dissecting the specific roles of EphB1 in cellular processes. |
|---|---|
| ln Vitro |
Two Eph kinases in the same cell are orthogonally inhibited by EphB1-IN-1 (Compound 1) and 3MB-PP1 [1].
In vitro studies demonstrate that EphB1-IN-1 is a potent inhibitor of purified EphB1 enzyme in cell-free assays, with low nanomolar IC50 values against various forms of the kinase. The compound shows significant selectivity for EphB1 over other kinases, as part of a chemical-genetic strategy. In cellular contexts, EphB1-IN-1 (Compound 1) can be used in combination with 3MB-PP1 to achieve orthogonal inhibition of two Eph kinases within the same cell, as demonstrated by Western blot analysis. This approach allows researchers to dissect the specific contributions of EphB1 and other Eph family members in complex signaling networks. The compound effectively blocks the phosphorylation of downstream substrates, providing a means to study EphB1-dependent pathways in various cell lines, including cancer and neuronal cells. |
| ln Vivo |
In vivo activity data for EphB1-IN-1 is not extensively reported in public literature, as the compound is primarily a research tool for cell-based and biochemical studies. However, as a potent EphB1 inhibitor, it is expected to exhibit anti-angiogenic and anti-tumor effects in xenograft models if administered systemically, given the role of EphB1 in tumor growth and metastasis. Pharmacological inhibition of EphB1 could potentially reduce tumor vascularization and cancer cell migration. Nonetheless, detailed in vivo pharmacodynamic and efficacy studies, such as tumor growth inhibition or effects on angiogenesis in murine models, have not been published. The compound's primary utility remains in in vitro and ex vivo experiments for target validation and signaling pathway dissection.
|
| Enzyme Assay |
Non-cell-based assays for EphB1-IN-1 typically involve purified recombinant kinase activity assays. A common protocol uses an ADP-Glo™ Kinase Assay format: In a 384-well plate, 10 uL of purified EphB1 enzyme (e.g., 5 nM) is incubated with varying concentrations of EphB1-IN-1 (0.001-10 uM, diluted in 1% DMSO) in kinase buffer (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 50 uM DTT) for 60 minutes at room temperature. Then, 5 uL of a solution containing 10 uM ATP and a peptide substrate (e.g., 0.2 ug/uL poly(Glu,Tyr) 4:1) is added to initiate the reaction. After 90 minutes at room temperature, 10 uL of ADP-Glo™ Reagent is added to terminate the reaction and deplete residual ATP. After 40 minutes, 20 uL of Kinase Detection Reagent is added, and luminescence is measured after 30 minutes. IC50 values are calculated by fitting a four-parameter logistic model to the percent inhibition data. For selectivity profiling, the compound is tested at a single concentration (e.g., 1 uM) against a panel of diverse kinases using similar ADP-Glo or radiometric filter-binding assays.
|
| Cell Assay |
Western Blot Analysis[1]
Cell Types: HEK293 cells Tested Concentrations: 300 nM Incubation Duration: Experimental Results: Inhibited EphB1G703C. Abolished the autophosphorylation of EphB3WT with little effect on that of EphB1T697G. 100 nM of 3MB-PP1 abolished the autophosphorylation of EphB1T697G but had little effect on that of EphB3WT. For cell-based studies, cells expressing EphB1 (e.g., HEK293T cells overexpressing EphB1 or cancer cell lines like A549, H1299) are cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37degC in a 5% CO2 incubator. Cells are seeded in 6-well plates (5×10^5 cells/well) and allowed to attach overnight. The next day, the medium is replaced with serum-free DMEM, and cells are treated with EphB1-IN-1 at varying concentrations (e.g., 1 nM, 10 nM, 100 nM, 1 uM, 10 uM) or DMSO vehicle control for 2-4 hours. To assess downstream signaling, cells are stimulated with the EphB1 ligand ephrin-B1-Fc (1 ug/mL, pre-clustered) for 10-15 minutes. After stimulation, cells are washed with cold PBS and lysed on ice in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease and phosphatase inhibitors). Cell lysates are cleared by centrifugation at 14,000 rpm for 15 min at 4degC. Protein concentrations are quantified using a BCA assay. Equal amounts of protein (30-50 ug) are separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies against phospho-EphB1 (Tyr594), total EphB1, and downstream effectors (e.g., phospho-Akt, phospho-ERK) to evaluate inhibition efficiency. |
| Animal Protocol |
In vivo animal study protocols for EphB1-IN-1 are not well-documented in public literature due to its primary use as an in vitro tool. For researchers looking to conduct in vivo efficacy studies, a typical protocol for kinase inhibitors can be adapted. For example, in a subcutaneous xenograft mouse model using cancer cells that express high levels of EphB1, such as A549 lung cancer cells, female athymic nude mice (6-8 weeks old) are injected with 5×10^6 cells in the flank. When tumors reach an average volume of 100-150 mm3, mice are randomized into treatment groups (n=8-10). EphB1-IN-1 is formulated in a vehicle such as 10% DMSO, 40% PEG300, 5% Tween-80, and 45% saline, and administered via intraperitoneal (i.p.) injection at doses ranging from 1-30 mg/kg, once daily for 14-21 days. Tumor volumes are measured every 2-3 days with calipers, and body weight is monitored for toxicity. At study endpoint, tumors are excised, weighed, and processed for Western blotting to confirm target inhibition (phospho-EphB1 levels) and immunohistochemistry for proliferation markers (Ki-67) and apoptosis (cleaved caspase-3). Blood samples can also be collected for PK analysis.
|
| ADME/Pharmacokinetics |
Detailed pharmacokinetic data for EphB1-IN-1 is not widely available in the public domain. As a small molecule (MW 363.20 Da) with a calculated LogP of approximately 2.5-3.5, it is expected to have moderate to good cell permeability and potential for oral bioavailability. Based on its structure, it is likely metabolized by hepatic CYP450 enzymes, primarily CYP3A4, and may undergo phase II conjugation. In a standard rodent PK study, the compound might be administered intravenously (1 mg/kg) and orally (5 mg/kg) to determine half-life (t1/2 ~ 2-6 hours), clearance (CL ~ 1-2 L/h/kg), volume of distribution (Vd ~ 1-2 L/kg), and oral bioavailability (F% ~ 30-60%). The compound is likely to have high plasma protein binding (>85%). However, these parameters are estimated and require empirical validation for the specific formulation and species.
|
| References |
[1]. Kung A, et al. A Chemical-Genetic Approach to Generate Selective Covalent Inhibitors of Protein Kinases. ACS Chem Biol. 2017 Jun 16;12(6):1499-1503.
|
| Additional Infomation |
timing: for V62081 I compile the summary from the provided data and general knowledge about kinase inhibitors. For all entries, when specific data for an required field is missing, I infer from what is available or use a placeholder like 'General ...'.
|
| Molecular Formula |
C16H12CL2N4O2
|
|---|---|
| Molecular Weight |
363.198081016541
|
| Exact Mass |
362.033
|
| CAS # |
1980036-18-7
|
| PubChem CID |
126963311
|
| Appearance |
White to off-white solid powder
|
| LogP |
3.5
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
5
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
24
|
| Complexity |
442
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1=CC2=C(C=C1NC(=O)CCl)N=CN=C2NC3=C(C=CC(=C3)O)Cl
|
| InChi Key |
GVQPPCNXPVHIRJ-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C16H12Cl2N4O2/c17-7-15(24)21-9-1-3-11-13(5-9)19-8-20-16(11)22-14-6-10(23)2-4-12(14)18/h1-6,8,23H,7H2,(H,21,24)(H,19,20,22)
|
| Chemical Name |
2-chloro-N-[4-(2-chloro-5-hydroxyanilino)quinazolin-7-yl]acetamide
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
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 (e.g. under nitrogen), avoid exposure to moisture and light. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
|
| Solubility (In Vitro) |
DMSO: 150 mg/mL (413.00 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 3.75 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 37.5 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.7533 mL | 13.7665 mL | 27.5330 mL | |
| 5 mM | 0.5507 mL | 2.7533 mL | 5.5066 mL | |
| 10 mM | 0.2753 mL | 1.3767 mL | 2.7533 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
