| Targets |
VEGFR2
VEGFR-2-IN-39 targets vascular endothelial growth factor receptor 2 (VEGFR-2), a receptor tyrosine kinase that is the primary mediator of VEGF-induced angiogenesis. VEGFR-2 is expressed primarily on endothelial cells and plays a critical role in endothelial cell proliferation, migration, survival, and tube formation. As a PROTAC molecule, VEGFR-2-IN-39 not only inhibits VEGFR-2 activity but also induces its degradation. PROTACs are bifunctional molecules that recruit an E3 ubiquitin ligase to the target protein, leading to ubiquitination and subsequent proteasomal degradation. By degrading VEGFR-2, VEGFR-2-IN-39 provides a more sustained inhibition compared to traditional kinase inhibitors. The compound exhibits an IC50 of 208.6 nM for VEGFR-2 inhibition, indicating potent activity. Its low toxicity profile further supports its utility in research applications. |
|---|---|
| ln Vitro |
VEGFR-2-IN-39 (0-60 μM; 0-72 h) can effectively reduce the protein level of VEGFR-2 in a dose-dependent manner[1]. VEGFR-2-IN-39 (10-40 μM; 72 h) prolongs the S phase of the HUVEC cell cycle[1]. VEGFR-2-IN-39 (0.1-40 μM; 72 h) induces apoptosis of HUVEC cells in a dose-dependent manner[1].
In vitro, VEGFR-2-IN-39 demonstrates potent activity against VEGFR-2 with an IC50 of 208.6 nM. The compound effectively inhibits the proliferation of EA.hy926 cells, a human umbilical vein endothelial cell line, in a concentration-dependent manner with an IC50 of 38.65 μM. VEGFR-2-IN-39 extends the S phase of the HUVEC cell cycle at concentrations of 10-40 μM for 72 hours and induces apoptosis in HUVEC cells at 0.1-40 μM for 72 hours. As a PROTAC molecule, VEGFR-2-IN-39 concentration-dependently inhibits VEGFR-2 activity. The compound's dual mechanism of action—both enzymatic inhibition and protein degradation—may contribute to its potent anti-proliferative effects on endothelial cells. The low toxicity profile suggests that the compound has a favorable therapeutic window for research applications. |
| ln Vivo |
In vivo activity of VEGFR-2-IN-39 has not been extensively reported in the available literature. However, as a VEGFR-2 inhibitor with anti-proliferative and pro-apoptotic effects on endothelial cells in vitro, the compound would be expected to inhibit tumor angiogenesis and tumor growth in vivo. The low toxicity profile suggests potential for in vivo applications. As a PROTAC molecule, VEGFR-2-IN-39 may offer advantages over traditional kinase inhibitors in vivo, including sustained target degradation and potentially improved efficacy. However, specific in vivo data, including animal models used, dosing regimens, and quantitative outcomes such as tumor growth inhibition, have not been reported. Further in vivo studies would be required to characterize the compound's efficacy, pharmacokinetics, and safety profile in animal models of cancer and angiogenesis-dependent diseases.
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| Enzyme Assay |
In vitro enzyme or receptor binding assay protocols for VEGFR-2-IN-39 would typically involve measuring its inhibition of VEGFR-2 kinase activity using biochemical kinase assays. A standard protocol involves incubating recombinant VEGFR-2 kinase with varying concentrations of VEGFR-2-IN-39 (typically 0.1 nM to 10 μM), ATP, and a peptide substrate in kinase assay buffer (e.g., HEPES, pH 7.5, with MgCl2, DTT, and other components) at 30°C for a defined period (e.g., 30-60 minutes). The reaction is terminated, and the extent of substrate phosphorylation is measured using methods such as radioactive 33P-ATP incorporation, time-resolved fluorescence resonance energy transfer (TR-FRET), or luminescent kinase assays (e.g., ADP-Glo). IC50 values are determined from concentration-response curves. For PROTAC activity assessment, assays measuring VEGFR-2 protein degradation in cells (e.g., Western blot) are employed in addition to kinase activity assays.
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| Cell Assay |
Western Blot Analysis[1]
Cell Types: HUVECs Tested Concentrations: 0-60 μM Incubation Duration: 0-72 h Experimental Results: VEGFR-2 levels by up to 60% at a concentration of 40 μmol/L, with rapid degradation visible as early as 24 hours and nearly complete by 48 hours. Cell Cycle Analysis[1] Cell Types: HUVECs Tested Concentrations: 10-40 μM Incubation Duration: 0-72 h Experimental Results: Resulted in a significant increase in the proportion of cells in the S phase, indicating a halt or delay in the progression of the cell cycle. Correspondingly, there was a notable reduction in the number of cells in the G1 phase. Apoptosis Analysis[1] Cell Types: HUVECs Tested Concentrations: 0.1-40 μM Incubation Duration: 0-72 h Experimental Results: Resulted in a significant dose-dependent increase in the percentage of apoptotic cells. The percentage of late apoptotic cells increased as follows: control (untreated) : 5.34% 10 μM: 6.94% 20 μM: 8.32% 30 μM: 15.6% 40 μM: 48.7% In vitro cell-based assay protocols for VEGFR-2-IN-39 typically involve assessing its effects on endothelial cell proliferation, cell cycle progression, and apoptosis. A standard protocol for proliferation assays uses EA.hy926 cells (a human umbilical vein endothelial cell line) or HUVECs seeded in 96-well plates and treated with varying concentrations of VEGFR-2-IN-39 (typically 0.1 to 100 μM) for 72 hours. Cell proliferation is measured using CCK-8, MTT, or BrdU incorporation assays. IC50 values are calculated from concentration-response curves. For cell cycle analysis, cells are treated with VEGFR-2-IN-39 (10-40 μM; 72 hours), fixed, stained with propidium iodide, and analyzed by flow cytometry to determine cell cycle distribution. For apoptosis assays, cells are treated with 0.1-40 μM VEGFR-2-IN-39 for 72 hours and stained with Annexin V-FITC and propidium iodide for flow cytometric analysis. Appropriate controls include vehicle-treated cells and positive controls such as known VEGFR-2 inhibitors. |
| Animal Protocol |
In vivo animal experimental protocols for VEGFR-2-IN-39 have not been extensively reported in the available literature. Based on its mechanism as a VEGFR-2 inhibitor and PROTAC molecule, potential studies would involve administering the compound to tumor-bearing mice to assess its anti-tumor and anti-angiogenic effects. A hypothetical protocol might involve implanting tumor cells (e.g., xenograft models) subcutaneously in immunodeficient mice, allowing tumors to reach a certain size, and then administering VEGFR-2-IN-39 via oral gavage or intraperitoneal injection at doses determined from preliminary pharmacokinetic and tolerability studies. Treatment would typically be administered daily or every other day for 2-4 weeks. Endpoints would include tumor volume measurement, tumor weight at necropsy, assessment of tumor vascular density (e.g., CD31 staining), evaluation of VEGFR-2 degradation in tumor tissues by Western blot, and assessment of apoptosis in tumor sections. Appropriate controls would include vehicle-treated groups and groups treated with a standard VEGFR-2 inhibitor.
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| ADME/Pharmacokinetics |
Pharmacokinetic properties of VEGFR-2-IN-39 have not been extensively characterized in published studies. The compound has a high molecular weight of 1000.23, which may limit oral bioavailability and cell permeability due to its size and the presence of multiple functional groups (PROTAC architecture). However, as a PROTAC, the compound is designed for intracellular activity, and its cellular uptake may be facilitated by its lipophilic properties. The compound exhibits low toxicity, suggesting that it may have a favorable safety profile. However, specific PK parameters such as half-life, Cmax, AUC, bioavailability, volume of distribution, and clearance have not been reported. The compound's protein binding, metabolism, and routes of elimination remain uncharacterized. Further pharmacokinetic studies would be required to understand its absorption, distribution, metabolism, and excretion profile.
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| Toxicity/Toxicokinetics |
Toxicological data for VEGFR-2-IN-39 indicate that the compound has low toxicity. However, comprehensive toxicology studies have not been reported. No acute toxicity (LD50), subchronic toxicity, genotoxicity, or reproductive toxicity data are available. As a VEGFR-2 inhibitor, potential on-target toxicities could include effects on normal angiogenesis, such as impaired wound healing, hypertension, and cardiovascular effects, which are known class effects of VEGFR inhibitors. However, these have not been evaluated specifically for VEGFR-2-IN-39. The compound is intended for research use only and is not intended for human use. Researchers should follow standard safety precautions when handling the compound, including working in a fume hood, wearing appropriate personal protective equipment, and avoiding inhalation, ingestion, or skin contact.
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| References | |
| Additional Infomation |
VEGFR-2-IN-39 is a research-grade compound that functions as a potent inhibitor of VEGFR-2 with an IC50 of 208.6 nM. It is a PROTAC molecule that induces targeted degradation of VEGFR-2. The compound inhibits proliferation of EA.hy926 cells (IC50 = 38.65 μM), extends the S phase of the HUVEC cell cycle, and induces apoptosis in HUVEC cells. It exhibits low toxicity, making it suitable for research in angiogenesis and vascular biology. VEGFR-2-IN-39 has not entered clinical trials and is not approved for any therapeutic indication. Its mechanism of action involves both inhibition of VEGFR-2 kinase activity and degradation of the receptor protein through the ubiquitin-proteasome pathway. The compound is available exclusively for research purposes and is not intended for diagnostic, therapeutic, or human applications. It is stored at 2-8°C.
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| Molecular Formula |
C55H66FN9O6S
|
|---|---|
| Molecular Weight |
1000.23
|
| Exact Mass |
999.484
|
| CAS # |
2353417-86-2
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| PubChem CID |
141744224
|
| Appearance |
White to off-white solid powder
|
| Hydrogen Bond Donor Count |
6
|
| Hydrogen Bond Acceptor Count |
11
|
| Rotatable Bond Count |
21
|
| Heavy Atom Count |
72
|
| Complexity |
1780
|
| Defined Atom Stereocenter Count |
3
|
| SMILES |
CC1=CC(=C(C=C1)F)NC(=O)NC2=CC=C(C=C2)C3=C4C(=CC=C3)N(N=C4N)C(=O)CCCCCCCCCCC(=O)N[C@H](C(=O)N5C[C@@H](C[C@H]5C(=O)NCC6=CC=C(C=C6)C7=C(N=CS7)C)O)C(C)(C)C
|
| InChi Key |
YZGNCBAFOZBYKS-SHPBXJAASA-N
|
| InChi Code |
InChI=1S/C55H66FN9O6S/c1-34-19-28-42(56)43(29-34)61-54(71)60-39-26-24-37(25-27-39)41-15-14-16-44-48(41)51(57)63-65(44)47(68)18-13-11-9-7-6-8-10-12-17-46(67)62-50(55(3,4)5)53(70)64-32-40(66)30-45(64)52(69)58-31-36-20-22-38(23-21-36)49-35(2)59-33-72-49/h14-16,19-29,33,40,45,50,66H,6-13,17-18,30-32H2,1-5H3,(H2,57,63)(H,58,69)(H,62,67)(H2,60,61,71)/t40-,45+,50-/m1/s1
|
| Chemical Name |
(2S,4R)-1-[(2S)-2-[[12-[3-amino-4-[4-[(2-fluoro-5-methylphenyl)carbamoylamino]phenyl]indazol-1-yl]-12-oxododecanoyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[[4-(4-methyl-1,3-thiazol-5-yl)phenyl]methyl]pyrrolidine-2-carboxamide
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO :~200 mg/mL (~199.95 mM; with sonication)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.9998 mL | 4.9989 mL | 9.9977 mL | |
| 5 mM | 0.2000 mL | 0.9998 mL | 1.9995 mL | |
| 10 mM | 0.1000 mL | 0.4999 mL | 0.9998 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.
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