Physicochemical Properties
| Molecular Formula | C31H29F3N8O2 |
| Molecular Weight | 602.609575986862 |
| Exact Mass | 602.236 |
| CAS # | 2259657-48-0 |
| PubChem CID | 146556121 |
| Appearance | Typically exists as solid at room temperature |
| LogP | 4.4 |
| Hydrogen Bond Donor Count | 2 |
| Hydrogen Bond Acceptor Count | 11 |
| Rotatable Bond Count | 7 |
| Heavy Atom Count | 44 |
| Complexity | 953 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | JCCUAVDBGXASEC-UHFFFAOYSA-N |
| InChi Code | InChI=1S/C31H29F3N8O2/c1-19-5-6-20(14-26(19)44-30-24-17-36-40-28(24)38-27(39-30)21-4-3-9-35-16-21)29(43)37-23-8-7-22(25(15-23)31(32,33)34)18-42-12-10-41(2)11-13-42/h3-9,14-17H,10-13,18H2,1-2H3,(H,37,43)(H,36,38,39,40) |
| Chemical Name | 4-methyl-N-[4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl]-3-[(6-pyridin-3-yl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)oxy]benzamide |
| 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 |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| ln Vitro | RET-IN-16 (Compound 9x) (0.005-10 μM; 48 hours) demonstrates strong efficacy against KIF5B-RET-Ba/F3 and CCDC6-RET-Ba/F3, with GI50 values of 17 nM and 9 nM, respectively[1]. The growth of CCDC6-RET fusion cells is specifically inhibited by RET-IN-16 (1 μM; 48 hours) [1]. RET-IN-16 (50 and 100 μM; 4 h) strongly suppressed RET autophosphorylation in KIF5B-RET and KIF5B-RETV804M Ba/F3 cells. Additionally, there was a dose-dependent inhibition of phosphorylation of the adaptor protein SHC. [1]. |
| ln Vivo | RET-IN-16 (1 mg/kg; IV; single dose) revealed good drug exposure (AUC0-t = 6959 ± 762 ng·h/mL) and moderate half-life (T1/2 = 4.28 ± 0.43 h) [1]. RET-IN-16 (10 mg/kg; PO; single dose) revealed reduced maximum plasma concentration (Cmax = 194 ± 47 ng·h/mL) and drug exposure (AUC0-t = 2112 ± 117 ng·h /mL) [1]. RET-IN-16 (30 and 50 mg/kg; i.v.; daily; for 8 days) decreased tumor development in a dose-dependent manner and dramatically inhibited p-RET and p- in KIF5B-RET and KIF5B-RETV804M SHC is in tumor tissue and strongly promotes apoptosis in vivo [1]. Pharmacokinetic characteristics of RET-IN-16 in male Sprague-Dawley rats [1]. IV (1 mg/kg) PO (10 mg/kg) T1/2 (h) 4.28 ± 0.43 7.59 ± 1.02 Tmax (h) 0.083 ± 0 0.75 ± 0.43 Cmax (ng/mL) 6097 ± 623 194 ± 47 AUC0- t (ng/mL·h) 6959 ± 762 2112 ± 117 AUC0-∞ (ng/mL·h) 7014 ± 753 2343 ± 157 F (%) 3.0 |
| Cell Assay |
Cell proliferation assay Cell Types: LC-2/ad, A549, H3122, MDA-MB-231 and A375, SKGT4, HepG2, KYSE450 and BGC823 cells [1] Tested Concentrations: 1 μM Incubation Duration: 48 hrs (hours) Experimental Results: Effective inhibition of proliferation Contains CCDC6-RET fusion in LC-2/ad NSCLC cells, but Dramatically diminished efficacy against other RET-negative cancer cells. Western Blot Analysis Cell Types: KIF5B-RET and KIF5B-RETV804M Ba/F3 cells [1] Tested Concentrations: 50 and 100 μM Incubation Duration: 4 hrs (hours) Experimental Results: Dramatically blocked the autophosphorylation of RET and the phosphorylation of the adapter protein SHC also suppressed. Inhibited in a dose-dependent manner. |
| Animal Protocol |
Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rat[1] Doses: 1 mg/kg IV, 10 mg/kg PO Dosing: IV and PO; Single (pharmacokinetic/PK/PK Analysis) Experimental Results: Demonstrated good drug exposure at 1 mg/kg IV (AUC0-t = 6959 ± 762 ng·h/mL) and intermediate half-life (T1/2 = 4.28 ± 0.43 h); lower maximum plasma concentrations (Cmax = 194 ± 47 ng·h/mL) and Drug exposure (AUC0-t = 2112 ± 117 ng·h/mL). Animal/Disease Models: Half and half male and half female BALB/c-nu (nude) mice (6-8 weeks; injection of KIF5B-RET Ba/F3 and KIF5B-RETV804M Ba/F3) [1] Doses: 30 and 50 mg/kg Administration Route of Administration: IV; injection daily; 8 days Experimental Results: Inhibited tumor growth in a dose-dependent manner, Dramatically inhibited p-RET and p-SHC in KIF5B-RET and KIF5B-RETV804M in tumor tissues, and Dramatically induced cells in vivo Apoptosis. |
| References |
[1]. Discovery of 4-methyl-N-(4-((4-methylpiperazin- 1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-((6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-oxy)benzamide as a potent inhibitor of RET and its gatekeeper mutant. Eur J Med Chem. |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| 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 (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *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). 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)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL 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). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6594 mL | 8.2972 mL | 16.5945 mL | |
| 5 mM | 0.3319 mL | 1.6594 mL | 3.3189 mL | |
| 10 mM | 0.1659 mL | 0.8297 mL | 1.6594 mL |