| Size | Price | |
|---|---|---|
| 500mg | ||
| 1g | ||
| Other Sizes |
| Targets |
BRafV600E:14 nM (Kd); Braf:36 nM (Kd); CRAF:39 nM (Kd); c-Kit:2 nM (Kd); Ret:2 nM (Kd); LCK:2 nM (Kd); Abl-1:3 nM (Kd); VEGFR-2:8 nM (Kd); CSF-1R:9 nM (Kd); EPHA2:14 nM (Kd) EGFR:22 nM (Kd); c-Met:513 nM (Kd); JAK-2:4700 nM (Kd); MEK-1:7100 nM (Kd); MEK-2:8300 nM (Kd) |
|---|---|
| ln Vitro |
High potency against multiple BRAFV600E-dependent cell lines is demonstrated by Agerafenib (CEP-32496), which also demonstrates preferential cytotoxicity towards tumor cell lines expressing mutant BRAFV600E in contrast to those having wild-type BRAF. In the proliferation experiment, agerafenib (CEP-32496) demonstrates strong binding (BRAFV600E Kd=14 nM) and cellular activity (pMEK IC50=82 nM and A375 proliferation IC50=78 nM). With determined IC50 values greater than 10 μM against the CYP1A2, CYP2C9, CYP2D6, and CYP3A4 isoforms and an IC50=3.4 μM against CYP2C19, CEP-32496 also demonstrates a favorable CYP450 inhibition profile[1].
In vitro, Agerafenib hydrochloride inhibits BRAFV600E with a Kd of 14 nM and demonstrates potent antiproliferative activity against BRAF-mutant cancer cell lines. It also inhibits wild-type BRAF (Kd = 36 nM) and c-Raf (Kd = 39 nM), as well as c-Kit and c-RET. The compound blocks MEK/ERK phosphorylation and induces apoptosis in sensitive cancer cells. It is used as a reference inhibitor in RAF kinase assays. |
| ln Vivo |
Agerafenib (CEP-32496) significantly inhibits pMEK in tumor cell lysates when given orally to mice with Colo-205 tumor xenografts. Agerafenib (CEP-32496), for example, exhibits a 50 and 75% inhibition of normalized pMEK in tumor lysates at the 2 and 6 h postdose time point, respectively (p<0.03), following a single 30 mg/kg (po) dose. Conversely, a 55 mg/kg (po) dose produced a 75% to 57% (p <0.03) inhibition of pMEK at the 2 through 10 h post administration, with a 24 h normalization to baseline. The PK profile of agerafenib (CEP-32496) administration in mice, dogs, and cynomolgus monkeys is remarkable. beagle dogs (single dose of 1 mg/kg iv and 10 mg/kg po) produces good bioavailability (%F=100) and low clearance (CL=5.0 (mL/min)/kg). Similar to this, Agerafenib (CEP-32496) administration results in high oral exposure in cynomolgus monkeys because of its good bioavailability (% F=100) and poor clearance (CL=6.7 mL/min/kg) at a single dosage of 1 mg/kg po and 1 mg/kg iv[1].
In vivo, Agerafenib hydrochloride is orally bioavailable and demonstrates antitumor activity in preclinical xenograft models of BRAF-mutant cancers. Oral administration of the compound inhibits tumor growth in a dose-dependent manner and reduces ERK phosphorylation in tumor tissues. It is used as a tool for validating RAF kinases as therapeutic targets and for overcoming resistance to first-generation BRAF inhibitors. |
| Enzyme Assay |
For non-cellular kinase assays, recombinant BRAFV600E, wild-type BRAF, or c-Raf is incubated with a peptide substrate and ATP in kinase buffer. Serial dilutions of Agerafenib hydrochloride are added, and the reaction is incubated at 30°C for 30-60 minutes. ADP production is measured using the ADP-Glo Kinase Assay kit or a fluorescence polarization-based assay. Kd and IC50 values are determined from dose-response curves.
|
| Cell Assay |
A375 cells are seeded at 10,000 cells per well in DMEM with 10%
fetal calf serum and allowed to attach. The cells are washed with PBS
and switched to DMEM with 0.5% of serum and incubated overnight. The
test compounds (e.g., Agerafenib (CEP-32496); 10 μM) are then added at
various concentrations with a final DMSO concentration of 0.5% and
incubated for 72 h. At the end of incubation, a Cell Titer Blue is added
per instructions, and incubation is continued for 3 h. Remaining viable
cells are quantified by measuring the strength of the fluorescence
signal using SoftMax Pro (excitation at 560 nm and emission at 590 nm).
IC50 values are derived using a 9-point curve and are presented as mean
values from experiments performed in duplicate[1].
For cell-based assays, BRAF-mutant cancer cell lines (e.g., A375, Colo205) are cultured and treated with serial dilutions of Agerafenib hydrochloride for 72 hours. Cell viability is measured by MTT or CellTiter-Glo assays. MEK/ERK phosphorylation is assessed by Western blot. Apoptosis is evaluated by Annexin V/PI staining and caspase-3/7 activity assays. |
| Animal Protocol |
Mice[1] Six to eight week old athymic nu/nu nude mice (20-25 g)
are inoculated subcutaneously with Colo-205 tumor cells (1×106/mouse) in
the right flank. Upon reaching an average tumor volume of 150-200 mm3
(10-12 days post implantation), animals are randomized into treatment
groups (n=10 mice/group). Each group is dosed orally for 14 days with
either vehicle only (22% HPβCD) or with Agerafenib (CEP-32496) at 10,
30, or 100 mg/kg twice daily (BID), and each dose of drug is given in a
volume of 0.1 mL per 20 g of body weight, adjusted for the body weight
of the animal. Tumor volumes are measured three times weekly using
vernier calipers, and volumes are calculated[1].
For in vivo experiments, Agerafenib hydrochloride is formulated in a suitable vehicle (e.g., DMSO/PEG300/saline or methylcellulose) and administered orally to immunodeficient mice bearing BRAF-mutant tumor xenografts. Tumor volume is measured regularly, and body weight is monitored for toxicity. At study termination, tumors are harvested for Western blot analysis of ERK phosphorylation and immunohistochemical assessment of apoptosis and proliferation markers. |
| References | |
| Additional Infomation |
See also: Agerafenib (note moved to).
|
| Molecular Formula |
C₂₄H₂₃CLF₃N₅O₅
|
|---|---|
| Molecular Weight |
553.92
|
| Exact Mass |
553.134
|
| CAS # |
1227678-26-3
|
| Related CAS # |
Agerafenib;1188910-76-0
|
| PubChem CID |
56954675
|
| Appearance |
Typically exists as solid at room temperature
|
| LogP |
6.148
|
| tPSA |
127.35
|
| Hydrogen Bond Donor Count |
3
|
| Hydrogen Bond Acceptor Count |
11
|
| Rotatable Bond Count |
7
|
| Heavy Atom Count |
38
|
| Complexity |
776
|
| Defined Atom Stereocenter Count |
0
|
| InChi Key |
XCZIYUDQUDWQHI-UHFFFAOYSA-N
|
| InChi Code |
InChI=1S/C24H22F3N5O5.ClH/c1-23(2,24(25,26)27)19-11-20(32-37-19)31-22(33)30-13-6-5-7-14(8-13)36-21-15-9-17(34-3)18(35-4)10-16(15)28-12-29-21;/h5-12H,1-4H3,(H2,30,31,32,33);1H
|
| Chemical Name |
1-[3-(6,7-dimethoxyquinazolin-4-yl)oxyphenyl]-3-[5-(1,1,1-trifluoro-2-methylpropan-2-yl)-1,2-oxazol-3-yl]urea;hydrochloride
|
| Synonyms |
CEP-32496 hydrochlorideRXDX-105CEP-32496AC-01377
|
| 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)
|
| 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
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 | 1.8053 mL | 9.0266 mL | 18.0531 mL | |
| 5 mM | 0.3611 mL | 1.8053 mL | 3.6106 mL | |
| 10 mM | 0.1805 mL | 0.9027 mL | 1.8053 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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