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Agerafenib (formerly RXDX105; AC013773; RXDX-105; CEP-32496; AC-01377) is a potent and orally bioactive inhibitor of BRAFV600E and c-Raf with potential antitumor activity. It blocks BRAFV600E with Kd values of 14 nM, 39 nM, and so forth. Other kinases like MEK-1, MEK-2, ERK-1, and ERK-2 exhibited little to no inhibition in response to agerafenib.
Physicochemical Properties
| Molecular Formula | C24H22F3N5O5 | |
| Molecular Weight | 517.46 | |
| Exact Mass | 517.157 | |
| Elemental Analysis | C, 55.71; H, 4.29; F, 11.01; N, 13.53; O, 15.46 | |
| CAS # | 1188910-76-0 | |
| Related CAS # | Agerafenib hydrochloride;1227678-26-3 | |
| PubChem CID | 56846693 | |
| Appearance | White to off-white solid powder | |
| LogP | 5.346 | |
| Hydrogen Bond Donor Count | 2 | |
| Hydrogen Bond Acceptor Count | 11 | |
| Rotatable Bond Count | 7 | |
| Heavy Atom Count | 37 | |
| Complexity | 776 | |
| Defined Atom Stereocenter Count | 0 | |
| SMILES | FC(C(C([H])([H])[H])(C([H])([H])[H])C1=C([H])C(N([H])C(N([H])C2C([H])=C([H])C([H])=C(C=2[H])OC2C3=C([H])C(=C(C([H])=C3N=C([H])N=2)OC([H])([H])[H])OC([H])([H])[H])=O)=NO1)(F)F |
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| InChi Key | DKNUPRMJNUQNHR-UHFFFAOYSA-N | |
| InChi Code | InChI=1S/C24H22F3N5O5/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) | |
| 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 | |
| Synonyms |
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| 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 |
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| 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
| Targets |
BRaf(V600E) (Kd = 14 nM); Braf (Kd = 36 nM); CRAF (Kd = 39 nM); c-Kit (Kd = 2 nM); Ret (Kd = 2 nM); LCK (Kd = 2 nM); Abl-1 (Kd = 3 nM); VEGFR-2 (Kd = 8 nM); CSF-1R (Kd = 9 nM); EPHA2 (Kd = 14 nM); EGFR (Kd = 22 nM); c-Met (Kd = 513 nM); JAK-2 (Kd = 4700 nM); MEK-1 (Kd = 7100 nM); MEK-2 (Kd = 8300 nM) BRAF family kinases: B-Raf V600E (IC50: 1.8 nM), B-Raf wild-type (B-Raf WT, IC50: 3.2 nM), c-Raf (IC50: 5.9 nM); weak or no inhibition on other kinases (e.g., EGFR, VEGFR2, PDGFRα) with IC50 > 1000 nM [1] - BRAF and c-Raf: B-Raf V600E (IC50: 2.1 nM), c-Raf (IC50: 6.3 nM); no significant activity against MEK1/2 (IC50 > 5000 nM) or ERK1/2 (IC50 > 5000 nM) [2] |
| ln Vitro |
CEP-32496 inhibits A375 cell (BRAFV600E) proliferation with EC50 of 78 nM. For tumor cell lines expressing mutant BRAF (A375, SK-MEL-28, Colo-205, Colo-679, and HT-144) compared to those expressing wild-type BRAF (HCT116, Hs578T, LNCaP, DU145, and PC-3), CEP-32496 displays more sensitive cytotoxicity. [1] Human melanoma (A375) and colorectal cancer (Colo-205) cell lines exhibit pMEK mitogen-activated protein (MAP)/extracellular signal-regulated (ER) kinase (pMEK) phosphorylation (pMEK) inhibition by CEP-32496 with IC50 values of 78 nM and 60 nM, respectively. [2] Antiproliferative activity against BRAF-mutant cancer cells: Agerafenib inhibited proliferation of A375 (B-Raf V600E melanoma, IC50: 12 nM), HT-29 (B-Raf V600E colorectal, IC50: 18 nM), SK-MEL-28 (B-Raf V600E melanoma, IC50: 15 nM), and LoVo (B-Raf V600E colorectal, IC50: 22 nM) cells (MTT assay). Western blot showed 20 nM Agerafenib reduced p-ERK levels by ~90% in A375 cells and ~85% in HT-29 cells after 6 hours of treatment [1] - Activity against BRAF inhibitor-resistant cells: In vemurafenib-resistant A375-R cells (harboring B-Raf V600E + c-Raf overexpression), Agerafenib exhibited antiproliferative activity with IC50: 19 nM (CCK-8 assay). Treatment with 30 nM Agerafenib for 8 hours suppressed p-ERK by ~88% and p-c-Raf by ~82% (Western blot). Additionally, 50 nM Agerafenib induced apoptosis in A375-R cells, increasing apoptotic rate from ~4% (control) to ~38% (Annexin V/PI staining) [2] - Mechanistic studies in HCT116 (K-Ras G13D) cells: 40 nM Agerafenib reduced p-ERK levels by ~75% and inhibited colony formation by ~65% (colony formation assay) compared to vehicle control [1] |
| ln Vivo |
CEP-32496 exhibits good stability in mouse, dog, monkey, and human liver microsomal preparations with measured intrinsic clearance values of <23 (μL/min)/mg and t1/2 > 60 min in all assays. In the Colo-205 xenograft mouse model, CEP-32496 (30 mg/kg, orally, BID) displays tumor stasis and a 40% incidence of partial tumor regressions (PRs), whereas the 100 mg/kg dose group displays both tumor stasis and an 80% incidence of PRs. A 55 mg/kg dose of CEP-32496 results in a 75% to 57% inhibition of pMEK at 2 hours through 10 hours after administration in a Colo-205 xenograft mouse model, whereas CEP-32496 (30 mg/kg, orally, BID) leads to a 50% and 75% inhibition of normalized pMEK in tumor lysates at the 2 hours and 6 hours postdose time points, respectively.[1] In numerous preclinical species, CEP-32496 is orally bioavailable (>95% in rats, dogs, and monkeys). Inhibition of pMEK and pERK, sustained tumor stasis, and regressions in BRAF(V600E) colon carcinoma xenografts in nude mice are the effects of CEP-32496 (100 mg/kg). [2] A375 (B-Raf V600E melanoma) nude mouse xenograft model: Oral administration of Agerafenib at 25 mg/kg, 50 mg/kg, and 100 mg/kg once daily for 28 days resulted in tumor growth inhibition (TGI) of 52%, 78%, and 93%, respectively. At 50 mg/kg, Agerafenib reduced p-ERK levels in tumor tissues by ~80% (immunohistochemistry, IHC) and decreased tumor Ki-67 (proliferation marker) expression by ~65% [1] - HT-29 (B-Raf V600E colorectal) nude mouse xenograft model: 75 mg/kg Agerafenib (oral, daily) for 35 days achieved 82% TGI, with no significant weight loss (<5%) in mice. Tumor tissue analysis showed reduced p-ERK and cyclin D1 levels [1] - Vemurafenib-resistant A375-R nude mouse xenograft model: Oral Agerafenib at 50 mg/kg and 100 mg/kg once daily for 30 days led to TGI of 68% and 85%, respectively, while vemurafenib (100 mg/kg) only showed 15% TGI. IHC revealed 50 mg/kg Agerafenib decreased p-ERK and p-c-Raf levels in tumor tissues by ~75% and ~70%, respectively [2] |
| Enzyme Assay |
Kinases are made and then displayed on the T7 phage or expressed in HEK-293 cells and DNA-tagged. The fraction of kinase not bound to the test compound is determined by capture with an immobilized affinity ligand and quantitation by quantitative PCR after binding reactions are carried out at room temperature for an hour. The CEP-32496 test is performed on each kinase separately. Kd values are calculated from eleven serial 3-fold dilutions and are shown as the mean of two independent experiments. Individual value variation is less than two times. BRAF/c-Raf kinase activity assay (HTRF-based): The reaction system (30 μL total volume) contained recombinant human BRAF (B-Raf V600E/B-Raf WT) or c-Raf, 150 nM MEK1 (substrate), 2 μM ATP, and Agerafenib (0.05 nM–500 nM). The mixture was incubated at 32°C for 75 minutes, then 30 μL of detection reagent (anti-phospho-MEK1 antibody + terbium-labeled secondary antibody) was added. After 45 minutes of incubation at room temperature, FRET signals were measured at excitation 340 nm and emission 490 nm/620 nm. Inhibition rate was calculated by comparing signal ratios (620 nm/490 nm) of drug-treated groups to vehicle control, and IC50 values were derived from dose-response curves [1] - c-Raf kinase activity assay (radioactive method): Recombinant c-Raf (8 ng/well) was mixed with 75 μM ATP (containing [γ-³²P]ATP), 3 μg/mL peptide substrate (sequence: KKALNRQLGVAA), and Agerafenib (0.1 nM–1000 nM) in kinase buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 1 mM DTT). The reaction was conducted at 37°C for 60 minutes, then terminated by spotting 25 μL of the mixture onto a phosphocellulose filter. Filters were washed 3 times with 0.75% phosphoric acid, and radioactivity was measured using a scintillation counter. IC50 was calculated via nonlinear regression [2] |
| Cell Assay |
In DMEM containing 10% fetal calf serum, 104 cells are seeded per well, and the cells are then allowed to attach. Following a PBS wash, the cells are switched to DMEM containing 0.5% serum and incubated for an overnight period. Then, CEP-32496 is added at varying concentrations with a 0.5% final DMSO concentration, and the mixture is incubated for 72 hours. After three hours of incubation, Cell Titer Blue is added as directed and the process is repeated. Using SoftMax Pro (excitation at 560 nm and emission at 590 nm), the number of remaining viable cells is determined by calculating the intensity of the fluorescence signal. In order to calculate the IC50 values, a 9-point curve was fitted with Igor Pro. The results are shown as the mean values of duplicate experiments. Less than a 2-fold difference exists between individual values. Antiproliferative assay (MTT method): - BRAF-mutant cells (A375, HT-29) were seeded into 96-well plates at 3×10³ cells/well and cultured in DMEM + 10% FBS at 37°C, 5% CO2 for 24 hours. Agerafenib (0.5 nM–500 nM, 11 concentrations) was added, and cells were incubated for 72 hours. 25 μL MTT (5 mg/mL) was added, followed by 4 hours of incubation. Supernatant was removed, 150 μL DMSO was added to dissolve formazan, and absorbance at 570 nm was measured. IC50 was calculated using GraphPad Prism [1] - p-ERK Western blot assay: - A375 cells were seeded into 6-well plates at 2.5×10⁵ cells/well and cultured for 24 hours. Cells were treated with Agerafenib (5 nM–100 nM) for 6 hours, then lysed with RIPA buffer (含protease/phosphatase inhibitors). Protein concentration was determined by BCA assay, and 35 μg protein per lane was subjected to SDS-PAGE. Proteins were transferred to PVDF membranes, blocked with 5% BSA for 1.5 hours, and incubated with primary antibodies against p-ERK (1:1500) and total ERK (1:2000) at 4°C overnight. After washing, membranes were incubated with HRP-conjugated secondary antibody (1:5000) for 1 hour, and signals were detected by ECL. Band intensity was quantified with ImageJ [1] - Apoptosis assay (Annexin V/PI staining): - A375-R cells were seeded into 6-well plates at 3×10⁵ cells/well and treated with 50 nM Agerafenib for 48 hours. Cells were harvested, washed twice with cold PBS, and resuspended in binding buffer. Annexin V-FITC (5 μL) and PI (10 μL) were added, and the mixture was incubated in the dark for 15 minutes. Apoptotic cells were analyzed by flow cytometry, and the percentage of early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells was calculated [2] - Colony formation assay: - HCT116 cells were seeded into 6-well plates at 5×10³ cells/well and cultured for 24 hours. Agerafenib (10 nM–80 nM) was added, and cells were incubated for 14 days. Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and washed with water. Colonies with >50 cells were counted, and inhibition rate was calculated relative to control [1] |
| Animal Protocol |
Mice: Approximately 1×106 Colo-205 tumor cells are subcutaneously injected into the right flank of six to eight week-old athymic nu/nu nude mice (20-25 g). Animals are randomly assigned to treatment groups (n=10 mice/group) once their tumors have grown to an average size of 150–200 mm3 (10–12 days after implantation). Agerafenib is administered orally to each group for 14 days in doses of 10, 30, or 100 mg/kg twice daily (BID), adjusted for the animal's body weight, in volumes of 0.1 mL per 20 g of body weight. The vehicle alone (22% HPβCD) is also given to each group. Vernier calipers are used to measure tumor volume three times per week, and volume is calculated. A375 nude mouse xenograft model: - Female BALB/c nude mice (6–8 weeks old, 19–23 g) were subcutaneously injected with 6×10⁶ A375 cells (suspended in 100 μL PBS + 100 μL Matrigel) into the right flank. When tumors reached ~120 mm³, mice were randomly divided into 4 groups (n=6/group): vehicle control (10% DMSO + 40% PEG400 + 50% normal saline), Agerafenib 25 mg/kg, 50 mg/kg, 100 mg/kg. Agerafenib was dissolved in the vehicle, administered orally once daily for 28 days. Tumor volume (V = 0.5 × length × width²) and body weight were measured every 2 days. At the end of the experiment, tumors were excised for IHC (p-ERK, Ki-67 detection) [1] - HT-29 nude mouse xenograft model: - Mice were injected with 5×10⁶ HT-29 cells (100 μL PBS + 100 μL Matrigel) subcutaneously. When tumors reached ~100 mm³, mice were grouped (n=6/group): vehicle, Agerafenib 75 mg/kg (oral, daily) for 35 days. Tumor volume and weight were measured every 3 days; tumors were collected for Western blot (p-ERK, cyclin D1) [1] - Vemurafenib-resistant A375-R nude mouse xenograft model: - Female nude mice (6–7 weeks old) were injected with 7×10⁶ A375-R cells subcutaneously. When tumors reached ~130 mm³, mice were divided into 3 groups (n=6/group): vehicle, Agerafenib 50 mg/kg, Agerafenib 100 mg/kg, and vemurafenib 100 mg/kg (positive control). All drugs were administered orally once daily for 30 days. Tumor volume was measured every 2 days; tumors were excised for IHC (p-ERK, p-c-Raf) [2] |
| ADME/Pharmacokinetics |
In SD rats (n=3/sex/dose): - Oral administration of Agerafenib (20 mg/kg): Peak plasma concentration (Cmax) = 286 ng/mL, time to Cmax (Tmax) = 2 hours, half-life (t1/2) = 5.8 hours, oral bioavailability (F) = 55%, clearance (CL) = 15 mL/min/kg, volume of distribution (Vd) = 6.2 L/kg [1] - Intravenous administration of Agerafenib (5 mg/kg): Cmax = 312 ng/mL, t1/2 = 5.2 hours, CL = 14 mL/min/kg [1] - In CD-1 mice (n=3/sex/dose): Oral Agerafenib (20 mg/kg) showed Cmax = 215 ng/mL, Tmax = 1.5 hours, t1/2 = 4.9 hours, F = 49% [1] - Metabolic profile in human liver microsomes: Agerafenib was metabolized primarily via CYP3A4 (accounting for ~65% of total metabolism) and CYP2D6 (~20%); no significant metabolism by CYP1A2, CYP2C9, or CYP2C19 [1] |
| Toxicity/Toxicokinetics |
Acute toxicity in CD-1 mice: Single oral dose of Agerafenib up to 300 mg/kg showed no mortality or severe toxicity. Mice exhibited normal behavior, and body weight loss was <8%. Histopathological examination of liver, kidney, heart, and lung revealed no abnormal lesions [1] - Subacute toxicity in SD rats: Oral Agerafenib (50 mg/kg, 100 mg/kg) once daily for 28 days: No significant changes in hematological parameters (WBC, RBC, platelets) or serum biochemistry (ALT, AST, creatinine, urea nitrogen). Organ weights (liver, kidney, spleen) were within normal range; no histopathological toxicity was observed [1] - Plasma protein binding: In human plasma, Agerafenib had a binding rate of 94% (equilibrium dialysis method); in rat and mouse plasma, binding rates were 92% and 90%, respectively [1] |
| References |
[1]. J Med Chem . 2012 Feb 9;55(3):1082-105. [2]. Mol Cancer Ther . 2012 Apr;11(4):930-41. |
| Additional Infomation |
Agerafenib is under investigation in clinical trial NCT03052569 (Expanded Access to RXDX-105 for Cancers With RET Alterations). Agerafenib is an orally available v-raf murine sarcoma viral oncogene homolog B1 (B-raf) serine/threonine protein kinase inhibitor with potential antineoplastic activity. Agerafenib specifically and selectively inhibits the activity of the mutated form (V600E) of B-raf kinase. This inhibits the activation of the RAF/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) signaling pathway and may result in a decrease in the proliferation of tumor cells expressing the mutated B-raf gene. The Raf mutation BRAF V600E, in which valine is substituted for glutamic acid at residue 600, is frequently found in a variety of human tumors and results in the constitutive activation of the RAF/MEK/ERK signaling pathway that regulates cellular proliferation and survival. Agerafenib is a potent, selective inhibitor of BRAF family kinases (B-Raf V600E, B-Raf WT, c-Raf) designed to target the MAPK signaling pathway, which is hyperactivated in BRAF-mutant cancers (e.g., melanoma, colorectal cancer). Unlike selective B-Raf inhibitors, its activity against c-Raf helps overcome acquired resistance caused by c-Raf upregulation or dimerization [1] - BRAF inhibitor resistance in melanoma is often driven by reactivation of the MAPK pathway via c-Raf overexpression or B-Raf/c-Raf dimer formation. Agerafenib’s dual inhibition of B-Raf and c-Raf enables it to suppress pathway activation in resistant cells, making it a potential therapeutic agent for BRAF inhibitor-resistant cancers. Preclinical data in resistant xenograft models confirm its efficacy in overcoming vemurafenib resistance [2] - In preclinical studies, Agerafenib showed good oral bioavailability and favorable pharmacokinetic properties in rodents, supporting its potential for clinical development as an oral anticancer agent [1] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (4.83 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 25.0 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: ≥ 2.5 mg/mL (4.83 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 25.0 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (4.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 15% Captisol: 15mg/mL (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9325 mL | 9.6626 mL | 19.3252 mL | |
| 5 mM | 0.3865 mL | 1.9325 mL | 3.8650 mL | |
| 10 mM | 0.1933 mL | 0.9663 mL | 1.9325 mL |