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Purity: ≥98%
| Targets |
BRAF V600E (IC50 = 7 nM); CRAF (IC50 = 5 nM); B-Raf (IC50 = 56 nM)
BRAF (pan‑Raf inhibitor). No IC50, Ki, EC50, or DC50 values are reported in this paper. Belvarafenib targets the RAF family kinases, including B-RAF, B-RAF(V600E), and C-RAF, with IC₅₀ values of 56 nM, 7 nM, and 5 nM, respectively. It inhibits the MAPK signaling pathway through pan-RAF inhibition. |
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| ln Vitro |
Belvarafenib exhibits high selectivity toward BRAF mutant and CRAF kinases when biochemically analyzed for over 120 kinases. For the BRAFWT, BRAFV600E, and CRAF kinases, the half maximal inhibitory concentrations (IC50) of HM95573 are 41 nM, 7 nM, and 2 nM, respectively. It appears that CSF1R (44 nM), DDR1 (77 nM), and DDR2 (182 nM) are the next kinases to be severely inhibited after RAF kinases. Mutant melanoma cell lines for BRAF and NRAS, including A375 (IC50: 57 nM) and SK-MEL-28 (69 nM), as well as SK-MEL-2 (53 nM) and SK-MEL-30 (24 nM), both exhibit potent growth inhibition when treated with HM95573. In addition, treatment with HM95573 effectively inhibits the phosphorylations of MEK and ERK downstream kinases linked to cell proliferation in mutant BRAF and mutant NRAS melanoma cells. Even in the presence of HGF, which is known to be a mediator of innate resistance to RAF inhibitors, HM95573 inhibits downstream signaling in melanoma cells[1].
Treatment of SUM‑159 and MDA‑MB‑231 cells with Belvarafenib significantly reduced mRNA levels of PD‑L1 and PD‑L2 as determined by qRT‑PCR. [1] Protein levels of PD‑L1 were markedly decreased after BRAF inhibition, as shown by Western blot and immunofluorescence staining. [1] BRAF inhibition blocked ERK phosphorylation (p‑ERK), indicating that ERK is downstream of BRAF. [1] Belvarafenib treatment also reduced ETV4 mRNA and protein levels, consistent with the role of BRAF in ETV4 transcription. [1] In cell-free kinase assays, belvarafenib shows potent inhibition of RAF kinase activity. IC₅₀ values are 56 nM for wild-type B-RAF, 7 nM for the V600E mutant, and 5 nM for C-RAF. The compound demonstrates selectivity for RAF kinases over other kinases in panel screening. |
| ln Vivo |
Belvarafenib shows the excellent antitumor activity in mouse models that were xenografted with cell lines that had both BRAF mutations (such as A375 and SK-MEL-28) and NRAS mutations (such as SK-MEL-2 and SK-MEL-30)[1].
In cellular models, belvarafenib inhibits RAF-mediated signaling, leading to reduced cell proliferation in cancer cell lines with RAF pathway activation. The compound shows potent antiproliferative activity in BRAF-mutant and RAS-mutant cancer cell lines. Mechanistic studies demonstrate inhibition of ERK phosphorylation downstream of RAF. |
| Enzyme Assay |
The in vitro kinase assay for belvarafenib uses recombinant B-RAF, B-RAF(V600E), or C-RAF proteins incubated with a substrate peptide and ATP in the presence of varying compound concentrations. Kinase activity is measured by quantifying phosphorylated substrate using either radioactive (³³P-ATP) or luminescent (ADP-Glo) detection methods. IC₅₀ values are calculated from dose-response curves.
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| Cell Assay |
For 48 hours, the pan-Raf inhibitor belvarafenib was used to treat SUM-159 cells.
SUM‑159 and MDA‑MB‑231 cells were seeded and allowed to adhere for 24 hours, then treated with the pan‑Raf inhibitor Belvarafenib (purchased from Selleck Chemicals) for 48 hours at indicated doses (exact concentrations not specified in the text). After treatment, cells were harvested for RNA isolation and qRT‑PCR to measure PD‑L1, PD‑L2, and ETV4 mRNA levels. Protein lysates were subjected to Western blotting with antibodies against PD‑L1, phosphorylated ERK, total ERK (loading control not shown), ETV4, and HSP90 or HSP70 as loading controls. Immunofluorescence staining was performed on cells grown on glass slides, fixed with paraformaldehyde, permeabilized, blocked, and incubated with anti‑PD‑L1 primary antibody followed by Alexa Fluor‑conjugated secondary antibodies; nuclei were stained with Hoechst. Fluorescence signals were captured by microscopy. [1] Cancer cell lines (e.g., BRAF-mutant melanoma or colorectal cancer cells) are cultured in appropriate media and treated with belvarafenib at concentrations ranging from 0.1 nM to 10 µM for 48-72 hours. Cell viability is assessed by CellTiter-Glo or MTT assays. Signaling inhibition is confirmed by Western blot analysis of phosphorylated ERK and other downstream effectors. |
| Animal Protocol |
In vivo efficacy studies use immunocompromised mice bearing subcutaneous xenografts of BRAF-mutant or RAS-mutant tumors. Belvarafenib is administered orally once or twice daily at doses ranging from 1-100 mg/kg. Tumor volume is measured regularly, and endpoints include tumor growth inhibition, regression, and pharmacodynamic assessment of pathway inhibition in tumor tissue.
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| ADME/Pharmacokinetics |
Belvarafenib (C₂₃H₁₆ClFN₆OS, MW 478.93) is orally bioavailable. It is soluble in DMSO and has a purity of ≥98%. The compound is typically stored at -20°C as a powder. Detailed PK parameters including half-life, Cmax, and bioavailability have been characterized in preclinical studies.
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| Toxicity/Toxicokinetics |
The toxicity of Belvarafenib was not evaluated in this study. The compound was used as a pharmacological tool to inhibit BRAF; no adverse effects or toxicity data were reported.
In preclinical toxicology studies, belvarafenib was generally well-tolerated at therapeutic doses. No significant body weight loss or overt toxicity was observed in mouse xenograft studies at efficacious doses. The compound is for research use only and is not yet approved for clinical use. |
| References | |
| Additional Infomation |
Bevafenib is an orally administered Raf family serine/threonine protein kinase inhibitor with potential antitumor activity. After administration, bevafenib binds to and inhibits the activity of B-Raf mutants V600E and C-Raf. This inhibits the signal transduction pathways mediated by B-Raf V600E and C-Raf, thereby suppressing the growth of susceptible tumor cells. Furthermore, bevafenib may also inhibit mutant Ras proteins. Raf protein kinases play a crucial role in the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway, which is frequently aberrantly activated in human cancers and plays a vital role in tumor cell proliferation and survival. The Raf mutant B-Raf V600E, in which valine residue 600 is replaced by glutamate, is frequently overexpressed in various human tumors, leading to constitutive activation of the Raf/MEK/ERK signaling pathway.
Belvarafenib is referred to as a pan‑Raf inhibitor (belvarafenib) in the study. It was used to demonstrate that BRAF is an upstream kinase required for ERK activation and subsequent ETV4‑mediated PD‑L1/L2 expression via the PD‑L1L2‑SE. Inhibition of BRAF by this compound rendered cancer cells susceptible to T cell‑mediated killing in vitro, suggesting that targeting BRAF could be a potential strategy to disrupt SE‑driven immune evasion. [1] Belvarafenib is a pan-RAF inhibitor being developed for the treatment of RAF- and RAS-mutant cancers. It has been studied in preclinical models of melanoma, colorectal cancer, and other solid tumors. The compound represents a promising approach to overcome resistance to first-generation BRAF inhibitors. |
| Molecular Formula |
C23H16CLFN6OS
|
|---|---|
| Molecular Weight |
478.9291
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| Exact Mass |
478.08
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| Elemental Analysis |
C, 57.68; H, 3.37; Cl, 7.40; F, 3.97; N, 17.55; O, 3.34; S, 6.69
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| CAS # |
1446113-23-0
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| Related CAS # |
Belvarafenib TFA;2443966-84-3
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| PubChem CID |
89655386
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| Appearance |
Off-white to yellow solid powder
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| LogP |
4.9
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
8
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
33
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| Complexity |
708
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
KVCQTKNUUQOELD-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C23H16ClFN6OS/c1-11-5-6-13-12(7-8-27-22(13)30-16-4-2-3-15(24)17(16)25)18(11)31-23(32)14-9-33-20-19(14)28-10-29-21(20)26/h2-10H,1H3,(H,27,30)(H,31,32)(H2,26,28,29)
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| Chemical Name |
4-amino-N-[1-(3-chloro-2-fluoroanilino)-6-methylisoquinolin-5-yl]thieno[3,2-d]pyrimidine-7-carboxamide
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| Synonyms |
Belvarafenib; GDC-5573; GDC 5573; GDC5573; HM 95573; HM-95573; HM95573; RG-6185; RG 6185; RG6185
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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: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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: 12.5~96 mg/mL (26.1~200.5 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (2.61 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 12.5 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 | 2.0880 mL | 10.4399 mL | 20.8799 mL | |
| 5 mM | 0.4176 mL | 2.0880 mL | 4.1760 mL | |
| 10 mM | 0.2088 mL | 1.0440 mL | 2.0880 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.
| NCT Number | Recruitment | interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT04835805 | Recruiting | Drug: Belvarafenib Drug: Cobimetinib |
Melanoma | Genentech, Inc. | May 13, 2021 | Phase 1 |
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