Belvarafenib (GDC-5573, HM95573, RG6185) is a novel, potent and selective inhibitor of the RAF (Rapidly Accelerated Fibrosarcoma) family kinases, with IC50 values for B-RAF, B-RAFv600E, and C-RAF of 56 nM, 7 nM, and 5 nM, respectively. The MAPK pathway, which is frequently activated in human tumors and promotes tumor growth, was intended to be inhibited by the drug.

Physicochemical Properties

Molecular Formula	C23H16CLFN6OS
Molecular Weight	478.9291
Exact Mass	478.08
Elemental Analysis	C, 57.68; H, 3.37; Cl, 7.40; F, 3.97; N, 17.55; O, 3.34; S, 6.69
CAS #	1446113-23-0
Related CAS #	Belvarafenib TFA;2443966-84-3
PubChem CID	89655386
Appearance	Off-white to yellow solid powder
LogP	4.9
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	4
Heavy Atom Count	33
Complexity	708
Defined Atom Stereocenter Count	0
InChi Key	KVCQTKNUUQOELD-UHFFFAOYSA-N
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)
Chemical Name	4-amino-N-[1-(3-chloro-2-fluoroanilino)-6-methylisoquinolin-5-yl]thieno[3,2-d]pyrimidine-7-carboxamide
Synonyms	Belvarafenib; GDC-5573; GDC 5573; GDC5573; HM 95573; HM-95573; HM95573; RG-6185; RG 6185; RG6185
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 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)

Biological Activity

Targets	BRAF V600E (IC50 = 7 nM); CRAF (IC50 = 5 nM); B-Raf (IC50 = 56 nM)
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].
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].
Cell Assay	For 48 hours, the pan-Raf inhibitor belvarafenib was used to treat SUM-159 cells.
References	[1]. AACR Cancer Res. 2015, 75(15 Suppl):Abstract nr 2606. [2]. Cancer Biol Med . 2021 Oct 9;19(5):669-684. [3]. Cancer Res (2015) 75 (15_Supplement): 2606.
Additional Infomation	Belvarafenib is an orally available inhibitor of members of the Raf family of serine/threonine protein kinases, with potential antineoplastic activity. Upon administration, belvarafenib binds to and inhibits the B-Raf mutant V600E and C-Raf. This inhibits B-Raf V600E- and C-Raf-mediated signal transduction pathways, thereby inhibiting tumor cell growth of susceptible tumor cells. In addition, belvarafenib may also inhibit mutated Ras proteins. Raf protein kinases play a key role in the Raf/mitogen-activated protein kinase kinase (MEK)/extracellular signal-related kinase (ERK) signaling pathway, which is often dysregulated in human cancers and plays a key role in tumor cell proliferation and survival. The Raf mutation B-Raf V600E, where the valine at residue 600 is substituted for glutamic acid, is frequently overexpressed in a variety of human tumors and results in the constitutive activation of the Raf/MEK/ERK signaling pathway.

Solubility Data

Solubility (In Vitro)

DMSO: 12.5~96 mg/mL (26.1~200.5 mM)

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.