B-Raf IN 1 is a novel, potent and selective B-Raf inhibitor with IC50 of 24 nM; it is equally potent against c-Raf with IC50 of 25 nM. Moderate selectivity was observed for compound 10n versus p38a (IC50: 0.216 lM) and CAMKII (IC50: 0.822 lM), while high selectivity was observed versus CDK2, CDK4, PKCa, IKKb, JNK1, MK2, PKA, Src, MKK6, PLK1, p70S6K, PI3 Ka, and PDK1 (IC50s: >2 lM). It binds to B-Raf kinase without forming a hinge-binding hydrogen bond. With basic amine residues appended to C-3 aryl residues, cellular activity and solubility were enhanced over previously described compounds of this class.

Physicochemical Properties

Molecular Formula	C29H24F3N5O
Molecular Weight	515.53
Exact Mass	515.193
CAS #	950736-05-7
Related CAS #	950736-05-7
PubChem CID	24884503
Appearance	Light yellow to yellow solid powder
Density	1.3±0.1 g/cm3
Index of Refraction	1.621
LogP	4.39
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	38
Complexity	787
Defined Atom Stereocenter Count	0
InChi Key	AIWJVLQNYNCDSL-UHFFFAOYSA-N
InChi Code	InChI=1S/C29H24F3N5O/c1-36(2)18-19-9-11-20(12-10-19)25-17-34-37-26(13-14-33-27(25)37)21-5-4-8-24(16-21)35-28(38)22-6-3-7-23(15-22)29(30,31)32/h3-17H,18H2,1-2H3,(H,35,38)
Chemical Name	N-[3-[3-[4-[(dimethylamino)methyl]phenyl]pyrazolo[1,5-a]pyrimidin-7-yl]phenyl]-3-(trifluoromethyl)benzamide
Synonyms	B-Raf-IN-1; B-Raf-IN 1; B-Raf-IN1
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

Targets	B-Raf IN 1 (compound 10n) targets Proto-Oncogene Proteins B-raf (B-Raf kinase) [1]
ln Vitro	WM 266-4 and HT29 cells are inhibited by B-Raf IN 1 (Compound 10n) at IC50s of 0.92 and 0.78 μM, respectively[1]. 1. B-Raf IN 1 (compound 10n), a C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamide, was identified as a potent B-Raf kinase inhibitor [1] 2. X-ray crystallography analysis demonstrated that B-Raf IN 1 bound to B-Raf kinase without forming a hinge-binding hydrogen bond, which is a distinct binding mode compared with conventional B-Raf kinase inhibitors [1] 3. The introduction of basic amine residues to the C-3 aryl residues of pyrazolo[1,5-a]pyrimidines (the structural class of B-Raf IN 1) enhanced cellular activity and solubility relative to previously reported compounds of the same class [1]
ln Vivo
Enzyme Assay	1. X-ray crystallography assay for B-Raf kinase binding mode: The binding interaction between B-Raf IN 1 and B-Raf kinase was characterized using X-ray crystallography. The experiment was designed to determine the structural basis of the inhibitor's binding to the kinase, focusing on whether hydrogen bonds were formed at the hinge region of B-Raf kinase. The results confirmed that B-Raf IN 1 bound to B-Raf kinase without forming a hinge-binding hydrogen bond [1] 2. Structure-activity relationship (SAR) assay for pyrazolo[1,5-a]pyrimidines: A series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides (including B-Raf IN 1) were synthesized and evaluated for their B-Raf kinase inhibitory activity. The impact of structural modifications (e.g., appending basic amine residues to C-3 aryl residues) on the potency, cellular activity and solubility of the compounds was analyzed [1]
Animal Protocol
References	[1]. Non-hinge-binding pyrazolo[1,5-a]pyrimidines as potent B-Raf kinase inhibitors. Bioorg Med Chem Lett. 2009 Dec 1;19(23):6519-23.
Additional Infomation	1. B-Raf IN 1 belongs to a series of C-3 substituted N-(3-(pyrazolo[1,5-a]pyrimidin-7-yl)phenyl)-3-(trifluoromethyl)benzamides, which are designed as B-Raf kinase inhibitors [1] 2. The non-hinge-binding mode of B-Raf IN 1 to B-Raf kinase is a key structural feature that distinguishes it from traditional B-Raf kinase inhibitors [1] 3. Structural optimization of pyrazolo[1,5-a]pyrimidines (including B-Raf IN 1) by adding basic amine residues to C-3 aryl residues addresses the limitations (e.g., low cellular activity, poor solubility) of earlier compounds in this class [1]

Solubility Data

Solubility (In Vitro)

DMSO:≥ 53 mg/mL Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 3.25 mg/mL (6.30 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 + to the above solution and mix evenly; then add 450 μL of 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	1.9398 mL	9.6988 mL	19.3975 mL
	5 mM	0.3880 mL	1.9398 mL	3.8795 mL
	10 mM	0.1940 mL	0.9699 mL	1.9398 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.