GNF-2 (GNF 2; GNF2) is a highly potent, selective and allosteric/non-ATP competitive inhibitor of Bcr-Abl with potential anticancer activity. It shows no activity against Flt3-ITD, Tel-PDGFR, TPR-MET and Tel-JAK1 transformed tumor cells. GNF-2 acts by allosterically binding the myristate-binding site of ABL and inhibits the proliferation of BCR-ABL positive cell and induces cell apoptosis. GNF-2 eliminated transplanted-CML-T315I-mutants in vivo and dose dependently sensitized primary cells from CML T315I patients to GNF-2-induced proliferation inhibition and apoptosis

Physicochemical Properties

Molecular Formula	C18H13F3N4O2
Molecular Weight	374.32
Exact Mass	374.099
CAS #	778270-11-4
Related CAS #	778270-11-4
PubChem CID	5311510
Appearance	White to off-white solid powder
Density	1.4±0.1 g/cm3
Boiling Point	536.4±50.0 °C at 760 mmHg
Flash Point	278.2±30.1 °C
Vapour Pressure	0.0±1.4 mmHg at 25°C
Index of Refraction	1.611
LogP	3.68
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	8
Rotatable Bond Count	5
Heavy Atom Count	27
Complexity	498
Defined Atom Stereocenter Count	0
InChi Key	WEVYNIUIFUYDGI-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H13F3N4O2/c19-18(20,21)27-14-6-4-13(5-7-14)25-16-9-15(23-10-24-16)11-2-1-3-12(8-11)17(22)26/h1-10H,(H2,22,26)(H,23,24,25)
Chemical Name	3-(6-((4-(trifluoromethoxy)phenyl)amino)pyrimidin-4-yl)benzamide
Synonyms	GNF-2; GNF 2; GNF2;
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

ln Vitro	GNF-2 inhibits Bcr-abl-dependent cell proliferation in a specific manner. GNF-2 (0.005-10 μM; 48 hours) does not exhibit any cytotoxic effects at concentrations up to 10 μM on nontransformed cells, but it specifically inhibits the proliferation of Bcr-abl-expressing cells with an IC50 of 138 nM. The Bcr-abl-positive cell lines exhibit a dose-dependent growth inhibition in response to GNF-2 (0.005-10 μM; 48 hours), with IC50 values of 273 nM (K562) and 268 nM (SUP-B15). E255V and Y253H mutant Bcr-abl cell growth is inhibited by GNF-2 (0.005-10 μM; 48 hours) (IC50 values of 268 and 194 nM, respectively)[1]. Bcr-abl-transformed cells undergo apoptosis when exposed to GNF-2 (1–10 μM) for 48 hours[1]. With an IC50 of 267 nM, GNF-2 (0.1–10 μM; 90 minutes) inhibits Bcr-abl's cellular tyrosine phosphorylation in a dose-dependent manner[1].
ln Vivo	In mice, GNF-2 (10 mg/kg; ip for 8 days) prevents bone degradation caused by LPS (5 mg/kg). GNF-2 prevents LPS-induced bone loss and reverses LPS-induced reductions in the BV/TV (bone volume/tissue volume) of mice exposed to LPS[2]. GNF-2 inhibits the increases in N.Oc/B.Pm, Oc.S/BS, and ES/BS that are brought on by LPS[2].
Cell Assay	Cell Proliferation Assay[1] Cell Types: Ba/F3.p210, Ba/F3.p210E255V and Ba/F3.p185Y253H cells Tested Concentrations: 0.005, 0.01, 0.1, 1, 10 μM Incubation Duration: 48 hrs (hours) Experimental Results: Inhibited Bcr-abl-transformed cells proliferation. Apoptosis Analysis[1] Cell Types: Ba/F3.p210 and Ba/F3.p210E255V cells Tested Concentrations: 1, 10 μM Incubation Duration: 48 hrs (hours) Experimental Results: Increased number of Ba/F3 .p210 cells underwent apoptosis at 1 μM for 48 h. Ba/F3.p210E255V underwent apoptotic death after 48 h incubation in the presence of 1 μM or higher concentration. Western Blot Analysis[1] Cell Types: Ba/F3.p210 and Ba /F3.p210E255V cells Tested Concentrations: 0.1, 1, 10 μM Incubation Duration: 90 minutes Experimental Results: diminished the autophosphorylation levels at a concentration of 1 μM and were barely detectable at 10 μM, whereas the level of total Bcr-abl remained unchanged. Induced a significant decrease in the levels of p-Stat5 (at Y694) at 1 μM in Ba/F3.p210 and Ba/F3.p210E255V cells.
Animal Protocol	Animal/Disease Models: Eightweeks old C57/BL6 black mouse were administered ip injections of LPS (5 mg/kg)[2] Doses: 10 mg/kg Route of Administration: Ip injections for 8 days; 1 day before and every day after the LPS injection Experimental Results: Prevented inflammatory bone destruction in vivo.
References	[1]. Allosteric inhibitors of Bcr-abl-dependent cell proliferation. Nat Chem Biol. 2006 Feb;2(2):95-102. [2]. The tyrosine kinase inhibitor GNF-2 suppresses osteoclast formation and activity. J Leukoc Biol. 2013 Oct 15.
Additional Infomation	3-[6-[4-(trifluoromethoxy)anilino]-4-pyrimidinyl]benzamide is a member of pyrimidines.

Solubility Data

Solubility (In Vitro)

DMSO: 74 mg/mL (197.69 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.68 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 (6.68 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 (6.68 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6715 mL	13.3576 mL	26.7151 mL
	5 mM	0.5343 mL	2.6715 mL	5.3430 mL
	10 mM	0.2672 mL	1.3358 mL	2.6715 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.