G-749 (G749) is a novel and potent FLT3 inhibitor with potential antineoplastic activity. With IC50 values of 0.4 nM, 0.6 nM, and 1 nM, respectively, it inhibits FLT3 (WT), FLT3 (D835Y), and Mer. Both in vitro and in vivo antitumor efficaciousness of G-749 are demonstrated, along with strong anti-proliferative activity. In a number of drug-resistant environments, including patient plasma, FLT3 ligand surge, and stromal protection, G-749 maintained its inhibitory efficacy.With IC50 values of 0.4 nM, 0.6 nM, and 1 nM, respectively, it inhibits FLT3 (WT), FLT3 (D835Y), and Mer. Both in vitro and in vivo antitumor efficaciousness of G-749 are demonstrated, along with strong anti-proliferative activity. In a number of drug-resistant environments, including patient plasma, FLT3 ligand surge, and stromal protection, G-749 maintained its inhibitory efficacy.

Physicochemical Properties

Molecular Formula	C25H25BRN6O2
Molecular Weight	521.41
Exact Mass	520.122
Elemental Analysis	C, 57.59; H, 4.83; Br, 15.32; N, 16.12; O, 6.14
CAS #	1457983-28-6
Related CAS #	1457983-28-6
PubChem CID	78357765
Appearance	White to off-white solid powder
Density	1.487±0.06 g/cm3
Index of Refraction	1.701
LogP	2.86
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	6
Heavy Atom Count	34
Complexity	716
Defined Atom Stereocenter Count	0
SMILES	O=C1C2=C(N=C(NC3CCN(C)CC3)N=C2NC4=CC=C(OC5=CC=CC=C5)C=C4)C(Br)=CN1
InChi Key	SXWMIXPJPNCXQQ-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H25BrN6O2/c1-32-13-11-17(12-14-32)29-25-30-22-20(26)15-27-24(33)21(22)23(31-25)28-16-7-9-19(10-8-16)34-18-5-3-2-4-6-18/h2-10,15,17H,11-14H2,1H3,(H,27,33)(H2,28,29,30,31)
Chemical Name	8-bromo-2-[(1-methylpiperidin-4-yl)amino]-4-(4-phenoxyanilino)-6H-pyrido[4,3-d]pyrimidin-5-one
Synonyms	denfivontinib; Denfivontinib; G-749; G 749; G749
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	FLT3 (IC50 = 0.4 nM); FLT3 (IC50 = 0.6 nM); Mer (IC50 = 1 nM); Aurora B (IC50 = 6 nM); RET (IC50 = 9 nM) Colony-Stimulating Factor 1 Receptor (CSF1R) [1]
ln Vitro	G-749 can effectively prevent FLT3 autophosphorylation in FLT3-WT-bearing RS4-11 cells, FLT3-ITD-bearing MV4-11, and Molm-14 cells, with an IC50 of less than 8 nM. G-749 induces apoptosis in leukemia cells to demonstrate antiproliferative activity. G-749 demonstrates potent inhibitory activity with an IC50 of less than 10 nM in BaF3 cell lines that stably express FLT3-ITD/N676D, FLT3-ITD/F691L, FLT3-D835Y, or FLT3-D835Y/N676D, thereby surmounting drug resistance. G-749 also shows strong antileukemia activity in blasts from AML patients.[1] Denfivontinib (G-749) inhibited CSF1R phosphorylation in CSF1-stimulated human monocytes and tenosynovial giant cell tumor (TGCT)-derived cells, which abrogated downstream AKT and ERK1/2 signaling pathways [1] It exhibited concentration-dependent antiproliferative effects on CSF1R-positive cells, with no significant growth inhibition observed in CSF1R-negative cell lines [1] Western blot analysis confirmed reduced phosphorylation of CSF1R and its downstream effectors in treated cells compared to untreated controls [1]
ln Vivo	G-749 (30 mg/kg p.o.) significantly reduces tumor growth and inhibits the FLT3 pathway in MV4-11 xenograft mice. Using Molm-14 cells in an orthogonal model of bone marrow engraftment, G-749 (20 mg/kg p.o.) also reduces tumor growth and improves survival.[1] Oral administration of Denfivontinib (G-749) induced dose-dependent tumor regression in patient-derived TGCT xenograft models in nude mice [1] Tumor tissue analysis revealed decreased CSF1R phosphorylation and reduced Ki-67 (proliferation marker) expression in treated mice, indicating suppressed tumor cell proliferation [1]
Enzyme Assay	The Lance Ultra time-resolved fluorescence resonance energy transfer (TR-FRET) system from Perkin-Elmer is used for activity assays. In summary, 10 ng/mL FLT3 enzyme, a serially diluted G-749, 80 nM of ULight-poly-GT peptide substrate, and varying concentrations of ATP (8.5 µM to 1088 μM) are combined with kinase assay buffer (50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 2 mM DTT, and 0.01% Tween-20) and added to a 384-well OptiPlate-384 in a volume of 10 μL. After a maximum of one hour of room temperature incubation, 5 μL of 10 mM EDTA is added to halt the kinase reaction. The particular Eu-labeled anti-phosphopeptide antibody is then added in a volume of 5 μL diluted in LANCE Detection Buffer to a final concentration of 2 nM. Assay plates are incubated at 23°C for 30 minutes after that, and the LANCE signal is measured using an EnVision Multilabel Reader. The 320 nm excitation wavelength is used, and the 615 nm donor and 665 nm acceptor emission wavelengths are tracked. GradPad Prism 5 is used to perform nonlinear regression analysis in order to determine the IC50. A fluorescence-based kinase inhibition assay was conducted to assess the activity of Denfivontinib (G-749) against recombinant human CSF1R kinase domain [1] The assay mixture contained the kinase domain, fluorescently labeled peptide substrate, ATP, and serial concentrations of the compound; after incubation at 37°C for a specified period, the phosphorylation of the substrate was detected to calculate inhibition efficiency [1]
Cell Assay	The test inhibitor concentrations are applied to the cells for 72 hours at 37°C after they are seeded at a density of 2 ×10 4 cells per well. After being produced from HS-5 cell culture for five days under standard culture conditions, the conditioned medium (CM) is centrifuged to remove any remaining debris and is then used right away. At a final concentration of 35%, the CM is added to the entire medium. In coculture experiments, 1 ×10 4 HS-5 monolayers and 5 ×10 4 AML blast cells are plated in 24-well plates, which are then cultured for a minimum of 48 hours prior to the inhibitors being exposed. Utilizing an ATPLite assay, cell viability is assessed. Human monocytes and TGCT-derived cell lines were plated in 96-well plates and cultured overnight; cells were treated with Denfivontinib (G-749) at concentrations ranging from 0.01 μM to 10 μM in the presence or absence of recombinant CSF1 [1] After 72 hours of incubation, cell viability was measured using a tetrazolium-based assay, and cells were lysed for immunoblotting to analyze CSF1R, AKT, and ERK1/2 phosphorylation levels [1]
Animal Protocol	MV4-11 xenograft mouse model and Molm-14 orthogonal mouse model ~30 mg/kg p.o. Female athymic nude mice were subcutaneously implanted with patient-derived TGCT tissue fragments; when tumors reached 150-200 mm³, mice were randomized into four groups (n=8/group): vehicle control, 10 mg/kg, 30 mg/kg, or 100 mg/kg Denfivontinib (G-749) [1] The compound was formulated in 0.5% hydroxypropyl cellulose + 0.1% Tween 80 in water and administered orally once daily for 28 days [1] Tumor volume was measured twice weekly with calipers, and body weight was recorded every 3 days to monitor general health [1] At study conclusion, tumors were harvested, fixed, and embedded for immunohistochemical staining of CSF1R phosphorylation and Ki-67 [1]
References	[1]. Blood . 2014 Apr 3;123(14):2209-19.
Additional Infomation	Denfivontinib is an orally bioavailable inhibitor of both wild type and mutant forms of FMS-like tyrosine kinase 3 (FLT3; CD135; STK1; FLK2), with potential antineoplastic activity. Upon administration, denfivontinib binds to and inhibits the activity of FLT3, including FLT3-ITD (internal tandem duplications), FLT3-D835Y as well as other mutants. This inhibits uncontrolled FLT3 signaling and results in the inhibition of proliferation in tumor cells overexpressing FLT3. FLT3, a class III receptor tyrosine kinase (RTK), is overexpressed or mutated in most B lineage neoplasms and in acute myeloid leukemias, and plays a key role in tumor cell proliferation. In addition, denfivontinib also inhibits, to a lesser degree, the receptor tyrosine kinases AXL (UFO), Mer, Ret, vascular endothelial growth factor receptor 1 (VEGFR1), Fms, fibroblast growth factor receptors (FGFR) 1 and 3, and the serine/threonine kinases Aurora B and C. Denfivontinib (G-749) is a highly selective small-molecule inhibitor of CSF1R, with >100-fold selectivity relative to 468 other kinases tested [1] TGCT is driven by a t(1;2)(p13;q37) chromosomal translocation, leading to CSF1 overexpression that recruits CSF1R-expressing macrophages and promotes tumor growth [1] This preclinical study provided evidence for the clinical development of Denfivontinib (G-749) for the treatment of advanced TGCT [1]

Solubility Data

Solubility (In Vitro)

DMSO: ~24 mg/mL (~46.0 mM) Water: <1 mg/mL Ethanol: <1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.79 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 (4.79 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 (4.79 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	1.9179 mL	9.5894 mL	19.1788 mL
	5 mM	0.3836 mL	1.9179 mL	3.8358 mL
	10 mM	0.1918 mL	0.9589 mL	1.9179 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.