FLT3/CDK4-IN-1 is a potent, selective and orally bioactive dual FLT3/CDK4 inhibitor (IC50 of FLT3 and CDK4 of 7 and 11 nM). FLT3/CDK4-IN-1 has anti-proliferation activity against certain cancer/tumor cells and has good anti-tumor effects in vivo.

Physicochemical Properties

Molecular Formula	C25H28F2N8
Molecular Weight	478.540230751038
Exact Mass	478.24
CAS #	2762296-44-4
PubChem CID	163321905
Appearance	Typically exists as solid at room temperature
LogP	3.6
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	9
Rotatable Bond Count	6
Heavy Atom Count	35
Complexity	678
Defined Atom Stereocenter Count	0
InChi Key	LGEMUEDLVOKLKH-UHFFFAOYSA-N
InChi Code	InChI=1S/C25H28F2N8/c1-4-33-7-9-34(10-8-33)22-6-5-18(12-19(22)26)31-25-29-14-20(27)24(32-25)17-11-23-21(28-13-17)15-30-35(23)16(2)3/h5-6,11-16H,4,7-10H2,1-3H3,(H,29,31,32)
Chemical Name	N-[4-(4-ethylpiperazin-1-yl)-3-fluorophenyl]-5-fluoro-4-(1-propan-2-ylpyrazolo[4,3-b]pyridin-6-yl)pyrimidin-2-amine
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	Compound 23k, FLT3/CDK4-IN-1, exhibits superior cellular anti-proliferative action on MV4-11 and HCT-116 cells, with IC50 values of 70 and 100 nM, respectively, at varying doses during a 72-hour period [1]. The cell cycle is arrested in the G1 phase in a concentration-dependent manner by FLT3/CDK4-IN-1 (12.5-200 nM; 24 hours) [1]. In MV4-11 and HCT-116 cells, FLT3/CDK4-IN-1 (200–3200 nM; 48 hours) causes apoptosis in a concentration-dependent manner; it is more effective in MV4-11 than in HCT-116 [1]. The phosphorylation of FLT3 Tyr589/591 is dose-dependently inhibited by FLT3/CDK4-IN-1 (0-100 nM; 2 hours) [1].
ln Vivo	FLT3/CDK4-IN-1 (100 and 200 mg/kg; oral; once daily for 14 days) effectively suppressed tumor growth at the 200 mg/kg dose [1].
Cell Assay	Cell proliferation experiment Cell Types: MV4-11, HCT-116, MDA-MB-436[1] Tested Concentrations: various concentrations Incubation Duration: 72 hrs (hours) Experimental Results: FLT3/CDK4-IN-1 is better for MV4-11 and HCT Antiproliferative activity in -116 cells with IC50 of 70 and 100 nM respectively. Cell cycle analysis Cell Types: MV4-11, HCT-116[1] Tested Concentrations: 12.5, 25, 50, 100 and 200 nM Incubation Duration: 24 hrs (hours) Experimental Results: Cell cycle arrest in G1 phase in a concentration-dependent manner. Apoptosis analysis Cell Types: MV4-11, HCT-116[1] Tested Concentrations: 200, 400, 800, 1600 and 3200 nM Incubation Duration: 48 hrs (hours) Experimental Results: Induction of MV4-11 and HCT-116 cells in a concentration-dependent manner apoptosis, and was more capable in MV4-11 than HCT-116. Western Blot Analysis Cell Types: MV4-11[1] Tested Concentrations: 0, 5, 10, 20, 40, 100 nM Incubation Duration: 2 hrs (hours) Experimental Results: Inhibited phosphorylation of FLT3 Tyr589/591 in a dose-dependent manner.
Animal Protocol	Animal/Disease Models: Female nu/nu (nude) mice (injected with MV4-11) [1] Doses: 100 and 200 mg/kg Route of Administration: oral; 14 days, one time/day Experimental Results: Dramatically inhibited tumor growth at the 200 mg/kg dose , there is no obvious anti-tumor effect at the dose of 100mg/kg.
References	[1]. Synthesis and biological evaluation of 6-(pyrimidin-4-yl)-1H-pyrazolo[4,3-b]pyridine derivatives as novel dual FLT3/CDK4 inhibitors. Bioorg Chem. 2022;121:105669.

Solubility Data

Solubility (In Vitro)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Solubility (In Vivo)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *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. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0897 mL	10.4484 mL	20.8969 mL
	5 mM	0.4179 mL	2.0897 mL	4.1794 mL
	10 mM	0.2090 mL	1.0448 mL	2.0897 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.