FLT3/D835Y-IN-1 (compound 13a) is an orally potent and specific inhibitor of FLT3 and FLT3/D835Y with IC50s of 0.26 nM and 0.18 nM, respectively. FLT3/D835Y-IN-1 can also block tumor growth, has anti-cancer effects, and may be utilized to study acute myeloid leukemia.

Physicochemical Properties

Molecular Formula	C22H21N5O3
Molecular Weight	403.433844327927
Exact Mass	403.164
CAS #	2648799-49-7
PubChem CID	156443742
Appearance	Typically exists as solid at room temperature
LogP	2.2
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	30
Complexity	705
Defined Atom Stereocenter Count	0
SMILES	C1CN(CCN1)C(=O)CO/N=C/2\C3=CC=CC=C3N=C2C4=C(NC5=CC=CC=C54)O
InChi Key	DTTMVAWVXDJYSD-LHLOQNFPSA-N
InChi Code	InChI=1S/C22H21N5O3/c28-18(27-11-9-23-10-12-27)13-30-26-20-15-6-2-4-8-17(15)24-21(20)19-14-5-1-3-7-16(14)25-22(19)29/h1-8,23,25,29H,9-13H2/b26-20+
Chemical Name	2-[(E)-[2-(2-hydroxy-1H-indol-3-yl)indol-3-ylidene]amino]oxy-1-piperazin-1-ylethanone
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	FLT3/D835Y-IN-1 (compound 13a) substantially reduces the proliferation of AML cells as well as the Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD/D835Y, and Ba/F3-FLT3-ITD-F691L cell lines at 100 nM for three hours[1]. The FLT3, AKT, ERK, and STAT5 pathways are all markedly inhibited by FLT3/D835Y-IN-1 (3–30 nM, 16 hours) [1].
ln Vivo	On MOLM14-ITD/D835Y cells, FLT3/D835Y-IN-1 (10 mg/kg, intraperitoneal injection, daily, six days a week) can effectively suppress tumor growth and show anti-tumor activity [1]. The intravenous or oral FLT3/D835Y-IN-1 (10 mg/kg) dose exhibits a very low AUC and a high clearance [1]. FLT3/D835Y-IN-1 pharmacokinetic parameters in ICR mice [1]. 1360 ± 110 CL (L/h/kg) 6.96 ± 0.66 Vss (L/kg) 14.8 ± 0.7 T1/2 (h) 1.5 ± 0.1 Parameter 13a AUClast (ng*h/mL)
Cell Assay	Cell proliferation assay Cell Types: Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD/D835Y and Ba/F3-FLT3-ITD-F691L cell lines, AML cells [1] Tested Concentrations: 100 nM Incubation Duration: 3 h Experimental Results: Inhibited the proliferation of Ba/F3-FLT3-ITD, Ba/F3-FLT3-ITD/D835Y, Ba/F3-FLT3-ITD-F691L, MV4-11, MOLM14 and MOLM14-ITD/D835Y, with GI50 values of 0.59 respectively. , 0.73, 5.54, 1.30, 6.20 and 4.58 nM. Western Blot Analysis of Cell Types: MOLM14-ITD/D835Y and MOLM14-ITD/F691L cells [1]. Tested Concentrations: 3, 10 and 30 nM Incubation Duration: 16 hrs (hours) Experimental Results: Significant inhibition of FLT3, AKT, ERK and STAT5 pathways at lower doses.
Animal Protocol	Animal/Disease Models: NOD/SCID (severe combined immunodeficient) mouse (6 weeks, male, 9 mice per group) [1] Doses: 10 mg/kg Route of Administration: IP, daily, 6 days per week, from day 7 to day 29 Experimental Results: Dramatically inhibited tumor growth. Animal/Disease Models: ICR mice (7-8 weeks, male) [1] Doses: 10 mg/kg in solution (10% DMSO, 40% PEG400, and 50% PBS) Route of Administration: intravenously (iv) (iv)(iv) or orally, single Second (pharmacokinetic/PK/PK analysis) Experimental Results: demonstrated extremely low AUC and high clearance.
References	[1]. Discovery of indirubin-3'-aminooxy-acetamide derivatives as potent and selective FLT3/D835Y mutant kinase inhibitors for acute myeloid leukemia. Eur J Med Chem. 2022 Apr 21;237:114356.

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	2.4787 mL	12.3937 mL	24.7874 mL
	5 mM	0.4957 mL	2.4787 mL	4.9575 mL
	10 mM	0.2479 mL	1.2394 mL	2.4787 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.