TNIK-IN-3 (compound 21k) is a specific and orally bioactive TNIK inhibitor (antagonist) with IC50 of 0.026 μM. TNIK-IN-3 also inhibits Flt4 (IC50=0.030 μM), Flt1 (IC50=0.191 μM) and DRAK1 (IC50=0.411 μM). TNIK-IN-3 may be used in colorectal cancer research.

Physicochemical Properties

Molecular Formula	C23H18FN3O2
Molecular Weight	387.41
Exact Mass	387.138
CAS #	2754265-25-1
PubChem CID	162641720
Appearance	Off-white to light yellow solid powder
LogP	3.7
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	29
Complexity	583
Defined Atom Stereocenter Count	0
SMILES	O1C2=CC(C3=CN=C4NC=CC4=C3)=CC=C2C(=O)N(CC2=CC=C(F)C=C2)CC1
InChi Key	UVLOISNMRLZTPF-UHFFFAOYSA-N
InChi Code	InChI=1S/C23H18FN3O2/c24-19-4-1-15(2-5-19)14-27-9-10-29-21-12-16(3-6-20(21)23(27)28)18-11-17-7-8-25-22(17)26-13-18/h1-8,11-13H,9-10,14H2,(H,25,26)
Chemical Name	4-[(4-fluorophenyl)methyl]-8-(1H-pyrrolo[2,3-b]pyridin-5-yl)-2,3-dihydro-1,4-benzoxazepin-5-one
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	IC50: 0.026 μM (TNIK)[1], 0.030 μM (Flt4)[1], 0.191 μM (Flt1)[1], 0.411 μM (DRAK1)[1]
ln Vitro	Compound 21k, TNIK-IN-3, inhibits GCK, MLK3, and Aurora-A with IC50 values of 3.657 μM, 4.552 μM, and 0.517 μM, respectively[1]. The viability of HCT116 and DLD-1 cells is inhibited by TNIK-IN-3 (0.1-100 μM; 3 days), with IC50s of 4.26 μM and 8.00 μM, respectively[1]. HCT116 and DLD-1 cell colony formation is dose-dependently inhibited by TNIK-IN-3 (2.5–40 μM; 10 days)[1]. TNIK-IN-3 (5–20 μM; 48 h) prevents DLD-1 and HCT116 cell migration[1]. In HCT116 cells, TNIK-IN-3 (5–40 μM; 48 h) dose-dependently suppresses the expression of the Wnt target genes AXIN2 and c-Myc, as well as the LRP5 and LRP6 proteins[1]. In Hela cells, TNIK-IN-3 (5–20 μM; 48 h) dramatically reduces JNK1/2 phosphorylation [1].
ln Vivo	TNIK-IN-3 (compound 21k) suppresses tumor development in a dose-dependent manner (100–150 mg/kg; po twice daily for 18 days)[1].
Cell Assay	Cell Viability Assay[1] Cell Types: HCT116 and DLD-1 cells Tested Concentrations: 0.1-100 μM Incubation Duration: 3 days Experimental Results: Inhibited cell viability in a dose-dependent manner. Cell Viability Assay[1] Cell Types: HCT116 cells Tested Concentrations: 5, 10, 20, 40 μM Incubation Duration: 48 hrs (hours) Experimental Results: Inhibited the expression of Wnt target genes AXIN2 and c-Myc, LRP5 and LRP6 proteins.
Animal Protocol	Animal/Disease Models: Sixweeks old female NOD-SCID (severe combined immunodeficient) mouse were injected with HCT116 cells[1] Doses: 100, 150 mg/kg Route of Administration: Po twice (two times) daily for 18 days Experimental Results: Dramatically inhibited tumor growth at a dose of 150 mg/kg. No obvious weight loss and no other side effects were observed.
References	[1]. Discovery of 3,4-Dihydrobenzo[ f][1,4]oxazepin-5(2 H)-one Derivatives as a New Class of Selective TNIK Inhibitors and Evaluation of Their Anti-Colorectal Cancer Effects. J Med Chem. 2022 Jan 5.

Solubility Data

Solubility (In Vitro)

DMSO : 83.33 mg/mL (215.10 mM)

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	2.5812 mL	12.9062 mL	25.8124 mL
	5 mM	0.5162 mL	2.5812 mL	5.1625 mL
	10 mM	0.2581 mL	1.2906 mL	2.5812 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.