THK01 is a potent ROCK2 inhibitor (antagonist) with IC50s of 5.7 and 923 nM for ROCK2 and ROCK1, respectively. THK01 inhibits breast cancer metastasis through the ROCK2-STAT3 signaling pathway. THK01 may be utilized in breast cancer research.

Physicochemical Properties

Molecular Formula	C20H13N3O2
Molecular Weight	327.34
Exact Mass	327.1
CAS #	2226941-26-8
PubChem CID	139589939
Appearance	Off-white to gray solid powder
LogP	3.3
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	2
Rotatable Bond Count	0
Heavy Atom Count	25
Complexity	583
Defined Atom Stereocenter Count	0
SMILES	O=C1NCC2=C3C4=CC=CC=C4NC3=C3NC4=CC=C(C=C4C3=C21)O
InChi Key	GXZFDXZCJALHRF-UHFFFAOYSA-N
InChi Code	InChI=1S/C20H13N3O2/c24-9-5-6-14-11(7-9)16-17-12(8-21-20(17)25)15-10-3-1-2-4-13(10)22-18(15)19(16)23-14/h1-7,22-24H,8H2,(H,21,25)
Chemical Name	7-hydroxy-3,13,23-triazahexacyclo[14.7.0.02,10.04,9.011,15.017,22]tricosa-1,4(9),5,7,10,15,17,19,21-nonaen-12-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	ROCK2 5.7 nM (IC50) ROCK1 923 nM (IC50)
ln Vitro	THK01 (1.25-10 μM; 48 h) dose-dependently suppresses MDA-MB-231 cells' capacity for migration and invasion [1]. Using the STAT signaling pathway, THK01 (5 μM; 24 h; MDA-MB-231 cells) prevents breast cancer metastasis [1]. In a dose-dependent manner, THK01 (1.25-10 μM; 24 h; MDA-MB-231 cells) modestly increases the level of STAT3S727 while downregulating the phosphorylation of STAT3Y705 [1].
ln Vivo	THK01 (15 mg/kg; iv; female BALB/c nude mice) prevents the in vivo spread of breast cancer cells [1].
Cell Assay	Western Blot Analysis[1] Cell Types: MDA-MB-231 cells Tested Concentrations: 5 μM Incubation Duration: 24 hrs (hours) Experimental Results: Down-regulated the phosphorylation levels (p-STAT) of STAT1, STAT2, and STAT3, up-regulated of p-STAT5A and STAT6. Western Blot Analysis[1] Cell Types: MDA-MB-231 cells Tested Concentrations: 1.25, 2.5, 5, 10 μM Incubation Duration: 24 hrs (hours) Experimental Results: decreased the level of p-STAT3Y705 and increased the expression of p-STAT3S727 in MDA-MB-231 cells.
Animal Protocol	Animal/Disease Models: Female BALB/c nude mice with MDAMB-231-Luc-mcherry xenografts (6 weeks old, weight about 20 g)[1] Doses: 15 mg/kg Route of Administration: intravenous (iv)injection; one time/day for 43 days Experimental Results: diminished the pulmonary metastasis of MDA-MB-231 in vivo with negligible toxicity.
References	[1]. Discovery of unglycosylated indolocarbazoles as ROCK2 isoform-selective inhibitors for the treatment of breast cancer metastasis. Eur J Med Chem. 2023 Mar 15;250:115181.
Additional Infomation	3-hydroxy-K252c is an indolocarbazole. 3-hydroxy-K252c has been reported in Streptomyces with data available.

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	3.0549 mL	15.2746 mL	30.5493 mL
	5 mM	0.6110 mL	3.0549 mL	6.1099 mL
	10 mM	0.3055 mL	1.5275 mL	3.0549 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.