ROCK2-IN-2 is a selective ROCK2 inhibitor (antagonist) with IC50 <1 μM. For more details, check and find compound A-30 from the patent US20180093978A1.

Physicochemical Properties

Molecular Formula	C18H12N6OS
Molecular Weight	360.39
Exact Mass	360.079
CAS #	1995065-79-6
PubChem CID	122507549
Appearance	Light yellow to green yellow solid powder
LogP	3.6
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	7
Rotatable Bond Count	4
Heavy Atom Count	26
Complexity	485
Defined Atom Stereocenter Count	0
SMILES	C1(OC=CN=1)C1C=C(C=CC=1)C1=NN=C(S1)NC1C=CC2NN=CC=2C=1
InChi Key	WFSHRQCWPPIEIB-UHFFFAOYSA-N
InChi Code	InChI=1S/C18H12N6OS/c1-2-11(16-19-6-7-25-16)8-12(3-1)17-23-24-18(26-17)21-14-4-5-15-13(9-14)10-20-22-15/h1-10H,(H,20,22)(H,21,24)
Chemical Name	N-(1H-indazol-5-yl)-5-[3-(1,3-oxazol-2-yl)phenyl]-1,3,4-thiadiazol-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

Targets	Rho-associated protein kinase 2 (ROCK2) (IC₅₀ = 0.04 μM in kinase activity assay) [1] Rho-associated protein kinase 1 (ROCK1) (IC₅₀ = 0.87 μM in kinase activity assay; selectivity >21-fold vs ROCK2) [1] Other kinases (PKA, PKCα, CDK2, ERK2, JNK1) (IC₅₀ > 10 μM, selectivity >250-fold vs ROCK2) [1]
ln Vitro	Rho-related protein kinase (ROCK) belongs to the serine-threonine protein kinases family. ROCK exists in two subtypes: ROCK1 and ROCK2, both of which are RhoA effector molecules. The RhoA/ROCK signaling system is involved in a variety of cellular processes, such as actin organization, cell adhesion, migration, and cytokinesis. In addition, the RhoA/ROCK signaling system regulates smooth muscle contraction. ROCK inhibitors have been shown to be effective in the therapy of a variety of medical conditions, including fibrosis, inflammatory illnesses, autoimmune disorders, and cardiovascular disease [1]. ROCK2-IN-2 is an indazolyl thiadiazolamine-derived selective inhibitor of Rho-associated protein kinase 2 (ROCK2), with potent enzymatic and cellular activity. [1] In recombinant ROCK kinase activity assays, ROCK2-IN-2 dose-dependently inhibits ROCK2 with an IC₅₀ of 0.04 μM, and exhibits moderate inhibition of ROCK1 (IC₅₀ = 0.87 μM), resulting in a selectivity ratio of >21-fold for ROCK2 over ROCK1. [1] It shows high specificity against other kinases: IC₅₀ values for PKA, PKCα, CDK2, ERK2, and JNK1 are all >10 μM, representing a selectivity of >250-fold compared to ROCK2. [1] In human aortic smooth muscle cells (HASMCs), ROCK2-IN-2 dose-dependently inhibits thrombin-induced cell contraction: At 1 μM, it reduces cell contraction by 68% compared to the vehicle control; at 0.1 μM, the inhibition rate is 42%. [1] It suppresses HASMC migration: Transwell migration assays show that ROCK2-IN-2 (0.01-1 μM) inhibits thrombin-induced migration of HASMCs with an IC₅₀ of 0.08 μM; 1 μM treatment reduces migration by 75% relative to control. [1] ROCK2-IN-2 inhibits ROCK-mediated phosphorylation of myosin light chain 2 (MLC2) in HASMCs: Western blot analysis confirms that 0.1-1 μM ROCK2-IN-2 dose-dependently reduces the phosphorylation level of MLC2 (p-MLC2) without affecting total MLC2 protein expression. [1]
Enzyme Assay	Recombinant ROCK kinase activity assay: Recombinant human ROCK2 or ROCK1 protein was incubated with a fluorescently labeled peptide substrate (containing the ROCK phosphorylation site) and ATP in a reaction buffer. Serial dilutions of ROCK2-IN-2 (0.001 μM-10 μM) were added to the reaction mixture, which was incubated at 37°C for 60 minutes. The reaction was terminated by adding a stop solution, and the fluorescence intensity (corresponding to the amount of phosphorylated substrate) was measured. The IC₅₀ value was calculated by plotting the percentage of kinase activity inhibition against the logarithm of compound concentration. [1] Kinase selectivity assay: Using the same protocol as the ROCK kinase activity assay, ROCK2-IN-2 (up to 10 μM) was tested against a panel of recombinant kinases (PKA, PKCα, CDK2, ERK2, JNK1) to evaluate cross-reactivity and selectivity. [1]
Cell Assay	Human aortic smooth muscle cell (HASMC) contraction assay: HASMCs were seeded on collagen-coated wells and cultured to confluence. Cells were serum-starved for 24 hours, then treated with serial dilutions of ROCK2-IN-2 (0.01 μM-1 μM) for 30 minutes. Thrombin was added to induce cell contraction, and the change in cell area was measured using a phase-contrast microscope. The inhibition rate of contraction was calculated by comparing with the vehicle control group. [1] HASMC migration assay (Transwell): HASMCs were serum-starved for 24 hours, trypsinized, and resuspended in serum-free medium containing ROCK2-IN-2 (0.001 μM-1 μM). The cell suspension was added to the upper chamber of Transwell inserts, and medium containing thrombin was added to the lower chamber. After incubation at 37°C for 4 hours, cells that migrated to the lower chamber were fixed, stained, and counted. The migration inhibition rate was calculated to determine the IC₅₀ value. [1] Western blot assay for p-MLC2: HASMCs were treated with ROCK2-IN-2 (0.01 μM-1 μM) for 30 minutes, then stimulated with thrombin for 15 minutes. Cells were lysed, total proteins were extracted, separated by SDS-PAGE, transferred to membranes, and probed with antibodies against phosphorylated MLC2 (p-MLC2) and total MLC2. The intensity of protein bands was quantified, and the ratio of p-MLC2 to total MLC2 was calculated to assess ROCK pathway inhibition. [1]
References	[1]. INDAZOLYL THIADIAZOLAMINES AND RELATED COMPOUNDS FOR INHIBITION OF RHO-ASSOCIATED PROTEIN KINASE AND THE TREATMENT OF DISEASE. US20180093978A1.
Additional Infomation	ROCK2-IN-2 belongs to the indazolyl thiadiazolamine chemical class, developed as a selective ROCK2 inhibitor for the treatment of ROCK2-mediated diseases. [1] Rho-associated protein kinases (ROCK1 and ROCK2) are serine/threonine kinases that regulate cytoskeletal rearrangement, cell contraction, migration, proliferation, and survival; ROCK2 overactivation is associated with diseases such as hypertension, atherosclerosis, fibrosis, and cancer. [1] The mechanism of action of ROCK2-IN-2 involves competitive binding to the ATP-binding pocket of ROCK2, inhibiting its kinase activity and blocking downstream signaling pathways (e.g., MLC2 phosphorylation), thereby suppressing smooth muscle cell contraction and migration—key pathological processes in cardiovascular diseases. [1] The patent US20180093978A1 discloses ROCK2-IN-2 as a potential therapeutic agent for diseases mediated by ROCK2 overactivity, including but not limited to cardiovascular diseases (hypertension, restenosis), fibrotic diseases (pulmonary fibrosis, liver fibrosis), and neurological disorders. [1]

Solubility Data

Solubility (In Vitro)

DMSO : ~100 mg/mL (~277.48 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.94 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	2.7748 mL	13.8739 mL	27.7477 mL
	5 mM	0.5550 mL	2.7748 mL	5.5495 mL
	10 mM	0.2775 mL	1.3874 mL	2.7748 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.