| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg |
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| 100mg |
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| 250mg | |||
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| 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] |
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| 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 | |
| Additional Infomation |
ROCK2-IN-2 belongs to the indazole thiadiazole amine class of compounds and is a selective ROCK2 inhibitor used to treat ROCK2-mediated diseases. [1] Rho-related protein kinases (ROCK1 and ROCK2) are serine/threonine kinases that regulate cytoskeleton reorganization, cell contraction, migration, proliferation and survival; excessive activation of ROCK2 is associated with diseases such as hypertension, atherosclerosis, fibrosis and cancer. [1] The mechanism of action of ROCK2-IN-2 is to competitively bind to the ATP-binding pocket of ROCK2, inhibit its kinase activity and block downstream signaling pathways (such as MLC2 phosphorylation), thereby inhibiting smooth muscle cell contraction and migration—key pathological processes in cardiovascular diseases. [1] Patent US20180093978A1 discloses ROCK2-IN-2 as a potential therapeutic agent for treating diseases mediated by ROCK2 overactivation, including but not limited to cardiovascular diseases (hypertension, restenosis), fibrotic diseases (pulmonary fibrosis, liver fibrosis) and neurological diseases. [1]
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| Molecular Formula |
C18H12N6OS
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|---|---|
| Molecular Weight |
360.39
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| Exact Mass |
360.079
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| CAS # |
1995065-79-6
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| PubChem CID |
122507549
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| Appearance |
Light yellow to green yellow solid powder
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| LogP |
3.6
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| Hydrogen Bond Donor Count |
2
|
| Hydrogen Bond Acceptor Count |
7
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
26
|
| Complexity |
485
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| Defined Atom Stereocenter Count |
0
|
| SMILES |
C1(OC=CN=1)C1C=C(C=CC=1)C1=NN=C(S1)NC1C=CC2NN=CC=2C=1
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| InChi Key |
WFSHRQCWPPIEIB-UHFFFAOYSA-N
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| 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
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ~100 mg/mL (~277.48 mM)
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|---|---|
| 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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