ROCK-IN-4 is a potent ROCK inhibitor that can maintain NO release capacity. ROCK-IN-4 reversibly depolymerizes F-actin and inhibits mitochondrial respiration in human trabecular meshwork (HTM) cells. ROCK-IN-4 can be used for research on glaucoma or ocular hypertension.

Physicochemical Properties

Molecular Formula	C20H26CLFN4O7S
Molecular Weight	520.959446430206
Exact Mass	520.119
CAS #	2488395-07-7
PubChem CID	164946733
Appearance	Typically exists as solid at room temperature
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	8
Heavy Atom Count	34
Complexity	775
Defined Atom Stereocenter Count	1
SMILES	C[C@H]1CN(CCCN1S(=O)(=O)C2=CC=CC3=CN=CC(=C32)F)C(=O)OCCCCO[N+](=O)[O-].Cl
InChi Key	PWJPEYUHWFWQON-RSAXXLAASA-N
InChi Code	InChI=1S/C20H25FN4O7S.ClH/c1-15-14-23(20(26)31-10-2-3-11-32-25(27)28)8-5-9-24(15)33(29,30)18-7-4-6-16-12-22-13-17(21)19(16)18;/h4,6-7,12-13,15H,2-3,5,8-11,14H2,1H3;1H/t15-;/m0./s1
Chemical Name	4-nitrooxybutyl (3S)-4-(4-fluoroisoquinolin-5-yl)sulfonyl-3-methyl-1,4-diazepane-1-carboxylate;hydrochloride
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	Reversible F-actin depolymerization is induced and p-MLC levels are decreased by ROCK-IN-4 (RNO-6) (10 μM; 1 h) [1]. In human trabecular meshwork (HTM) cells, ROCK-IN-4 (10 μM; 24 hours) contributes to cGMP activation and raises cGMP concentration [1]. Significantly lowering basal respiration, peak respiration, and ATP generation, ROCK-IN-4 (10 μM; 1 h) suppresses mitochondrial respiration by releasing NO [1].
ln Vivo	In a glaucoma mouse model, ROCK-IN-4 (0.26% w/v, 10 μL; op in right eye; single dose) exerts protective activity of retinal ganglion cells (RGC) and shows significant effects on IOP-lowering [1]. In New Zealand white rabbits, ROCK-IN-4 (0.579% w/v, 25 μL; left eye procedure; single dose) showed hypocongestive effects and ocular irritation [1].
Cell Assay	Western Blot analysis [1] Cell Types: Human trabecular meshwork (HTM) Cell Tested Concentrations: 10 μM Incubation Duration: 1 hour Experimental Results: Inhibited MLC phosphorylation and diminished p-MLC levels. Immunofluorescence [1] Cell Types: Human trabecular meshwork (HTM) Cell Tested Concentrations: 10 μM Incubation Duration: 1 hour Experimental Results: F-actin demonstrated obvious depolymerization. After 4 hrs (hours) of recovery, F-actin returned to its aggregated form, indicating reversible decomposition. aggregation effect and will not cause permanent damage to cells.
Animal Protocol	Animal/Disease Models: Microbead-induced chronic ocular hypertension mouse model [1] Doses: 0.26% (w/v), 10 μL Route of Administration: eye drops; single dose; administration to the right eye 7 days after modeling, and IOP was measured before instillation and 1, 2, 3, 4 and 6 hrs (hrs (hours)) after instillation. Experimental Results: IOP (mmHg) diminished to 3.68 ± 0.5 mmHg (19.9%) and 1.36 ± 0.6 mmHg at 1 hour and 4 hrs (hrs (hours)) after instillation, respectively. is (7.4%). Animal/Disease Models: New Zealand white rabbit[1] Doses: 0.579% (w/v); 25 μL Route of Administration: eye drops; single dose; before the first infusion and 1, 2 and 4 hrs (hrs (hours)) after the first infusion Results of administration in the left eye: demonstrated lower side effects in terms of conjunctival hyperemia.
References	[1]. Identification of Nitric Oxide-Donating Ripasudil Derivatives with Intraocular Pressure Lowering and Retinal Ganglion Cell Protection Activities. J Med Chem. 2022 Aug 25.

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	1.9195 mL	9.5977 mL	19.1953 mL
	5 mM	0.3839 mL	1.9195 mL	3.8391 mL
	10 mM	0.1920 mL	0.9598 mL	1.9195 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.