Rho-Kinase-IN-2 (Compound 23) is an orally bioactive, selective, and central nervous system (CNS)-permeable/penetrable Rho kinase (ROCK) inhibitor (ROCK2 IC50=3 nM). Rho-Kinase-IN-2 may be utilized in Huntington's disease study.

Physicochemical Properties

Molecular Formula	C20H25FN4O2
Molecular Weight	372.436507940292
Exact Mass	372.196
CAS #	2573071-18-6
PubChem CID	155749520
Appearance	Light yellow to yellow solid powder
LogP	2.5
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	5
Rotatable Bond Count	4
Heavy Atom Count	27
Complexity	495
Defined Atom Stereocenter Count	2
SMILES	N1(C(N[C@@H](C2=CC=CC(OC)=C2)C)=O)CCN(C2C=CN=CC=2F)C[C@H]1C
InChi Key	CIPXFTLGPVQJKN-HUUCEWRRSA-N
InChi Code	InChI=1S/C20H25FN4O2/c1-14-13-24(19-7-8-22-12-18(19)21)9-10-25(14)20(26)23-15(2)16-5-4-6-17(11-16)27-3/h4-8,11-12,14-15H,9-10,13H2,1-3H3,(H,23,26)/t14-,15-/m1/s1
Chemical Name	(2R)-4-(3-fluoropyridin-4-yl)-N-[(1R)-1-(3-methoxyphenyl)ethyl]-2-methylpiperazine-1-carboxamide
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	Rho-Kinase-IN-2 (0–10 mM, 1 hour) therapy resulted in decreased MYPT1 phosphorylation and enhanced AKT phosphorylation [1].
ln Vivo	Rho-Kinase-IN-2 (orally given; 10 mg/kg; 6 times; 0.5, 1, 2, 4, 8, and 12 hours) therapy exhibited dose- and time-dependent ROCK1 and ROCK2 target engagement [1]. Rho-Kinase-IN-2 (oral; 10 or 20 mg/kg; QD or BID; 2 weeks) treatment revealed excellent tolerance assessment [1]. Treatment with Rho-Kinase-IN-2 (oral; 1-20 mg/kg; once) indicates a direct dose- and time-dependent link between brain exposure and MYPT1 phosphorylation status [1]. Treatment with Rho-Kinase-IN-2 (oral; 10 or 20 mg/kg; once) decreases mean arterial pressure, systolic blood pressure, diastolic blood pressure, and heart rate [1]. Treatment with Rho-Kinase-IN-2 (orally; 10 mg/kg; twice daily; 90 days) resulted in lower than predicted brain concentrations [1].
Cell Assay	Western Blot Analysis[1] Cell Types: A7r5 and PANC1 Cell Tested Concentrations: 0-10 mM Incubation Duration: 1 hour Experimental Results: demonstrated concentration-dependent effects resulting in increased phosphorylation of AKT (EC50=28 nM) and diminished phosphorylation of MYPT1 (IC50 =14 nM).
Animal Protocol	Animal/Disease Models: 3 to 4 months old heterozygous Q175DN KI and wild-type littermate mice [1] Doses: 10 or 20 mg/kg Route of Administration: po (po (oral gavage)) 10 or 20 mg/kg; one time/day or twice a day; 2-week Experimental Results: Neurologic indices were normal at all doses, although body weight diminished slightly (∼2%) in the 20 mg/kg treatment group. Animal/Disease Models: Heterozygous HTT zQ175DN knock-in mice [1] Doses: 1-20 mg/kg Route of Administration: Oral; 1-20 mg/kg; Experimental Results: Maintained over MYPT1 in free brain at 10 mg/kg dose IC50 exceeded 2 hrs (hrs (hours)), and dose- and time-dependent inhibition of MYPT1 phosphorylation in the striatum was observed after acute in vivo administration. Animal/Disease Models: CD1 mice [1] Doses: 10 and 20 mg/kg Route of Administration: Oral; 10 or 20 mg/kg; Experimental Results: Mean arterial pressure (maximum change from baseline 61.0 ± 8.5 mmHg), Systolic blood pressure (maximum change from baseline was 59.5 ± 8.4 mmHg), diastolic blood pressure (maximum change from baseline was 56.4 ± 9.0 mmHg), heart rate wi
References	[1]. Identification of a Potent, Selective, and Brain-Penetrant Rho Kinase Inhibitor and its Activity in a Mouse Model of Huntington's Disease. J Med Chem. 2022 Jul 11.

Solubility Data

Solubility (In Vitro)

DMSO : ~50 mg/mL (~134.25 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2 mg/mL (5.37 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 20.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2 mg/mL (5.37 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 2 mg/mL (5.37 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 20.0 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	2.6850 mL	13.4250 mL	26.8500 mL
	5 mM	0.5370 mL	2.6850 mL	5.3700 mL
	10 mM	0.2685 mL	1.3425 mL	2.6850 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.