SR-3677 (SR3677) is a potent and selective Rho-kinase/ROCK2 inhibitor with anticancer activity. In enzyme-based assays for ROCK-II and ROCK-I, it inhibits ROCK with IC50 values of 3 and 56 nM, respectively. With respect to 353 kinases, SR-3677's off-target hit rate was 1.4%, and it only inhibited three of the seventy nonkinase enzymes and receptors. Pharmacological investigations demonstrated the effectiveness of SR-3677 in both cases, enhancing the outflow of aqueous humor in vivo in the eyes of pigs and preventing the phosphorylation of the myosin light chain.

Physicochemical Properties

Molecular Formula	C22H24N4O4
Molecular Weight	408.45
Exact Mass	408.179
Elemental Analysis	C, 64.69; H, 5.92; N, 13.72; O, 15.67
CAS #	1072959-67-1
Related CAS #	1072959-67-1
PubChem CID	25093235
Appearance	White to off-white solid
Density	1.3±0.1 g/cm3
Boiling Point	646.9±55.0 °C at 760 mmHg
Flash Point	345.0±31.5 °C
Vapour Pressure	0.0±1.9 mmHg at 25°C
Index of Refraction	1.632
LogP	2.34
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	7
Heavy Atom Count	30
Complexity	563
Defined Atom Stereocenter Count	0
SMILES	CN(C)CCOC1=C(NC(C2OC(C=CC=C3)=C3OC2)=O)C=CC(C4=CNN=C4)=C1
InChi Key	OQWZIAVXCYIZNN-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H24N4O4/c1-26(2)9-10-28-20-11-15(16-12-23-24-13-16)7-8-17(20)25-22(27)21-14-29-18-5-3-4-6-19(18)30-21/h3-8,11-13,21H,9-10,14H2,1-2H3,(H,23,24)(H,25,27)
Chemical Name	N-[2-[2-(dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl]-2,3-dihydro-1,4-benzodioxine-3-carboxamide
Synonyms	SR-3677; SR3677; SR 3677
HS Tariff Code	2934.99.03.00
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	ROCK-II (IC50 = 3 nM); ROCK-I (IC50 = 56 nM)
ln Vitro	SR-3677 has an off-target hit rate of 1.4% against 353 kinases, an IC50 of approximately 3 nM in enzyme and cell-based assays, and only 3 out of 70 nonkinase enzymes and receptors that it inhibits. For ROCK-I, SR-3677's IC50 value is 56±12 nM. The primary cause of SR-3677's high potency is the hydrophobic interaction between the benzodioxane phenyl ring and the hydrophobic pocket surface.
ln Vivo	SR-3677 is effective in both, as demonstrated by pharmacology studies, which also show that it inhibits myosin light chain phosphorylation and increases ex vivo aqueous humor outflow in in pig eyes. The outflow facility is increased by 60% at one hour of perfusion with continuous exposure to 25 µM SR-3677, and by 70–80% at two to five hours.
Enzyme Assay	The STK2 kinase system from Cisbio is used for assays. The wells are filled with a 5 μL mixture of ATP (ROCK-I: 4 μM; ROCK-II: 20 μM) and a 1 μM STK2 substrate in STK-buffer. Test compounds (SR-3677) are dispensed in 20 nL. To initiate the reaction, add 5 μL of either 0.5 nM ROCK-II or 2.5 nM ROCK-I in STK-buffer. Addition of 10 μL of 1x antibody and 62.5 nM Sa-XL in detection buffer stops the reaction after 4 hours at room temperature[1].
Animal Protocol	NA ExVivo
References	[1]. Discovery of substituted 4-(pyrazol-4-yl)-phenylbenzodioxane-2-carboxamides as potent and highly selective Rho kinase (ROCK-II) inhibitors. J Med Chem. 2008 Nov 13;51(21):6642-5.

Solubility Data

Solubility (In Vitro)

DMSO: ≥ 43 mg/mL Water: <1 mg/mL Ethanol: N/A

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.12 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 25.0 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: 2.5 mg/mL (6.12 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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 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: ≥ 2.5 mg/mL (6.12 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	2.4483 mL	12.2414 mL	24.4828 mL
	5 mM	0.4897 mL	2.4483 mL	4.8966 mL
	10 mM	0.2448 mL	1.2241 mL	2.4483 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.