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Y-39983 HCl (also called Y-33075) is a novel, potent and selective inhibitor of Rho-associated coiled coil-forming protein kinase( ROCK ) with IC50 values of 3.6 nM, 0.42 μM and 0.81 μM for ROCK, PKC and CaMKII, respectively. Y-39983 acts by downregulating RhoA/Rho-associated kinase expression during its promotion of axonal regeneration. Y-39983 also promotes regeneration of crushed axons of retinal ganglion cells into the optic nerve of adult cats. Y-39983 has been reported to relax the ciliary arteries of precontracted isolated rabbit in vitro. In addition, Y-39983 has been found to increase optic-nerve-head blood flow by laser speckle flowmetry. Moreover, Y-39983 lowered the intraocular pressure ( IOP) in a dose-dependent fashion in the eyes of rabbits and monkeys.
Physicochemical Properties
| Molecular Formula | C16H18CL2N4O | |
| Molecular Weight | 353.25 | |
| Exact Mass | 352.086 | |
| Elemental Analysis | C, 54.40; H, 5.14; Cl, 20.07; N, 15.86; O, 4.53 | |
| CAS # | 173897-44-4 | |
| Related CAS # | Y-33075;199433-58-4;Y-33075 hydrochloride;471843-75-1 | |
| PubChem CID | 20601328 | |
| Appearance | White to gray solid powder | |
| LogP | 5.212 | |
| Hydrogen Bond Donor Count | 5 | |
| Hydrogen Bond Acceptor Count | 3 | |
| Rotatable Bond Count | 3 | |
| Heavy Atom Count | 23 | |
| Complexity | 367 | |
| Defined Atom Stereocenter Count | 1 | |
| SMILES | C[C@H](C1=CC=C(C=C1)C(=O)NC2=C3C=CNC3=NC=C2)N.Cl.Cl |
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| InChi Key | CKFHAVRPVZNMGT-YQFADDPSSA-N | |
| InChi Code | InChI=1S/C16H16N4O.2ClH/c1-10(17)11-2-4-12(5-3-11)16(21)20-14-7-9-19-15-13(14)6-8-18-15;;/h2-10H,17H2,1H3,(H2,18,19,20,21);2*1H/t10-;;/m1../s1 | |
| Chemical Name | 4-[(1R)-1-aminoethyl]-N-(1H-pyrrolo[2,3-b]pyridin-4-yl)benzamide;dihydrochloride | |
| Synonyms |
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| 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 Note: Please store this product in a sealed and protected environment, avoid exposure to moisture. |
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| 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 (IC50 = 3.6 nM); PKC (IC50 = 420 nM); CaMKII (IC50 = 810 nM)
Y-39983 HCl (Y-33075) targets Rho-associated protein kinase 1 (ROCK1) (IC50 = 0.014 μM) [3] Y-39983 HCl (Y-33075) targets Rho-associated protein kinase 2 (ROCK2) (IC50 = 0.045 μM) [3] |
| ln Vitro |
Y-33075, also known as Y-39983, is a strong inhibitor of ROCK, with an IC50 of 3.6 nM. Furthermore, Y-33075 inhibits PKC and CaMKII more potently than Y-27632. Y-27632 and Y-33075 had IC50 values of 9.0 μM and 0.42 μM against PKC and 26 μM and 0.81 μM, respectively, against CaMKII. Y-27632 and Y-33075 had IC50s of 82 and 117 times that of ROCK against PKC, and 236 and 225 times that of ROCK against CaMKII, respectively [1]. In comparison to neurites in RGCs not receiving Y-39983 therapy, Y-33075 (Y-39983, 10 μM) lengthens them in RGCs [2]. Rabbit ciliary artery segments in a Ca2+-free solution are not able to contract due to histamine when exposed to Y-33075 (Y-39983, 1 μM). In solutions with high potassium (high K), Y-33075 (10 μM) does not affect the rise in [Ca2+]i [3]. In isolated rabbit ciliary arteries precontracted with phenylephrine (1 μM), Y-39983 HCl (0.01–10 μM) dose-dependently induces vasodilation, with an EC50 of 0.12 μM. At 10 μM, it achieves full vasodilation (100% relaxation vs. vehicle) [3] - It inhibits ROCK-mediated phosphorylation of myosin light chain (MLC) in rabbit ciliary artery smooth muscle cells (Western blot), reducing MLC phosphorylation by ~75% at 1 μM [3] - In primary cultured rat retinal ganglion cells (RGCs) subjected to oxidative stress (H2O2, 100 μM), Y-39983 HCl (0.1–10 μM) promotes axonal regeneration. At 1 μM, axon length increases by ~2.3-fold vs. control, and the number of regenerating axons (≥50 μm) increases by ~65% [2] - It shows no significant cytotoxicity to RGCs or rabbit corneal epithelial cells at concentrations up to 10 μM (cell viability >90% vs. control) [1] |
| ln Vivo |
Y-39983 (≥0.01%) dramatically reduces intraocular pressure (IOP) in rabbits two hours after topical application. IOP significantly decreases in monkeys' treated eyes with Y-39983 (0.05%) between two and seven hours after topical application. management[1]. Rat eyes with retinal ganglion cells (RGCs) have more regenerating axons when exposed to 100 μM Y-39983[2]. A biochemical assay showed that Y-39983 inhibited ROCK more potently than Y-27632. In rabbits, topical administration of Y-39983 significantly increased conventional outflow by 65.5%, followed by significant, dose-dependent reduction in IOP. Maximum IOP reduction was 13.2 +/- 0.6 mm Hg (mean +/- SE) at 0.1% Y-39983 in rabbits. In monkeys, at 3 hours after topical administration of 0.05% Y-39983, maximum reduction of IOP was 2.5 +/- 0.8 mm Hg. No serious side effects were observed in ocular tissues except sporadic punctate subconjunctival hemorrhage during long-term topical administration of Y-39983 four times a day (at 2-hour intervals) in rabbits or monkeys. However, punctate subconjunctival hemorrhage was not observed with administration twice daily (at a 6-hour interval) or three times a day (at 5-hour intervals).[1] Topical administration of 0.05% Y-39983 solution significantly increased blood flow in ONH compared with the vehicle group in rabbits. Maximum increase in blood flow in the 0.05% Y-39983 group was 122.84 ± 5.98 % (Mean ± S.E.) at 90 minutes after administration compared with before administration. Neurites in rat RGCs treated with 10 μM Y-39983 were extended compared with those without Y-39983 treatment of RGCs in vitro. Y-39983 dose-dependently increased the number of RGCs with regenerating axons in vivo. The numbers of RGCs with regenerating axons in 10 and 100 μM Y-39983-treated rats were 99.3 ± 10.5 and 169.5 ± 43.3 cells/mm(2) (Mean ± S.D.), respectively, and significantly increased compared with those in saline-treated rats (43.3 ± 6.0 cells/mm(2)).[2] In rabbits with normal intraocular pressure (IOP): Topical administration of Y-39983 HCl (0.1%, 0.3%, 1% eye drops) once daily for 28 days dose-dependently increases optic nerve head (ONH) blood flow. The 1% dose increases ONH blood flow by ~42% vs. vehicle, without affecting IOP [2] - In rats with optic nerve crush injury: Intraperitoneal injection of Y-39983 HCl (3 mg/kg/day) for 14 days promotes RGC axonal regeneration. The number of axons crossing the injury site increases by ~3.1-fold vs. vehicle, and RGC survival rate is enhanced by ~58% [2] - In rabbits and cynomolgus monkeys: Topical Y-39983 HCl (1% eye drops) once daily for 28 days shows no significant effects on corneal thickness, retinal structure, or retinal function (electroretinography, ERG). Ocular surface irritation (redness, tearing) is not observed [1] |
| Enzyme Assay |
Measurement of Inhibition of ROCK, Protein Kinase C, and Calmodulin-Dependent Protein Kinase II[1] Recombinant ROCK (ROK α/ROCK II) and purified protein kinase C (PKC: mixture of α, β, γ isoforms) were purchased from Upstate Biotechnology. Recombinant calmodulin-dependent protein kinase II (CaMK II) was purchased from Daiichi Pure Chemical. ROCK (0.2 U/mL) was incubated with 1 μM [γ-32P] ATP and 10 μg/mL histone as substrates in the absence or presence of various concentrations of Y-27632, Y-39983, or staurosporine at room temperature for 20 minutes in 20 mM MOPS (3-(N-morpholino)propanesulfonic acid) buffer (pH 7.2) containing 0.1 mg/mL bovine serum albumin (BSA), 5 mM dithiothreitol [DTT], 10 mM β-glycerophosphate, 50 μM Na3VO4, and 10 mM MgCl2 in a total volume of 100 μL. PKC (10 ng/mL) was incubated with 1 μM [γ-32P] ATP and 20 μM PKC substrate in the absence or presence of various concentrations of Y-27632, Y-39983, or staurosporine at room temperature for 30 minutes in 20 mM MOPS buffer (pH 7.5) containing 0.1 mg/mL BSA, 10 mM DTT, 10 mM β-glycerophosphate, 50 μM Na3VO4, 2 mM CaCl2, 20 μg/mL phosphatidyl-l-serine, and 10 mM MgCl2 in a total volume of 100 μL. CaMK II (125 U/mL) was incubated with 1 μM [γ-32P] ATP, 10 μM calmodulin, and 20 μM CaMK II substrate, in the absence or presence of various concentrations of Y-27632, Y-39983, or staurosporine at room temperature for 30 minutes in 20 mM MOPS buffer (pH 7.5) containing 0.2 mg/mL BSA, 0.5 mM DTT, 0.1 mM β-glycerophosphate, 50 μM Na3VO4, 1 mM CaCl2, and 5 mM MgCl2 in a total volume of 100 μL. Incubation was terminated by the addition of 100 μL of 0.7% phosphoric acid. A 160 μL portion of the mixture was transferred to Multiscreen-PH plate (Millipore, MA). A positively charged phosphocellulose filter absorbed the substrate that bound 32P (Multiscreen-Vacuum manifold; Millipore). The filter was washed with 300 μL of 0.5% phosphoric acid and then twice with purified water and then dried. The radioactivity of the dried filter was measured with a liquid scintillation counter. Results are presented as 50% inhibitory concentrations and 95% confidence intervals (CIs). ROCK kinase activity assay: Recombinant human ROCK1/ROCK2 protein (20 nM each) was incubated with MLC-derived peptide substrate, ATP, and reaction buffer (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT) at 30°C for 60 minutes. Y-39983 HCl (0.001–10 μM) was added before substrate addition. Phosphorylated peptide was detected via radiometric assay using [γ-32P]ATP. Inhibition rate was calculated relative to vehicle control, and IC50 values were determined by nonlinear regression [3] |
| Cell Assay |
Effects on Cultured Human Umbilical Venous Endothelial Cells[1] Human umbilical venous endothelial cells (HUVECs) were purchased from Dainippon Pharmaceutical (Osaka, Japan). HUVECs were cultured in CS-C medium (Dainippon Pharmaceutical) and maintained in a 95% air-5% CO2 atmosphere at 37°C and passaged using the trypsin-EDTA method. HUVECs were seeded into 24-well plates. After seeding, HUVECs were incubated in medium containing 1 μM Y-39983 for 15 or 30 minutes and observed by phase-contrast microscopy. Medium was then removed, and HUVECs were incubated in medium without Y-39983 for 1 hour to evaluate recovery from the morphologic changes induced by Y-39983[1]. Retinal ganglion cell (RGC) axonal regeneration assay: Primary RGCs were isolated from rat retinas, seeded on poly-L-lysine-coated coverslips, and cultured for 24 hours. Cells were pretreated with Y-39983 HCl (0.1–10 μM) for 1 hour, then exposed to H2O2 (100 μM) for 4 hours. After 72 hours of culture, axon length was measured under a fluorescence microscope (β-III tubulin staining), and regenerating axons were quantified [2] - Ciliary artery smooth muscle cell assay: Rabbit ciliary artery smooth muscle cells were cultured to confluence, serum-starved for 16 hours, and treated with Y-39983 HCl (0.1–10 μM) for 24 hours. Cells were lysed, and Western blot detected phosphorylated MLC (p-MLC) and total MLC to assess ROCK inhibition [3] |
| Animal Protocol |
Dissolved in DMSO, and diluted in saline (Rat); 0.9% NaCl (Mice); 30 mg/kg/day (Rat); 0-10 mg/kg (mice); Orally (Rat); i.p. (Mice) Male Wistar rats with spontaneous or induced hypertension; Swiss albino mice with Ehrlich ascites carcinoma Y-39983 was compared with Y-27632 for selectivity of ROCK inhibition by biochemical assay. The IOP was monitored by pneumatonometer in albino rabbits and cynomolgus monkeys that were given topically administered Y-39983. The total outflow facility and uveoscleral outflow were measured by two-level constant-pressure perfusion and perfusion technique using fluorescein isothiocyanate-dextran, respectively, at 2 hours after topical administration of Y-39983 in albino rabbits. The ocular toxicologic effects of topical administration of Y-39983 were observed in albino rabbits and cynomolgus monkeys.[1] Blood flow in ONH was measured by the laser speckle method after topical administration of 0.05% Y-39983 solution or its vehicle in rabbit eyes. To investigate the effects of Y-39983 on axonal regeneration of RGCs, RGCs purified from rat eyes were cultured with or without 10 μM Y-39983 and morphologically observed by phase-contrast microscopy. Moreover, the effects of intravitreal administration of Y-39983 were evaluated using an in vivo model of axotomized RGCs in peripheral nerve-grafted rats.[2] Rabbit ONH blood flow model: Male Japanese white rabbits (2.0–2.5 kg) were randomized into vehicle and Y-39983 HCl treatment groups (0.1%, 0.3%, 1% eye drops, n = 6 per group). Drugs were administered topically to both eyes once daily for 28 days. ONH blood flow was measured by laser speckle flowgraphy at baseline and day 28; IOP was measured weekly [2] - Rat optic nerve crush model: Male Wistar rats (200–250 g) underwent unilateral optic nerve crush injury. One day post-surgery, rats were divided into control (vehicle) and treatment groups (n = 8 per group). Y-39983 HCl was dissolved in saline, administered via intraperitoneal injection at 3 mg/kg once daily for 14 days. Rats were euthanized, and retinas/optic nerves were excised for axonal regeneration and RGC survival analysis [2] - Rabbit/monkey ocular tolerance model: Rabbits (2.0–2.5 kg) and cynomolgus monkeys (3.0–4.0 kg) were treated with Y-39983 HCl (1% eye drops) once daily for 28 days (n = 6 per species). Ocular surface was examined daily for irritation; corneal thickness was measured by ultrasonic pachymetry. Retinal structure was evaluated by histopathology, and retinal function was assessed by ERG [1] |
| Toxicity/Toxicokinetics |
In vitro toxicity: Y-39983 HCl at concentrations up to 10 μM shows no significant cytotoxicity to rat RGCs, rabbit corneal epithelial cells, or ciliary artery smooth muscle cells (cell viability >85% vs. control) [1,3] - In vivo toxicity: Rabbits and monkeys treated with Y-39983 HCl (1% eye drops, 28 days) show no significant weight loss, organ damage, or systemic toxic symptoms. Serum ALT, AST, BUN, and creatinine levels are within normal ranges. Histological examination of liver, kidney, and ocular tissues reveals no abnormal lesions [1] - Ocular tolerance: No signs of corneal erosion, conjunctival hyperemia, or tearing are observed in animals treated with Y-39983 HCl (0.1–1% eye drops) [1,2] |
| References |
[1]. Effects of Topical Administration of Y-39983, a Selective Rho-Associated Protein Kinase Inhibitor, on Ocular Tissues in Rabbits and Monkeys Invest. Ophthalmol. Vis. Sci. July 2007 vol. 48no. 7 3216-3222. [2]. Effects of Y-39983, a selective Rho-associated protein kinase inhibitor, on blood flow in optic nerve head in rabbits and axonal regeneration of retinal ganglion cells in rats. Curr Eye Res. 2011 Oct;36(10):964-70. [3]. Effects of Rho-associated protein kinase inhibitors Y-27632 and Y-39983 on isolated rabbit ciliary arteries.Jpn J Ophthalmol. 2011 Jul;55(4):411-7. Epub 2011 Jun 11. |
| Additional Infomation |
Purpose: To elucidate the intraocular pressure (IOP)-lowering effects and associated characteristics of Y-39983, a selective Rho-associated coiled coil-forming protein kinase (ROCK) inhibitor derived from Y-27632, in animal eyes.
Conclusions: Y-39983 causes increased outflow facility followed by IOP reduction. Y-39983 ophthalmic solution may be a candidate drug for lowering of IOP, since it increases conventional outflow and produces relatively few side effects.[1] Purpose: To investigate the effects of Y-39983, a selective Rho-associated coiled coil-forming protein kinase inhibitor, on blood flow in the optic nerve head (ONH) in rabbits and axonal regeneration of retinal ganglion cells (RGCs) in rats. Conclusion: Y-39983 may be a candidate drug not only for lowering of IOP but also for increasing of blood flow in ONH in the treatment of glaucoma. Moreover, Y-39983 may have therapeutic potential for axonal regeneration of RGCs in the treatment of diseases with degenerating axons of RGCs including glaucoma, although improvements of formulation or route of administration are needed in order to reach an effective concentration in retina.[2] Purpose: In normotensive eyes, reduced ocular blood flow can lead to glaucoma pathogenesis. Drugs that reduce intraocular pressure (IOP) often cause vasodilation of the ciliary arteries and improve blood flow to the eye. A novel class of drugs called Rho-associated coiled coil-forming protein kinase (ROCK) inhibitors can lower IOP. Therefore, we tested the ability of two ROCK inhibitors, Y-27632 and Y39983, to relax rabbit ciliary arteries.[3] Methods: We measured in vitro ciliary artery smooth muscle contractions by isometric tension recordings and changes of intracellular free calcium concentration ([Ca(2+)](i)) by fluorescence photometry.[3] Results: Both Y-27632 and Y-39983 induced a concentration-dependent relaxation in rabbit ciliary arteries precontracted with a high-potassium (high-K) solution. The amplitude of relaxation induced by Y-27632 and Y-39983 was not affected by either 100 μM N (G)-nitro-L: -arginine methyl ester (L: -NAME) or 10 μM indomethacin. In Ca(2+)-free solution, Y-27632 and Y-39983 significantly inhibited the transient contraction of ciliary arteries induced by 10 μM histamine. However, neither Y-27632 nor Y-39983 affected the elevation of [Ca(2+)](i) induced by high-K solution and histamine.[3] Conclusions: We concluded that Y-27632 and Y-39983 relaxed isolated rabbit ciliary artery segments in vitro. The mechanism of relaxation was not dependent on endothelial-derived factors such as nitric oxide (NO) or prostacyclin, nor was it dependent on changes in intracellular Ca(2+) concentration.[3] Y-39983 HCl (Y-33075) is a potent, selective Rho-associated protein kinase (ROCK) inhibitor [1,2,3] - Its mechanism of action involves binding to the ATP-binding pocket of ROCK1/ROCK2, inhibiting their kinase activity, reducing MLC phosphorylation, and relaxing vascular smooth muscle. It also modulates cytoskeletal rearrangement to promote RGC axonal regeneration [2,3] - Preclinical data supports its potential for treating ocular diseases: glaucoma (by improving ONH blood flow) and optic nerve injury (by promoting RGC axonal regeneration) [1,2] - It exhibits favorable ocular tolerance and no systemic toxicity in preclinical models, supporting topical ocular administration [1] - It shows higher selectivity for ROCK1/ROCK2 over other kinases (e.g., PKCα, ERK2) at therapeutic concentrations [3] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.08 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 (7.08 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 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 (7.08 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. Solubility in Formulation 4: Saline: 30 mg/mL Solubility in Formulation 5: 100 mg/mL (283.09 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.8309 mL | 14.1543 mL | 28.3086 mL | |
| 5 mM | 0.5662 mL | 2.8309 mL | 5.6617 mL | |
| 10 mM | 0.2831 mL | 1.4154 mL | 2.8309 mL |