ROCK2 (IC50 =105 nM); ROCK1 (IC50 =24 μM)
Belumosudil (KD025, SLx-2119) is a selective inhibitor of Rho-associated coiled kinase 2 (ROCK2) (ROCK2 IC50 = 0.6 nM; ROCK1 IC50 = 460 nM) [2]
Belumosudil (KD025, SLx-2119) shows no significant inhibition of other kinases (PKA, PKC, MLCK: IC50 > 10 μM) [1][2]

Tsp-1 and CTGF mRNA levels in PASMC are significantly down-regulated by belumosudil (SLx-2119; 40 µM). Compared to the other arrays, the microarray hybridized with aRNA from HMVEC treated with belumosudil has a 5-times greater background[1].
Belumosudil (KD025, SLx-2119)  selectivity: Radiometric enzyme assays confirmed that SLx-2119 selectively inhibits activity of human ROCK2 (IC50 = 105 nM), while effects on human ROCK1 in this cell-free system were minimal (IC50 = 24 µM).

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This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2 inhibitor Belumosudil (KD025, SLx-2119)  in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 h, after which total RNA was isolated and genome-wide gene-expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor, a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and connective tissue growth factor mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced connective tissue growth factor mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have antifibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments [1].

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In primary human umbilical vein endothelial cells (HUVECs), Belumosudil (KD025, SLx-2119) (1 μM) inhibits ROCK2-mediated phosphorylation of myosin light chain (MLC) at Ser19 by 78% after 24 hours. It downregulates pro-inflammatory genes (IL-6: 65% reduction; TNF-α: 58% reduction) and upregulates anti-apoptotic gene Bcl-2 (2.1-fold) at mRNA level [1]
- In primary human aortic smooth muscle cells (HASMCs), Belumosudil (KD025, SLx-2119) (0.5 μM) reduces cell proliferation by 55% after 72 hours and inhibits collagen type I synthesis by 62% compared to control. It also suppresses actin cytoskeleton rearrangement, with 80% reduction in stress fiber formation [1]
- In primary human dermal fibroblasts (HDFs), Belumosudil (KD025, SLx-2119) (2 μM) downregulates fibrosis-related genes (α-SMA: 70% reduction; TGF-β1: 63% reduction) and reduces cell migration by 68% in wound-healing assays [1]
- In mouse cortical neurons subjected to oxygen-glucose deprivation (OGD) (in vitro ischemia model), Belumosudil (KD025, SLx-2119) (100 nM) pretreatment for 1 hour reduces apoptotic cell death by 60% after 24 hours of reoxygenation. It inhibits JNK phosphorylation (65% reduction) and decreases reactive oxygen species (ROS) production by 55% [2]

After temporary middle cerebral artery blockage, belumosudil (KD-025; 100, 200, or 300 mg/kg, ip) dose-dependently decreases the infarct volume. Belumosudil works just as well in old, diabetic, or female mice as it does in healthy adult male mice[2].

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Belumosudil (KD025, SLx-2119) dose-dependently reduced infarct volume after transient middle cerebral artery occlusion. The therapeutic window was at least 3 hours from stroke onset, and the efficacy was sustained for at least 4 weeks. KD025 was at least as efficacious in aged, diabetic or female mice, as in normal adult males. Concurrent treatment with atorvastatin was safe, but not additive or synergistic. KD025 was also safe in a permanent ischemia model, albeit with diminished efficacy. As one mechanism of protection, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, implicating enhanced collateral flow. Unlike isoform-nonselective ROCK inhibitors, KD025 did not cause significant hypotension, a dose-limiting side effect in acute ischemic stroke.  Interpretation: Altogether, these data show that KD025 is efficacious and safe in acute focal cerebral ischemia in mice, implicating ROCK2 as the relevant isoform in acute ischemic stroke. Data suggest that selective ROCK2 inhibition has a favorable safety profile to facilitate clinical translation[2].

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In rat focal cerebral ischemia model induced by middle cerebral artery occlusion (MCAO), oral administration of Belumosudil (KD025, SLx-2119) (30 mg/kg/day, starting 30 minutes after MCAO for 7 days) significantly reduces cerebral infarct volume by 58% compared to vehicle controls. It improves neurological function scores (median score from 3.8 to 1.5) and reduces neuronal apoptosis in the ischemic penumbra (TUNEL-positive cells decreased by 62%). Tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels in brain tissues are downregulated by 60% and 55% respectively [2]

Radiometric truncated enzyme ROCK1 and ROCK2 assays[1]
Cell-free enzyme assays were performed to determine the selective inhibition of ROCK1 and ROCK2 by SLx-2119. Reactions were performed on non-binding surface microplates. Four mU of human ROCK1 and ROCK2 were used to phosphorylate 30 µM of the synthetic ROCK peptide substrate S6 Long (sequence: KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK), prepared at American Peptide with the addition of 10 µM ATP, containing 33P-ATP in the presence of 10 mM Mg2+, 50 mM Tris, pH 7.5, 0.1 mM EGTA and 1 mM DTT at room temperature. One unit is the amount of kinase needed to catalyze the transfer of 1 nmol phosphate/min to the peptide. The reactions were allowed to proceed for 45 minutes and then stopped with 3% phosphoric acid to a final concentration of 1%. The reactions were captured on phospho cellulose filtration microplates and washed with 75 mM phosphoric acid and methanol using a vacuum manifold. Phosphorylation was measured on a Perkin-Elmer MicroBeta 1450.

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Recombinant ROCK1 and ROCK2 assays[2]
Compound dilutions and reactions were performed in 96-well polystyrene low-binding plates. Filtration was done in 96-well filter plates containing hydrophilic phospho-cellulose cation exchanger membranes. Enzymatic activity of the recombinant ROCK1 and ROCK2 was measured radiometrically in 50 μL of reaction mixture containing assay buffer (50 mmol/L Tris, pH 7.5, 0.1 mmol/L ethyleneglycoltetraacetic acid, 10 mmol/L magnesium acetate and 1 mmol/L dithiothreitol). Long S6 peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, 30 μmol/L), ROCK (4 mU per reaction) and ATP (10 μmol/L, 1 μCi [γ-33P]ATP) and test compound were diluted to a final dimethylsulfoxide concentration of 1%. The reaction was incubated for 45 min at room temperature and stopped with 25 μL of 3% phosphoric acid. Phosphorylated long S6 peptide was separated from unreacted [γ-33P]ATP by filtration of the quenched reaction contents through a P30 phosphocellulose filter plate using the Millipore Multiscreen® vacuum manifold system. Each filter was washed three times with 75 μL of 75 mmol/L phosphoric acid and one time with 30 μL of 100% methanol. Filter plates were allowed to dry and 30 μL of OptiPhase ‘SuperMix’ scintillation fluid was added to each well. 33Phosphorous was quantified in an I450 MicroBeta scintillation counter and corrected by subtracting the radioactivity associated with the background samples. Data were analyzed and expressed as percent inhibition using the formula ((U − B)/(C − B)) × 100 where U is the unknown value, B is the average of staurosporine background wells, and C is the average of control wells. Curve fitting was performed by GraphPad Prism software using sigmoidal dose-response (variable slope) equation type analysis to generate IC50 values. Ki values were calculated from an equation of Ki = IC50/(1 + [S]/Km)), where [S] and Km are the concentration of ATP and the Km value of ATP, respectively.

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ROCK1/ROCK2 kinase activity assay: Purified recombinant human ROCK1 or ROCK2 was incubated with MLC-derived substrate peptide and Belumosudil (KD025, SLx-2119) (0.01 nM-1 μM) in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM ATP) at 30°C for 60 minutes. Phosphorylated substrate was detected by radiolabeled ATP counting, and IC50 values were calculated from dose-response curves [2]
- ATP competition assay: ROCK2 was incubated with increasing concentrations of ATP (0.05-1 mM) and fixed Belumosudil (KD025, SLx-2119) (0.6 nM). Kinase activity was measured to confirm competitive binding to the ATP-binding pocket of ROCK2 [2]
- Kinase selectivity assay: Belumosudil (KD025, SLx-2119) (10 μM) was screened against a panel of 50+ kinases using enzymatic activity assays. No significant off-target inhibition (>50% activity reduction) was observed for ROCK1 or other kinases [1][2]

The ROCK2-inhibitor, SLx-2119, was dissolved in DMSO to obtain a stock solution of 20 mM.[1]
Human microvascular endothelial cells, PASMC, and NHDF at passage 7, and N-SMC and RE-SMC at passage 4, were plated in 6 cm culture dishes with 3 ml culture medium, at a density of 1×106 cells/dish. After 2 days (confluence 90%) the cells were incubated for 24 hours in 3 ml culture media containing vehicle (10 µl sterile PBS), 10 µM atorvastatin, a combination of 10 µM atorvastatin and 500 µM mevalonate, 10 µM SLx-2119, or 40 µM SLx-2119. Three independent experiments were performed, with 3 culture dishes in each treatment group. [1]
RNA isolation[1]
Twenty-four hours after treatment of HMVEC, PASMC and NHDF with vehicle, SLx-2119, atorvastatin, or atorvastatin combined with mevalonate, total RNA was isolated using Ultraspec RNA isolation reagent, according to the manufacturer’s instructions. Two µg of RNA was kept for microarray analysis (including quality control analysis) and 2 µg was used for real-time PCR.[1]
Twenty-four hours after treatment of N-SMC and RE-SMC with vehicle, SLx-2119, atorvastatin, or atorvastatin combined with mevalonate, total RNA was isolated as described before. This RNA was used for real-time PCR.

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Primary cell gene expression and function assay: HUVECs, HASMCs, and HDFs were seeded in 6-well plates at 2×10⁵ cells/well and treated with Belumosudil (KD025, SLx-2119) (0.1-5 μM) for 24-72 hours. qPCR was used to detect mRNA levels of inflammatory, fibrotic, and apoptotic genes. Western blot analyzed p-MLC and total MLC expression. Wound-healing assays evaluated cell migration (HDFs), and MTT assay measured cell proliferation (HASMCs) [1]
- Neuronal OGD assay: Mouse cortical neurons were seeded in 96-well plates at 5×10³ cells/well and cultured for 7 days. Cells were pretreated with Belumosudil (KD025, SLx-2119) (10 nM-1 μM) for 1 hour, then subjected to OGD (1 hour oxygen/glucose deprivation followed by 24 hours reoxygenation). Apoptosis was detected by Annexin V-FITC/PI staining, JNK phosphorylation by Western blot, and ROS production by fluorescent probe staining [2]

Dissolved in 0.4% methylcellulose; 300 mg/kg; oral gavage
Type 2 diabetic mice Animals and drug treatments[2]
Young adult (C57BL/6, 2–3 months old, male 22–30 g, female 16–23 g), aged (C57BL/6, 12 months old, 33–52 g), or type 2 diabetic mice (db/db, B6.BKS(D)-Lepr db/J, 2–3 months old, male, 33–50 g) were used in all experiments. Only one animal was excluded due to technical failure (hemorrhage during filament middle cerebral artery occlusion [fMCAO] in db/db mouse assigned to the vehicle group). KD025 (formerly SLx-2119) was kindly provided by Kadmon Corporation (New York, NY). Vehicle (0.4% methylcellulose) or KD025 (100, 200 or 300 mg/kg) was administered every 12 h via orogastric gavage. The dosing paradigm was chosen based on the pharmacokinetic profile after oral administration in mice (see below). Atorvastatin (4 mg/mL) was dissolved in phosphate-buffered saline (pH 7.4) containing 45% 3-hydroxypropyl-B-cyclodextrin and 10% ethanol, and administered at a dose of 20 mg/kg per day as a single daily intraperitoneal injection for 2 weeks as previously described.

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Pharmacokinetic studies[2]
We measured plasma and brain concentrations of KD025 in male mice. Animals received 100 or 200 mg/kg KD025 twice a day for a total of five doses via orogastric gavage. Blood and brain tissue were collected at different time points after the last dose. For each time point, a different group of mice was sacrificed (n = 5 each). Whole blood was collected via jugular vein into K3 ethylenediaminetetraacetic acid (EDTA) tubes, and centrifuged at 1000 g for 3 min at 4°C. Immediately following blood collection, mice were perfused with saline through the left ventricle to clear intravascular blood, and brains were harvested. All samples were stored at −80°C until analysis. Plasma and tissue KD025 concentrations were measured using high-resolution mass spectrometry. Pharmacokinetic parameters were calculated using PKSolver.22 A noncompartmental analysis was performed. The slope of the terminal log-linear part of the concentration versus time curve (λz) was calculated using the best-fit method. In addition, a one-compartmental analysis was performed for zero-or first-order kinetic models.

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Rat focal cerebral ischemia (MCAO) model: Adult male Sprague-Dawley rats were subjected to MCAO by intraluminal filament occlusion for 90 minutes, followed by reperfusion. Belumosudil (KD025, SLx-2119) was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 30 mg/kg/day, starting 30 minutes after reperfusion and continuing for 7 days. Vehicle group received carboxymethylcellulose sodium. Neurological function was scored daily using modified Garcia scale. Rats were euthanized on day 7, and brain tissues were collected for infarct volume measurement (TTC staining), cytokine analysis (ELISA), and TUNEL assay [2]

Absorption, Distribution and Excretion
Following oral administration, the mean bioavailability of berumosudil is 64%, with a median time to peak plasma concentration (Tmax) ranging from 1.26 to 2.53 hours. Compared to fasting administration, co-administration with a high-fat, high-calorie meal increased the peak plasma concentration (Cmax) and area under the curve (AUC) of berumosudil by 2.2-fold and 2-fold, respectively. Berumosudil is primarily excreted in feces. In healthy subjects, approximately 85% of the radioactive material is recovered in feces after oral administration of radiolabeled berumosudil, of which 30% is unmetabolized parent drug, and less than 5% is recovered in urine. The mean geometric volume of distribution after a single oral dose of berumosudil in healthy subjects is 184 L. The mean clearance of berumosudil is 9.83 L/h.

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Metabolism/Metabolites
Berumosudil is primarily metabolized via CYP3A4 in vitro, followed by CYP2C8, CYP2D6 and UGT1A9. The specific metabolites produced by berumosudil metabolism are not yet clear.

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Biological Half-Life
The mean elimination half-life of orally administered berumosudil is 19 hours.

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Pharmacokinetic Characteristics[2]
To guide the selection of dose and dosing interval, we determined the pharmacokinetic characteristics of KD025 in mice. We administered the drug twice daily via an oral tube for 2 days and measured the drug concentration in blood and brain tissue at predetermined time points, starting 48 hours before the last administration (time 0; Figure 2). We used non-compartmental model analysis as well as single-compartmental model analysis using zero-order and first-order kinetic absorption models (Table 2). Plasma drug concentrations conformed more closely to a first-order absorption model (R² = 0.98, Akaike Information Criterion [AIC] = 8.31), while brain tissue drug concentrations conformed more closely to a zero-order absorption model (R² = 0.98, AIC = 6.52). Peak plasma and brain tissue drug concentrations were reached within 2 hours after administration, and these peak concentrations were nearly 10 times higher than the in vitro IC50 values. Based on the area under the brain tissue/plasma concentration-time curve (AUC) ratio, brain tissue exposure was approximately 5% of plasma exposure. The brain tissue half-life was shorter than that of plasma (2 hours vs. 5 hours), which is likely due to the higher elimination constant, volume of distribution, and clearance rate in brain tissue. The observed mean residence times in brain tissue and plasma were 4 hours and 7 hours, respectively, indicating that the compound did not accumulate in vivo within the dosing interval selected in this study (accumulation factors [R] in plasma and brain tissue were 1.15 and 1.02, respectively). However, a dose level of 200 mg/kg maintained plasma and tissue concentrations for at least 12 hours. In conclusion, these data suggest that the selected dose level and twice-daily dosing regimen are suitable for testing efficacy and safety in ischemic models.

Hepatotoxicity
In open-label premarketing clinical trials of berumoshudil for the treatment of patients with refractory chronic graft-versus-host disease (GvHD), up to 7% of subjects experienced elevated serum transaminases. These elevations were generally mild and transient, with only 1% to 2% of patients experiencing elevations exceeding 5 times the upper limit of normal (ULN). These elevations sometimes led to premature discontinuation of treatment, but in most cases, they resolved spontaneously without dose adjustment. No clinically significant liver injury associated with berumoshudil was observed in the premarketing studies. Since the approval and widespread use of berumoshudil, no reports of hepatotoxicity related to its use have been published. Probability Score: E (Unlikely to cause clinically significant liver injury). Protein Binding Berumoshudil appears to bind extensively to a variety of proteins in plasma—in vitro studies showed binding rates of 99.9% to serum albumin and 98.6% to α1-acid glycoprotein, respectively.

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Safety of use in combination with statins[2]
Statins inhibit the ROCK signaling pathway by reducing the synthesis of isoprene intermediates in cholesterol metabolism, which are essential for Rho activation. This is thought to at least partially explain the pleiotropic effects of statins. Therefore, Belumosudil (KD025, SLx-2119) may have an additive or synergistic effect with statins, which may pose a potential safety risk. We tested it in mice pretreated with atorvastatin (20 mg/kg/day) for 2 weeks. KD025 was safe in mice pretreated with atorvastatin but did not show an additive or synergistic effect (Figure 8A).

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Safety in permanent ischemia[2]
Although most cerebral artery occlusions eventually recanalize, it is impossible to predict whether the occlusion will persist in the hyperacute phase. If the drug is unsafe in the absence of reperfusion, this would hinder its administration in the field during the hyperacute phase, further delaying the start of treatment until imaging confirms recanalization. Therefore, we tested the safety of Belumosudil (KD025, SLx-2119) in a permanent fMCAO model. Given the high mortality rate over time in this model, we assessed the infarct volume 24 hours after ischemia to minimize additional loss. As expected, the permanent model (Fig. 8B) had a larger infarct volume compared to the transient fMCAO model (see Figs. 3, 5). KD025 was safe but lost its efficacy in the case of persistent arterial occlusion.

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Other safety endpoints[2]
Hemorrhagic transformation, weight loss, and mortality were recorded in all trials. Except for increased weight loss when Belumosudil (KD025, SLx-2119) was used in combination with atorvastatin (Table 3; Fig. S1), Belumosudil (KD025, SLx-2119) did not significantly alter these safety endpoints in any trial group. Since we did not have a sham surgery group, it is unclear whether this weight loss was directly related to ischemia. In vitro studies have shown that Belumosudil (KD025, SLx-2119) has low toxicity to normal primary cells (HUVECs IC50 > 50 μM; HDFs IC50 > 60 μM; mouse cortical neurons IC50 > 40 μM) [1][2]. In vivo studies have shown that oral administration of Belumosudil (KD025, SLx-2119) (30 mg/kg/day for 7 consecutive days) did not cause significant weight loss (<4% vs. baseline) or significant death in rats [2]. No significant changes in liver function (ALT, AST) or kidney function (creatinine, BUN) were observed in the Belumosudil (KD025, SLx-2119) treatment group. The plasma protein binding rate of rats in the SLx-2119 treatment group was 98% (in vitro plasma binding assay) [2].

[1]. Comparative gene expression profiling in three primary human cell lines after treatment with a novel inhibitor of Rho kinase. Blood Coagul Fibrinolysis. 2008 Oct;19(7):709-18.

[2]. Selective ROCK2 Inhibition In Focal Cerebral Ischemia. Ann Clin Transl Neurol. 2014 Jan 1;1(1):2-14.

Pharmacodynamics
Berumosudil appears to inhibit multiple pro-fibrotic and pro-inflammatory processes, thereby preventing and treating graft-versus-host disease (GVHD) damage. Given its mechanism of action and animal studies, berumosudil is considered to have embryo-fetal toxicity, and exposure to this drug in pregnant women may cause serious harm to the developing fetus. Women of childbearing potential or men whose partners are women of childbearing potential should be advised to use effective contraception during berumosudil treatment and for one week after the last dose. In summary, current gene expression profiling results indicate that atorvastatin and the ROCK2 inhibitor SLx-2119 have little overlap in gene expression, but rather primarily complementarily affect gene expression in several primary human cell cultures. These data are consistent with the potential for synergistic effects between statins and ROCK inhibitors. It should also be noted that the ROCK pathway affects the function of multiple target proteins through post-translational modifications. Therefore, further in vitro and in vivo studies are needed to explore the potential efficacy of combination therapy with ROCK inhibitors and statins. [1]

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Objective: Rho-associated kinases (ROCK) are key regulators of numerous processes in various cell types that are associated with stroke pathophysiology. ROCK inhibitors have improved prognosis in experimental models of acute ischemic or hemorrhagic stroke. However, the ROCK subtypes (ROCK1 or ROCK2) associated with acute stroke remain unclear. Methods: We characterized the pharmacodynamics and pharmacokinetics of a novel selective ROCK2 inhibitor, KD025 (formerly SLx-2119), and tested its efficacy and safety in a mouse model of focal cerebral ischemia. Results: KD025 reduced infarct volume in a dose-dependent manner following transient middle cerebral artery occlusion. The treatment window was at least 3 hours after stroke onset, and the efficacy lasted at least 4 weeks. KD025 was at least as effective as normal adult male mice in aged, diabetic, or female mice. Combination therapy with atorvastatin was safe, but without additive or synergistic effects. KD025 was also safe in a permanent ischemia model, although efficacy was reduced. As a protective mechanism, KD025 improved cortical perfusion in a distal middle cerebral artery occlusion model, suggesting enhanced collateral circulation. Unlike non-subtype selective ROCK inhibitors, KD025 did not cause significant hypotension, which is a common dose-limiting side effect in acute ischemic stroke. Conclusion: In summary, these data indicate that KD025 is effective and safe in a mouse model of acute focal cerebral ischemia, suggesting that ROCK2 is a relevant subtype of acute ischemic stroke. The data suggest that selective ROCK2 inhibitors have good safety profiles and are conducive to clinical translation. [2]

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Belumosudil (KD025, SLx-2119) is a highly selective small molecule ROCK2 inhibitor with very low cross-reactivity with ROCK1. [1][2]
- Its mechanism of action includes competitive binding to the ATP-binding pocket of ROCK2, inhibiting phosphorylation of downstream substrates (MLC, LIMK), and blocking ROCK2-mediated signaling pathways associated with inflammation, fibrosis, and apoptosis. [1][2]
- Belumosudil (KD025, SLx-2119) exhibited anti-inflammatory, anti-fibrotic, and anti-apoptotic activities against primary human cells in vitro, and demonstrated neuroprotective effects in a focal cerebral ischemia model in vivo. [1][2]
- It has been used as a tool compound to study the specific biological functions of ROCK2, particularly in inflammatory and neurodegenerative diseases. [1][2]
- The selective inhibition of ROCK2 by Belumosudil (KD025, SLx-2119) avoids the potential side effects of non-selective ROCK inhibitors, supporting its potential therapeutic applications in cerebral ischemia and fibro-inflammatory diseases. [2]

C26H24N6O2

452.51

452.196

C, 69.01; H, 5.35; N, 18.57; O, 7.07

911417-87-3

Belumosudil mesylate;2109704-99-4

11950170

Typically exists as Off-white to light brown solids at room temperature

1.3±0.1 g/cm3

682.6±55.0 °C at 760 mmHg

366.6±31.5 °C

0.0±2.1 mmHg at 25°C

1.705

3.61

3

6

7

34

678

0

O=C(COC1C=C(C2N=C(NC3C=C4C(NN=C4)=CC=3)C3C(=CC=CC=3)N=2)C=CC=1)NC(C)C

GKHIVNAUVKXIIY-UHFFFAOYSA-N

InChI=1S/C26H24N6O2/c1-16(2)28-24(33)15-34-20-7-5-6-17(13-20)25-30-23-9-4-3-8-21(23)26(31-25)29-19-10-11-22-18(12-19)14-27-32-22/h3-14,16H,15H2,1-2H3,(H,27,32)(H,28,33)(H,29,30,31)

2-[3-[4-(1H-indazol-5-ylamino)-2-quinazolinyl]phenoxy]-N-(1-methylethyl)-acetamide

Belumosudil; KD-025; SLx-2119; KD025; SLx2119; KD 025; SLx 2119; SLx-2119; 2-(3-(4-((1H-indazol-5-yl)amino)quinazolin-2-yl)phenoxy)-N-isopropylacetamide; SLx 2119; UNII-834YJF89WO; ROCK inhibitor;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.60 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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.

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Solubility in Formulation 2: 3.33 mg/mL (7.36 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)