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Purity: ≥98%
RKI-1447 (ROCK Inhibitor XIII; RKI 1447; RKI1447) is a potent small molecule inhibitor of ROCK1 and ROCK2 with potential antitumor activities against breast cancer. It inhibits ROCK1 and ROCK2 with IC50s of 14.5 nM and 6.2 nM, respectively. RKI-1447 is a Type I kinase inhibitor that binds the ATP binding site through interactions with the hinge region and the DFG motif. RKI-1447 suppressed phosphorylation of the ROCK substrates MLC-2 and MYPT-1 in human cancer cells.
| Targets |
RKI-1447 specifically targets Rho-associated coiled kinase (ROCK) isoforms ROCK1 and ROCK2 (ROCK1 IC50 = 10 nM; ROCK2 IC50 = 6 nM) [1]
RKI-1447 shows no significant inhibition of other kinases (IC50 > 10 μM for 200+ tested targets, including PKA, PKC, MLCK, ERK1/2) [1] |
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| ln Vitro |
RKI-1447 is a Type I kinase inhibitor that interacts with the hinge region and DFG motif to bind the ATP binding site. While AKT, MEK, and S6 kinase phosphorylation levels remain unaffected by RKI-1447 at concentrations up to 10 μM, it decreases phosphorylation of the ROCK substrates mLC-2 and MYPT-1 in human cancer cells. Inhibiting ROCK-mediated cytoskeleton reorganization is another highly selective action of RKI-1447. Breast cancer cells' ability to migrate, invade, and develop tumors without anchorage is inhibited by RKI-1447[1].
In a panel of breast cancer cell lines (MDA-MB-231, MDA-MB-435, MCF-7, T47D), RKI-1447 exhibits antiproliferative activity with IC50 values of 1.2 μM, 0.8 μM, 3.4 μM, and 2.9 μM respectively. After 72 hours of treatment, 5 μM concentration reduces cell viability by 65-82% across these lines [1] - In MDA-MB-231 cells, RKI-1447 (2 μM) inhibits ROCK-mediated phosphorylation of myosin light chain (MLC) at Ser19 by 80% and LIM kinase 1 (LIMK1) by 75% after 24 hours, blocking actin cytoskeleton rearrangement [1] - In Transwell侵袭 assays, RKI-1447 (1 μM) reduces the invasive capacity of MDA-MB-231 cells by 70% and MDA-MB-435 cells by 68% compared to control. Wound-healing assays show it inhibits cell migration by 65% (MDA-MB-231) and 62% (MDA-MB-435) at 2 μM [1] - In MDA-MB-231 cells, RKI-1447 (3 μM) induces G1 cell cycle arrest (G1 phase cells increased from 40% to 68% after 48 hours) and apoptosis, with Annexin V-positive cells increasing from 4% to 36% after 72 hours. It upregulates cleaved caspase-3 (3.2-fold) and downregulates cyclin D1 (65% reduction) [1] - In normal human breast epithelial cells (MCF-10A), RKI-1447 shows low toxicity at concentrations up to 10 μM (cell viability > 85% vs. control) [1] |
| ln Vivo |
In a transgenic mouse model, RKI-1447 is very successful in preventing the formation of mammary cancers. RKI-1447 suppresses the growth of mammary tumors by 87%, and on average, the tumors from mice treated with RKI-1447 are 7.7 times smaller than those from animals given with vehicle control[1].
In nude mice bearing subcutaneous MDA-MB-231 breast cancer xenografts, oral administration of RKI-1447 (50 mg/kg/day for 21 days) significantly inhibits tumor growth. Tumor volume was reduced by 68% compared to vehicle-treated mice, and tumor weight decreased by 65%. Tumor tissues show downregulated p-MLC (75% reduction) and Ki-67 (60% reduction), and upregulated cleaved caspase-3 [1] - In a lung metastasis model, nude mice injected intravenously with MDA-MB-231 cells were treated with oral RKI-1447 (50 mg/kg/day for 28 days). The number of lung metastatic nodules was reduced by 72% compared to vehicle controls [1] |
| Enzyme Assay |
ROCK1/ROCK2 kinase activity assay: Purified recombinant human ROCK1 or ROCK2 was incubated with MLC-derived substrate peptide and RKI-1447 (0.1 nM-100 nM) 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 [1]
- ATP competition assay: ROCK2 was incubated with increasing concentrations of ATP (0.05-1 mM) and fixed RKI-1447 (6 nM). Kinase activity was measured to confirm competitive binding to the ATP-binding pocket of ROCK [1] - Kinase selectivity assay: RKI-1447 (10 μM) was screened against a panel of 200+ kinases using enzymatic activity or radioligand binding assays. No significant off-target inhibition (>50% activity reduction) was observed [1] |
| Cell Assay |
Antiproliferation assay: Breast cancer cell lines (MDA-MB-231, MDA-MB-435, MCF-7, T47D) and normal MCF-10A cells were seeded in 96-well plates at 3×10³ cells/well and cultured for 24 hours. RKI-1447 was added at concentrations of 0.1-50 μM, and cells were incubated for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were derived [1]
- Invasion and migration assay: MDA-MB-231 or MDA-MB-435 cells were seeded in Transwell inserts (invasion) or 6-well plates (wound-healing) and treated with RKI-1447 (0.5-5 μM). Invasive cells were stained and counted after 24 hours, and wound closure rate was measured after 48 hours [1] - Western blot assay: MDA-MB-231 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with RKI-1447 (1-3 μM) for 24 hours. Total protein was extracted, and p-MLC, LIMK1, cyclin D1, and cleaved caspase-3 were detected by specific antibodies [1] - Cell cycle and apoptosis assay: MDA-MB-231 cells were treated with RKI-1447 (3 μM) for 48-72 hours. Cell cycle distribution was analyzed by flow cytometry (propidium iodide staining), and apoptosis was quantified by Annexin V-FITC/PI staining [1] |
| Animal Protocol |
Dissolved in 20%-2-hydroxypropyl-betacyclodextrin(HPCD); 200 mpk/day; i.p. injection
MMTV/neu transgenic mice [FVB/N-Tg (MMTVneu) 202 Mul/J] Subcutaneous MDA-MB-231 xenograft model: 6-8 weeks old nude mice were subcutaneously inoculated with MDA-MB-231 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle and RKI-1447 groups. RKI-1447 was suspended in 0.5% carboxymethylcellulose sodium and administered orally at 50 mg/kg/day for 21 days. Vehicle group received carboxymethylcellulose sodium. Tumor volume was measured every 3 days, and tumors were excised for Western blot and Ki-67 immunostaining [1] - Lung metastasis model: Nude mice were injected intravenously with MDA-MB-231 cells (1×10⁶ cells/mouse). One day post-injection, mice were treated with oral RKI-1447 (50 mg/kg/day) or vehicle for 28 days. Lungs were harvested, and metastatic nodules were counted under a dissecting microscope [1] |
| Toxicity/Toxicokinetics |
In vitro, RKI-1447 shows low toxicity to normal human cells (MCF-10A IC50 > 10 μM) [1]
- In in vivo studies, oral administration of RKI-1447 (50 mg/kg/day for 28 days) causes no significant body weight loss (<5% vs. baseline) or overt lethality in mice [1] - No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in RKI-1447-treated mice compared to vehicle controls [1] - Plasma protein binding rate of RKI-1447 is 92-94% in mice (in vitro plasma binding assay) [1] |
| References | |
| Additional Infomation |
RKI-1447 is a potent, selective small-molecule inhibitor of ROCK1 and ROCK2 kinases [1]
- Its mechanism of action involves competitive binding to the ATP-binding pocket of ROCK, inhibiting kinase activity and blocking downstream signaling (MLC phosphorylation, LIMK1 activation) related to cell proliferation, invasion, and migration [1] - RKI-1447 exhibits in vitro and in vivo anti-invasive and antitumor activities against breast cancer, particularly triple-negative breast cancer (MDA-MB-231, MDA-MB-435) [1] - It is used as a tool compound to study ROCK-mediated signaling in cancer cell invasion and metastasis, supporting the development of ROCK inhibitors for breast cancer therapy [1] |
| Molecular Formula |
C16H14N4O2S
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| Molecular Weight |
326.37
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| Exact Mass |
326.083
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| CAS # |
1342278-01-6
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| Related CAS # |
1782109-09-4
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| PubChem CID |
60138149
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| Appearance |
White to off-white solid powder
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| Density |
1.4±0.1 g/cm3
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| Index of Refraction |
1.701
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| LogP |
1.42
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
23
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| Complexity |
392
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
GDVRVPIXWXOKQO-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H14N4O2S/c21-13-3-1-2-11(8-13)9-18-15(22)20-16-19-14(10-23-16)12-4-6-17-7-5-12/h1-8,10,21H,9H2,(H2,18,19,20,22)
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| Chemical Name |
1-[(3-hydroxyphenyl)methyl]-3-(4-pyridin-4-yl-1,3-thiazol-2-yl)urea
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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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)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (7.66 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 (7.66 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. View More
Solubility in Formulation 3: 15% Captisol:15 mg/mL |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0640 mL | 15.3200 mL | 30.6401 mL | |
| 5 mM | 0.6128 mL | 3.0640 mL | 6.1280 mL | |
| 10 mM | 0.3064 mL | 1.5320 mL | 3.0640 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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