R-268712 is a potent, orally active inhibitor of activin receptor-like kinase 5 (ALK-5), which is the type I receptor of transforming growth factor-beta (TGF-β). Its in vitro half-maximal inhibitory concentration (IC₅₀) against ALK-5 is 2.5 nM [1].

In HFL-1 cells, R-268712 (3, 10, 30, 100, 300 nM; 1 h) suppresses Smad3 phosphorylation in a dose-dependent manner with an IC50 of 10.4 nM [1]. Without affecting the proliferation of HFL-1 cells, R-268712 (3, 10, 30, 100, and 300 nM; 72 h) suppresses the myofibroblast transdifferentiation (MTD) of fibroblasts in a dose-dependent manner [1].

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Inhibition of Smad3 Phosphorylation: In HFL-1 human lung fibroblasts stimulated with TGF-β1 (5 ng/mL), R-268712 inhibits the phosphorylation of Smad3 in a dose-dependent manner. The IC₅₀ for this inhibition is 10.4 nM [1].
Inhibition of Myofibroblast Transdifferentiation (MTD): In HFL-1 human lung fibroblasts, stimulation with TGF-β1 (5 ng/mL) for 72 hours increases the population of alpha-smooth muscle actin (αSMA)-positive cells, a marker for myofibroblasts. R-268712 inhibits this TGF-β1-induced myofibroblast transdifferentiation in a dose-dependent manner without affecting cell proliferation [1].

In the UUO model, R-268712 (1, 3, 10 mg/kg; oral; once daily for 3 days) inhibits renal luciferase activity in a dose-dependent manner [2]. In a Thy1 nephritis model, R-268712 (0.3, 1, 3, 10 mg/kg; oral; single dose) demonstrated renoprotective effects (improvement and maintenance of renal function and inhibition of glomerular function) at 1 mg/kg [2].

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Efficacy in Bleomycin-Induced Lung Fibrosis Model (Short-Term): In a bleomycin (BLM)-induced lung fibrosis model using Col1a1-IRES-Luc knock-in (KI) mice, oral administration of R-268712 significantly suppresses the increase in lung luciferase activity (a reporter for type I collagen expression) measured 7 days after BLM instillation. At a dose of 30 mg/kg, it reduces luciferase activity by 79% compared to the vehicle-treated group [1].
Efficacy in Bleomycin-Induced Lung Fibrosis Model (Long-Term): In a 21-day BLM-induced lung fibrosis model using C57BL/6 mice, oral administration of R-268712 significantly attenuates the increase in lung hydroxyproline content (a measure of collagen deposition). Significant effects are observed at doses of 30 mg/kg and 100 mg/kg [1].

The in vitro inhibitory activity of R-268712 against ALK-5 was previously determined. It is a potent ALK-5 inhibitor developed through optimization of SB-431542, with an IC₅₀ of 2.5 nM, making it 40 times more potent than SB-431542 (IC₅₀ = 96 nM) [1].

Cell Viability Assay[1]
Cell Types: HFL-1 Cell
Tested Concentrations: 3, 10, 30, 100, 300 nM
Incubation Duration: 1 or 72 hrs (hours)
Experimental Results: Inhibited the phosphorylation of Smad3 in a dose-dependent manner with an IC50 of 10.4 incubation 1 hrs (hours) are nM. Inhibited myofibroblast transdifferentiation (MTD), but not cell growth, of fibroblasts in a dose-dependent manner after 72 hrs (hours) of incubation.

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Smad3 Phosphorylation Assay in HFL-1 Cells: HFL-1 human lung fibroblasts were seeded in 96-well culture plates. After overnight culture, cells were starved in serum-free medium for 4 hours. They were then treated with 5 ng/mL of recombinant human TGF-β1 and various concentrations of R-268712. After 1 hour of incubation, cells were harvested. Phosphorylated Smad3 (pSmad3) levels were measured using a Smad3 PhosphoTracer ELISA kit and normalized to GAPDH protein levels measured with a GAPDH PhosphoTracer ELISA kit [1].
Myofibroblast Transdifferentiation (MTD) Assay in HFL-1 Cells: HFL-1 cells were seeded in 96-well clear-bottom black microplates. After overnight culture, cells were starved and then treated with 5 ng/mL TGF-β1 and various concentrations of R-268712 for 72 hours. Cells were then fixed with cold acetone-methanol, blocked, and incubated with an anti-αSMA antibody, followed by an Alexa Fluor 488-conjugated secondary antibody and Hoechst 33342 for nuclear staining. Immunofluorescence was measured using an IN Cell Analyzer 6000. The αSMA-positive area was quantified and normalized to the cell number (based on nuclei count) [1].

Animal/Disease Models: Male WKY/Hos rat [2]. Strength: 0.3, 1, 3 and 10 mg/kg
Route of Administration: Oral; single.
Experimental Results: 1.19 pharmacokinetic/PK/PK parameters of R-268712 in male WKY/Hos rats (n=4)[2]. PO (0.3 mg/kg) PO (1 mg/kg) PO (3 mg/kg) PO (10 mg/kg) AUC0-24 (μg·h/mL) 0.075 0.28 1.6 8.2 Animal/Disease Models: Male Col1a1-Luc Tg Rats (10 to 14 weeks old; UUO model; n=5-6) [2].
Doses: 1, 3, 10 mg/kg
Doses: po (po (oral gavage)) one time/day for 3 days.
Experimental Results: Inhibition of renal luciferase activity in a dose-dependent manner.

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Animal/Disease Models: Male WKY/Hos rats (4 weeks old; Thy1 nephritis model; n=7) [2].
Doses: 0.3, 1 mg/kg
Route of Administration: Oral; one time/day for 33 days.
Experimental Results: Proteinuria on day 21 (suppression continued until day 28) and serum creatinine levels (dose 1 mg/kg) were Dramatically diminished. At 1 mg/kg, it Dramatically inhibits glomerulosclerosis by 28% and reduces the increase in hydroxyproline content. 1 mg/kg inhibits the activation of mesangial parenchymal cells and the damage of podocytes on the basis

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BLM-Induced Lung Fibrosis Model in KI Mice (Short-Term Efficacy):** Male Col1a1-IRES-Luc KI mice were instilled intratracheally with bleomycin (BLM) to induce lung fibrosis. R-268712 was suspended in 0.5% methylcellulose 400 and administered orally once daily at various doses (e.g., 30 mg/kg) in a volume of 10 mL/kg body weight. Dosing started on the day of BLM injection and continued until the day before dissection. The control and vehicle groups received an equal volume of 0.5% methylcellulose. Luciferase activity in the lung was measured on day 7 after BLM instillation [1].
* **BLM-Induced Lung Fibrosis Model in C57BL/6 Mice (Long-Term Efficacy):** Male C57BL/6 mice were instilled intratracheally with BLM. R-268712 was suspended in 0.5% methylcellulose and administered orally once daily at doses of 30 and 100 mg/kg (10 mL/kg) for 21 days. Hydroxyproline content in the lung was measured on day 21 after BLM instillation [1].

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BLM-Induced Lung Fibrosis Model in KI Mice (Short-Term Efficacy): Male Col1a1-IRES-Luc KI mice were instilled intratracheally with bleomycin (BLM) to induce lung fibrosis. R-268712 was suspended in 0.5% methylcellulose 400 and administered orally once daily at various doses (e.g., 30 mg/kg) in a volume of 10 mL/kg body weight. Dosing started on the day of BLM injection and continued until the day before dissection. The control and vehicle groups received an equal volume of 0.5% methylcellulose. Luciferase activity in the lung was measured on day 7 after BLM instillation [1].
BLM-Induced Lung Fibrosis Model in C57BL/6 Mice (Long-Term Efficacy): Male C57BL/6 mice were instilled intratracheally with BLM. R-268712 was suspended in 0.5% methylcellulose and administered orally once daily at doses of 30 and 100 mg/kg (10 mL/kg) for 21 days. Hydroxyproline content in the lung was measured on day 21 after BLM instillation [1].

R-268712 is described as an orally active ALK-5 inhibitor [1].

A preliminary study indicated that oral administration of R-268712 at a dose of 10 mg/kg suppressed weight gain in the heminephrectomized anti-Thy1 glomerulonephritis model. Consequently, doses of 0.3 and 1 mg/kg were selected for efficacy evaluation in this model to avoid potential side effects related to the pleiotropic actions of TGF-β [2].

[1]. Attenuation of pulmonary fibrosis in type I collagen-targeted reporter mice with ALK-5 inhibitors. Pulm Pharmacol Ther. 2019 Feb;54:31-38.

[2]. R-268712, an orally active transforming growth factor-β type I receptor inhibitor, prevents glomerular sclerosis in a Thy1 nephritis model. Eur J Pharmacol. 2014 Jul 5;734:60-6.

[3]. Development of small molecule inhibitors targeting TGF-β ligand and receptor: Structures, mechanism, preclinical studies and clinical usage. Eur J Med Chem. 2020 Apr 1;191:112154.

R-268712 is a potent and orally active inhibitor of ALK-5, developed through optimization of the earlier ALK-5 inhibitor SB-431542. Its primary mechanism of action is the inhibition of TGF-β signaling by blocking the phosphorylation of Smad3. In models of lung fibrosis, it demonstrates strong antifibrotic efficacy by inhibiting myofibroblast transdifferentiation and subsequent collagen production. The compound's activity was effectively evaluated using a novel Col1a1-IRES-Luc KI mouse model, which allows for the rapid (7-day) assessment of antifibrotic effects by measuring luciferase activity as a surrogate for type I collagen expression [1].

C20H18FN5O

363.39

363.149

879487-87-3

11703284

White to light yellow solid powder

1.3±0.1 g/cm3

576.8±50.0 °C at 760 mmHg

302.7±30.1 °C

0.0±1.7 mmHg at 25°C

1.675

2.79

2

5

5

27

483

0

JQGOCCALXFSRHZ-UHFFFAOYSA-N

InChI=1S/C20H18FN5O/c1-13-3-2-4-19(24-13)20-17(11-22-25-20)14-5-6-18(21)16(9-14)15-10-23-26(12-15)7-8-27/h2-6,9-12,27H,7-8H2,1H3,(H,22,25)

2-[4-[2-fluoro-5-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]phenyl]pyrazol-1-yl]ethanol

R-268712 R268712 R 268712

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)

DMSO : ~125 mg/mL (~343.98 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.72 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 20.8 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: ≥ 2.08 mg/mL (5.72 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 20.8 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (5.72 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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