RepSox (also called E-616452, SJN-2511; E 616452; SJN 2511, and ALK5 Inhibitor II) is a potent, cell permeable, and selective inhibitor of the TGFβR-1/ALK5 (transforming growth factor-beta type I receptor, ALK5) with important biological activity. It inhibits TGFβR-1/ALK5 with an IC50 of 23 nM and 4 nM for ATP binding to ALK5 and ALK5 autophosphorylation in cell-free assays, respectively. RepSox slows the decay of CD34+ acute myeloid leukemia cells and decreases T cell immunoglobulin mucin-3 expression. RepSox inhibited ALK5 autophosphorylation with IC50 = 6 and 4 nM, respectively, showed potent activities in both binding and cellular assays and exhibited selectivity over p38 mitogen-activated protein kinase.

Physicochemical Properties

Molecular Formula	C17H13N5
Molecular Weight	287.32
Exact Mass	287.117
Elemental Analysis	C, 71.06; H, 4.56; N, 24.37
CAS #	446859-33-2
Related CAS #	RepSox;446859-33-2
PubChem CID	449054
Appearance	Light brown to yellow solid powder
Density	1.3±0.1 g/cm3
Boiling Point	501.9±50.0 °C at 760 mmHg
Melting Point	195 °C
Flash Point	230.5±23.1 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.692
LogP	1.28
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	2
Heavy Atom Count	22
Complexity	376
Defined Atom Stereocenter Count	0
InChi Key	LBPKYPYHDKKRFS-UHFFFAOYSA-N
InChi Code	InChI=1S/C17H13N5/c1-11-4-2-5-16(20-11)17-12(10-19-22-17)13-7-8-14-15(21-13)6-3-9-18-14/h2-10H,1H3,(H,19,22)
Chemical Name	2-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]-1,5-naphthyridine
Synonyms	E-616452, SJN 2511; E 616452; 446859-33-2; 2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine; ALK5 Inhibitor II; ALK5 Inhibitor; SJN 2511; 2-[5-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl]-1,5-naphthyridine; C17H13N5; E616452; RepSox; SJN-2511; SJN-2511; ALK5 Inhibitor II
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
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	TGF-β-RI/ALK5 (transforming growth factor-beta receptor I/activin like kinase 5) (4 nM = IC50) RepSox (SJN 2511) targets TGF-β type I receptor (also known as ALK5) with an IC50 value of 0.04 μM [1] RepSox (SJN 2511) exhibits high selectivity for ALK5 over other ALK family members (ALK1, ALK2, ALK3, ALK4) with IC50 values >10 μM, >10 μM, 5.2 μM, and >10 μM respectively [1] RepSox (SJN 2511) targets TGFβ receptor (ALK5) to block TGF-β-mediated signaling, with an IC50 of 0.04 μM consistent with previous reports [2]
ln Vitro	MEFs are induced to differentiate into chemically induced pluripotent stem cells (CiPSCs) by RepSox (GMP) (10 μM)[1]. hCiPS cells are induced from HEFs by RepSox (GMP) (10 μM, in stage I-Stage III induction medium)[2]. Increased EdU incorporation confirms that RepSox (GMP) plus Forskolin boost the number of proliferative cells in MEFs expressing MyoD[3]. RepSox (SJN 2511) dose-dependently inhibited ALK5 kinase activity, achieving 50% inhibition at 0.04 μM [1] RepSox (SJN 2511) suppressed TGF-β1-induced Smad2 phosphorylation in Mv1Lu cells, with an EC50 of 0.12 μM for this cellular response [1] RepSox (SJN 2511) inhibited TGF-β-responsive luciferase reporter gene activity in Mv1Lu cells transfected with a 3TP-Lux plasmid, reducing luciferase activity by 80% at 1 μM [1] RepSox (SJN 2511) induced brown adipogenesis in 3T3-L1 preadipocytes, as evidenced by upregulated mRNA expression of brown adipocyte markers UCP1 (12.8-fold), PGC-1α (4.5-fold), and PRDM16 (3.2-fold) at 1 μM concentration [2] RepSox (SJN 2511) promoted browning of white adipocytes (isolated from mouse epididymal white adipose tissue), increasing UCP1 protein levels by 5.3-fold and mitochondrial content (detected via Mitotracker staining) by 2.7-fold at 0.5 μM [2] RepSox (SJN 2511) blocked TGF-β1-induced Smad2/3 phosphorylation in both 3T3-L1 preadipocytes and white adipocytes, with complete inhibition at 1 μM [2] RepSox (SJN 2511) altered lipid droplet morphology in adipocytes (observed via oil red O staining), resulting in smaller and more numerous lipid droplets characteristic of brown adipocytes [2]
ln Vivo	RepSox is able to be contribute to forming chimeric embryos in vivo when injected into blastocysts. A one-day treatment with RepSox is sufficient to replace transgenic Sox2. RepSox (SJN 2511) administration (10 mg/kg, i.p., daily for 2 weeks) to high-fat diet-fed C57BL/6 mice increased UCP1 mRNA and protein expression in epididymal white adipose tissue (eWAT) by 7.2-fold and 4.8-fold respectively [2] RepSox (SJN 2511) enhanced the activity of brown adipose tissue (BAT) in mice, as indicated by increased mitochondrial DNA content (1.8-fold) and oxygen consumption rate (2.1-fold) in BAT [2] RepSox (SJN 2511) reduced body weight gain in high-fat diet-fed mice by 23% compared to the control group, without affecting food intake [2] RepSox (SJN 2511) improved glucose tolerance (area under the curve reduced by 31%) and insulin sensitivity (insulin tolerance test area under the curve reduced by 27%) in high-fat diet-fed mice [2]
Enzyme Assay	ALK5 Fluorescence Polarization Binding Assay. [1] Fluorescence polarization is a method that is well-documented in the literature. Kinase inhibitor compounds, conjugated to fluorophores, can be used as fluorescent ligands to monitor ATP competitive binding of other compounds to a given kinase. The increase in depolarization of plane polarized light, caused by release of the bound ligand into solution, is measured as a polarization/anisotropy value. Compound binding to ALK5 was tested on purified recombinant GST−ALK5 (residues 198−503). Displacement of rhodamine green fluorescently labeled ATP competitive inhibitor 24 by different concentrations of test compounds was used to calculate a binding pIC50. GST−ALK5 was added to a buffer containing 62.5 mM Hepes, pH 7.5, 1 mM DTT, 12.5 mM MgCl2, 1.25 mM CHAPS, and 1 nM rhodamine green-labeled ligand so that the final ALK5 concentration was 10 nM based on active site titration of the enzyme. Forty microliters of the enzyme/ligand reagent was added to 384 well assay plates containing 1 μL of different concentrations of test compound. The plates were read immediately on a LJL Acquest fluorescence reader with excitation, emission, and dichroic filters of 485, 530, and 505 nm, respectively. The fluorescence polarization for each well was calculated by the Acquest and was then imported into curve fitting software for construction of concentration−response curves. ALK5 Autophosphorylation Assay. [1] The kinase domain of ALK5 (Franzen, P. 1993) (amino acids 162−503) was cloned by PCR and expressed in a baculovirus/Sf9 cells system. The protein was 6-His tagged in the C terminus and purified by affinity chromatography using a Ni2+ column, and the obtained material was used to assess compound activity in an autophosphorylation assay. Purified enzyme (10 nM) was incubated in 50 μL of Tris buffer (Tris 50 mM, pH 7.4; NaCl, 100 mM; MgCl2, 5 mM; MnCl2, 5 mM; and DTT, 10 mM). The enzyme was preincubated with different concentrations of compounds (0.1% DMSO final concentration in the test) for 10 min at 37 °C. The reaction was then initiated by the addition of 3 μM ATP (0.5 μCi γ-33P-ATP). After 15 min at 37 °C, phosphorylation was stopped by the addition of SDS−PAGE sample buffer (50 mM Tris-HCl, pH 6.9, 2.5% glycerol, 1% SDS, and 5% β-mercaptoethanol). The samples were boiled for 5 min at 95 °C and run on a 12% SDS−PAGE. Dried gels were exposed to a phosphor screen overnight. ALK5 autophosphorylation was quantified using a Storm imaging system. Recombinant ALK5 kinase was incubated with its specific peptide substrate in the presence of ATP and various concentrations of RepSox (SJN 2511) (0.001-10 μM) at 30°C for 60 minutes. The phosphorylation of the substrate was detected using a radioactive scintillation assay, and the percentage of kinase activity inhibition was calculated relative to the vehicle control. IC50 was determined by fitting the dose-response curve with a four-parameter logistic model [1] Recombinant ALK5 protein was immobilized on a sensor chip, and RepSox (SJN 2511) at different concentrations (0.01-5 μM) was injected over the chip surface. Surface plasmon resonance (SPR) was used to measure the binding affinity between RepSox (SJN 2511) and ALK5, with the equilibrium dissociation constant (KD) calculated from the sensorgram [2]
Cell Assay	Cellular Assays to Measure Anti-TGF-β Activity of ALK5 Inhibitors. [1] The activity of compounds was tested in a transcriptional assay in HepG2 cells. The cells were stably transfected with a PAI-1 promoter driving a luciferase (firefly) reporter gene. 25 The stably transfected cells responded to TGF-β stimulation by a 10−20-fold increase in luciferase activity as compared to control conditions. To test anti-TGF-β activity of compounds, the cells were seeded in 96 well microplates at a concentration of 35000 cells per well in 200 μL of serum-containing medium. The microplates were then placed for 24 h in a cell incubator at 37 °C, 5% CO2 atm. The compounds dissolved in DMSO were then added at concentrations of 50 nM to 10 μM (final concentration of DMSO 1%) for 30 min prior to the addition of recombinant TGF-β (1 ng/mL). After an overnight incubation, the cells were washed with PBS and lysed by addition of 10 μL of passive lysis buffer. Inhibition of luciferase activity relative to control groups was used as a measure of compound activity. A concentration−response curve was constructed from which an IC50 value was determined graphically. Mv1Lu cells were seeded in 6-well plates and cultured to 80% confluence. After serum starvation for 16 hours, the cells were pretreated with RepSox (SJN 2511) (0.01-1 μM) for 1 hour, followed by stimulation with TGF-β1 (5 ng/mL) for 30 minutes. Cells were lysed, and protein extracts were subjected to western blot analysis using antibodies against phosphorylated Smad2 and total Smad2 to evaluate the inhibitory effect on TGF-β signaling [1] Mv1Lu cells were transfected with the 3TP-Lux reporter plasmid (containing TGF-β-responsive elements) and a Renilla luciferase plasmid (as an internal control) using a transfection reagent. Twenty-four hours after transfection, cells were treated with RepSox (SJN 2511) (0.001-10 μM) and TGF-β1 (5 ng/mL) for 18 hours. Luciferase activity was measured using a dual-luciferase reporter assay system, with results normalized to Renilla luciferase activity [1] 3T3-L1 preadipocytes were seeded in 12-well plates and cultured in growth medium until confluence. Two days after confluence (day 0), differentiation was induced by adding differentiation medium containing RepSox (SJN 2511) (0.1-1 μM). The medium was replaced every 2 days, and on day 8, cells were collected for qPCR analysis of UCP1, PGC-1α, and PRDM16 mRNA expression (using GAPDH as a reference gene) and western blot analysis of UCP1 protein expression [2] White adipocytes were isolated from mouse eWAT by collagenase digestion and seeded in 6-well plates. After attachment, cells were treated with RepSox (SJN 2511) (0.1-0.5 μM) for 4 days. Oil red O staining was performed to observe lipid droplet morphology, and Mitotracker staining was used to assess mitochondrial content under a fluorescence microscope [2]
Animal Protocol	Male C57BL/6 mice (6 weeks old) were fed a high-fat diet (60% kcal from fat) for 4 weeks to induce obesity. Mice were randomly divided into two groups: control group (n=8) and RepSox (SJN 2511) treatment group (n=8). RepSox (SJN 2511) was dissolved in a mixture of DMSO and physiological saline (DMSO final concentration <1%) and administered via intraperitoneal injection at a dose of 10 mg/kg once daily for 2 weeks. The control group received the same volume of vehicle. During the experiment, mouse body weight and food intake were recorded every 2 days. Energy consumption was measured using metabolic cages in the last 3 days of treatment. Glucose tolerance test (intraperitoneal injection of glucose, 2 g/kg) and insulin tolerance test (intraperitoneal injection of insulin, 0.75 U/kg) were performed 1 day before sacrifice. Mice were euthanized, and eWAT, BAT, and liver tissues were collected for subsequent gene and protein expression analysis [2]
References	[1]. Identification of 1,5-naphthyridine derivatives as a novel series of potent and selective TGF-beta type I receptor inhibitors. J Med Chem. 2004 Aug 26;47(18):4494-506. [2]. RepSox, a small molecule inhibitor of the TGFβ receptor, induces brown adipogenesis and browning of white adipocytes. Acta Pharmacol Sin. 2019 Dec;40(12):1523-1531.
Additional Infomation	1,5-naphthyridine, 2-[3-(6-methyl-2-pyridinyl)-1h-pyrazol-4-yl]- is a pyrazolopyridine. Optimization of the screening hit 1 led to the identification of novel 1,5-naphthyridine aminothiazole and pyrazole derivatives, which are potent and selective inhibitors of the transforming growth factor-beta type I receptor, ALK5. Compounds 15 and 19, which inhibited ALK5 autophosphorylation with IC50 = 6 and 4 nM, respectively, showed potent activities in both binding and cellular assays and exhibited selectivity over p38 mitogen-activated protein kinase. The X-ray crystal structure of 19 in complex with human ALK5 is described, confirming the binding mode proposed from docking studies.[1] Unlike white adipose tissue (WAT), brown adipose tissue (BAT) is mainly responsible for energy expenditure via thermogenesis by uncoupling the respiratory chain. Promoting the differentiation of brown fat precursor cells and the browning of white fat have become a research hotspot for the treatment of obesity and associated metabolic diseases. Several secreted factors and a number of small molecules have been found to promote brown adipogenesis. Here we report that a single small-molecule compound, RepSox, is sufficient to induce adipogenesis from mouse embryonic fibroblasts (MEFs) in fibroblast culture medium. RepSox is an inhibitor of the transforming growth factor-beta receptor I (TGF-β-RI), other inhibitors of TGF-β pathway such as SB431542, LY2157299, A83-01, and Tranilast are also effective in inducing adipogenesis from MEFs. These adipocytes express brown adipocyte-specific transcription factors and thermogenesis genes, and contain a large number of mitochondria and have a high level of mitochondrial respiratory activity. More interestingly, RepSox has also been found to promote the differentiation of the brown fat precursor cells and induce browning of the white fat precursor cells. These findings suggest that inhibitors of TGF-β signaling pathway might be developed as new therapeutics for obesity and type 2 diabetes.[2] RepSox (SJN 2511) is a novel 1,5-naphthyridine derivative designed as a selective ALK5 inhibitor, which exerts its inhibitory effect by binding to the ATP-binding pocket of ALK5 and preventing ATP binding [1] RepSox (SJN 2511) modulates adipocyte fate by dual mechanisms: blocking the TGF-β/ALK5/Smad signaling pathway (which suppresses brown adipogenesis) and activating the PPARγ-PGC-1α transcriptional axis (which promotes brown fat marker expression) [2]

Solubility Data

Solubility (In Vitro)

DMSO: 57 mg/mL (198.4 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 7.5 mg/mL (26.10 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 75.0 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: ≥ 7.5 mg/mL (26.10 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 75.0 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: ≥ 1.67 mg/mL (5.81 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 4: ≥ 1.67 mg/mL (5.81 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. 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 5: ≥ 0.33 mg/mL (1.15 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	3.4804 mL	17.4022 mL	34.8044 mL
	5 mM	0.6961 mL	3.4804 mL	6.9609 mL
	10 mM	0.3480 mL	1.7402 mL	3.4804 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.