ALK5-IN-8 - ≥98% , CAS No.2705900-81-6

CAS: 2705900-81-6 Cat. No.: A646723 Formula: C₁₉H₁₅FN₄O₂ Molecular Weight: 350.35

GRADE & PURITY ≥98%

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Overview

ALK5-IN-8 is a potent inhibitor of TGFβRI ( ALK5 ). ALK5-IN-8 Inhibits the phosphorylation of ALK5 on its downstream signaling proteins (Smad2 or Smad3) by blocking the binding of TGFβRI to ligands, thereby affecting or blocking TGF-β signaling. ALK5-IN-8 has the potential for the research of various ALK5-mediated related diseases (extracted from patent WO2021190425A1, compound 1).

Form:Solid

Specifications

Specifications & Purity

≥98%

Biochemical and Physiological Mechanisms

ALK5-IN-8 is a potent inhibitor of TGFβRI ( ALK5 ). ALK5-IN-8 Inhibits the phosphorylation of ALK5 on its downstream signaling proteins (Smad2 or Smad3) by blocking the binding of TGFβRI to ligands, thereby affecting or blocking TGF-β signaling. ALK5-IN-8

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

This product requires cold chain shipping. Ground and other economy services are not available.

Action Type

INHIBITOR

Purity

≥98%

Names and Identifiers

Molecular Weight

350.35

Application Protocols

Item-specific validated applications and dilution guidelines: Not specified for this item; refer to CoA/Spec Sheet.

General example protocols (literature/general, not item-specific)

• Biochemical kinase IC50 assay
  • Prepare 10 mM DMSO stock. Perform 3-fold serial dilutions to cover ~0.1 nM–30 µM range.
  • Incubate ALK5 kinase domain with substrate and ATP (at Km) in assay buffer; add inhibitor dilutions, final DMSO ≤1%.
  • Incubate 30–60 min at room temperature; detect activity via ADP-Glo or equivalent. Fit dose–response with a 4PL model.
• Cellular p-SMAD2/3 inhibition
  • Seed responsive cells (e.g., HaCaT, A549, NIH/3T3) 24 h prior. Pre-treat with inhibitor (0.01–10 µM) for 45 min; stimulate with TGF-β1 (e.g., 2 ng/mL) for 45 min.
  • Lyse and immunoblot for p-SMAD2 (Ser465/467) and p-SMAD3 (Ser423/425); normalize to total SMAD2/3 or housekeeping protein.
• Reporter gene assay (CAGA-luc)
  • Transfect cells with CAGA12-luc and Renilla control. After 24 h, treat with inhibitor series and TGF-β1. Measure luciferase after 16–24 h; calculate % inhibition relative to vehicle.

Notes

• Always include vehicle controls, confirm target engagement with orthogonal readouts, and verify solubility at top test concentrations.

Biological Roles

Item-specific biological targeting data are not provided for this SKU.

Literature/general biology for ALK5 (TGFBR1) inhibitors (not item-specific)

• Target: ALK5 (also known as TGF-β type I receptor, TGFBR1) is a serine/threonine kinase that propagates canonical TGF-β signaling by phosphorylating SMAD2/3 after ligand-induced receptor complex formation.
• Pathway context: TGF-β ligands bind TGFBR2, which recruits and phosphorylates ALK5. Activated ALK5 phosphorylates SMAD2/3, which complex with SMAD4 and translocate to the nucleus to regulate transcription of genes involved in extracellular matrix production, epithelial–mesenchymal transition, and immune modulation.
• Research utility: Small-molecule ALK5 inhibitors serve as chemical probes to acutely suppress canonical SMAD signaling, enabling temporal dissection of TGF-β responses in diverse systems (fibroblasts, epithelial cells, organoids). They aid in distinguishing ALK5-dependent from ALK4/7-mediated effects when used with selective controls.
• Selectivity considerations: Many inhibitors show activity on closely related receptors (ALK4/ALK7) or off-target kinases at higher concentrations. Profiling against kinome panels and using orthogonal readouts (e.g., p38/ERK phosphorylation) help attribute phenotypes to ALK5 blockade.
• Biomarkers/readouts: Rapid reduction in p-SMAD2 (Ser465/467) and p-SMAD3 (Ser423/425), decreased CAGA-luc reporter activity, and modulation of EMT markers (E-cadherin up, vimentin down) are typical indicators of on-target activity in cells.

Buffer Applications

This product is not a buffering agent. No classical buffer applications (pH control, electrophoresis buffers) apply.

Assay solution preparation (general guidance, not item-specific)

• Vehicle: Prepare concentrated DMSO stock; dilute into physiological buffers (e.g., PBS, HBSS) or complete culture media. Keep final DMSO typically ≤0.1–0.5% v/v in cell-based assays.
• Order of addition: Add the DMSO stock slowly to vigorously mixing buffer/media to minimize local supersaturation and precipitation.
• Filtration: If sterility is required, pass working solutions through a 0.22 µm filter using low-binding membranes (PTFE, PES, or RC), recognizing potential adsorption losses for hydrophobic compounds.

Green Alternatives

This product functions as a bioactive tool compound; “green solvent” substitution is not typically applicable. Environmental impact is dominated by small-scale assay use and DMSO-based stocks.

General sustainability guidance (literature/general)

• Minimize solvent use: Prepare concentrated DMSO stocks (e.g., 10–50 mM) to reduce solvent volumes in assays; aliquot to avoid repeated freeze–thaw and waste.
• Waste segregation: Collect DMSO-containing aqueous waste separately if institutional policy requires. Avoid chlorinated solvent rinses unless analytically necessary.
• Greener vehicles (where compatible): For discovery animal method development, consider PEG400/saline or aqueous cyclodextrin formulations instead of high DMSO percentages, subject to compound solubility and model constraints.
• Procurement and documentation: Batch experiments to fully utilize each aliquot; document stability to prevent premature disposal due to degradation.

Trade-offs

• Lower DMSO content reduces cytotoxicity and environmental load but may increase risk of compound precipitation; verify solubility at working concentrations before scaling experiments.

Pharmaceutical Uses

Item-specific pharmacopeial or excipient status: Not specified for this item; refer to CoA/Spec Sheet.

General notes (not item-specific; no therapeutic claims)

• Role in R&D: ALK5 inhibitors are widely employed as reference standards and tool compounds in discovery biology and preclinical method development to interrogate TGF-β signaling. They may be used as internal controls for assay qualification or for PK bioanalytical method development when authentic standards are available.
• Formulation in research: For non-clinical studies, solutions/suspensions may be prepared in DMSO-based vehicles, PEG-containing systems, or cyclodextrin complexes to enhance apparent solubility. Compound-specific compatibility and stability should be established experimentally.
• Regulatory status: No pharmacopeial monograph or GMP status is implied. This product is supplied strictly for research use only, not for human or veterinary applications.

Physical Properties

Item-specific values

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Purity/grade: Not specified for this item; refer to CoA/Spec Sheet.
• Melting point (MP): Not specified for this item; refer to CoA/Spec Sheet.
• Boiling point (BP): Not applicable/unknown for solid small molecules; specific value not specified for this item; refer to CoA/Spec Sheet.
• Density: Not specified for this item; refer to CoA/Spec Sheet.
• LogP/logD: Not specified for this item; refer to CoA/Spec Sheet.
• Refractive index: Not applicable for solids; not specified for this item.
• Solubility: Not specified for this item; refer to CoA/Spec Sheet.

General guidance for small-molecule kinase inhibitors (literature/general, not item-specific)

• Physical state: Typically crystalline solid, often off-white to light yellow.
• Solubility profile: Frequently soluble in polar aprotic organics (DMSO, DMF) at 10–50 mM; limited aqueous solubility unless formulated with cosolvent or cyclodextrin. Actual solubility for ALK5-IN-8 is not provided here.
• pKa/logP trends: Many ATP-competitive inhibitors exhibit weakly basic heterocycles (pKa 1–8) and logP 2–5 to balance permeability and solubility.
• Stock solution practice: 10 mM in anhydrous DMSO is a common working stock for screening; filter-sterilize if used in cell assays (0.22 µm PTFE/RC). Always verify compound-specific stability before preparing stocks.

Quality & Grades

Item-specific quality information

• Grade/purity: Not specified for this item; refer to CoA/Spec Sheet.

Context and expectations (general, not item-specific)

• Research grade small molecules are typically supplied at ≥95% purity (by HPLC/UPLC), with identity confirmed by MS and/or NMR. Chromatographic purity does not guarantee bioassay purity if co-eluting isomers exist; check spectral data on the CoA.
• Lot-specific documentation: For assay-critical work (enzymology, cellular phenotyping), request lot-specific CoA with test methods (HPLC method, solvent system, detection wavelength), identity spectra (1H NMR, LC–MS), water content (KF), and residual solvent profile.
• Stabilizers: Not specified for this item. If present in any lot, stabilizers or counterions should be disclosed on the CoA due to potential assay interference.
• UV behavior: If HPLC-grade attributes are relevant (low baseline drift), confirm UV cutoffs/absorptivity on the CoA. Not specified for this item.

Reaction & Applications

This product is a bioactive reference compound, not a synthetic reagent. No preparative organic reaction applications are specified for this item.

General research applications (literature/general, not item-specific)

• Pathway interrogation: Used to inhibit TGF-β type I receptor kinase (ALK5/TGFBR1) activity in biochemical and cellular assays, enabling dissection of canonical SMAD2/3 signaling.
• Phenotypic studies: Applied in studies of EMT induction, fibrosis signaling, stromal–epithelial crosstalk, and extracellular matrix regulation in cell models.
• Assay types: Enzymatic kinase assays (IC50 determination), Western blot/ELISA readouts of p-SMAD2/3, reporter gene assays (CAGA-luc), and high-content imaging of EMT markers (E-cadherin, vimentin).
• Combination studies: Often co-dosed with TGF-β ligands or pathway modulators (e.g., ALK4/7 inhibitors) to delineate receptor-specific contributions.

Practical tips (general)

• Verify compound identity and purity prior to critical experiments (LC–MS, HPLC). Establish dose–response curves across ≥8 concentrations with technical replicates.
• Include vehicle (DMSO) controls matched to the highest DMSO content in treated wells.
• Confirm on-target pathway inhibition by measuring p-SMAD2/3 within 30–60 min post-treatment in responsive cells.

Reaction Conditions

Not applicable. This product is not supplied as a reagent for synthetic transformations, and no item-specific reaction conditions are provided.

General assay conditions for ALK5 pathway studies (literature/general, not item-specific)

• Enzymatic assays: ALK5 kinase domain with peptide substrate; ATP near Km; inhibitor dose–response (10–12 points). Incubation 30–60 min at room temperature; readout by radiometric transfer, ADP-Glo, or mobility shift.
• Cellular assays: Pre-stimulate cells with TGF-β1 (e.g., 1–10 ng/mL) following pretreatment with inhibitor (30–60 min). Assess p-SMAD2/3 by Western blot 30–60 min post-stimulation or reporter activity after 12–24 h. Maintain final DMSO ≤0.1–0.5%.
• Stability checks: Confirm compound integrity in assay buffer over the assay period (LC–MS) and assess nonspecific binding/adsorption by plasticware where relevant.

Safety & Handling

Item-specific hazard data

• Signal word (GHS): Not specified for this item; refer to SDS.
• Hazard statements (H-codes): Not specified for this item; refer to SDS.
• GHS classification/pictograms: Not specified for this item; refer to SDS.

General laboratory safety guidance (literature/general, not item-specific)

• Likely hazards: Many kinase inhibitor research compounds may cause skin/eye irritation or respiratory irritation and can be harmful if swallowed. Treat as a potentially hazardous organic compound.
• PPE: Lab coat, safety glasses, and appropriate chemically resistant gloves (nitrile). Handle powders in a fume hood to avoid dust inhalation; minimize aerosolization when weighing.
• Handling: Avoid contact with strong oxidizers and strong acids/bases unless compatibility is known. When preparing DMSO stocks, avoid skin contact; DMSO enhances dermal absorption of solutes.
• First aid (overview; defer to SDS): Skin/eye contact—rinse with water for ≥15 min; remove contaminated clothing. Inhalation—move to fresh air. Ingestion—rinse mouth; seek medical attention.
• Spill response: Contain solids with damp disposable towels; for solutions, absorb with inert material (vermiculite). Dispose via appropriate hazardous waste streams.
• Stability notes: Store at the recommended temperature. Protect from moisture and prolonged light exposure unless stability data indicate otherwise. Always consult the product’s SDS for authoritative safety information.

Solvent Selection

Item-specific solvent data

• Solubility and preferred solvents: Not specified for this item; refer to CoA/Spec Sheet.

General guidance for ALK5-class small molecules (literature/general)

• Primary stock solvent: Anhydrous DMSO is commonly used due to strong solvating power for heteroaromatic inhibitors. Typical stock concentrations: 10–50 mM, depending on compound solubility.
• Aqueous working solutions: Prepare by diluting the DMSO stock into buffer (e.g., PBS, HBSS, cell culture media) with final DMSO ≤0.1–0.5% v/v to limit cytotoxicity. Add stock slowly with vortexing to minimize precipitation.
• Alternative solvents: DMF or ethanol can be used for initial dissolution; however, DMSO generally affords higher solubility and stability. For in vivo method development (non-clinical), ternary vehicles (DMSO/PEG400/saline or captisol) are used in discovery settings; confirm compatibility for your model system.
• Solubility troubleshooting: Warm gently (≤37 °C), brief sonication, or addition of minimal co-solvent (e.g., PEG400) can aid dissolution. Avoid high pH adjustments unless compound stability is documented.

Comparison (general)

• DMSO: Highest solvency, best for stocks; may affect cells at >0.5%.
• Ethanol: Biologically tolerated up to ~0.1–0.5% in some assays; lower solvency for many kinase inhibitors.
• Aqueous buffers: Generally poor solubility unless the compound carries ionizable groups.

Storage & Reconstitution

Item-specific storage and shipping

• Storage conditions: Store at -20°C (from Product Data). Keep tightly closed in a desiccated, light-protected container.
• Shipping: Shipped in an ice chest with ice pads (from Product Data).

General reconstitution guidance for small-molecule inhibitors (not item-specific)

• Reconstitution solvent: Anhydrous DMSO is commonly used to prepare concentrated stock solutions (e.g., 10–50 mM). Actual solubility for this item is not specified; determine experimentally or refer to the CoA.
• Aliquoting: Divide stock into single-use aliquots in low-bind tubes to avoid repeated freeze–thaw cycles. Purge headspace with inert gas if oxidation-sensitive.
• Storage of solutions: Store DMSO stocks at -20°C or -80°C, protected from light. Typical stability for many inhibitors is months when frozen and desiccated, but this is not specified for this item; verify by LC–MS/HPLC.
• Thaw/use: Thaw quickly at room temperature, mix thoroughly, and immediately dilute into pre-warmed media/buffer. Avoid extended bench exposure.
• Aqueous working solutions: Prepare fresh on the day of use. If sterility is required, filter through 0.22 µm. Discard if precipitation or discoloration occurs.

Research use notice

• For research use only (from Product Data). No medical, diagnostic, or therapeutic use is intended or implied.

Structure & Identity

Item-specific identifiers (from Product Data)

• SKU: A646723
• Product Name: ALK5-IN-8
• CAS: 2705900-81-6
• Molecular formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.

General/literature description (not item-specific)

• Compound type: Small-molecule kinase inhibitor probe targeting the TGF-β type I receptor kinase (ALK5/TGFBR1) (literature/general).
• Structural features (typical for ALK5 inhibitors): Flat heteroaromatic/biaryl cores with H-bond acceptors oriented to the hinge region, and hydrophobic substituents occupying the adenine pocket and selectivity pockets (literature/general). Without supplied structure for ALK5-IN-8, specific functional groups or stereochemistry cannot be described for this item.
• 2D structural motifs (class-level): Kinase hinge-binding motif (e.g., amide/urea/aza-heteroaryl), possible solubilizing tertiary amine or polar tail to improve DMSO solubility (literature/general).

Synthetic Utility

This product is a finished bioactive small molecule and is not intended as a synthetic building block. No item-specific synthetic transformations or reagent roles are provided.

General comment (not item-specific)

• If chemical modification is planned (e.g., probe derivatization, biotinylation, or photoaffinity tag installation), obtain structural information and IP clearance, and confirm positions amenable to substitution via SAR literature for ALK5 inhibitors. Purification and verification should include LC–MS and NMR to ensure that activity is retained post-derivatization.

Target Specificity

Item-specific targeting data are not provided for this SKU. No affinity/IC50, kinome selectivity, or off-target profile is specified for this item; refer to the CoA/Spec Sheet and primary literature if available.

General statement (not item-specific)

• “ALK5-IN-8” nomenclature indicates a putative inhibitor of ALK5 (TGFBR1) by convention, but without supplied characterization data, target, potency, and selectivity for this specific item cannot be confirmed here.

Frequently Asked Questions

What is the purity of this product?

This product is supplied at ≥98% purity (chemical assay). Lot-specific values are stated on the Certificate of Analysis.

How should this product be stored?

Store at ?20 °C. Freezer storage is required to maintain the specified shelf life.

How is this product shipped?

This product ships in an insulated container with ice pads. Unpack on arrival and transfer it to the storage condition stated above.

What are the CAS number, molecular formula and molecular weight?

The CAS Number is 2705900-81-6, the molecular formula is C19H15FN4O2, and the molecular weight is 350.35 g/mol.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.