hCAIX-IN-24 , CAS No.3038222-64-6

CAS: 3038222-64-6 Cat. No.: H1438862 Summenformel: C₂₃H₂₄N₂O₄STe Molekulargewicht: 552.11

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Übersicht

hCAIX-IN-24 is a potent and selectively hCA IX inhibitor with an K I value of 32.1 nM. hCAIX-IN-24 shows anti-proliferative activity.

Specifications

Storage

Store at -20°C

Verschickt in

Ice chest + Ice pads

Dieses Produkt erfordert Kühlkettenversand. Grundversand und andere Economy-Optionen sind nicht verfügbar.

Namen und Kennungen

Molekulargewicht

552.11

Application Protocols

No manufacturer-validated protocols are provided in the Product Data for this item. The following are general, non-validated suggestions for initial evaluation; adapt to your laboratory’s standards.

Biochemical inhibition assay (general): • Prepare a 10 mM stock in DMSO. Perform serial half-log dilutions to generate 8–10 concentrations. Keep final DMSO ≤1% v/v. • Assay buffer: 25–50 mM HEPES or TRIS, pH 7.4–8.0, 150 mM NaCl; add 0.01% Tween-20 if aggregation is suspected. • Enzyme: recombinant human CA IX at a concentration yielding robust initial rates. • Readout: stopped-flow CO2 hydration or surrogate esterase assay; collect triplicate wells per concentration. • Analysis: Fit to a four-parameter logistic model for IC50; if feasible, determine Ki via appropriate kinetic analysis.

Thermal shift (DSF) binding assay (general): • Mix CA IX protein with SYPRO Orange (or similar) and varying inhibitor concentrations. • Record melt curves; calculate ΔTm vs vehicle control to infer binding.

Cell-based assessment (general): • Culture cells expressing CA IX (hypoxia-induced) with a dilution series of the compound for 24 h. • Measure target-relevant endpoints (e.g., extracellular acidification proxies) and include cytotoxicity counterscreens.

Note: These protocols are generic templates. Optimize protein concentration, substrate, temperature, and timing for your specific setup.

Biological Roles

Context (general, literature): • Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of CO2 to bicarbonate and a proton, contributing to pH homeostasis, CO2 transport, and ion balance. CA IX is a transmembrane isoform with an extracellular catalytic domain and a proteoglycan-like region; its expression is typically upregulated under hypoxia via HIF-1–dependent pathways. • CA IX participates in extracellular acidification in hypoxic microenvironments, facilitating proton extrusion and supporting cellular adaptation to low oxygen by modulating pH at the cell surface.

Relevance to inhibitor studies (research use): • Small-molecule CA IX inhibitors are used to interrogate the biochemical and biophysical properties of the CA IX active site, dissect isoform-specific contributions to pH regulation, and explore structure–activity relationships (SAR) around zinc-binding groups (ZBGs) and hydrophobic pocket occupancy. • Selectivity profiling against cytosolic CA I/II and membrane-associated CA XII is commonly performed to map isoform preferences. Inhibitors with different ZBGs (e.g., primary sulfonamide vs non-classical motifs) exhibit distinct binding modes and pH dependence (literature/general).

Assay endpoints (non-clinical): • Enzymology: kcat/Km modulation, Ki, and mechanism (competitive/uncompetitive/mixed) from initial-rate analyses. • Biophysical: thermal stabilization (ΔTm), binding kinetics (kon/koff) by SPR, and target engagement in cells by CETSA or DARTS.

Note: The specific molecular interactions of hCAIX-IN-24 are not provided in the Product Data; all above points are general background to guide experimental planning.

Buffer Applications

This product is a small-molecule screening compound and is not itself a buffering agent. Therefore, standard buffer system design (pKa-defined buffering capacity) is not applicable to the compound per se.

Practical guidance for using buffers with this compound in assays (general): • Common assay buffers for carbonic anhydrase enzymology include HEPES, TRIS, or phosphate buffers (typically pH 7.2–8.0) at 25–150 mM ionic strength. Select buffer components that do not chelate Zn2+ or otherwise interfere with CA catalysis. • Maintain consistent pH and ionic strength across inhibitor dilutions; include identical DMSO percentages in control and test wells to avoid solvent artifacts. • When using esterase surrogate assays (e.g., p-nitrophenyl acetate), ensure buffer composition does not interfere with detection (e.g., avoid strong UV-absorbing components at the readout wavelength). • For cell-based work, use physiologically compatible buffers/media (e.g., HEPES-buffered DMEM) and confirm compound stability in the chosen medium over the assay timeframe by LC–MS.

Item-specific buffering parameters (pKa, solubility vs pH): Not specified for this item; refer to CoA/Spec Sheet or determine experimentally.

Green Alternatives

Green chemistry considerations are generally aimed at solvent and process choices. As hCAIX-IN-24 is a screening compound rather than a process solvent or reagent, “green alternatives” pertain primarily to formulation and assay design rather than replacement of the compound itself.

Strategies to improve sustainability (general): • Solvent minimization: Prepare the highest practical DMSO stock concentration to reduce total organic solvent use across assays. Dispense using acoustic or low-volume pin tools to minimize waste. • Greener cosolvents: When DMSO is problematic, consider ethanol or water–ethanol mixtures where compatible with assay biology. Avoid DMF where possible due to disposal burdens. • Plate format: Prefer 384- or 1536-well miniaturized assays to reduce reagent consumption, plastic usage, and hazardous waste generation. • Buffer selection: Favor phosphate, HEPES, or TRIS buffers at moderate ionic strength; avoid volatile amines if MS detection is planned. • Transport and storage: Consolidate cold shipments and maintain efficient cold-chain logistics to limit dry ice/ice pack use.

Comparison (general guidance): • DMSO vs ethanol for stock solutions: DMSO typically affords higher solubility and stability but has higher concerns for skin permeation and presents challenges in some cell systems; ethanol is greener and more biocompatible at low percentages but often limits solubility.

Note: There is no direct “green substitute” for the active chemical entity in a screening context; sustainability gains come from operational choices.

Pharmaceutical Uses

This product is supplied strictly for research use only. No clinical use, diagnostic use, or therapeutic claims are made or implied.

Formulation and development context (general, non-clinical): • In early discovery, small-molecule inhibitors are commonly formulated as DMSO stock solutions for in vitro biochemical and cellular assays. • For in vivo exploratory studies in animals conducted under appropriate ethical approvals (outside the scope of this product listing), solubilization strategies may include co-solvent systems (e.g., PEG400/saline, Captisol, Cremophor EL variants) or nanosuspensions; such use requires compound-specific safety and stability data and is not covered by this listing. • Pharmacopeial status: Not applicable; no monograph.

Excipients and compatibility: • Not specified for this item; refer to CoA/Spec Sheet for any stabilizers or residual solvents that may influence formulation choices.

Quality considerations: • For any downstream development, additional testing typically includes polymorph/solid-state characterization, impurity profiling, and stability under ICH conditions—none of which are specified here and would need to be generated de novo.

Physical Properties

Item-specific physical data are not provided in the Product Data. Do not assume values; consult the CoA/SDS.

Provided: • Molecular weight: Not specified for this item; refer to CoA/Spec Sheet.

Not provided (do not infer): • Appearance (solid/oil), melting point, boiling point, density, refractive index, water content, residual solvents, logP/logD, pKa, UV–vis profile, and solubility in water/organic media.

General guidance for small-molecule screening inhibitors (literature/general): • Many CA IX inhibitors are moderately lipophilic solids with limited aqueous solubility at neutral pH, often formulated as DMSO stock solutions (e.g., 10–50 mM) for biochemical assays. This is general practice and may not reflect this specific item. • If a sulfonamide motif is present (literature trend), pKa of the sulfonamide N–H can be ~9–10; deprotonation facilitates Zn2+ binding. This is generic; verify for the actual structure.

Practical recommendations: • Determine solubility experimentally in intended assay media (e.g., PBS, HEPES, Tris) with and without cosolvent (DMSO 0.1–1% v/v) and surfactant (0.01% Tween-20) if assay-compatible. • Record UV–vis absorbance to identify potential interference in spectrophotometric readouts; if unknown, preferentially use fluorescence-independent or MS-based detection for initial screening. • For purity, assess by analytical HPLC with diode-array detection and LC–MS; compare retention time and mass to CoA.

Quality and Grades

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

How to interpret grades for screening compounds (general): • Research-grade small molecules for biochemical/cellular screening are commonly supplied at ≥95% HPLC purity (literature/industry practice). However, acceptance criteria vary by application; confirm numerical purity and analytical methods on the CoA for this specific item. • HPLC purity alone does not guarantee identity; corroborate with LC–MS and NMR when critical decisions (SAR, potency ranking) depend on the result.

What to check on receipt: • Verify CAS (3038222-64-6), lot number, appearance, and mass balance. • Review the CoA for: assay method (HPLC/UPLC conditions), detection wavelength(s), purity value, residual solvents, water content (KF), and identity confirmation (MS/NMR). • If working in light-sensitive assays or long-term screens, request data on stability (solid and in DMSO at room temperature/4°C/−20°C) and known degradants.

Stabilizers/inhibitors: • Not specified for this item; refer to CoA/Spec Sheet. If stabilizers are present, consider their assay compatibility (e.g., BHT, ascorbate) and adjust controls accordingly.

Fit-for-purpose qualification: • For enzymology: confirm single, sharp HPLC peak at assay wavelength; verify no absorbance overlap with assay reporters. • For cell assays: screen for endotoxin only if required (rare for small molecules) and ensure residual solvent levels meet your lab’s criteria.

Reaction and Applications

This product is intended as a small-molecule library member for biochemical or cellular screening, not as a synthetic reagent. Accordingly, classical organic reactions (e.g., cross-couplings, condensations) are not its primary application.

Relevant applications (research use): • Enzyme inhibition assays targeting human carbonic anhydrase IX (CA IX): Evaluate concentration–response curves (e.g., 8–10 point, half-log dilutions) to derive IC50/Ki using appropriate kinetic models. Use a validated biochemical assay (e.g., esterase surrogate substrate or CO2 hydration-coupled readouts) or a biophysical platform (SPR, DSF, nanoDSF, CETSA, thermal shift) to confirm binding. • Cellular assays: If compound permeability and stability are acceptable, assess effects on CA IX–dependent readouts (e.g., extracellular acidification rate, pH regulation markers) under hypoxic conditions where CA IX expression is induced. Include counter-screens against off-target CA isoforms (CA I/II) if available to profile selectivity. • Orthogonal profiling: Use CA IX-containing membrane fractions or recombinant extracellular domains in combination with Zn2+-binding motif controls to verify target engagement.

Assay controls and tips: • Include positive controls (literature CA inhibitors such as acetazolamide/sulfonamide standards) and negative controls (vehicle only). • Validate DMSO tolerance and check for compound aggregation (0.01% Triton X-100 control, dynamic light scattering) to avoid artifactual inhibition. • Confirm chemical stability in assay media by LC–MS time-course to correlate potency with exposure.

Note: No item-specific potency/selectivity data are provided; generate de novo under your conditions.

Reaction Conditions

This item is not intended for use as a reagent in chemical synthesis; therefore, classical reaction conditions (solvent, catalysts, temperatures, yields) are not applicable.

Assay conditions for inhibitor evaluation (general guidance): • Biochemical CA IX inhibition assays: Conduct at controlled temperature (e.g., 25°C), with buffers such as 25–50 mM HEPES or TRIS, pH 7.4–8.0, containing 0.01–0.05% Tween-20 if needed to mitigate aggregation. Maintain constant DMSO (≤1% v/v). Use 8–10 point dilution series to generate concentration–response curves. • Stopped-flow CO2 hydration assays (literature): Provide precise enzyme and substrate concentrations; acquire initial rates within milliseconds to seconds. Analyze with appropriate kinetic models to derive Ki or IC50. • Surrogate substrate assays (e.g., p-NPA esterase assay): Monitor absorbance change at the relevant wavelength; verify the compound does not absorb strongly at the detection wavelength or include spectral deconvolution controls. • Cell-based assays: Culture cells under normoxic vs hypoxic conditions to modulate CA IX expression; test multiple time points (e.g., 4–48 h) and concentrations spanning at least three logs. Include cytotoxicity counterscreens (e.g., resazurin/CellTiter) to distinguish target effects from general viability loss.

Data quality tips: • Pre-incubate inhibitor with enzyme (e.g., 10–30 min) if slow-binding is suspected. • Confirm assay linearity and validate Z′-factor for HTS. • Verify chemical stability of the test article in the assay matrix by LC–MS.

Safety and Handling

GHS/SDS information not provided in the Product Data. Always consult the official SDS for authoritative hazard classifications and response measures.

Provided hazard fields (all not specified): • Signal Word: Not specified for this item; refer to CoA/Spec Sheet. • H-Statements: Not specified for this item; refer to CoA/Spec Sheet. • GHS Classification and Pictograms: Not specified for this item; refer to CoA/Spec Sheet.

General laboratory precautions (good practice): • Handle in a chemical fume hood; avoid inhalation, ingestion, and skin/eye contact. • PPE: lab coat, safety glasses or goggles, and appropriate chemical-resistant gloves (e.g., nitrile). Change gloves frequently when handling DMSO solutions due to skin permeation potential of DMSO. • Avoid aerosol generation when preparing dry powders or weighing small quantities. • Storage incompatibilities: segregate from strong oxidizers and strong acids/bases until specific compatibility is known. Keep away from light and moisture as a precaution for small organic molecules.

First-aid overview (defer to SDS): • Skin/eye contact: rinse with water for at least 15 minutes; remove contaminated clothing; seek medical attention if irritation persists. • Inhalation: move to fresh air; seek medical attention for symptoms. • Ingestion: rinse mouth; do not induce vomiting; seek medical attention.

Waste disposal: • Collect solutions and solids as organic laboratory waste per institutional and local regulations. Do not dispose to drain.

Note: This product is for research use only.

Solvent Selection

Item-specific solubility and polarity data are not provided; adopt a stepwise dissolution strategy commonly used for small-molecule inhibitors.

Recommended approach (general, practical): • Primary solvent: anhydrous DMSO for preparing concentrated stocks (e.g., 10–50 mM), then dilute into aqueous assay buffer ensuring final DMSO ≤0.1–1% v/v, consistent with assay tolerance. • Alternative solvents for pre-dissolution if DMSO limits apply: DMF, ethanol, or 1:1 DMSO:ethanol. Filter sterilize (0.22 µm PTFE) if needed for cell work. • Aqueous media: If precipitation occurs upon dilution, add compound to buffer slowly with vigorous mixing, warm gently (≤37°C), and consider including 0.01–0.05% non-ionic surfactant (if assay-compatible). Adjust pH only if the compound’s ionization is known; otherwise avoid pH-driven solubilization until characterized.

Miscibility and compatibility (general): • DMSO is fully miscible with water and most polar aprotic solvents; ethanol is miscible with water and many organics; DMF is miscible with water but may complicate certain biological assays—validate controls accordingly.

Selection vs alternatives: • Use DMSO when maximum solubility and chemical stability are priorities. • Use ethanol when minimizing DMF/DMSO is required for specific cells/assays. • For high-throughput screens, pre-aliquoted DMSO stocks in 96/384-well plates reduce freeze–thaw cycles and variability.

Note: Determine actual solubility and stability empirically for this item and document in your method.

Storage and Reconstitution

Storage (from Product Data): • Store at −20°C. • Shipped in an ice chest with ice pads to maintain a cold chain.

General handling recommendations (small-molecule inhibitors): • Keep container tightly closed in a dry, inert atmosphere. Protect from light and moisture. Allow the vial to equilibrate to room temperature in a desiccator before opening to minimize condensation. • On first use, subdivide into single-use aliquots to avoid repeated freeze–thaw cycles, especially for DMSO solutions.

Reconstitution (general guidance): • Prepare a concentrated stock solution in dry DMSO (e.g., 10–50 mM). Vortex and, if needed, briefly warm to 25–37°C to aid dissolution. If particulates persist, sonicate briefly or filter through 0.22 µm PTFE. • Record the exact concentration by weight and volume; optionally verify by UV–vis or quantitative 1H NMR if a suitable standard and chromophore are available. • For working solutions, dilute into assay buffer or media to the desired final concentration while maintaining consistent DMSO percentage across controls.

Stability: • Item-specific shelf-life and solution stability: Not specified for this item; refer to CoA/Spec Sheet. As a precaution, store DMSO stocks at −20°C and avoid more than 3–4 freeze–thaw cycles.

Research use note: For research use only.

Structure and Identity

• Product name: hCAIX-IN-24 (SKU: H1438862) • CAS: 3038222-64-6 • 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. • Synonyms: Not specified for this item; refer to CoA/Spec Sheet.

Structural features (general): • hCAIX-IN-24 is categorized as a small-molecule inhibitor in a screening library. Specific ring systems, heteroatoms, and functional groups are not provided in the product data and should be confirmed from the CoA/SDS. • Without structure, stereochemistry (chiral centers, E/Z geometry) cannot be ascertained; consult the CoA/Spec Sheet.

How to interpret 2D structure (guidance): • For carbonic anhydrase IX (CA IX) inhibitors in general (literature): many incorporate sulfonamide/sulfamate zinc-binding motifs or alternative ZBGs that coordinate the active-site Zn2+ via deprotonated nitrogen/oxygen donors. Aromatic scaffolds are common for hydrophobic pocket occupancy. This description is generic and may not apply to this specific item; verify against the actual structure when available.

Identity checks (practical): • Confirm by LC–MS (exact mass), 1H/13C NMR, and HPLC purity per CoA. • Match CAS number 3038222-64-6 to internal records before use in screening campaigns.

Synthetic Utility

hCAIX-IN-24 is offered as a screening compound, not as a building block or reagent. As such, it is not typically employed as a synthon in multistep organic synthesis.

General remarks (contextual, literature): • Many CA inhibitor chemotypes are assembled via amide couplings, aryl–aryl bond formations (e.g., Suzuki–Miyaura), and sulfonamide installations. If the structure of hCAIX-IN-24 contains such motifs, analog generation for SAR would likely use these tactics. This is general information and may not reflect the exact structure of this item. • If a zinc-binding group (e.g., primary sulfonamide) is present, late-stage introduction is often preferred to minimize protection/deprotection steps and maintain integrity of sensitive motifs.

Practical lab perspective: • Consider purchasing analogs or related chemotypes for rapid SAR rather than repurposing this item as a reagent. • If derivatization is desired (e.g., for probe development), first obtain and review the full structural information, functional group compatibility, and stability data from the CoA/SDS; then design transformations compatible with presumed sensitive groups (avoid strong acids/bases, high-temperature conditions that can desulfonylate or hydrolyze amides).

Conclusion: • Use this compound primarily for biological evaluation and target engagement studies. For synthetic campaigns, obtain starting materials or intermediates tailored for chemical modification.

Target Specificity

Item-specific target data are not provided in the Product Data. Based on the name, hCAIX-IN-24 is presumably designed for research on human carbonic anhydrase IX, but no binding constants, selectivity over other CA isoforms, or validated targets are supplied here.

Provided from Product Data: • Target, antigen/epitope, species reactivity, clone/isotype (where applicable): Not specified for this item; refer to CoA/Spec Sheet.

General guidance for establishing specificity (research use): • Profile against a CA isoform panel (e.g., CA I, II, IV, IX, XII) using the same assay platform to obtain comparable IC50/Ki values. • Use orthogonal methods (SPR, thermal shift/CETSA, native MS) to confirm target engagement. • Test for aggregation-based inhibition (detergent sensitivity, DLS) and redox cycling (horseradish peroxidase/Amplex Red assay) to rule out assay artifacts. • In cells, correlate activity with CA IX expression levels (siRNA/CRISPR knockdown, overexpression) to support on-target effects.

Note: Report any measured selectivity ratios and kinetic mechanisms alongside experimental conditions to ensure reproducibility.