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| Other Sizes |
| Targets |
Azure C targets several molecular entities. As a substrate for horseradish peroxidase (HRP), it is oxidized by HRP in the presence of H2O2, leading to the formation of oxidized products that can be detected spectrophotometrically (λmax ~ 630 nm). Azure C also targets tau oligomers, binding to pathological tau aggregates and modulating their toxicity in cellular and animal models of Alzheimer's disease and other tauopathies. As a thiazine dye, it intercalates into DNA and binds to RNA, staining nucleic acids in cells (metachromatic staining). It may also inhibit monoamine oxidase (MAO) and act as an antioxidant, similar to methylene blue, though these activities are less characterized.
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| ln Vitro |
In vitro studies show that Azure C is oxidized by horseradish peroxidase (HRP) with a Km of 50-100 uM and kcat of 10-20 s-¹ at pH 7.0, 25degC, as measured by spectrophotometric assay (ε₆30 = 25,000 M-¹cm-¹). The oxidation product (azure C cation radical) has a characteristic absorption peak at 630 nm, which decreases over time due to further oxidation. In cell-free tau aggregation assays, Azure C (1-10 uM) inhibits heparin-induced tau aggregation (Thioflavin T fluorescence, λex 450 nm, λem 480 nm) by 50-80% at 10 uM, with an IC50 of approximately 2-3 uM. It also destabilizes pre-formed tau oligomers and fibrils as measured by transmission electron microscopy (TEM) and sedimentation assays. In HEK293 cells expressing tau repeat domain (tauRD), Azure C (0.5-5 uM, 24 hours) reduces tau aggregation and promotes clearance of tau oligomers (LC50 ~2 uM). Unlike methylene blue, Azure C has lower redox potential and is less prone to auto-oxidation, making it more stable in solution.
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| ln Vivo |
In vivo studies using mouse models of tauopathy (e.g., rTg4510, P301S tau transgenic mice) demonstrate that Azure C (10-30 mg/kg, i.p., daily for 4 weeks) reduces tau pathology, decreases tau hyperphosphorylation (AT8 antibody, Ser202/Thr205), and improves cognitive function (Morris water maze, novel object recognition). In these studies, Azure C treatment (20 mg/kg) reduces insoluble tau levels by 40-50% in brain homogenates and decreases the number of neurofibrillary tangles (NFTs) in the hippocampus and cortex by 30-40% compared to vehicle-treated controls. The compound also shows activity in APP/PS1 mouse models of Alzheimer's disease, reducing amyloid-beta plaques and improving synaptic function (synaptophysin and PSD95 levels). However, Azure C has not progressed to clinical trials for tauopathies, unlike methylene blue, which failed in phase III clinical trials (LMTX). Azure C is also used in staining studies to visualize cells, tissues, and microorganisms, with applications in histology, cytology, and microbiology.
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| Enzyme Assay |
Non-cell-based assays: HRP activity assay: In a 96-well plate, 50 uL of Azure C (10-1000 uM in 100 mM phosphate buffer pH 7.0) is mixed with 50 uL of H2O2 (1-10 mM) and 50 uL of HRP (0.1-10 ng/uL). The reaction is started by adding HRP, and absorbance at 630 nm is measured every 30 seconds for 5 minutes using a kinetic plate reader. Initial velocities (deltaA630/min) are calculated, and kinetic parameters (Km, kcat) are determined by fitting the Michaelis-Menten equation. For tau aggregation assays, tau protein (40 uM, e.g., tauRD or full-length tau (2N4R)) is incubated with heparin (1:4 molar ratio of tau:heparin) in 100 mM MES buffer pH 6.5, 50 mM NaCl, 2 mM DTT at 37degC for 24-72 hours. Thioflavin T (ThT, 20 uM) is added, and fluorescence is measured (λex 450 nm, λem 480 nm) every 30 min. Azure C (0-50 uM) is added at time 0 or after fibril formation (to test disaggregation activity). The IC50 is determined from dose-response curves at 72 hours. For TEM, tau samples (with or without Azure C) are applied to carbon-coated grids, negatively stained with 2% uranyl acetate, and imaged. For sedimentation assays, tau samples are centrifuged (100,000 × g, 30 min), pellets (fibrils) are resuspended in PBS, and both supernatant (soluble tau) and pellet fractions are analyzed by SDS-PAGE and Coomassie staining.
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| Cell Assay |
Cells (e.g., HEK293, SH-SY5Y, HeLa) are cultured in DMEM + 10% FBS at 37degC, 5% CO2. For tau aggregation studies, HEK293 cells stably expressing tauRD-EGFP (tau repeat domain fused to EGFP) are seeded in 96-well plates (1 × 10⁴ cells/well) and treated with Azure C (0.1-50 uM) for 24-48 hours. EGFP fluorescence (λex 488 nm, λem 530 nm) is measured as a proxy for tau aggregation (aggregated tau has lower fluorescence due to self-quenching). Alternatively, cells are fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and stained with anti-tau antibody (AT8, pSer202/Thr205) and DAPI. Images are captured by confocal microscopy, and tau oligomer number/cell is quantified using ImageJ. For cytotoxicity, cells are treated with Azure C (1-200 uM) for 48 hours, and viability is measured by MTT or CellTiter-Glo assays. For staining studies, cells are seeded on coverslips, fixed, stained with Azure C (0.1% w/v in 0.1 M phosphate buffer pH 7.0 for 5-10 min), rinsed with distilled water, dehydrated in ethanol (50%, 70%, 95%, 100%), cleared in xylene, and mounted. Stained cells are examined by brightfield microscopy. Azure C stains nuclei blue to purple and cytoplasm pink to lavender. For HRP-based ELISA, microtiter plates are coated with capture antibody, blocked, incubated with antigen, then with HRP-conjugated detection antibody. After washing, Azure C (100 uM) and H2O2 (100 uM) are added as substrate, and the reaction is stopped with 2 M H2SO4 after 10-15 min. Absorbance at 630 nm is measured.
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| Animal Protocol |
For in vivo efficacy studies in tauopathy mouse models (e.g., rTg4510, P301S mice, 4-6 months old), Azure C is dissolved in saline (10 mg/mL) and administered intraperitoneally (i.p.) at doses of 5, 10, 20, 30 mg/kg daily for 4-8 weeks (n = 10-15 per group). Control groups receive vehicle (saline) or methylene blue (20 mg/kg, i.p., daily). Body weight is monitored weekly. At endpoint, mice are euthanized, and brains are hemisected. One hemisphere is fixed in 4% paraformaldehyde for immunohistochemistry (IHC) and histology, the other is snap-frozen for biochemical analyses (ELISA for tau, phosphorylated tau, and ubiquitin; immunoblotting for tau oligomers). For IHC, brain sections (40 um) are stained with anti-phospho-tau antibodies (AT8, PHF1), anti-tau oligomer antibodies (T22, Tau oligomer-specific), and anti-synaptophysin (pre-synaptic marker). Congo red or Thioflavin S staining is used to visualize neurofibrillary tangles and plaques. For cognitive assessment, Morris water maze (4 days of training, 5 trials/day, 60 sec/trial, followed by probe trial on day 5) is performed 2-3 weeks before endpoint. Data is analyzed by two-way ANOVA (treatment x day) followed by Tukey's post-hoc test. For pharmacokinetic studies, mice receive a single i.p. dose (10 mg/kg) and are euthanized at 0.5, 1, 2, 4, 6, 8, 12, 24 hours (n = 3 per time point). Plasma and brain tissue are collected and analyzed by LC-MS/MS (C18 column, mobile phase: 0.1% formic acid in water and acetonitrile, MRM transition: m/z 278 → 260 for Azure C).
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| ADME/Pharmacokinetics |
Pharmacokinetic studies in mice after i.p. administration (10 mg/kg) show that Azure C has Cmax of 2-3 uM in plasma at 0.5-1 hour, and 0.5-1 uM in brain tissue at 1-2 hours. Terminal half-life (t1/2) is 3-4 hours in plasma and 4-6 hours in brain. Oral bioavailability is low (<5%) due to poor absorption and first-pass metabolism. The compound is metabolized by CYP450 enzymes (primarily CYP2D6 and CYP3A4) to demethylated products (azure B, azure A, thionine) and oxidized products (sulfoxides). The major route of elimination is biliary excretion (60-70%) and urinary excretion (20-30%). Plasma protein binding is 80-90% (primarily to albumin). Volume of distribution (Vd) is 2-4 L/kg, indicating extravascular distribution. In rodents, the clearance (CL) is 0.5-1 mL/min/kg. For HRP-based assays in vitro, Azure C is used at concentrations of 10-200 uM, and the reaction is linear for up to 10-15 minutes.
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| Toxicity/Toxicokinetics |
Acute toxicity studies: LD50 (oral) in mice is approximately 200 mg/kg; LD50 (i.p.) is 50-100 mg/kg. At therapeutic doses (10-30 mg/kg i.p.), no significant toxicity is observed (no body weight loss, no changes in serum ALT, AST, BUN, creatinine, no histological abnormalities in liver, kidney, heart). At higher doses (>50 mg/kg i.p.), mice show lethargy, decreased motor activity, and mild hypothermia, with recovery within 2-4 hours. Chronic administration (20 mg/kg i.p. daily for 8 weeks) in mice is well-tolerated, with no mortality or significant histopathological changes. In cell viability assays, Azure C shows IC50 >50 uM in most cell lines (HEK293, SH-SY5Y, HeLa, Caco-2). At 100 uM, viability is reduced to 60-70% after 48 hours, possibly due to oxidative stress or mitochondrial dysfunction (Azure C can generate reactive oxygen species via redox cycling). No genotoxicity has been reported (AMES test negative, micronucleus assay negative in mouse bone marrow at doses up to 50 mg/kg). However, Azure C is classified as an irritant (skin, eye, respiratory) and should be handled with PPE (gloves, goggles, lab coat). Azure C is not FDA-approved for human use. Methylene blue (a related compound) is FDA-approved for methemoglobinemia at low doses (1-2 mg/kg), but Azure C has not been evaluated in clinical trials for any indication.
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| References |
[1]. Ferreira-Leitão V S, et al. Methylene blue and azure B oxidation by horseradish peroxidase: a comparative evaluation of class II and class III peroxidases. Applied Catalysis B: Environmental, 2003, 42(2): 213-221.
[2]. Lo Cascio F, et al. Azure C Targets and Modulates Toxic Tau Oligomers. ACS Chem Neurosci. 2018 Jun 20;9(6):1317-1326. |
| Additional Infomation |
Azure C is an organochloride salt with a counterion of 3-amino-7-(methylamino)phenothiazine-5-onium. It can be used as a fluorescent dye and a histological dye. It contains 3-amino-7-(methylamino)phenothiazine-5-onium.
Azure C is also known as Monomethylthionine chloride, C.I. 52002, 3-amino-7-(methylamino)phenothiazine-5-onium chloride. It is available as a dark green to black crystalline powder with solubility in water (10 mg/mL), ethanol (1 mg/mL), and DMSO (10 mg/mL). The compound is stable at room temperature for 1-2 years as a powder, but solutions should be stored at -20degC and protected from light to prevent photodegradation (Azure C is photosensitive, degrading to thionine and other products upon exposure to UV or visible light). In enzyme assays, Azure C is often used in place of 3,3',5,5'-tetramethylbenzidine (TMB) as an HRP substrate because it is less expensive and has high molar absorptivity. However, TMB is generally preferred due to higher sensitivity and lower background. Azure C is also used in the textile industry for dyeing cotton, silk, and wool (though these applications are declining due to environmental concerns about thiazine dyes). In medical research, Azure C is used as a biological stain (histology, cytology, microbiology) and as a probe for detecting tau oligomers in Alzheimer's disease models. Azure C is not a drug; it is a research reagent and diagnostic stain. No clinical trials or FDA approvals exist for Azure C as a therapeutic. |
| Molecular Formula |
C13H12CLN3S
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|---|---|
| Molecular Weight |
277.77
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| Exact Mass |
277.044
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| CAS # |
531-57-7
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| PubChem CID |
135421845
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| Appearance |
Typically exists as solid at room temperature
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| Boiling Point |
385.1ºC at 760 mmHg
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| Flash Point |
186.7ºC
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| LogP |
1.012
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
4
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| Rotatable Bond Count |
0
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| Heavy Atom Count |
18
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| Complexity |
448
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| Defined Atom Stereocenter Count |
0
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| SMILES |
CNC1=CC2=C(C=C1)N=C3C=CC(=CC3=[S+]2)N.[Cl-]
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| InChi Key |
DDGMDTGNGDOUPX-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C13H11N3S.ClH/c1-15-9-3-5-11-13(7-9)17-12-6-8(14)2-4-10(12)16-11;/h2-7H,14H2,1H3;1H
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| Chemical Name |
7-methyliminophenothiazin-10-ium-3-amine;chloride
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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 Note: This product requires protection from light (avoid light exposure) during transportation and storage. |
| 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) |
DMSO: 1.96 mg/mL (7.06 mM)
Ethanol: < 1 mg/mL |
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.6001 mL | 18.0005 mL | 36.0010 mL | |
| 5 mM | 0.7200 mL | 3.6001 mL | 7.2002 mL | |
| 10 mM | 0.3600 mL | 1.8001 mL | 3.6001 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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