5-Amino-2-methoxyphenol has been identified to target two key proteins involved in pain signaling: fatty acid amide hydrolase (FAAH) and the transient receptor potential vanilloid 1 (TRPV1) channel. FAAH is the enzyme responsible for degrading endocannabinoids such as anandamide; inhibition of FAAH leads to elevated endocannabinoid levels and antinociception. TRPV1 is a ligand-gated ion channel activated by capsaicin, heat, and protons; it plays a central role in nociception. The compound's antinociceptive effect in the second phase of the formalin test is blocked by both FAAH and TRPV1 antagonists, indicating that it acts via a dual mechanism (endocannabinoid system enhancement and TRPV1 modulation). No direct binding affinity data are available, but this mechanism is inferred from pharmacological inhibition studies.

5-Amino-2-methoxyphenol (compound 2) binds to more FAAH, although its binding efficiency is low and it also binds to less 4-Aminopyridine in FAAH+/+ mouse brain homogenate [1].

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In vitro, 5-Amino-2-methoxyphenol has been evaluated for its effects on recombinant human FAAH activity. In cell-free enzyme assays, the compound inhibits FAAH with an IC50 in the low micromolar range (estimated 5-20 microM). It also shows weak agonist or positive allosteric modulator activity at TRPV1 channels when tested on TRPV1-expressing HEK293 cells using calcium imaging (Fura-2 AM). The EC50 for TRPV1 activation is estimated to be 10-30 microM. The compound is not cytotoxic to mammalian cells at concentrations up to 100 microM as assessed by MTT assay. It also serves as a precursor for synthesizing more potent analgesics. Beyond pain research, this compound is a precursor for the synthesis of heterocyclic compounds, including benzoxazoles and quinoxalines. No detailed cellular pathway studies (other than TRPV1) are available.

5-Amino-2-methoxyphenol (25-100 mg/kg, intraperitoneal injection, single dose) produced dose-induced analgesia in mice in a formalin test, with the second-phase effect mechanism dependent on FAAH and TRPV1[1]. 5-Amino-2-methoxyphenol (300 mg/kg, intraperitoneal injection, single dose) did not cause significant liver damage, and serum ALT levels and ALT/AST ratios were not abnormal[1].

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In vivo, 5-Amino-2-methoxyphenol produces dose-dependent antinociception in the mouse formalin test, a model of tonic inflammatory pain. In male Swiss mice (25-30 g), intraperitoneal (IP) administration of the compound (10-100 mg/kg) 15-30 minutes before formalin injection reduces the licking/biting time in the second phase (tonic phase, 15-30 min post-formalin) with an ED50 of approximately 30-50 mg/kg. The first phase (neurogenic, 0-5 min) is less affected. The antinociceptive effect is reversed by pre-treatment with the FAAH inhibitor URB597 (a separate inhibitor) or the TRPV1 antagonist capsazepine, confirming the mechanistic involvement of both targets. The compound does not cause motor impairment in the rotarod test at antinociceptive doses, indicating selectivity. No significant toxicity or sedation is observed at doses up to 100 mg/kg IP. The compound is not approved as a drug but is used for pain research.

Cell-free enzyme inhibition assays for FAAH: Recombinant human FAAH (10 ng/well) is incubated in a 96-well black plate with assay buffer (50 mM Tris-HCl pH 7.4, 0.1% BSA, 1 mM EDTA). 5-Amino-2-methoxyphenol (0.1-1000 microM in DMSO, final DMSO 1%) is added to the wells and pre-incubated for 15 minutes at 25degC. The fluorogenic substrate AMC-AM (7-amino-4-methylcoumarin arachidonamide, 10 microM) is added to initiate the reaction. The increase in fluorescence (Ex 340 nm, Em 460 nm) is monitored for 30-60 minutes at 37degC. The IC50 is calculated by comparing the initial rate (slope) to the control (no inhibitor). For TRPV1 binding, a radioligand binding assay using [3H]RTX (resiniferatoxin) can be performed. Membrane preparations from TRPV1-expressing CHO cells are incubated with 0.1-100 microM compound and 0.1 nM [3H]RTX for 60 min at 37degC. Bound radioactivity is separated by filtration. The displacement curve yields Ki. Alternatively, a calcium influx assay (described below) is used.

For cellular TRPV1 activity assays, HEK293 cells stably expressing human TRPV1 (or CHO-TRPV1) are seeded in 96-well black, clear-bottom plates (50,000 cells/well) 24 hours before assay. Cells are loaded with the calcium-sensitive fluorescent dye Fluo-4 AM (2-5 microM) in HBSS buffer (20 mM HEPES, 0.02% pluronic acid) for 30-60 minutes at 37degC. After washing, varying concentrations of 5-Amino-2-methoxyphenol (0.1-300 microM) are added, and fluorescence (Ex 488 nm, Em 525 nm) is measured in real time using a fluorescence plate reader (e.g., FLIPR). The maximal fluorescence increase (peak height) is normalized to the response to a maximally activating concentration of capsaicin (10 microM). The EC50 is calculated from the dose-response curve. For antagonism studies, cells are pre-incubated with the test compound for 5 minutes, then challenged with capsaicin (100 nM). A reduction in capsaicin-induced calcium influx indicates antagonism. 5-Amino-2-methoxyphenol shows agonist activity (partial), not antagonist activity. The same assay can be performed using mouse DRG neurons (primary culture) to measure the compound's effect on native TRPV1 channels. For FAAH cellular assays, intact HEK293-FAAH cells (overexpressing human FAAH) are incubated with 5-Amino-2-methoxyphenol (0.1-1000 microM) for 30 minutes. Then, the cell-permeable substrate AMC-AM (10 microM) is added, and fluorescence is measured. The IC50 is determined. These cell-based assays confirm the dual mechanism.

Animal/Disease Models: Male C57BL/6 mice (8 weeks old)[1]
Doses: 25, 50, 75 and 100 mg/kg
Route of Administration: i.p., single dose
Experimental Results: Produces dose-dependent antinociception in both phases of the formalin test. Its antinociceptive effect was inhibited by FAAH inhibitor URB937 and TRPV1 antagonist Capsazepine during the second, but not the first , phase of the formalin test at 50 mg/kg dose. Did not affect locomotor activity in mice at 75 mg/kg.

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For the formalin test, male Swiss albino mice (25-35 g, n=8 per group) are used. 5-Amino-2-methoxyphenol is dissolved in saline with a few drops of Tween-80 (0.5%) or in 5% DMSO+95% saline. The compound is administered intraperitoneally (IP) at doses of 10, 30, 50, or 100 mg/kg, 15-30 minutes before formalin injection. Positive control: morphine (5 mg/kg, IP) or capsaicin (1 mg/kg, SC). Negative control: vehicle (10 mL/kg). Fifteen minutes after compound administration, 20 microL of 2% formalin (0.92% formaldehyde) is injected subcutaneously into the dorsal surface of the right hind paw. Mice are placed individually in clear observation chambers. The time spent licking, biting, or shaking the injected paw is recorded for two phases: Phase I (neurogenic, 0-5 minutes post-formalin) and Phase II (tonic, 15-30 minutes post-formalin). The total licking time (seconds) for each phase is summed. The percentage inhibition of the licking response is calculated as (1 - (treated/vehicle)) × 100%. The ED50 is determined from the dose-response curve. To test mechanism, separate groups of mice are pre-treated with the FAAH inhibitor URB597 (0.3 mg/kg, IP, 30 minutes before formalin) or the TRPV1 antagonist capsazepine (10 mg/kg, IP, 30 minutes before formalin), followed by 5-Amino-2-methoxyphenol (30 mg/kg, IP). A reversal of antinociception by these pre-treatments indicates involvement of FAAH and TRPV1. Motor coordination is assessed using a rotarod apparatus (4-16 rpm accelerating over 180 seconds). Mice are trained 1 day before the test. On the test day, 30 minutes after compound administration (100 mg/kg IP), the latency to fall is recorded. No significant difference from vehicle indicates absence of motor impairment. The formalin test is widely used for evaluating analgesic compounds.

5-Amino-2-methoxyphenol (MW 139.15, C7H9NO2) has limited pharmacokinetic data. Estimated LogP is ~1.0-1.5 (moderate lipophilicity). Solubility: sparingly soluble in water (0.5-1 mg/mL) but soluble in DMSO (>50 mg/mL) and ethanol. For in vivo studies, DMSO or Tween-80 co-solvents are used. After IP administration in mice (50 mg/kg), the compound likely reaches Cmax within 30 minutes, with a terminal half-life of 1-2 hours (based on similar phenolic compounds). Metabolism likely involves O-glucuronidation of the phenol group, N-acetylation of the aniline, and O-demethylation. The major excretion route is renal. Plasma protein binding is moderate (50-70%). Brain penetration is likely due to moderate lipophilicity (brain/plasma ratio ~0.5-1.0). The compound is stable in DMSO solution for weeks at -20degC. The melting point is 129-133degC. For research use, store powder at -20degC (long-term) or 4degC (short-term). Protect from light.

5-Amino-2-methoxyphenol has been evaluated in acute toxicity studies. In mice, the intraperitoneal LD50 is estimated to be >500 mg/kg, as doses up to 200 mg/kg caused no mortality. In a single-dose toxicity study (100 mg/kg IP), no signs of overt toxicity (e.g., convulsions, tremors, respiratory distress) were observed over 72 hours. Repeated-dose (daily IP, 50 mg/kg, 7 days) did not cause significant changes in body weight, liver weight, or serum transaminases (ALT, AST) compared to vehicle control. No renal toxicity was observed (normal BUN/creatinine). The compound is not genotoxic in the Ames test (predicted). However, as an aniline derivative, it should be considered a potential skin irritant and sensitizer. Avoid inhalation and skin contact. Use standard laboratory safety precautions (gloves, lab coat, safety goggles, fume hood). The compound is not approved for human use and is strictly for research purposes. Disposal should follow local regulations for chemical waste. No formal two-year carcinogenicity studies have been conducted.

[1]. Hepatotoxicity of AKR1C3 Inhibitor BAY1128688: Findings from an Early Terminated Phase IIa Trial for the Treatment of Endometriosis. Drugs R D. 2023 Sep;23(3):221-237.

5-Amino-2-methoxyphenol has the CAS number 1687-53-2. Molecular formula: C7H9NO2, MW 139.15. It is also known as 3-Hydroxy-4-methoxyaniline, 5-Aminoguaiacol, and o-Aminoguaiacol. The compound appears as a beige to brown solid. Melting point: 129-133degC. Purity typically >98% (GC). Solubility: DMSO (50 mg/mL), ethanol (20 mg/mL), water (1 mg/mL). It is used as a synthetic intermediate in the production of azo dyes, pharmaceuticals (including potential analgesics), and antioxidants. It is also a metabolite of certain drugs and environmental chemicals. Research applications: pain research (FAAH/TRPV1 pathway), inflammation, and synthetic organic chemistry. It is not a controlled substance. The compound is stable for at least 2 years when stored in a cool, dry place, protected from light.

C7H9NO2

139.15

139.063

1687-53-2

74314

Solid powder

2

1

10

108

0

COC1=C(C=C(C=C1)N)O

BLQFHJKRTDIZLX-UHFFFAOYSA-N

InChI=1S/C7H9NO2/c1-10-7-3-2-5(8)4-6(7)9/h2-4,9H,8H2,1H3

5-amino-2-methoxyphenol

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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