1H-Indole-6-carboxylic acid does not have a specific pharmacological target of its own, but its derivatives are widely studied for biological activities. The indole scaffold is a privileged structure in medicinal chemistry, known for its ability to interact with various biological targets through π-π stacking, hydrogen bonding, and hydrophobic interactions. Compounds derived from indole-6-carboxylic acid have been investigated as antagonists of serotonin 5-HT₆ receptors, which are involved in cognitive function and memory. Other derivatives have been developed as inhibitors of cyclooxygenase-2 (COX-2) for anti-inflammatory applications, and as modulators of the aryl hydrocarbon receptor (AhR) with potential immunomodulatory effects. The carboxylic acid group can be derivatized to form amides and esters that penetrate cell membranes and interact with intracellular targets such as protein kinases, including CDK2, CDK9, and GSK-3β. In addition, indole-6-carboxylic acid-based compounds have been explored as antiviral agents (e.g., inhibitors of HCV NS5B polymerase) and as ligands for the melatonin receptor. The compound's structural similarity to the amino acid tryptophan allows it to interact with tryptophan-related enzymes and transporters.

In cell-free biochemical assays, 1H-indole-6-carboxylic acid itself is generally considered inactive at pharmacologically relevant concentrations. In enzyme inhibition screens, the compound shows no significant activity against common targets such as acetylcholinesterase, monoamine oxidase, or carbonic anhydrase at concentrations up to 100 μM. In kinase inhibition assays (e.g., EGFR, CDK2, MAPK), the compound does not inhibit activity at 10 μM. The compound shows weak antioxidant activity in DPPH (IC50 ~200 μM) and ABTS (IC50 ~150 μM) assays, attributed to the indole ring's ability to donate electrons. In antimicrobial testing using broth microdilution, 1H-indole-6-carboxylic acid shows moderate activity against Gram-positive bacteria such as Staphylococcus aureus with MIC of 64-128 μg/mL, but no activity against Gram-negative bacteria or fungi at concentrations up to 256 μg/mL. The compound does not inhibit cyclooxygenase (COX-1/COX-2) at concentrations up to 100 μM. In metal chelation studies, the indole nitrogen and carboxylate group can coordinate to metal ions (Cu²⁺, Fe³⁺, Zn²⁺) with moderate affinity, but this is not biologically significant at physiological conditions. The compound is structurally related to tryptophan metabolites that act as AhR ligands, and it may show weak AhR binding (EC50 >10 μM) in reporter assays. Its primary utility is as a synthetic intermediate for more potent derivatives.

No in vivo pharmacological activity has been reported for 1H-indole-6-carboxylic acid itself, as it is primarily used as a chemical intermediate. When administered to animal models, the compound is metabolized primarily by conjugation (glucuronidation and sulfation) of the carboxylic acid group and by oxidation of the indole ring. In rodent studies, intraperitoneal administration at doses up to 100 mg/kg produces no observable behavioral changes, effects on body temperature, or alterations in locomotor activity. Oral administration results in rapid absorption and clearance, with low systemic exposure to the parent compound due to extensive first-pass metabolism. The compound's carboxylic acid group limits its ability to cross biological membranes, reducing its bioavailability and brain penetration. Derivatives of indole-6-carboxylic acid, particularly those with reduced carboxylate polarity or amide-containing conjugates, have been evaluated in animal models for various activities. For example, 5-HT₆ receptor antagonists derived from indole-6-carboxylic acid have shown pro-cognitive effects in Morris water maze and novel object recognition tests in rats at doses of 1-10 mg/kg. COX-2 inhibitors based on this scaffold have demonstrated anti-inflammatory activity in carrageenan-induced paw edema models. However, the parent compound shows no in vivo activity in these models.

For in vitro enzyme-binding or receptor-binding studies, 1H-indole-6-carboxylic acid is typically used as a fragment-like starting compound or a negative control. The compound is dissolved in DMSO (10-50 mM stock) and diluted in assay buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, 0.1% BSA). For enzyme inhibition assays, enzyme (0.5-10 nM) is pre-incubated with compound (0.1-500 μM) for 15-30 minutes at 25°C, then substrate is added, and activity is monitored using spectrophotometric, fluorometric, or luminescent detection. For serine protease inhibition, trypsin or chymotrypsin (1-5 nM) is used with fluorogenic peptide substrates (e.g., Boc-Gln-Ala-Arg-AMC for trypsin, Suc-Ala-Ala-Phe-AMC for chymotrypsin), monitoring fluorescence at 380 nm excitation/460 nm emission. For kinase assays, the compound is tested at 0.1-100 μM using a panel of purified kinases (e.g., EGFR, CDK2, MAPK, AKT) with ATP (10-100 μM) and appropriate peptide substrates. Phosphorylation is detected by ELISA, fluorescence polarization, or radiometric methods. For receptor binding studies, membrane preparations (20-50 μg) from cells expressing the receptor of interest are incubated with radioligand (0.5-5 nM) and test compound (0.01-100 μM) in binding buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 1 mM EDTA, 0.1% BSA) for 1-2 hours at 25-37°C. Non-specific binding is determined using 10 μM unlabeled ligand. Bound radioactivity is separated by vacuum filtration through GF/B or GF/C filters and counted by liquid scintillation. Data analysis for IC₅₀ and Kᵢ values is performed using GraphPad Prism with one-site or two-site binding models. Positive controls (e.g., reference inhibitors) and vehicle controls are included in all experiments. For assays involving AhR binding, the compound is tested in ligand-binding domain (LBD) assays or using reporter cell lines with luciferase readout.

For in vitro cell-based assays, 1H-indole-6-carboxylic acid is evaluated for cytotoxicity, cellular effects, and potential bioactivity. Mammalian cell lines including HEK293, HeLa, MCF-7, A549, and HepG2 are cultured in DMEM or RPMI-1640 supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin at 37°C with 5% CO₂. Cells are seeded in 96-well plates (5,000-20,000 cells/well) and allowed to attach overnight. Test compound is dissolved in DMSO (10 mM stock) and diluted in culture medium to final concentrations of 0.1, 0.5, 1, 5, 10, 25, 50, 100, 250, and 500 μM (DMSO ≤0.5%). After 24-72 hours of exposure, cell viability is measured using MTT assay (0.5 mg/mL MTT, 4 hours, absorbance at 570 nm) or CellTiter-Glo (luminescent ATP quantification). The compound typically shows moderate cytotoxicity with IC50 values of 50-150 μM in various cell lines, likely due to its ability to induce oxidative stress and mitochondrial dysfunction. For apoptosis detection, cells are stained with annexin V-FITC/propidium iodide and analyzed by flow cytometry. Caspase-3/7 activity is measured using a fluorogenic substrate (Ac-DEVD-AMC) with fluorescence monitored at 380 nm excitation/460 nm emission. For cell cycle analysis, fixed cells are stained with propidium iodide (50 μg/mL) and analyzed by flow cytometry. Reactive oxygen species (ROS) generation is measured using DCFH-DA (2',7'-dichlorofluorescein diacetate), where cells are incubated with 10 μM DCFH-DA for 30 minutes and fluorescence is measured (excitation 485 nm, emission 530 nm). For assessment of mitochondrial membrane potential, cells are stained with JC-1 dye (10 μg/mL) and the ratio of red (aggregates, 590 nm) to green (monomers, 530 nm) fluorescence is measured. Cellular uptake of the compound (10-100 μM, 0.5-6 hours) is determined by extracting cell lysates with acetonitrile and analyzing by LC-MS/MS. Positive controls include staurosporine (1 μM) for apoptosis and doxorubicin (1-10 μM) for cytotoxicity. All experiments are performed in triplicate, and data are expressed as mean ± SEM.

For in vivo studies with 1H-indole-6-carboxylic acid, standard protocols are used for pharmacokinetic and toxicological characterization. For oral administration, male Sprague-Dawley rats (250-300 g, n=4-5 per time point) are fasted overnight and administered the compound by gavage as a suspension in 0.5% methylcellulose or a solution in 10% DMSO/40% PEG-400 at doses of 10, 50, and 200 mg/kg. Blood samples (200-250 μL) are collected from the tail vein into heparinized tubes at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. Plasma is obtained by centrifugation (3,000 rpm, 10 minutes, 4°C) and stored at -80°C until analysis. For intravenous administration, the compound is dissolved in 10% DMSO/90% saline and injected at 1-5 mg/kg via tail vein. For tissue distribution, animals are euthanized by CO₂ asphyxiation at 0.5, 2, 6, and 24 hours, and tissues (liver, kidney, heart, lung, brain, spleen, muscle, adipose) are collected, weighed, and homogenized in 3 volumes of PBS using a tissue homogenizer. Bioanalysis of compound and metabolites in plasma, tissues, urine, and feces is performed by LC-MS/MS with a C18 column (50 × 2.1 mm, 1.7 μm), mobile phase of 0.1% formic acid in water/acetonitrile gradient, and electrospray ionization in positive ion mode, monitoring the transition m/z 162→116 for 1H-indole-6-carboxylic acid and appropriate internal standard (e.g., 1H-indole-5-carboxylic acid-d₄). Pharmacokinetic parameters are calculated using non-compartmental analysis in WinNonlin or Phoenix software. For metabolite identification, urine and bile samples are analyzed by high-resolution mass spectrometry (Q-TOF MS) with full scan MS and MS/MS fragmentation. For toxicity studies, repeated dose administration is performed for 14 or 28 days at 10, 50, and 200 mg/kg/day by oral gavage. Clinical signs (behavior, appearance, respiratory rate, salivation, piloerection), body weight, food and water consumption, hematology (CBC, WBC differential), clinical chemistry (ALT, AST, ALP, GGT, BUN, creatinine, glucose, total protein, albumin, bilirubin, electrolytes), and histopathology of all major organs (liver, kidney, heart, lung, spleen, brain, stomach, intestine, pancreas, gonads) are evaluated according to OECD Test Guideline 407. For efficacy studies, derivatives of 1H-indole-6-carboxylic acid (e.g., amides, esters) are evaluated in appropriate disease models. For anti-inflammatory activity, the carrageenan-induced paw edema model in rats is used: animals receive test compound (10-50 mg/kg, p.o.) 1 hour before subplantar injection of 1% carrageenan (100 μL), and paw volume is measured by plethysmometer at 0, 1, 2, 3, 4, 6 hours. For anticancer activity, tumor xenograft models are used with compound administration (5-50 mg/kg, i.p. or p.o.) daily for 14-21 days.

Pharmacokinetic properties of 1H-indole-6-carboxylic acid are characterized by moderate oral absorption and extensive metabolism. In rat studies, the compound exhibits oral bioavailability of 20-40% with Cmax achieved at 1-2 hours post-dose. The compound has low lipophilicity (clogP ~1.2) with a volume of distribution of 0

C9H7NO2

161.16

161.047

1670-82-2

595230

Light yellow to yellow solid powder

1.4±0.1 g/cm3

419.6±18.0 °C at 760 mmHg

249-253 °C(lit.)

207.6±21.2 °C

0.0±1.0 mmHg at 25°C

1.726

1.82

2

2

1

12

193

0

O=C(C1=CC2=C(C=C1)C=CN2)O

GHTDODSYDCPOCW-UHFFFAOYSA-N

InChI=1S/C9H7NO2/c11-9(12)7-2-1-6-3-4-10-8(6)5-7/h1-5,10H,(H,11,12)

1H-indole-6-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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.)