Catechol-O-methyltransferase (COMT) inhibitor [1]
Xanthine oxidase (competitive inhibitor, \(K_i = 8.8 \mu M\)) [1]

ROO-induced GSH (reduced glutathione) consumption is decreased by 11–38% and oxidation to oxidized glutathione (GSSG) is decreased by 32–45% when nitecapone (1–100 μM) is used[1].

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Scavenges peroxyl radicals (ROO•) in solution with a stoichiometry factor of 2 (determined by fluorescence method using AAPH as radical initiator and B-phycoerythrin as reporter) [1]
Scavenges peroxyl radicals in DOPC liposome membranes (luminol-enhanced chemiluminescence assay using AMVN as radical initiator; half inhibition at 0.5 μM) [1]
Inhibits AMVN-induced lipid peroxidation in rat heart microsomes (TBARS assay; concentration-dependent reduction of TBARS production) [1]
Scavenges superoxide anion radicals (O₂⁻•) with a second-order kinetic rate constant of \(1.0 \times 10^4 \mathrm{M}^{-1}\mathrm{s}^{-1}\) (determined by competition with cytochrome c reduction in DMSO/NaOH system) [1]
Scavenges hydroxyl radicals (HO•) (ESR spin trapping with DMPO; 50% suppression of DMPO-OH signal at 300 μM in Fenton reaction system) [1]
Interacts with ascorbate oxidation products to participate in vitamin E recycling: Does not directly reduce vitamin E radicals (chromanoxyl radicals) generated by UV irradiation, but can generate semidehydroascorbyl radicals from ascorbate oxidation products when ascorbate is depleted; ascorbate protects nitecapone from oxidative degradation under strong UV irradiation [1]

Rats given a spinal nerve ligation (30 mg/kg, intraperitoneal injection every day for 13 days) show a reduction in the development and symptoms of neuropathic pain [3].

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In spinal nerve ligation (SNL) model of neuropathic pain in male Wistar rats:
- Pretreatment (30 mg/kg, i.p., once daily, starting 1 h before surgery, continued for 14 days) significantly reduced mechanical allodynia (withdrawal thresholds 80-95% higher than control) and cold allodynia (fewer responses to acetone) on ipsilateral paw [3]
- Post-treatment (30 mg/kg, i.p., once daily, starting on day 2 after surgery, continued for 20 days) also significantly reduced mechanical allodynia (from day 6 onward) and cold allodynia compared to vehicle [3]
- A single dose of nitecapone (30 mg/kg, i.p.) on day 14 after surgery in nitecapone-naive animals acutely reduced mechanical allodynia (thresholds higher at 180 and 240 min post-dose), but did not affect cold allodynia; no acute effect was seen on day 2 after surgery [3]
- After discontinuation of 14-day pretreatment, the antiallodynic effect persisted for several days (until day 19 after surgery) [3]

Peroxyl radical scavenging in solution (fluorescence method): A water-soluble azo-initiator (AAPH) was used to generate peroxyl radicals. B-phycoerythrin fluorescence (excitation 540 nm, emission 575 nm) was monitored at 40°C in Tris-HCl buffer (pH 7.4). The lag period during which nitecapone protected fluorescence loss was measured to calculate the stoichiometry factor relative to Trolox (known factor of 2). [1]
- Peroxyl radical scavenging in membranes (chemiluminescence method): AMVN was incorporated into DOPC liposomes along with nitecapone. Luminol-enhanced chemiluminescence was monitored at 40°C. The concentration of nitecapone causing 50% inhibition of chemiluminescence was determined. [1]
- Lipid peroxidation (TBARS assay): Rat heart microsomes (2.5 mg protein/mL) were incubated in phosphate buffer (pH 7.4) with AMVN (2.5 mM) and various nitecapone concentrations at 42°C. Aliquots were mixed with TCA/TBA and butylated hydroxytoluene, heated at 100°C for 20 min, centrifuged, and absorbance at 535 nm was measured. A molar extinction coefficient of \(1.56 \times 10^{-5} \mathrm{M}^{-1}\mathrm{cm}^{-1}\) was used. [1]
- Xanthine oxidase activity assay: Xanthine oxidase activity was determined by monitoring urate formation at 295 nm at 25°C in phosphate buffer (pH 7.4). Michaelis constants in absence and presence of nitecapone (10 μM) were obtained from Lineweaver-Burk plots. Inhibition constant (\(K_i\)) was calculated using the equation \(K_m(\text{with inhibitor}) = K_m(\text{without inhibitor}) \times (1 + [I] \cdot K_i^{-1})\). [1]
- Superoxide scavenging activity (cytochrome c competition): Superoxide radicals were generated by alkaline DMSO (DMSO containing 1% water and 5 mM NaOH, incubated 30 min at 25°C). Different concentrations of nitecapone, 20 μM cytochrome c, and 100 μM EDTA in phosphate buffer (pH 8.6) were incubated at 4°C for 20 min, then alkaline DMSO was added. Cytochrome c reduction was measured at 550 nm. The bimolecular kinetic rate constant for nitecapone with O₂⁻• was calculated using a known rate constant for cytochrome c with O₂⁻• (\(2.6 \times 10^{5} \mathrm{M}^{-1}\mathrm{s}^{-1}\)). [1]
- Hydroxyl radical scavenging (ESR spin trapping): Hydroxyl radicals were generated by Fenton reaction (200 μM FeSO₄ + 2 mM H₂O₂) in phosphate buffer (pH 7.4). DMPO (150 μM) was used as spin trap. ESR spectra were recorded 3 min after reaction initiation. Hyperfine splitting constants for DMPO-OH adduct were \(a^H = 14.8\) G (both). Percentage suppression by nitecapone was determined. [1]
- Vitamin E recycling (ESR detection of chromanoxyl radicals): Chromanol-α-C6 (a vitamin E homologue, 20 mM) in DOPC liposomes (20 mg/mL) in phosphate buffer (pH 7.4) was exposed to UV light (1.5 mW/cm² at 310 nm) at 25°C. ESR spectra were recorded under oxygen. Nitecapone (2 mM) was added alone or with chromanol. To test interaction with ascorbate, ascorbate (20 μM) was added after chromanoxyl radical generation, and then nitecapone (20 μM) was added. Semidehydroascorbyl radical signals were monitored. [1]
- Nitecapone photooxidation (spectrophotometry): Nitecapone (15 μM) or ascorbate (15 μM) alone or together in phosphate buffer (pH 7.4) containing DETAPAC (20 μM) were exposed to UV light (29.4 mW/cm² at 310 nm) under oxygen saturation. Absorption spectra from 200 to 600 nm were recorded at various time points. [1]
COMT activity assay: Tissues (striatum, liver, prefrontal cortex, spinal cord segments L1-2, L3-4, L5-6, and lumbar sensory ganglia) were dissected and stored at -80°C. The enzyme preparation was incubated at 37°C in 100 mM phosphate buffer (pH 7.4) containing 5 mM MgCl₂, 200 μM S-adenosyl-L-methionine, and 500 μM 3,4-dihydroxybenzoic acid. Reaction products (vanillic and isovanillic acid) were analyzed by HPLC with electrochemical detection. Mobile phase: 0.1 M Na₂HPO₄ (pH 3.3), 0.15 mM EDTA, 25% methanol, flow rate 0.8 mL/min. COMT activity was expressed as pmol vanillic acid formed per min per mg protein. [3]

Animal/Disease Models: 86 male Wistar rats, weighing 140-350 g[3].
Doses: 30 mg/kg (3.3 ml/kg).
Doses: IP one time/day for 13 days.
Experimental Results: Selectively and specifically inhibits COMT in peripheral tissues and, to some extent, the central nervous system (CNS) for approximately 10 minutes. 3 hrs (hrs (hours)). Increases the threshold for mechanical stimulation, thereby reducing mechanical allodynia. The number of positive responses on the ipsilateral paw was diminished compared with baseline in rats that received nitcapone pretreatment.

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Spinal nerve ligation (SNL) model in rats: Male Wistar rats (140-350 g) were anaesthetized with isoflurane (4.5% for induction, 2-3% for maintenance) in air. The left L5 and L6 spinal nerves were exposed by removing a small piece of paravertebral muscle and part of the left spinous process of L5 vertebra, carefully isolated from L4 nerve, and tightly ligated with 6-0 silk. Muscle and fascia were closed with sutures, skin with metal clips. Animals showing motor complications or weight loss were sacrificed. [3]
- Drug administration – pretreatment study: Nitecapone (30 mg/kg) or vehicle (0.5% carboxymethylcellulose, CMC) was administered intraperitoneally (i.p.) in a volume of 3.3 mL/kg, starting 1 h before surgery, then once daily for 13 days. On day 14 after surgery, both groups received a single dose of nitecapone. In a subgroup, treatments were crossed over for additional 4 days (nitecapone→CMC, CMC→nitecapone). Behavioural tests were performed before surgery and every other day after surgery, 1 h before drug administration. [3]
- Drug administration – post-treatment study: Nitecapone (30 mg/kg, i.p.) or vehicle (0.5% CMC, i.p.) was administered once daily starting on day 2 after surgery, continued for 20 days. Behavioural tests were performed before surgery and every other day after surgery, 1 h before drug administration. [3]
- Acute effect assessment: On day 2 after surgery (in nitecapone-naive rats) and on day 14 after surgery (in both nitecapone-pretreated and nitecapone-naive rats), behavioural tests were performed before and at 1, 2, 3, and 4 h after a single nitecapone dose (30 mg/kg, i.p.). [3]
- Mechanical allodynia test (von Frey): Rats stood on a metal mesh under individual transparent plastic cages. A metal monofilament (tip diameter 0.5 mm) attached to a digital force gauge was applied to the plantar surface of the paw for 1 second. The force just sufficient to produce a response (brisk foot withdrawal, lifting, or licking) was recorded. Thresholds were measured from both paws twice with 1-min intervals. [3]
- Cold allodynia test (acetone test): A drop of acetone was applied to the plantar surface of the paw. The number of brisk foot withdrawal responses out of five trials was recorded. Measurements were made on both hindpaws with 1-min intervals. [3]

Partition coefficient (octanol/water): log P = 1.21 [1]
After intraperitoneal administration of 30 mg/kg nitecapone in male Wistar rats: COMT activity in liver and striatum was significantly decreased at 30, 60, and 180 min; inhibition peaked between 30-60 min. By 24 h after the last dose, COMT activity returned to control levels (no difference between treatment groups). [3]

[1]. Antioxidant properties of nitecapone (OR-462). Free Radic Biol Med. 1992 Nov;13(5):517-25.

[2]. Nitecapone: a nitric oxide radical scavenger. Biochemistry and Molecular Biology International, 01 Oct 1994, 34(3):531-541.

[3]. Nitecapone reduces development and symptoms of neuropathic pain after spinal nerve ligation in rats. Eur J Pain. 2011 Aug;15(7):732-40.

Nitecapone is a hydroxycinnamic acid.

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Nitecapone [3-(3,4-dihydroxy-5-nitrophenyl)methylene-2,4-pentanedione] (OR-462) is a novel catechol-O-methyltransferase (COMT) inhibitor with gastroprotective properties. Its antioxidant properties were discovered recently. [1]
- Nitecapone has a remarkable ability to act as an antioxidant: scavenges peroxyl radicals, inhibits lipid peroxidation, inhibits xanthine oxidase (competitive, \(K_i = 8.8 \mu M\)), scavenges superoxide and hydroxyl radicals, and participates in vitamin E recycling through interaction with ascorbate. These properties suggest it could be an effective therapeutic antioxidant, possibly in combination with other antioxidants. [1]
The antiallodynic effect of nitecapone in SNL model is long-lasting, far exceeding the duration of COMT inhibition (effect persists for days after discontinuation). This suggests mechanisms beyond COMT inhibition, possibly involving antioxidative properties or long-acting beneficial processes. Nitecapone’s close derivative entacapone is clinically used for Parkinson’s disease and is well tolerated with poor blood-brain barrier penetration. Further studies on COMT inhibitors for neuropathic pain are warranted. [3]

C12H11NO6

265.221

265.059

116313-94-1

5464105

Light yellow to yellow solid powder

1.451g/cm3

495.3ºC at 760 mmHg

211.6ºC

1.95E-10mmHg at 25°C

1.645

2.09

2

6

3

19

408

0

UPMRZALMHVUCIN-UHFFFAOYSA-N

InChI=1S/C12H11NO6/c1-6(14)9(7(2)15)3-8-4-10(13(18)19)12(17)11(16)5-8/h3-5,16-17H,1-2H3

3-(3,4-Dihydroxy-5-nitrobenzylidene)-2,4-pentanedione

Nitecapone OR 462 OR462OR-462

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)

DMSO : ~50 mg/mL (~188.52 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.84 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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