Synthesis of nicorandil
Nicorandil was synthesized as shown in Fig. 2. In summary, ester 1 was treated with ethanolamine under reflux, furnishing the N-(2-hydroxyethyl)nicotinamide (2). Compound 2 was then treated with fuming nitric acid at -5°C, leading to nicorandil (Scheme 1). The nicorandil was characterized by 1H and 13C NMR, infrared spectroscopy, melting point determination, and elemental analysis; the data are consistent with previous reports [8,15,16].
N-(2-Hydroxyethyl)nicotinamide (2) (Yield 64%): mp 88.0 oC. IR (ATR): 3321, 3173, 2876, 1659, 1550, 1422, 1296, 1057, 1028, 702. 1H NMR (200 MHz, DMSO-d6): 3.34–3.39 (m, 2H), 3.57 (t, 2H, J = 5.8 Hz), 4.83 (brs, 1H), 7.42–7.50 (m, 1H), 8.19 (d, 1H, J = 8.0 Hz), 8.66–8.68 (m, 2H), 9.02 (s, 1H). 13C NMR (50 MHz, DMSO-d6): 42.3, 59.8, 123.4, 130.1, 135.0, 148.5, 151.7, 165.1. Anal. Calcd for C8H10N2O2. Calcd (%): C, 57.82; H, 6.07; N, 16.86. Found (%): C, 56.94; H, 6.03; N, 16.88.
Nicorandil (Yield 40%): mp 91.0 oC. IR (ATR): 3241, 3073, 1717, 1627, 1590, 1554, 1372, 1361, 1319, 1286, 1012, 1000, 860, 824, 705. 1H NMR (200 MHz, DMSO-d6): 3.65 (q, 2H, J = 5.1 Hz), 4.67 (t, 2H, J = 5.1 Hz), 7.46–7.55 (m, 1H), 8.18 (d, 1H, J = 7.8 Hz), 8.71 (d, 1H, J = 4.4 Hz), 8.94–9.02 (m, 2H). 13C NMR (50 MHz, DMSO-d6): 36.8, 72.1, 123.5, 129.5, 135.0, 148.3, 152.0, 165.2. Anal. Calcd for C8H9N3O4. Calcd (%): C, 45.50; H, 4.30; N, 19.90. Found (%): C, 45.62; H, 4.31; N, 20.03.
Zebrafish
Adult wild-type zebrafish (Danio rerio) of both sexes (short-fin phenotype), aged 60–90 days, with sizes of 3.5 ± 0.5 cm and weights of 4.0 ± 0.1 g, were obtained from Agroquímica: Comércio de Produtos Veterinários LTDA, a supplier in Fortaleza (Ceará, Brazil). The acclimation and handling of the animals were conducted at the Experimental Biology Center of the University of Fortaleza – NUBEX/UNIFOR. The fish were housed in a glass aquarium at a temperature of 24°C, with tap water treated with a dechlorinator (ProtectPlus®). All experimental procedures were approved by the Animal Use Ethics Committee of the State University of Ceará (#05299177/2021).
Acute toxicity (96 h)
To determine the lethal concentration that kills 50% of the animals (LC50) LC50-96 h, zebrafish (n = 8/group) were treated (20 µL; p.o.) with nicorandil (5.0, 10, 15 mg/mL) or vehicle (0.9% saline + 5% Tween 80). Following the treatments, animal mortality was assessed every 24 hours. After 96 hours, the number of dead fish in each group was recorded [17].
Evaluation of locomotor activity (open field test)
The open field test was conducted to assess changes in motor coordination of the animals, whether due to sedation and/or muscle relaxation. Initially, the animals (n = 8/group) were treated (20 µL), orally (p.o.), with nicorandil (5.0, 10, or 15 mg/mL) or vehicle (0.9% saline + 5% Tween 80). A group of untreated animals was included (Naive; n = 8). One hour after the treatments, the animals were individually placed in Petri dishes (∅ = 15 cm) containing the same aquarium water, divided into quadrants, and the number of line crossings over 0–5 minutes was recorded [18].
Orofacial nociception induced by cinnamaldehyde
Nociception was induced with cinnamaldehyde (TRPA1 agonist; 0.33 µM; 5.0 µL; [19]), applied to the lip (n = 8/group) 1 hour after pretreatment (20 µL; p.o.) with nicorandil (5.0, 10, or 15 mg/mL) or vehicle (control; 0.9% saline + 5% Tween 80). A treatment-free group (Naive; n = 8) was included. After the final treatment, the animals were individually placed in Petri dishes (ø = 15 cm), and nociceptive response was quantified in terms of locomotor activity (as mentioned above) for 0–5 minutes.
In a subsequent experiment, the animals (n = 8) were pretreated (20 µL; p.o.) with vehicle, nicorandil (5 mg/mL), or with HC-030031 (TRPA1 antagonist; 0.1 mg/mL) prior to nicorandil (5 mg/mL). Orofacial nociception was induced with cinnamaldehyde as mentioned above. A Naive group (n = 8) was also included.
Orofacial nociception induced by capsaicin
Nociception was induced with capsaicin (TRPV1 agonist; 40.93 µM; 5.0 µL; [19]), applied to the lip (n = 8/group), 1 h after pretreatment (20 µL; p.o.) with nicorandil (5.0; 10 or 15 mg/mL) or vehicle (control; 0.9% Saline + 5% Tween 80). A treatment-free group (Naive; n = 8) was included. After the last treatment, the animals were individually placed in Petri dishes (∅ = 15 cm), and nociceptive response was quantified in terms of locomotor activity (as mentioned above) for 10–20 min.
In a subsequent experiment, the animals (n = 8) were pretreated (20 µL; p.o.) with vehicle, nicorandil (5 mg/mL), or nicorandil (5 mg/mL) 15 min after capsazepine (TRPV1 antagonist; 0.5 mg/mL). Orofacial nociception was induced with capsaicin as mentioned above. A Naive group (n = 8) was also included.
Orofacial nociception induced by menthol
Orofacial nociception was induced with menthol (TRPM8 agonist; 0.006%, 5.0 µL; [19]), applied to the lip (n = 8/group), 1 h after pretreatment (20 µL; p.o.) with nicorandil (5.0; 10, or 15 mg/mL) or vehicle (control; 0.9% Saline + 5% Tween 80). A treatment-free group (Naive; n = 8) was included. After the last treatment, the animals were individually placed in Petri dishes (∅ = 15 cm), and nociceptive response was quantified in terms of locomotor activity (as mentioned above) for 0–10 min.
In a subsequent experiment, the animals (n = 8) were pretreated (20 µL; p.o.) with vehicle, nicorandil (5 mg/mL), or AMTB (TRPM8 antagonist; 0.3 mg/mL) 15 min before nicorandil (5 mg/mL). Orofacial nociception was induced with menthol as mentioned above. A Naive group (n = 8) was also included.
Molecular docking
The interaction between nicorandil and TRPV1/TRPA1 channels was analyzed in silico using computational molecular docking simulation. The study involves the use of a computational algorithm with specific software for the docking of two molecules, aiming to form a stable complex. The three-dimensional structures of Nicorandil and TRPV1 and TRPA1 channels were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the Protein Data Bank (https://www.rcsb.org/) with the codes: 47528, 3J5P, and 3J9P, respectively.
Docking was performed using AutoDock Vina software (version 1.1.2), employing "3-way multithreading" and "Lamarckian Genetic Algorithm" [20]. The grid box was positioned at the center of the receptor, measuring 100 Å x 100 Å x 100 Å, and the algorithm searched for all possible binding sites based on the association energy and spatial compatibility between Nicorandil and TRPV1 and TRPA1 channels. The data obtained were analyzed using PyMol v1.4.7 software.
Statistical analysis
The LC50 was determined using the Trimmed Spearman-Karber mathematical method with a 95% confidence interval. Results from the open field test were expressed as mean values ± standard error of the mean for each group of 6 animals. After confirming data normality and homogeneity of variance, differences between groups were subjected to one-way analysis of variance (ANOVA), followed by Tukey's post hoc test. All analyses were performed using GraphPad Prism software v. 8.0. The level of statistical significance was set at 5% (p < 0.05).