Searching for Mechanisms of Analgesic Activity in the Group of 1H-Pyrrolo[3,4-c]pyridine-1,3(2H)-dione Derivatives—In Vitro and In Vivo Studies

Abstract

The present study was to evaluate the analgesic activity of two newly synthesized 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives, designated DSZ-13 and DSZ-19. To achieve the desired result, the in vitro XTT cell proliferation assay, serotonin 5-HT_1A receptor affinity and COX-1 and COX-2 enzyme inhibition potential of the compounds were conducted by real-time qPCR. Non-compartmental analysis was used to estimate the pharmacokinetic parameters of the compounds in serum and brain tissue. The analgesic activity was evaluated using various in vivo pain models, encompassing acute pain (hot plate test), tonic pain (formalin test), neurogenic pain (capsaicin test), carrageenan-induced acute inflammation, and neuropathic pain models. Both compounds showed moderate affinity for serotonin 5-HT_1A receptors, a lack of cytotoxic activity, desirable pharmacokinetic parameters and slightly reduced mRNA expression for COX-1 and COX-2. Only the DSZ-19 revealed central/supraspinal analgesic activity and did not affect movement. Both compounds attenuated tonic and neurogenic pain, in the formalin and capsaicin tests, respectively. In addition, the involvement of the 5-HT_1A receptors in the formalin test was confirmed. Both compounds also showed antiallodynic activity in the oxaliplatin- and streptozotocin-induced neuropathy models. Slightly weaker than indomethacin, DSZ-13 and DSZ-19 attenuated carrageenan-induced inflammation (edema) and hyperalgesia in rat models.

1. Introduction

The search for novel analgesic agents has increasingly focused on small molecules with defined structural features that confer potent pharmacological activity. Among these, derivatives of 3,4-pyridinedicarboximide have attracted attention due to their promising analgesic properties, which appear to depend on specific structural motifs, such as an alkoxy substituent at the 4-position of the pyridine ring, an arylamine moiety, and an alkyl linker connecting the core components of the molecule. Structural modifications, including variations in lipophilicity and linker length, can significantly influence pharmacological profiles, often leading to the formation of Mannich base-type products [1,2,3]. This nucleophilic addition–condensation reaction is widely used in the synthesis of biologically active compounds, including peptides, nucleotides, antibiotics, alkaloids, and therapeutic agents such as fluoxetine, tramadol, and rolitetracycline [3,4]. Compounds containing aminoalkyl chains are considered key pharmacophores in medicinal chemistry and exhibit diverse pharmacological activities, including anti-inflammatory, analgesic, anticancer, antibacterial, antifungal, anticonvulsant, antipsychotic, and antiviral effects [5,6,7,8,9,10,11,12,13,14]. It is worth noting that in recent years, selective 5-HT_1A receptor agonists, such as Befiradol [15] (also known as F13640 or NLX-112) [16], and ST171 [17] have attracted considerable interest, as they have demonstrated significant analgesic efficacy in preclinical studies, further confirming the key role of this receptor in the regulation of nociception.

Previous studies demonstrated that several 3,4-pyridinedicarboximide derivatives exhibited significant analgesic activity in the mouse writhing test, in some cases surpassing acetylsalicylic acid and approaching the efficacy of morphine [18].

Building upon these findings, the present study reports two novel 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives, DSZ-13 and DSZ-19, designed to evaluate their antinociceptive potential and further elucidate their mechanisms of action.

To address this, we applied a multi-level experimental approach. In vitro assays included receptor binding studies to assess affinity for the serotonin 5-HT_1A receptor and evaluation of cyclooxygenase inhibition (COX-1 and COX-2), using quantitative real-time PCR (qPCR) and in silico molecular docking to explore possible molecular interactions. Pharmacokinetic profiling and brain distribution analyses were conducted to assess CNS exposure, and an XTT assay was used to verify early-stage cytotoxicity.

The analgesic effects of DSZ-13 and DSZ-19 were then evaluated across a series of in vivo models targeting distinct types of nociception. The acute thermal pain model (hot plate test) assessed immediate, centrally mediated nociceptive responses, while the tonic pain model (formalin test) evaluated prolonged inflammatory pain and allowed the investigation of 5-HT_1A receptor involvement. Antiedematous (anti-inflammatory) activity was evaluated using the carrageenan-induced paw edema model, while streptozotocin- and oxaliplatin-induced neuropathy models were employed to assess efficacy in conditions mimicking diabetic and chemotherapy-induced neuropathy. Finally, spontaneous locomotor activity was monitored to exclude sedative or stimulatory effects that could confound behavioral outcomes. Together, these complementary models provide a comprehensive and translationally relevant assessment of the antinociceptive properties of DSZ-13 and DSZ-19.

2. Materials and Methods

2.1. Chemistry

The starting materials for the synthesis of the investigated compounds, DSZ-13 and DSZ-19, were 4- butoxy- and 4-ethoxy -6-methyl-1H-pyrrolo[3,4-c]pyridine-1,3(2H)diones (1,2) [3,18], condensed in a Mannich reaction with 33% formalin and suitable amines (m-fluorophenylpiperazine (DSZ-19); methoxyphenylpiperazine (DSZ-13) in THF solution (Scheme 1).

All results of the C, H, and N determinations, made using the Carlo Erba elemental analyzer model NA-1500 (Thermo Scientific, Waltham, MA, USA), were within ±0.4% of the theoretical values. All melting points are not corrected. The IR spectra of the KBr pellets were measured with the Specord M 80 model (Jena, Germany). ¹H NMR spectra were determined in CDCl₃ on a Bruker (Billerica, MA, USA) Ultra Shield 300 MHz, using tetramethylsilane as an internal standard (IS).

4-Butoxy- and 4-ethoxy-6-methyl-pyrrolo[3,4-c]pyridine-1,3-diones (1,2), which were the starting point for the synthesis, were obtained according to a protocol previously developed at the Department of Medicinal Chemistry, Wroclaw Medical University [3,18].

• DSZ-13: 4-butoxy-2-[[4-(2-ethoxyphenyl)piperazin-1-yl]methyl]-6-methyl-pyrrolo[3,4-c]pyridine-1,3-dione.

A 0.002 mol (0.47 g) of 4-butoxy-6-methyl-pyrrolo[3,4-c]pyridine-1,3-dione (1) was dissolved in 40 mL of tetrahydrofuran (THF) and to this solution was added 0.4 mol (1 mL) of 33% formalin. The mixture was heated at reflux of tetrahydrofuran (65–66 °C) for 0.5 h. After this time, 0.0022 mol (0.45 g) of 2-ethoxyphenylpiperazine was added and the mixture was heated at reflux again for 10 h. Then, the solvent was completely evaporated under reduced pressure. The crude precipitate was purified by crystallization from hexane. The properties of the compounds obtained are shown below.

Molecular Formula: C₂₅H₃₂N₄O₄; Formula Weight: 452.54597; Exact Mass: 452.2423; MP = 127–128 °C; yield: 78%; Composition: C: 66.4% H: 7.1% N: 12.4% O: 14.1%; PSA: 75.21; ALogP: 3.9286; Stereo Center Count: 0; Hydrogen Acceptor Count: 7; Hydrogen Donor Count: 0. FT-IR (cm⁻¹) 1730, 1770 (C=O), 695, 750 (disubstituted benzene); ¹H NMR/300 MHz/in CDCl₃ (ppm) ƍ = 0.94–0.98 (t-6H, J = 7.00, OCH₂CH₂CH₂CH₃ and OCH₂CH₃), 1.41–1.54 (t-2H, J = 7.1, OCH₂CH₂CH₂CH₃), 1.76–1.86 (t-2H, J = 7.00, OCH₂CH₂CH₂CH₃), 2.58 (s-3H, CH₃), 3.22–3.32 (t-4H, J = 18, 6.6, 5.5, -(CH₂)₂-N-C₆H₄-OC₂H₅), 3.68–3.82 (t-4H, J = 14, 6.6,2.5, -N-(CH₂)₂ of piperazine), 4.51–4.55 (t-4H, -OCH₂CH₃ and -OCH₂CH₂CH₃), 4.66 (s-2H, N-CH₂-N), 6.99–7.05 (m-4H, H of benzene), 7.16 (1H, H of pyridine).

InChI = 1S/C25H32N4O4/c1-4-6-15-33-23-22-19(16-18(3)26-23)24(30)29(25(22)31)17-27-11-13-28(14-12-27)20-9-7-8-10-21(20)32-5-2/h7-10,16H,4-6,11-15,17H2,1-3H3.

• DSZ-19: 4-ethoxy-2-[[4-(2-fluorophenyl)piperazin-1-yl]methyl]-6-methyl-pyrrolo[3,4-c]pyridine-1,3-dione.

A 0.002 mol (0.43 g) of 4-ethoxy-6-methyl-pyrrolo[3,4-c]pyridine-1,3-dione (2) was dissolved in 40 mL of tetrahydrofuran (THF) and to this solution was added 0.4 mol (1 mL) of 33% formalin. The mixture was heated at reflux of tetrahydrofuran (65–66 °C) for 0.5 h. After this time, 0.0022 mol (0.46 g) of 2-fluorophenylpiperazine was added and the mixture was again heated at reflux for 10 h. Then, the solvent was completely evaporated under reduced pressure. The crude precipitate was purified by crystallization from hexane.

The properties of the compound obtained are shown below.

Molecular Formula: C₂₁H₂₃FN₄O₃; Formula Weight: 398.4307; Exact Mass: 398.1754; MP = 120–122 °C, yield: 67%. Composition: C: 63.3% H: 5.8% F: 4.8% N: 14.1% O: 12%; PSA: 65.98, ALogP: 2.8218, Stereo Center Count: 0;

Hydrogen Acceptor Count: 6; Hydrogen Donor Count: 0.

FT-IR (cm⁻¹) 1720,1775 (C = O), 695, 745 (disubstituted benzene);

¹H NMR/300 MHz/(ppm) in CDCl_3, ƍ =1.45–1.50 (t-3H, J = 7.00, OCH₂CH₃), 2.61 (s-3H, CH₃), 2.81–2.84 (dd-4H, J= 18, 6.6, 2.5, -(CH₂)₂-N-C₆H₄-F), 3.05–3.08 (dd-4H, J= 13, 6.6, 2.5, -N-(CH₂)₂- of piperazine), 4.58–4.62 (t-2H, J = 7.00, -OCH₂CH₃), 4.67 (s-2H, N-CH₂-N), 6.90–7.01 (m-4H, H arom. of benzene), 7.26 (s1H, H of pyridine).

InChI = 1S/C21H23FN4O3/c1-3-29-19-18-15(12-14(2)23-19)20(27)26(21(18)28)13-24-8-10-25(11-9-24)17-7-5-4-6-16(17)22/h4-7,12H,3,8-11,13H2,1-2H3.

The chemical and physical properties of the obtained compounds are summarized in Table S3 (DSZ-13) and S4 (DSZ-19) in the Supplementary Materials. The ¹H NMR spectrum of the starting compound 1 and the ¹H NMR spectrum of the final product are also provided in the Supplementary Materials (Figures S7–S10, PDF format).

2.2. Animals and Housing Conditions

Animals were supplied by the Jagiellonian University Collegium Medicum Faculty of Pharmacy Animal Breeding Facility. Pharmacological assessments, including the formalin test, hot plate test, capsaicin test, as well as oxaliplatin- and streptozotocin-induced neuropathy models, were performed using male Swiss Albino CD-1 mice (18–25 g). Pharmacokinetic experiments were also conducted in male Swiss Albino CD-1 mice (18–25 g). In contrast, the carrageenan-induced model was performed using male Wistar rats (Krf: WI (WU) (180–250 g). Experiments were conducted between 9:00 a.m. and 2:00 p.m. Animals were group-housed (maximum 10 per cage) under standard laboratory conditions (20–24 °C, 45–65% humidity, 12 h light/dark cycle) with environmental enrichment, and had ad libitum access to food and water. All procedures were carried out by an experimenter blinded to drug administration and statistical analysis. Mice were maintained in accordance with the Guide for the Care and Use of Experimental Animals. All in vivo experimental procedures were approved by the Krakow Regional Ethics Committee of Jagiellonian University (Approval Nos. 44/2018, 218/2019, 440A/2020, 440B/2020, 441/2020, 495/2021) and conducted in full compliance with the relevant Polish and EU regulations (Directive 86/609/EEC).

2.3. Drugs and Doses

The doses of the test compounds used for the in vivo experiments were chosen based on the results of our previous preliminary studies [19,20] as well as the data available in the literature [21]. The compounds were suspended in 1% Tween 80 solution (Merck, Darmstadt, Germany) and administered by the intraperitoneal (i.p.) route 30 min before the pharmacological experiments, at a constant volume of 0.1 mL/10 g (mice) and 0.1 mL/100 g (rats). The following reagents and pharmacological agents were used in the in vivo experiments: formalin (37% w/w formaldehyde solution; P.O. Ch., Gliwice, Poland), capsaicin (Sigma-Aldrich, Darmstadt, Germany), λ-carrageenan (Sigma, Kawasaki, Japan), oxaliplatin (Activate Scientific, Prien am Chiemsee, Germany), and streptozotocin (Sigma-Aldrich, Darmstadt, Germany), Indometacin (Sigma-Aldrich, Darmstadt, Germany), ketoprofen (Sigma-Aldrich, Darmstadt, Germany).

2.4. In Vitro Experiments

2.4.1. Radioligand Binding Assay—Evaluation of 5-HT_1A Receptor Affinity

Radioligand binding was performed in duplicate using membranes from CHO-K1 cells stably expressing the human 5-HT_1A receptor (PerkinElmer, USA), as previously described [22]. Stock solutions of the tested ligands (10 mM in DMSO) were serially diluted in assay buffer to yield final concentrations ranging from 10⁻⁵ to 10⁻¹¹ M. Dilutions were performed using an automated epMotion 5070 liquid handling system (Eppendorf, Hamburg, Germany). Each compound was evaluated in 7 concentrations of 10⁻⁵ to 10⁻¹¹ M (final concentration). The reaction mixture in each well included 50 µL of the test compound solution, 50 µL of [³H]-8-OH-DPAT at a final concentration of 1 nM, and 150 µL of membrane suspension (containing 20 µg of protein per well), all prepared in assay buffer (50 mM Tris-HCl, pH 7.4, 10 mM MgSO₄, 0.5 mM EDTA, and 0.1% ascorbic acid). All components were added into 96-well polypropylene microplates using a Rainin Liquidator pipetting system (Mettler Toledo, Oakland, CA, USA). To find non-specific binding, 8-OH-DPAT at a concentration of 10 μM was added to the control wells. The plates were sealed, gently mixed, and incubated at 27 °C for 60 min. The reactions were ended via rapid filtration onto GF/B filter mats pretreated with 0.5% polyethyleneimine (30 min pre-soak). Filters were washed ten times with ice-cold Tris buffer (50 mM, pH 7.4) using a Harvester-96 MACH III FM (Tomtec, PA, USA).

Following filtration, filter mats were dried at 37 °C, and solid scintillator (MeltiLex, PerkinElmer, Waltham, MA, USA) was melted onto the filters at 90 °C for 4 min. Radioactivity levels were measured using a MicroBeta2 scintillation counter (PerkinElmer, Waltham, MA, USA). Data were fitted to a one-site curve-fitting equation with Prism 5.0 (GraphPad Software, San Diego, CA, USA) and the inhibitory constant (Ki) values were estimated from the Cheng−Prusoff equation.

2.4.2. XTT Assay—Assessment of Cell Culture Viability

The impact of DSZ-13 and DSZ-19 on RAW 264.7 cell proliferation was evaluated according to the protocol described previously [19,23]. Briefly, RAW 264.7 cells (Mus musculus monocyte/macrophages, TIB-71, American Type Culture Collection; cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS and 1% antibiotic solution: 100 IU/mL penicillin and 0.1 mg/mL streptomycin, ATTC, Manassas, VA, USA, in 96-well plates (2.5 × 10³ cells/well)) were seeded and incubated for 24 h. The medium was then removed and then 0.5, 1, 2.5, 5, 10, 50 and 100 µM/µL of DSZ-13 and DSZ-19 compounds were added to FCS-free medium and incubated for 24 h. After this time, XTT solution (50 μL) was added to each well and incubated for 4 h at 37 °C according to the manufacturer’s instructions (Sigma-Aldrich). The absorbance was measured at 475 and 630 nm in an FLUOstar Omega microplate reader with MARS Data Analysis Software v4.x (BMG LABTECH, Ortenberg, Germany) and the specific absorbance of the sample was expressed as follows: specific absorption = A475 nm (sample) A475 nm (blank) A660 nm (sample). Cell viability was expressed as the percentage of the control. RAW 264.7 cells (Mus musculus monocyte/macrophages, TIB-71, American Type Culture Collection) were activated with 10 ng/mL lipopolysaccharide (LPS from Escherichia coli 026:B6, Sigma-Aldrich, St. Louis, MO, USA) for 24 h and then treated for 24 h with 1, 2.5 and 5 µM/µL of DSZ-13 and DSZ-19 diluted with dimethylsulfoxide (DMSO), according to the method described previously [19,22].

2.4.3. Real-Time qPCR Assay

A PureLink RNA isolation kit (Thermo Fisher Scientific, Rockford, IL, USA) was used for total RNA isolation. The concentration of RNA was normalized to 20 ng/µL. A High-Capacity Reverse Transcription Kit (Thermo Fisher Scientific, USA) was used for reverse transcription. A qPCR was performed on 96-well fast reaction plates with samples, TaqMan Master mix, and TaqMan gene assays for Ptgs2 (Mm00478374_m1), Ptgs1 (Mm00477214_m1) on an Applied Biosystems 7500 Fast Real-Time PCR Instrument (Applied Biosystems, Foster City, CA, USA). The endogenous control was Gapdh (Mm99999915_g1). The delta delta Cq method was used to calculate relative expression.

2.5. Pharmacokinetic Assay

The experimental procedure employed in this study was following the method previously described in [24]. The experimental groups consisted of three mice each, and the compounds DSZ-13 or DSZ-19 suspended in 1% Tween were administered intraperitoneally at a dose of 5 mg/kg. Blood samples were collected after decapitation (under isoflurane anesthesia) in Eppendorf tubes 5, 15, 30, 60, 120, 240, 360 and 480 min after dosing. Plasma was harvested by centrifuging at 2500× g for 10 min; the brain was also dissected, and all samples were stored at −80 °C until bioanalysis.

The analysis of both compounds was performed using a Hitachi HPLC system (Japan) consisting of a pump (model L-2130), an autosampler (model L-2200), a column oven (model L-2350), a fluorescence detector operating at excitation and emission wavelengths of 327 and 420 nm, respectively (model L-2485), and a computer with EZChrom Elite Client/Server v. 3.2 Software for data collection and analysis. Chromatographic separation was achieved at room temperature (22 ± 1 °C) on the LiChroCART^® 250-4 LiChrosorb^® RP-18 (5 µm) analytical column. The mobile phase consisted of acetonitrile and water mixed in a 50:50 (v/v) ratio run at a rate of 1 mL/min. The brains were homogenized in distilled water (1:4, w/v) with a tissue homogenizer TH220 (Omni International, Inc., Warrenton, VA, USA). The analytical compounds were isolated from plasma and brain homogenates using liquid–liquid extraction. Namely, 100 µL of plasma was diluted with 100 µL of water, and 10 µL of IS solution (DSZ-13 or DSZ-19) was added. The samples were mixed and extracted with 1 mL of ethyl acetate on a shaker (VXR Vibrax, IKA, Königswinter, Germany). After centrifugation (Universal 32, Hettich, Tuttlingen, Germany) at 3000 rpm for 15 min, the organic layer was transferred to conical tubes and evaporated to dryness at 37 °C in a water bath under a gentle stream of nitrogen. Brain homogenates (500 µL for DSZ-19 or 1000 µL for DSZ-13) were transferred to glass tubes, and after the addition of IS, extracted with 3 or 5 mL of ethyl acetate in the case of DSZ-19 or DSZ-13, respectively. Dry residue (extraction of plasma or brain homogenates) was reconstituted in 100 µL of methanol. The calibration curves were constructed by plotting the ratio of the peak area of the studied drug to IS versus drug concentration, and were linear in the ranges of 5 to 250 ng/mL (or 2.5 to 50 ng/g of brain tissue) and 2 to 100 ng/mL (or 10 to 500 ng/g of brain tissue) for DSZ-13 and DSZ-19, respectively. The assays were reproducible with low intra- and inter-day variation (coefficient of variation less than 10%).

Pharmacokinetic parameters were calculated using a non-compartmental approach, using Monolix version 2019R1 (Antony, France: Lixoft SAS, 2019) software. The area under the mean plasma concentration versus time curve extrapolated to infinity (AUCINF) was estimated using the log-linear trapezoidal rule. The AUMCINF was estimated by calculating the total area under the first-moment curve by combining the trapezoid calculation of the AUMClast (from time zero to the last sampling point) and the extrapolated area. The mean residence time (MRT) was calculated from the AUMCINF/AUCINNF. The terminal rate constant (lambda_z) was calculated by log-linear regression of the drug concentration data in the terminal phase, and the terminal half-life (HL_lambda_z) was calculated as 0.693/k. Systemic clearance (CL) was estimated from the dose administered divided by the AUCINF. The volume of distribution in steady state (Vss) was calculated from D i.p. AUMCINF/AUCINF, where D i.p. is the dose administered intraperitoneally in mg per kg of body weight.

2.6. In Vivo Study

2.6.1. Hot Plate Test

The hot plate test was conducted according to the method described previously [25]. Animals received test compounds or the vehicle 30 min prior to placement on a hot plate apparatus with an electrically regulated surface maintained at 55 °C. The latency to the first nociceptive response (licking or jumping) was recorded using a stopwatch. A cut-off time of 60 s was implemented to prevent tissue damage to the paw, and no paw injuries or adverse effects were observed.

2.6.2. Formalin Test—Tonic Pain Model

The procedure was conducted according to the method described previously with specific modifications as detailed in [19,26]. A 20 μL volume of 5% formalin solution was administered intraplantarly (i.pl.) into the right hind paw of each mouse. Immediately following injection, subjects were individually placed on a glass beaker and observed continuously for 30 min. The cumulative duration (in seconds) that the animals engaged in licking or biting the injected paw was recorded at designated time intervals (0–5 and 15–30 min) and served as an indicator of nociceptive behavior.

2.6.3. Formalin Test—Assessment of 5HT_1A Receptor in Antinociceptive Activity

Mice were pretreated intraperitoneally (i.p.) with WAY 100635 (0.7 mg/kg) or vehicle 15 min prior to administration of DSZ-13 or DSZ-19 (10 mg/kg, i.p.) to investigate the potential modulatory role of 5-HT_1A receptors in the observed analgesic response. The pretreatment protocol was based on previously published studies [16] and preliminary data [19].

2.6.4. Capsaicin Test—Neurogenic Pain Model

To evoke a pain response, 20 μL of a capsaicin solution in saline (1.6 μg per right hind paw) was injected into the dorsal surface of the mouse paw, according to previously described procedures [19,27]. The tested compounds were administered via the same route 15 min prior to capsaicin injection. Each animal was then monitored individually for 5 min, and the duration of paw licking, measured with a stopwatch, was recorded as an indicator of nociceptive behavior.

2.6.5. Carrageenan-Induced Edema in Rats—Acute Inflammatory Pain Model

Paw edema (inflammation) was induced by subplantar injection of 0.1 mL of 1% carrageenan solution in PBS into the hind paw of rats, as previously described [19,28]. Paw volume was measured using a plethysmometer (Type A, 7140; UGO Basile) prior to carrageenan injection (V0) and at 1, 2, and 3 h post-injection (V1, V2, V3), with all measurements recorded for analysis.

DSZ-13 and DSZ-19 were administered intraperitoneally at their highest active dose (20 mg/kg) 30 min before carrageenan injection. Indomethacin (10 mg/kg, i.p.) served as a positive control, while the vehicle (1% Tween 80) was administered via the same route as the test compounds. Edema inhibition (%) was calculated using the following formula [19]: %inhibition = [(C − V)/C] × 100, where C and V represent the mean paw volume increases measured at 1, 2, or 3 h post-carrageenan injection in the control and treated groups, respectively.

2.6.6. Assessment of Mechanical Hyperalgesia in Carrageenan-Induced Edema in Rats—Randall–Selitto (Paw Pressure) Test

Mechanical hyperalgesia induced by carrageenan, resulting from a lowered nociceptive threshold to mechanical stimulation, was evaluated in the carrageenan-treated group using the Randall–Selitto paw pressure test, as previously described [29] with minor modifications [19]. In the same group of rats that had received a carrageenan injection into the right hind paw to induce edema (see Section 2.6.5), increasing pressure was gradually applied to the same site. The threshold for mechanical nociception was defined as the force (g) that elicited a paw withdrawal reflex, at which point pressure application was immediately stopped. A safety cut-off of 250 g was applied.

Mechanical hyperalgesia thresholds were assessed prior to carrageenan injection (0 h) and 3 h post-injection (i.e., 3.5 h after intraperitoneal pretreatment with the tested compounds). The percentage of analgesia was calculated using the following formula [19]: %inhibition of hyperalgesia = [(100 × T/C) − 100], where C represents the mean pressure (g) in the vehicle-treated group and T the mean pressure (g) in the drug-treated group.

2.6.7. Oxaliplatin-Induced Neuropathic Pain Assessment of Tactile Allodynia—Von Frey Test

Peripheral neuropathy was induced by a single intraperitoneal injection of oxaliplatin at a dose of 10 mg/kg, following previously described protocols [19,20,24,30].

Tactile (mechanical) allodynia was assessed using an electronic von Frey apparatus (Panlab, Cornellà de Llobregat, Spain) equipped with a flexible filament, the distal end of which was applied to the plantar surface of the right hind paw. The force required to elicit a withdrawal response was automatically recorded in grams. On the day of testing, three baseline measurements were taken after a 30 min habituation period. The procedure was repeated 3 h after oxaliplatin administration and again 30 min after administration of the tested compound. The antiallodynic effects were also evaluated 7 days after oxaliplatin injection to assess late-phase allodynia.

2.6.8. Streptozotocin-Induced Diabetic Neuropathy in Mice

To induce diabetes, mice received a single intraperitoneal (i.p.) injection of streptozotocin (200 mg/kg) dissolved in 0.1 N citrate buffer. Body weight was recorded before and after streptozotocin administration. Blood glucose levels were measured 1, 2, and 3 weeks post-injection using a blood glucose monitoring system (Accu-Chek Active, Roche, France). Blood samples (5 µL) were collected from the tail vein. Mice with blood glucose concentrations exceeding 200 mg/dL were classified as diabetic and subsequently included in further experiments, according to a previously described protocol [16].

2.6.9. Von Frey Test—Assessment of Tactile Allodynia in Streptozotocin-Induced Diabetic Neuropathy in Mice

The test was performed 21 days after streptozotocin administration in diabetic mice. Tactile allodynia was assessed using an electronic von Frey unit (Panlab, Cornellà de Llobregat, Spain), as described in Section 2.6.7 and in previous studies [16,20]. Measurements were taken before streptozotocin injection (in normoglycemic mice), 21 days post-injection (in diabetic mice), and 30 min after compound administration in diabetic mice exhibiting mechanical allodynia.

2.6.10. Hot Plate Test—Assessment of Heat Hyperalgesia in Streptozotocin-Induced Diabetic Neuropathy in Mice

Thermal hyperalgesia was evaluated using the hot plate test with a hot/cold plate apparatus (Panlab/Harvard Apparatus, Cornellà de Llobregat, Spain), as previously described [25]. Following determination of the baseline latency to a nociceptive response for each animal, the mice received the tested compounds and, 30 min later, were placed on a hot plate maintained at 55 °C. The latency to a nocifensive response (hind paw licking or jumping) was recorded. A cut-off time of 45 s was applied to prevent tissue damage.

2.6.11. Spontaneous Motor Behavior

The experimental procedure was carried out according to previously described methods [19]. Each mouse was individually placed in an actometer (40 × 40 × 31 cm, Ugo Basile, Gemonio, Italy) for a 30 min habituation period to acclimate to the testing environment. Immediately after habituation, spontaneous locomotor activity was recorded during a 30 min test session by counting the number of light beam interruptions, which served as an index of horizontal movement. To assess whether the tested compounds influenced baseline locomotor activity, animals received intraperitoneal (i.p.) injections of the vehicle or the respective compounds at doses of 5, 10, or 20 mg/kg. Locomotor activity was expressed as the total number of beam breaks recorded during the 30 min testing period.

2.6.12. Statistical Analysis

Data from the 5-HT_1A receptor binding assay were fitted to a one-site curve-fitting equation with Prism (Graph Pad Software, San Diego, CA, USA) and Ki values were estimated from the Cheng−Prusoff equation. All results are presented as mean ± SEM. They were analyzed using Graph Pad Prism 9.5.1 software. Statistically significant differences between groups were estimated using one- or two-way analysis of variance (ANOVA) followed by Dunnett or Šídák’s post hoc test, respectively. Significance is considered at the level of p < 0.05 level.

3. Results

3.1. 5-HT_1A Receptor Binding Analysis Using Radioligand Assay

The binding affinities of the tested compounds toward the 5-HT_1A receptor were evaluated using a radioligand displacement assay, and the results are summarized in

Table 1. The affinity (Ki) of DSZ-13 and DSZ-19 and reference compounds 8-OH-DPAT and 5-CT for serotonin 5-HT_1A receptor assessed in radioligand binding assay. The results are expressed as means ± SEM from at least three of independent experiments.

Compounds	5-HT1A/Ki [nM] ± SEM
DSZ-13	72.0 ± 2.6
DSZ-19	87.0 ± 2.1
8-OH-DPAT	1.0 ± 0.05
5-CT	0.2 ± 0.01

. Compounds DSZ-13 and DSZ-19 exhibited moderate affinity for the 5-HT_1A receptor, with Ki values of 72.0 ± 2.6 nM and 87.0 ± 2.1 nM, respectively. In contrast, the reference agonists 8-OH-DPAT (Ki = 1.0 ± 0.05 nM) and 5-CT (Ki = 0.2 ± 0.01 nM) showed high to very high affinity, which confirms the validity of the assay system.

3.2. Assessment of Cell Viability Using the XTT Assay

Treatment of RAW 264.7 macrophages with DSZ-13 and DSZ-19 in the 0.5–25 μmol range did not significantly affect cell viability compared with the untreated control (Figure 1). For both compounds, viability remained stable within approximately 95–100% of the baseline values. A slight but non-significant reduction was observed only at the highest concentrations (50 and 100 μmol), where viability still exceeded ~90% of the control. No differences in cytotoxicity were detected between DSZ-13 and DSZ-19 at any tested concentration.

3.3. Real-Time qPCR Assay

3.3.1. COX-1 mRNA (qPCR, Figure 2)

The expression of Ptgs1 increased significantly following LPS stimulation (p < 0.01). DSZ-13 and DSZ-19 decreased the COX-1 mRNA levels in LPS-activated macrophages; however, the reductions were moderate and did not fully return to baseline control values. The strongest effect was observed for DSZ-13 at 5 µmol in the presence of LPS.

Both compounds partially attenuated the LPS-induced upregulation of COX-1, suggesting a mild modulatory effect rather than complete suppression of this constitutive isoform.

Figure 2. Relative mRNA level of Ptgs1 in RAW 264.7 cells treated with DSZ-13 and DSZ-19 compounds. Values are presented as means ± SD, n = 6. Control cells (C) were not treated with the tested compounds nor LPS (untreated). Data are normalized to Gapdh. Ptgs1 prostaglandin-endoperoxide synthase 1. * p < 0.05, ** p < 0.01.

Figure 2. Relative mRNA level of Ptgs1 in RAW 264.7 cells treated with DSZ-13 and DSZ-19 compounds. Values are presented as means ± SD, n = 6. Control cells (C) were not treated with the tested compounds nor LPS (untreated). Data are normalized to Gapdh. Ptgs1 prostaglandin-endoperoxide synthase 1. * p < 0.05, ** p < 0.01.

3.3.2. COX-2 mRNA (qPCR, Figure 3)

The expression of Ptgs2 markedly increased after LPS stimulation (about 2.5–3-fold vs. control; p < 0.01). DSZ-13 and DSZ-19 significantly lowered the COX-2 mRNA levels in LPS-activated macrophages (p < 0.05 to p < 0.01), with the strongest effect observed at 5 µmol. However, the mRNA levels in the “compound + LPS” groups remained higher than in the untreated controls, indicating a partial but not complete suppression of Ptgs2 induction.

Both compounds exert anti-inflammatory effects associated with reduced COX-2 mRNA expression.

Figure 3. Relative mRNA level for Ptgs2 in RAW 264.7 cells treated with DSZ-13 and DSZ-19 compounds. Values are presented as means ± SD, n = 6. Control cells (C) were not treated with the tested compounds nor LPS (untreated). Data are normalized to Gapdh. Ptgs2 -prostaglandin-endoperoxide synthase 2. * p < 0.05, ** p < 0.01.

Figure 3. Relative mRNA level for Ptgs2 in RAW 264.7 cells treated with DSZ-13 and DSZ-19 compounds. Values are presented as means ± SD, n = 6. Control cells (C) were not treated with the tested compounds nor LPS (untreated). Data are normalized to Gapdh. Ptgs2 -prostaglandin-endoperoxide synthase 2. * p < 0.05, ** p < 0.01.

3.4. Pharmacokinetic Assay

Table 2. Pharmacokinetic parameters determined by non-compartmental analysis calculated from mean plasma concentration values (n = 3) of DSZ-13 or DSZ-19 compound after single intraperitoneal administration to mice at a dose of 5 mg/kg.

Pharmacokinetic Parameters	DSZ-13	DSZ-19
Cmax [µg/L]	231.44	87.25
tmax [h]	0.083	0.083
λz [h−1]	0.19	0.31
t1/2(λz) [h]	3.56	2.25
CL/F [L/h/kg]	36.5	83.39
AUC0-inf [µg∙h/L]	137.17	60.00
Vz/F [L/kg]	187.73	271.04
MRT [h]	2.91	2.85

C_max—maximum serum concentration; t_max—time to reach maximum serum concentration; λz—terminal elimination rate constant; t_1/2(λz)—half-life in the elimination phase; CL—clearance; AUC_0-inf—area under the concentration time curve extrapolated to infinity; Vz—volume of distribution in the elimination phase; MRT—mean residence time.

summarizes the pharmacokinetic (PK) parameters derived from the non-compartmental analysis of the serum concentration–time curves following a single i.p. administration of the investigated compounds.

Both compounds reached maximum concentration at the first sampling point (5 min after administration); however, the concentration achieved by DSZ-19 was much lower (87.25 vs. 231.44 µg/L). The compound DSZ-19 also has higher clearance (83.39 vs. 36.5 L/h/kg) and a larger volume of distribution (271.04 vs. 187.73 L/kg) compared to DSZ-13, although both seem to freely cross biological membranes. Surprisingly, the compound with the larger volume of distribution (DSZ-19) has a shorter half-life (2.25 vs. 3.56 h) that could show its ability to quickly redistribute from deep compartments into central circulation. We have also analyzed the brain concentrations of DSZ-19 and DSZ-13 and based on these results, the calculated pharmacokinetic parameters are presented in

Table 3. Pharmacokinetic parameters determined by non-compartmental analysis calculated from mean brain concentration values (n = 3) of DSZ-13 or DSZ-19 compound after single intraperitoneal administration to mice at a dose of 5 mg/kg.

Pharmacokinetic Parameters	DSZ-13	DSZ-19
Cmax [ng/g]	12.21	104.1
tmax [h]	0.083	0.083
t1/2(λz) [h]	3.28	5.76
AUC0-inf [ng∙h/g]	16.11	195.83
MRT [h]	4.3	8.11

C_max—maximum concentration; t_max—time to reach maximum concentration; t_1/2(λz)—half-life in the elimination phase; AUC_0-inf—area under the concentration time curve extrapolated to infinity; MRT—mean residence time.

.

In addition, in this case, the maximal brain concentration of DSZ-19 is much higher than DSZ-13 (104.1 vs. 12.21 ng/g). The brain/serum partitioning coefficient calculated using the AUC values extrapolated to infinity for DSZ-13 equaled 0.11; however, for the compound DSZ-19, it was equal to 3.26, which means that in contrast to the compound DSZ-13, DSZ-19 has a high ability to penetrate the blood–brain barrier into the central nervous system.

3.5. Evaluation of Antinociceptive Activity in the Hot Plate Test

The compounds DSZ-13 and DSZ-19 were evaluated in a dose range of 5 to 20 mg/kg (Figure 4). In this test, dose-dependent DSZ-19 extended the latency time to the pain reaction, but only at doses of 10 and 20 mg/kg was a statistically significant effect shown. The compound prolonged the time by 55.2%, p < 0.01 and 77.3%, p < 0.001, respectively. At the dose of 5 mg/kg, the latency time to the pain reaction was prolonged by 34%, p > 0.05 but the results were not statistically significant. The compound DSZ-13 did not show analgesic activity in the test, as a not statistically significant prolongation of the latency time to the pain response was observed.

3.6. Evaluation of Antinociceptive Activity in the Formalin-Treated Mice

In the formalin test, the compounds DSZ-13 and DSZ-19 in a dose range of 5 to 20 mg/kg after administration of i.p. were assessed [Figure 5A–C].

Intraperitoneal administration of DSZ-13 and DSZ-19 resulted in a marked and dose-dependent antinociceptive effect in both phases (neurogenic—early phase and inflammatory—second phase) of the formalin test.

In the neurogenic phase, DSZ-13 at doses of 5, 10, 15, and 20 mg/kg significantly decreased the licking time by 51.4% (p = 0.174 < 0.05), by 65% (p = 0.0025), by 69% (p = 0.0022) and by 78.3% (p = 0.0006), respectively.

In the inflammatory phase, DSZ-13 at doses of 5, 10, 15, and 20 mg/kg significantly reduced the licking time by 53.6% (p = 0.003), by 67.8% (p < 0.0001), by 80.5% (p < 0.0001), and 94% (p < 0.0001), respectively.

In the neurogenic phase, DSZ-19 at doses of 5, 10, 15, and 20 mg/kg decreased the time of nociceptive response by 20% (p = 0.1932), by 61.3% (p < 0.0001), by 69% (p < 0.0001), and by 80% (p < 0.0001), respectively.

In the inflammatory phase, DSZ-19 at doses of 5, 10, 15, and 20 mg/kg significantly decreased the time of nociceptive response by 32.5% (p = 0.0162), 80% (p < 0.0001), by 90% (p < 0.0001), and 98.5% (p < 0.001), respectively.

Ketoprofen, in the neurogenic phase, at the dose of 10 and 20 mg/kg, decreased the nociceptive behavior by 43% and by 57%, respectively (p = 0.0129 and p = 0.0042, respectively). A strong antinociceptive effect was seen in the inflammatory phase for ketoprofen administered at doses of 20 mg/kg and 50 mg/kg, as it decreased the duration of the response to licking by 73% (p = 0.0036) and by 82% (p = 0.0010), respectively.

Formalin Test—Blockade of the Antinociceptive Effect of DSZ-19 but Not DSZ-13 with WAY 100635

The involvement of 5-HT_1A receptors in the analgesic efficacy of DSZ-13 and DSZ-19 in the formalin test was evaluated using the selective 5-HT_1A antagonist WAY-100635 (0.7 mg/kg, i.p.) (Figure 6). DSZ-19 (10 mg/kg) effectively reduced nociceptive behaviors compared with the vehicle-treated group in both the early and late phases of the formalin test (p = 0.0154 and p = 0.0002, respectively). Pretreatment with WAY-100635 significantly attenuated the antinociceptive effect of DSZ-19 in the late phase (p = 0.0228) but not in the early phase of the test (p < 0.05).

DSZ-13 (10 mg/kg) also significantly reduced paw licking compared with the vehicle-treated group in both the early and late phases (p = 0.0256 and p = 0.0014, respectively). However, the antagonistic effect of WAY-100635 on DSZ-13 did not reach statistical significance. Furthermore, WAY-100635 alone did not affect nociceptive behaviors in either phase of the test.

3.7. Evaluation of Antinociceptive Activity in the Capsaicin-Treated Mice

In the capsaicin-induced pain model, pretreatment with DSZ-13 and DSZ-19 significantly attenuated the nociceptive response and in a dose-dependent manner. DSZ-13 at doses of 4, 8 and 16 μg/20 μL attenuated licking behavior by 31% (p = 0.0408), by 63% (p < 0.0001), and by 86% (p < 0.0001), respectively. DSZ-19 at doses of 4, 8 and 16 μg/20 μL decreased the nociceptive response by 35% (p = 0.0400), by 64% (p = 0.0002) and 85% (p < 0.0001), respectively (Figure 7).

3.8. Evaluation of Antiedematous Activity in Carrageenan-Induced Edema in Rats

Injection of carrageenan produced a marked increase in the volume of the injected rat hind paw, leading to the development of paw edema (Figure 8A–C). In the control groups, paw volume gradually increased beginning at the first hour of observation, reaching approximately 62% of the baseline value. The maximum degree of edema was observed 3 h after carrageenan administration, when the increase in paw volume ranged from 62% to 137%.

Treatment with DSZ-13, DSZ-19, and indomethacin reduced the formation of paw edema at the time of evaluation. For DSZ-13 (20 mg/kg), the two-way ANOVA showed a significant effect of drug F (1, 10) = 129.83, p < 0.0001 and time F (2.031, 20.31) = 355.4, p < 0.0001 as well as a significant time × drug interaction time: F (3, 30) = 30.2, p < 0.0001. Treatment with DSZ-13 reduced edema by 23% in the first h, by 28% in the second h, and by 30% in the third h of the test compared to the vehicle-treated group.

DSZ-19 (20 mg/kg) showed a weaker antiedema effect compared to DSZ-13 (20 mg/kg). For DSZ-19, the two-way ANOVA showed a significant effect of drug F (1, 10) = 18.29, p =0.0016 and time F (1.764, 17.64) = 31.67, p < 0.0001 but not time × drug interaction: F (3, 30) = 2.386, p > 0.05. Treatment with DSZ-19 reduced edema by 14% in the first h, by 21% in the second h, and by 27% in the third h of the test compared to the vehicle-treated group.

The most pronounced inhibition of paw edema was seen for indomethacin treatment (10 mg/kg), which reduced edema by 25%, 43% and 37% at time points 1, 2 and 3 h of inflammation development, respectively. The two-way ANOVA showed a significant effect of drug F (2, 15) = 85.11, p < 0.0001 and time F (1.874, 28.11) = 95.34, p < 0.0001 as well as a significant time × drug interaction time: F (6, 45) = 40.15, p < 0.0001.

Of the tested compounds, DSZ-13 in the first and third hours of the test showed antiedematous activity like indomethacin used at the same time of the test. Furthermore, the antiedematous effect of DSZ-19 at 3 h was comparable to that of indomethacin at the first hour.

3.9. Assessment of Mechanical Hyperalgesia in Carrageenan-Induced Edema in Rats

Injection of carrageenan significantly decreased the withdrawal threshold (mechanical hyperalgesia) with the most pronounced effect at 3 h after administration. Figure 8D shows that pretreatment with DSZ-13, DSZ-19 and indomethacin (10 mg/kg) resulted in the inhibition of mechanical inflammatory hyperalgesia observed as an increased withdrawal threshold of 20%, 24% and 44%, respectively, compared to the vehicle-treated rats. For DSZ-13, the bidirectional ANOVA showed a significant effect of the interaction drug x time effect F (4, 25) = 7.559, p = 0.0004, effect of the drug F (4, 25) = 13.02, p < 0.0001 and time effect F (1, 25) = 6.407, p = 0.0180.

For DSZ-19, the two-way ANOVA showed a significant interaction effect between drug x time, F (2, 15) = 15.48, p = 0.0002, effect of the drug: F (2, 15) = 4.310, p = 0.0332 but not a significant effect of time, F (1, 15) = 4.309, p = 0.0555.

For indomethacin, the two-way ANOVA showed a significant effect of interaction between drug x time F (2, 15) = 14.10, p = 0.0004, effect of the drug: F (2, 15) = 11.73, p = 0.0009 but not a significant effect of time, F (1, 15) = 0.5021, p = 0.4895.

3.10. Assessment of Tactile Allodynia in Oxaliplatin-Induced Neuropathy in Mice

In untreated mice, the mean force that caused pain withdrawal was found to be 3.01 ± 0.08 g. In the group of animals treated with oxaliplatin, a significant reduction in the pain threshold was observed and the value was found to be 1.78 ± 0.03 g after 3 h (by 69%) and 1.84 ± 0.05 g after 7 days (by 55%), respectively (p < 0.001 in all groups).

In the early phase of oxaliplatin-induced neuropathy, a general effect of treatment was observed on the mechanical nociceptive threshold: [DSZ-13, F (4, 35) = 75.72, p < 0.0001; DSZ-19, F (4, 35) = 97.24, p < 0.0001]. In this phase, the compound DSZ-13 at doses of 5 mg/kg and 10 mg/kg increased the pain threshold for mechanical stimulation by 28.5% (p < 0.0001) and 43.4% (p < 0.0001), respectively, while DSZ-19 at the same doses by 44.5% (p < 0.0001) and 26% (p < 0.0001), respectively, (Figure 9).

In the late phase of oxaliplatin-induced neuropathy, a general effect of treatment was observed on the mechanical nociceptive threshold: [DSZ-13, F (4, 35) = 71.65, p < 0.0001; DSZ-19, F (4, 35) = 63.14, p < 0.0001]. In this phase, the compound DSZ-13 at a dose of 5 mg/kg did not show antiallodynic properties (by 9.8%, p = 0.956) but at a dose of 10 mg/kg significantly increased the pain threshold, by 36.7% (p < 0.0001). The compound DSZ-19 at both doses significantly elevated the pain threshold for mechanical stimulation: by 14% (p = 0.0143) and 31% (p < 0.0001), respectively, (Figure 9).

3.11. Assessment of Tactile Allodynia and Heat Hyperalgesia in Streptozotocin-Induced Diabetic Neuropathy in Mice

In the von Frey test, the mean mechanical pain threshold in the non-diabetic control group was 4.51 ± 0.85 g. In contrast, the streptozotocin-treated mice exhibited a significant increase in pain sensitivity, reflected by a reduction in the mechanical nociceptive threshold to values ranging from 2.78 ± 0.89 g to 3.29 ± 0.21 g (Figure 10).

In the von Frey test, streptozotocin-treated mice receiving DSZ-13 and DSZ-19 at 10 mg/kg exhibited a significant increase in the mechanical pain threshold of 60.8% and 84.4%, respectively (Figure 10A). In the hot plate test, the baseline latency to a pain response in the normoglycemic control group was 25.96 ± 1.7 s. In the streptozotocin-treated mice, the latency was significantly reduced to 17.4 ± 2.6 s (p < 0.05). DSZ-13 and DSZ-19 did not produce a significant effect in this assay (Figure 10B).

3.12. Spontaneous Locomotor Behavior

The compounds DSZ-13 and DSZ-19 at doses of 5 and 10 mg/kg did not produce statistically significant effects on locomotor activity. At higher doses, DSZ-13 (15 and 20 mg/kg) significantly reduced the number of crossings by 59% and 56%, respectively. DSZ-19 at doses of 15 and 20 mg/kg also significantly decreased the number of crossings by 65% (p < 0.01) and 79% (p < 0.001), respectively.

4. Discussion

As a continuation of our research on compounds with antinociceptive activity [19], this study investigated two novel 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives, differing in substituents on the pyridine ring and phenyl group, to characterize their pharmacokinetic profiles and explore potential mechanisms underlying their analgesic effects. Both compounds readily cross biological membranes, with DSZ-19 showing higher bioavailability, faster brain distribution, and larger clearance and volume of distribution compared with DSZ-13. In vitro analyses revealed moderate affinity toward 5-HT_1A receptors. Both compounds partially attenuated LPS-induced COX-1 upregulation, suggesting a modulatory rather than suppressive effect on this constitutive isoform. In addition, treatment with both compounds decreased COX-2 mRNA expression levels.

The obtained molecular docking results showed the weak affinity of the tested compounds for cyclooxygenase, as presented in the Supplementary Materials (Figure S1–S6, Tables S1 and S2) [31,32]. In silico docking studies (Supplementary Materials) showed higher binding energies for DSZ-13 and DSZ-19 than meloxicam in COX-1, while interactions with COX-2 were weaker than with the reference compound. Both molecules formed hydrogen bonds with Arg120, Arg121, and Ser530 in the COX active sites, but steric conflicts with Tyr385 in COX-1 likely reduce the binding affinity (Tables S1 and S2, Figures S1–S6).

In the in vivo study, the antinociceptive properties of DSZ-13 and DSZ-19 were evaluated using well-established models of thermal and chemical nociception [33,34]. The hot plate test, reflecting primarily supraspinal responses, enabled the assessment of central analgesic activity, whereas the formalin test provided insight into both neurogenic and inflammatory mechanisms. Owing to its biphasic nature, the formalin model allows discrimination between direct nociceptor activation (early phase) and inflammation-driven nociception with central sensitization (late phase) [35]. Drugs effective in both phases are typically considered to act through both central and peripheral mechanisms. Formalin-evoked pain involves multiple ion channels (e.g., TRPA1, TRPV1), G-protein-coupled receptors, and pro-inflammatory signaling cascades [27,35,36,37,38], and leads to prolonged hypersensitivity and neuroplastic adaptations resembling neuropathic pain [39]. The ability of both tested compounds to reduce behaviors associated with this prolonged hypersensitivity highlights their potential utility not only in acute but also chronic and neuropathic conditions. Combining the hot plate and formalin tests provides a comprehensive assessment of antinociceptive activity and supports differentiation between central and peripheral mechanisms. This design strengthens the interpretability and consistency of the observed pharmacological effects. In the hot plate test, DSZ-19, administered at 10 and 20 mg/kg, significantly increased the latency to pain response, whereas DSZ-13 showed no significant central analgesic effect. Importantly, the 10 mg/kg dose of DSZ-19 did not impair locomotor activity, confirming that analgesia was not confounded by sedation. In the formalin test, both DSZ-13 and DSZ-19 attenuated tonic pain in a dose-dependent manner in both the early neurogenic and late inflammatory phases, indicating the involvement of both central and peripheral mechanisms. In contrast, the reference drug ketoprofen showed phase-specific activity: strong effects in the early phase at 10 and 20 mg/kg and activity in the late phase only at higher doses (20 and 50 mg/kg), consistent with the known pharmacodynamics of NSAIDs. The broader phase-spanning efficacy of DSZ-13 and DSZ-19—particularly at lower doses—suggests a wider spectrum of action than classical NSAIDs and supports the hypothesis that they may modulate multiple targets involved in nociception. These findings warrant further mechanistic studies.

Given the moderate affinity of the compounds for 5-HT_1A receptors, we further examined the involvement of this receptor in the formalin test. The literature indicates that 5-HT_1A agonists may exert either pro- or antinociceptive effects depending on experimental context [40,41,42], and serotonin is a key neuromodulator of nociception and neuroinflammation [42,43,44]. Previous studies have confirmed 5-HT_1A receptor involvement in the antinociceptive actions of 8-OH-DPAT and NLX-112 in the formalin model [16,45]. The observed moderate affinity of DSZ-19 for the 5-HT_1A receptor (Ki ≈ 87 nM) is not inconsistent with the finding that the selective 5-HT_1A antagonist WAY-100635 reduced, rather than enhanced, the analgesic effect of DSZ-19 in the formalin test. These findings indicate that 5-HT_1A receptors participate in mediating the antinociceptive action of the compound DSZ-19, although the specific receptor populations involved and their precise role in the signaling cascade remain unclear. Further receptor-specific functional assays are required to determine whether DSZ-19 acts as a direct (partial) 5-HT_1A agonist or indirectly modulates 5-HT_1A-dependent pathways. The observed discrepancy may be attributed to structural and pharmacokinetic differences between the compounds, which could influence their bioavailability, receptor affinity, or ability to penetrate the central nervous system [12,46].

Pharmacokinetic data support this interpretation: DSZ-19 achieved substantially higher brain concentrations compared with DSZ-13, indicating that effective receptor engagement can occur in vivo despite moderate affinity. Structural differences, particularly alkoxy substituents on the pyridine ring and phenyl moiety, likely contribute to divergent CNS penetration and antinociceptive profiles [12,21,46]. The substituent pattern in DSZ-19 (–OC₂H₅ on the pyridine ring and –F on the phenyl ring) versus DSZ-13 (–OC₄H₉ on the pyridine and –OC₂H₅ on the phenyl ring) appears to limit the CNS penetration of DSZ-13, resulting in lower brain exposure and correspondingly reduced central analgesic effects.

Both compounds attenuated neurogenic pain in a capsaicin-induced inflammation test. Capsaicin activates TRPV1 channels on primary afferent neurons, triggering nociceptive signaling via C and Aδ fibers and promoting the release of pronociceptive mediators such as substance P and glutamate, as well as oxidative stress, glutathione depletion, and potassium channel inhibition [47,48]. The dose-dependent reduction in capsaicin-induced nociceptive behaviors suggests a modulatory effect on TRPV1-related pathways.

Acute inflammation was further assessed using the carrageenan-induced paw edema model, a standard approach for evaluating anti-inflammatory and antiedematous effects [49]. At the highest analgesic doses, both significantly inhibited edema during the first–third hours, with DSZ-13 showing the strongest effect at hour 3. DSZ-13 matched indomethacin in the first and third hours, whereas DSZ-19 reached similar efficacy only at hour 3. In carrageenan-induced mechanical hyperalgesia, both compounds (20 mg/kg) reduced hyperalgesia at 3 h, albeit less effectively than indomethacin (10 mg/kg). These results indicate that COX-2 suppression contributes to their anti-inflammatory actions.

Compounds demonstrating significant activity in the late inflammatory formalin phase were subsequently evaluated in a murine oxaliplatin-induced peripheral neuropathy model. Oxaliplatin induces sensory neuropathy characterized by tactile and cold allodynia [50,51,52], with an acute early phase and a later chronic phase. DSZ-19 attenuated tactile allodynia dose-dependently in both phases, whereas DSZ-13 was active only in the acute phase and at higher doses in the late phase. Overall, both compounds were more effective in the acute phase, with DSZ-19 showing the strongest activity.

Finally, streptozotocin was used to induce a diabetic neuropathy model [53,54]. After 21 days, diabetic mice exhibited hyperglycemia and reduced nociceptive thresholds [55]. Under these conditions, both compounds reduced tactile allodynia. DSZ-13 also significantly attenuated thermal hyperalgesia, whereas DSZ-19 produced only modest effects.

Based on these findings, the underlying antinociceptive mechanisms in neuropathy as well as the differences in efficacy between the two compounds cannot be clearly determined and require further investigation. The divergent effects observed in the two neuropathic pain models likely reflect fundamental differences in their underlying pathophysiology. Chemotherapy-induced neuropathy is predominantly driven by neuroinflammation, mitochondrial dysfunction, and damage to peripheral nerve endings, whereas diabetic neuropathy involves chronic metabolic disturbances, oxidative stress, alterations in ion channel expression, and impaired nerve blood flow. These distinct mechanisms may differentially affect the accessibility, receptor engagement, or downstream signaling pathways of the tested compounds.

Moreover, structural and pharmacokinetic differences between DSZ-19 and DSZ-13 likely contribute to their model-dependent efficacy. DSZ-19 may act preferentially in conditions dominated by inflammatory or acute neurotoxic mechanisms, whereas DSZ-13 may better modulate pathways relevant to chronic metabolic neuropathy. Such model-specific variations in receptor distribution, sensitivity, and neurotransmitter involvement may explain the differential antiallodynic and antihyperalgesic profiles observed.

5. Conclusions

In this study, we synthesized and evaluated two novel 1H-pyrrolo[3,4-c]pyridine-1,3(2H)-dione derivatives, DSZ-13 and DSZ-19, which differ in their substitution patterns and pharmacokinetic profiles. DSZ-19 exhibited substantially higher bioavailability and brain penetration than DSZ-13. In vitro, the derivatives showed moderate affinity for the 5-HT₁A receptor, exerted a mild modulatory effect rather than complete suppression of the COX-1 isoform, and reduced COX-2 mRNA expression levels.

Across a broad set of in vivo pain assays, the compounds demonstrated antinociceptive, antiedematous, and antiallodynic activity in acute, tonic, neurogenic, and neuropathic pain models, indicating a robust and broad analgesic profile. Notably, only DSZ-19 produced clear supraspinal analgesia, and its effects in the tonic pain model were attenuated by the 5-HT_1A antagonist WAY-100635, suggesting a contribution of 5-HT_1A receptor signaling to its mechanism of action. Both derivatives attenuated carrageenan-induced inflammation and hyperalgesia, likely through the suppression of COX-2 mRNA expression, although their anti-inflammatory activity was slightly lower than that of indomethacin. Additionally, both compounds produced significant antiallodynic effects in oxaliplatin- and streptozotocin-induced neuropathy models.

Despite these promising results, the precise molecular mechanisms underlying the antinociceptive activity of DSZ-13 and DSZ-19 remain incompletely defined. Their moderate 5-HT_1A affinity, reduction in COX expression, and divergent pharmacokinetic characteristics suggest the involvement of multi-target and/or indirect mechanisms, particularly for DSZ-19. Future studies should include receptor-specific functional assays, extended pharmacokinetic–pharmacodynamic studies, and broader molecular target profiling to clarify the pathways responsible for their activity. Such efforts will be essential to fully define their therapeutic potential and guide further structural optimization.