Therapeutic potential of CMPP, a novel maleimide derivative, in a reserpine-induced mouse model of pain–depression dyad
Authors: Lorca Alzoubi, Yousra Bsieso, Omar Gammoh, Alaa A.A. Aljabali, Bader A. Salameh, Abdelrahim Alqudah, Esam Qnais, Ahmad M. Al-malkawi, Kayed Abu-Safieh
Published: 2026

ABSTRACT
BackgroundPain and depression often coexist through shared mechanisms including monoamine depletion, oxidative stress, and inflammation. Multi-target therapies remain limited.MethodsThe effects of 3-Chloro-4-morpholino-1-(4-(4-(pyridin-2-yl)piperazin-1-yl)but-2-yn-1-yl)−1H-pyrrole-2,5-dione (CMPP) were assessed in a reserpine-induced mouse model of pain–depression dyad. Mice received reserpine (0.5 mg/kg, s.c., 3 days) with CMPP (5, 10, or 20 mg/kg, p.o.) or gabapentin (10 mg/kg, p.o.) for behavioral evaluation. Based on the behavioral dose-screening results, the 10 mg/kg dose of CMPP was selected for subsequent biochemical analyses.ResultsReserpine reduced paw withdrawal thresholds in von Frey and PAM tests, increased immobility in the forced swim test, and prolonged escape latency in the Morris water maze. CMPP reversed these effects. Reserpine decreased brain GSH, increased TBARS, depleted serotonin and norepinephrine, elevated glutamate, and increased MAO-A activity. CMPP increased GSH, reduced TBARS, partially restored serotonin and norepinephrine, lowered glutamate, and inhibited MAO-A. It also suppressed serum TNF-α and IL-1β.ConclusionCMPP demonstrated antinociceptive, antidepressant-like, antioxidant, and anti-inflammatory effects, supporting its potential for treating pain–depression comorbidity.

Introduction

Depression is a common mental disorder characterized by recurrent disturbances in mood, cognition, and activity. The World Health Organization has reported that at least 300 million people worldwide are diagnosed with depression, with approximately 800,000 deaths resulting from suicide annually [1]. In addition to its emotional impact, depression is commonly associated with somatic symptoms such as chronic pain. Evidence increasingly supports the bidirectional interaction between pain and depression, with one condition contributing to the initiation, progression, and severity of the other [2,3]. Clinical overlap is supported by common underlying biology, such as monoaminergic dysregulation, inflammatory cytokine activation, oxidative stress, and neuroplastic changes [4,5].

One of the most reproducible neurochemical changes in depression and chronic pain is a decrease in monoamine neurotransmitters, including serotonin (5-HT) and norepinephrine (NE). These biogenic amines significantly contribute to the functioning of the descending inhibitory pain conduits, particularly in the spinal cord and brainstem [[6], [7], [8]]. Pharmacological treatments that modulate monoaminergic transmission, including serotonin–norepinephrine reuptake inhibitors, are effective in treating depressive and/or pain symptoms [9,10]. In addition, as well as modifications in neurotransmitter systems, pro-inflammatory cytokines (i.e., IL-1β, IL-6, and TNF-α) are important in the sensitization of peripheral and central nociceptive circuits [[11], [12], [13]]. Increased levels of circulating cytokines in patients with depression have also been observed, further supporting the “macrophage theory of depression,” which hypothesizes an inflammatory component in mood disorders [[14], [15], [16]]. These cytokines not only induce hyperalgesia but also activate the hypothalamic–pituitary–adrenal (HPA) axis and the reuptake of monoamines, linking inflammation, neurotransmitter depletion, and behavioral symptoms [5,17].

The mouse model of reserpine remains a reliable tool for studying the pain–depression dyad. VMAT-2 inhibitor reserpine causes irreversible inhibition of VMAT-2, followed by severe monoamine depletion and augmented oxidative distress, and exhibits fibromyalgia-like behaviors, such as hyperalgesia, depression-like behavior, and memory impairment [4,18]. Therefore, this model allows for the concurrent testing of the analgesic, antidepressant, and neuroprotective effects of novel compounds in vivo.

Fibromyalgia and related pain–depression syndromes are currently treated pharmacologically with gabapentin, pregabalin, or duloxetine. The α2δ calcium channel ligand gabapentin is commonly used to treat chronic pain, pain associated with fibromyalgia, and mood symptoms [19]. However, gabapentin and related medications are only partially effective and can cause side effects of sedation and dizziness, with abuse potential [20]. This highlights the need for new drugs with multi-target actions that can simultaneously target nociception, mood disturbances, and cognitive deficits in patients with fibromyalgia and pain–depression comorbidity.

Maleimides: a class of unreactive endocyclic unsaturated imides and their medicinal applications. Their structural framework has been used in various biomedical fields, such as in protein modification [21,22], as linkers in drug–protein conjugates [23] and as bioactive compounds with anticancer or antibacterial [21,24]. Aminoacetylenes analogs are also a competitive chemotype, with possible activities that extend from neuroprotection to anti-inflammatory and anticancer activities [[25], [26], [27]]. Compounds such as oxotremorine and tremorine, which feature aminoacetylene entities, have been evaluated as lead molecules for Alzheimer's disease and Parkinson's disease [28,29].

Recently, several new aminoacetylene–maleimide hybrids, such as CMPP, (3-Chloro-4-morpholino-1-(4-(4-(pyridin-2-yl)piperazin-1-yl)but-2-yn-1-yl)−1H-pyrrole-2,5-dione) (Fig. 1) have been prepared and assayed mainly as anticancer agents [30]. CMPP suppressed cancer cell proliferation, highlighting the multi-prognostic properties of maleimide-based compounds. However, to date, no studies have investigated the neuropsychiatric disorder-modulating activity of CMPP or its utility in diseases with comorbid pain and depression.Fig. 1Chemical structure of CMPP. The chemical structure of 3-Chloro-4-morpholino-1-(4-(4-(pyridin-2-yl) piperazin-1-yl) but-2-yn-1-yl)−1H-pyrrole-2,5-dione (CMPP), a newly synthesized maleimide–aminoacetylene hybrid, is presented.

Chemical structure of CMPP. The chemical structure of 3-Chloro-4-morpholino-1-(4-(4-(pyridin-2-yl) piperazin-1-yl) but-2-yn-1-yl)−1H-pyrrole-2,5-dione (CMPP), a newly synthesized maleimide–aminoacetylene hybrid, is presented.

Based on these findings, the current study aimed, for the first time, to evaluate the potential therapeutic effects of CMPP in a murine model of the pain–depression dyad elicited by reserpine administration. Specifically, we analyzed the effects of CMPP on nociceptive, affective, and cognitive behavioral parameters, as well as its modulation of oxidative stress, inflammatory cytokines, neurotransmitter levels, and MAO-A activity in whole-brain homogenates. By collecting both behavioral and biochemical data, we aimed to generate a comprehensive preclinical evidence base for CMPP, and hence, a candidate to treat complex pain–depression comorbidities.

Materials and methods

Study design

This controlled experimental study was conducted to assess the therapeutic effects of CMPP (3-Chloro-4-morpholino-1-(4-(4-(pyridin-2-yl)piperazin-1-yl)but-2-yn-1-yl)-1H-pyrrole-2,5-dione) in a reserpine-induced mouse model of pain–depression dyad. The study was performed in two sequential phases. In the first phase, CMPP was evaluated at three oral doses (5, 10, and 20 mg/kg) in behavioral assays to examine dose dependence and identify an effective dose range. Following the behavioral studies, during phase 2, biochemical analyses were performed on the 10 mg/kg group. All treatment groups were randomly assigned, all outcome measures had been pre-specified, and behavioral and biochemical assessments were carried out under conditions that minimized experimental bias (e.g., blinding) in accordance with standards for reporting animal research.

Mice were subject to behavioral assays that reflect the core manifestations of the pain–depression dyad, including mechanical sensitivity (electronic von Frey and PAM tests), depressive-like behavior (forced swim test) and cognitive performance (Morris water maze) as primary outcome measures. Secondary outcome measures included biochemical and neurochemical endpoints, namely brain GSH, TBARS, MAO-A activity, serum TNF-α and IL-1β, and brain neurotransmitter levels (serotonin, norepinephrine, and glutamate), which were analyzed to provide mechanistic support for the behavioral findings.

Animals and housing

Adult male Swiss albino mice (20–25 g, 8–10 weeks old) were used in this study. Animals were obtained from an approved supplier and were given a week to acclimatize before being used in the experiments. The mice were then caged in polycarbonate cages (five mice per cage) on corn cob bedding and housed at 22–25 °C, 40–60% relative humidity, and a 12 h light/dark cycle. Regular rodent chow and tap water were provided ad libitum to the animals. The cage contained environmental enrichment, such as paper bedding. Male mice were used in this initial study to provide a controlled proof-of-concept assessment of CMPP efficacy before undertaking sex-comparative investigations.

The study was approved by the Institutional Animal Ethics Committee under approval reference number 22/2/2024/2025 and was performed in accordance with national regulations and the ARRIVE 2.0 guidelines for reporting animal research.

Chemicals and reagents

Reserpine (≥98% purity, Cat. R0875) and carboxymethylcellulose (CMC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Gabapentin (Cat. No.0806) was purchased from Tocris Bioscience (Bristol, UK). CMPP was synthesized in-house and its structure and purity were confirmed as previously described [30]. Ketamine hydrochloride (Ketalar®) was purchased from Hikma Pharmaceuticals (Amman, Jordan), and xylazine obtained from InterChemie (Venray.

The Netherlands). Tween 80 was obtained from Loba Chemie (Mumbai, India).

Experimental protocol

Creation of the pain–depression dyad. The pain–depression dyad was induced by subcutaneous injection of reserpine (0.5 mg/kg) once daily for three consecutive days, as previously described. For the behavioral dose-screening phase, mice were randomly assigned into the following groups (n = 6 per group): vehicle control (0.1% CMC), reserpine only, reserpine + CMPP (5 mg/kg, p.o.), reserpine + CMPP (10 mg/kg, p.o.), reserpine + CMPP (20 mg/kg, p.o.), and reserpine + gabapentin (10 mg/kg, p.o.). CMPP or gabapentin was administered once daily for 5 days. On days 1–3, treatments were given 30 min before reserpine injection, and on days 4 and 5 treatments were continued without reserpine administration. Behavioral assessments were performed on days 4 and 6 of the experiment. The group size used in this study was based on common practice in comparable preliminary in vivo pharmacology studies and was intended to balance detection of treatment effects with ethical reduction of animal use; however, a formal a priori power analysis was not performed. Based on the behavioral findings, the 10 mg/kg dose of CMPP was selected for the biochemical phase because it showed greater efficacy than 5 mg/kg and produced effects comparable to those of 20 mg/kg. For biochemical analyses, mice were assigned into four groups (n = 6 per group): vehicle control, reserpine only, reserpine + CMPP (10 mg/kg, p.o.), and reserpine + gabapentin (10 mg/kg, p.o.). On day 6, animals were anesthetized with ketamine (50 mg/kg, i.p.), blood was collected by cardiac puncture, and mice were sacrificed for tissue collection.

Behavioral assessments

All behavioral tests were performed in a sound-attenuated, temperature-controlled (22–24 °C) room during the lights-on phase of a 12 h light/dark cycle (lights on at 09:00 h, lights off at 21:00 h). The animals were brought to the testing room at least 30 min before the experiments to acclimatize to the environment. All equipment was wiped with 70% ethanol and dried between applications to ensure that no olfactory cues remained in the experimental environment. The testing order was from least to most stressful (mechanical sensitivity → forced swim → Morris water maze tests).

Electronic von Frey (eVF) test

Mechanical allodynia was measured using an electronic von Frey Analgesimeter (Ugo Basile SRL, Italy; model 38450). The animals were individually transferred to plexiglass chambers (10 × 10 × 15 cm) with wire mesh floors and were allowed to adapt for 15 min before the experiment. A hard probe (diameter tip 0.5) was pressed perpendicularly into the plantar surface of the hind paw under constant pressure. The force required to elicit a fast withdrawal of the paw was automatically registered in gram-force (gf) using the Data Collection Application (DCA) software. The average of three measurements with an interval of >5 min was considered the hind paw withdrawal threshold. The same site was not stimulated repetitively to avoid any sensitization.

Pressure Application Measurement (PAM) test

Mechanical hyperalgesia was evaluated using the Pressure Application Measurement test (Ugo Basile SRL, Italy, model 38500). The hind paw of the mouse was lightly positioned with a flat force transducer (diameter, 5 mm) and a blunt-tip probe. The force gradually increased to approximately 30 gf/s until the response to withdrawal was identified. The upper cutoff of the force was set at 450 g to avoid tissue injury. The 3 measurements were taken from the hind paws, maintained for at least five intervals, and the mean values were used for calculations. Withdrawal thresholds were digitally recorded using the DCA software.

Forced swim test (FST)

Depressive-like behavior was assessed in a clear cylindrical glass tank (height: 30 cm, diameter: 25 cm) with 20 cm of water at 25 ± 1 °C, where mice were tested alone in the tank for a 6-min trial. The initial 2 min were regarded as habituation, and the immobility time was measured over the last 4 min of the test. Immobilization was defined as a state without active movement, with minimal movement of the limb to keep the head above the water. The tanks were emptied, wiped clean, and filled with fresh water at the same temperature between trials to minimize the stress.

Morris water maze (MWM) test

Spatial learning and memory were assessed using a circular water tank (diameter: 90 cm; height: 50 cm) filled with water to a depth of 30 cm. The water was maintained at 23–25 °C and rendered opaque using non-toxic white tempera. A circular escape platform (diameter: 9 cm) was placed approximately 1 cm below the water surface in a fixed quadrant throughout the experiment. Extra-maze visual cues, including geometric shapes and colored posters, were placed around the testing room and kept constant throughout all trials.

Training trials: Each mouse completed four training trials daily for five consecutive days. During training, each mouse was allowed a maximum of 60 s to locate the hidden platform. Mice that failed to locate the platform within 60 s were gently guided to it and allowed to remain on the platform for 10–15 s.

Testing: Escape latency, defined as the time required to locate the hidden platform, was assessed on days 4 and 6. For the testing sessions, a maximum cut-off time of 5 min was used. Trials exceeding this cut-off were excluded from the analysis. The longer cut-off during testing was used to ensure adequate assessment of escape latency in reserpine-treated animals with expected cognitive impairment. The water was replaced daily to maintain cleanliness and consistency across testing sessions.

Biochemical analyses

CMPP at 10 mg/kg was selected for biochemical analyses based upon the behavioral dose-screening results, demonstrating greater efficacy than 5 mg/kg and no significant loss of effect compared to 20 mg/kg. On day 6, after completion of behavioral testing, animals were anesthetized, blood was collected, and brain tissues were harvested for biochemical analyses. Blood was drawn by cardiac puncture under anesthesia using a 1 mL syringe. Serum was obtained after centrifugation at 3000 × g for 15 min at 4 °C and stored at −20 °C, and brains were quickly removed, washed in cold saline, blotted, and weighed before homogenization for neurochemical and oxidative stress assays.

Brain homogenization

Tissue homogenates were prepared in 10 vol (w/v) of ice-cold phosphate buffer (0.1 M, pH 7.4), containing 0.5% Triton X-100. After 10 min centrifugation at 3000 rpm and 4 °C, the supernatants were withdrawn from homogenates for further analysis.

Aliquots were stored on ice and measured within 24 h.

Reduced glutathione (GSH) and lipid peroxidation (TBARS)

Levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) were determined to assess antioxidant status and lipid peroxidation, respectively. GSH was measured using a commercially available kit (E-BC-K030-M, Elabscience), while TBARS levels were evaluated via a TBARS Colorimetric Assay Kit (E-BC-K298-M, Elabscience). Both assays were performed in accordance with the respective manufacturer's instructions.

Neurotransmitter estimation

Brain tissue was rinsed thoroughly with ice-cold PBS (pH 7.4) to remove excess blood and then homogenized in PBS supplemented with protease inhibitor. To further break down the cells, the suspension was subjected to a freeze–thaw cycle. The suspension was then centrifuged for 5 min at 5000 × g to obtain the supernatant. Serotonin (5-HT), norepinephrine (NE), and glutamate levels were assessed using commercially available ELISA kits (MyBioSource; 5-HT, MBS1601042; NE, MBS2600834; glutamate, MBS2601720).

MAO-A activity

MAO-A activity was measured in whole-brain homogenates using a colorimetric assay kit (XPX-5006; Cell Biolabs Inc.) on whole-brain homogenates. MAO substrate was incubated in the brain homogenates, and enzymatic reaction progression was measured (570 nm) with a microplate reader (BioTek Epoch). The enzymatic activities were determined in units (U) per length of assay and normalized to liters (L) using standard curves based upon the kit.

Serum cytokines

Serum TNF-α and IL-1β were measured using mouse ELISA kits. TNF-α was purchased from MyBioSource (MBS161138), and IL-1β was purchased from Abcam (ab197742). Both assays were performed according to the manufacturers’ instructions. The results were expressed as fold change normalized to control.

Statistical analysis

All data are expressed as mean ± SEM. Data distribution was assessed for normality prior to statistical testing. For behavioral assessments performed repeatedly in the same animals on days 4 and 6, data were analyzed using two-way repeated-measures ANOVA, with treatment group as the between-subject factor and time as the within-subject factor. When appropriate, this was followed by post hoc multiple-comparison testing. For single-time-point endpoints, including oxidative stress markers, MAO-A activity, serum cytokines, and neurotransmitter levels, data were analyzed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Differences were considered statistically significant at p < 0.05. All analyses were performed using GraphPad Prism version 7.0 (GraphPad Software, San Diego, CA, USA) (see Fig. 1).

Results

Effect of CMPP on reserpine-induced mechanical allodynia and hyperalgesia in mice

Electronic von Frey test

Analysis of paw withdrawal threshold using two-way repeated-measures ANOVA revealed a significant main effect of treatment (F(5,30) = 129.21, p < 0.0001) and a significant main effect of time (F(1,30) = 24.18, p < 0.0001), whereas the treatment × time interaction was not significant (F(5,30) = 2.15, p = 0.0871). Post hoc analysis showed that, on day 4, reserpine-treated mice exhibited a significantly lower withdrawal threshold than vehicle-treated controls (p < 0.0001). Compared with the reserpine group, gabapentin significantly increased withdrawal threshold (p = 0.0090), and CMPP at 10 mg/kg and 20 mg/kg also produced significant increases (both p < 0.0001), whereas 5 mg/kg did not (p = 0.4960). The effect of 10 mg/kg was significantly greater than that of 5 mg/kg (p < 0.0001), with no significant difference between 10 mg/kg and 20 mg/kg (p = 0.3118). On day 6, reserpine again significantly reduced withdrawal threshold relative to controls (p < 0.0001). Gabapentin (p < 0.0001), CMPP 10 mg/kg (p < 0.0001), and CMPP 20 mg/kg (p < 0.0001) significantly reversed this effect, whereas CMPP 5 mg/kg remained non-significant (p = 0.0584). CMPP 10 mg/kg was significantly more effective than 5 mg/kg (p < 0.0001) and did not differ significantly from gabapentin (p = 0.9976), although the 20 mg/kg group showed a modest but statistically significant increase compared with 10 mg/kg on day 6 (p = 0.0051) (Fig. 2A).Fig. 2Effects of CMPP on nociceptive and depressive-like behaviors in reserpine-treated mice.(A) Electronic von Frey test showing paw withdrawal threshold on days 4 and 6. Reserpine significantly reduced withdrawal threshold relative to the vehicle control group, indicating mechanical allodynia. CMPP at 10 mg/kg and 20 mg/kg significantly increased withdrawal threshold compared with the reserpine group, whereas 5 mg/kg showed limited efficacy. (B) Pressure application measurement (PAM) test showing mechanical withdrawal threshold on days 4 and 6. Reserpine significantly decreased withdrawal threshold, consistent with mechanical hyperalgesia. CMPP improved this response in a dose-dependent manner, with 10 mg/kg and 20 mg/kg producing the most consistent effects. (C) Forced swim test showing immobility time on days 4 and 6. Reserpine significantly increased immobility time compared with the vehicle control group, indicating depressive-like behavior. CMPP reduced immobility duration, with 10 mg/kg and 20 mg/kg showing stronger effects than 5 mg/kg. Gabapentin (10 mg/kg, p.o.) served as the positive control. Data are expressed as mean ± SEM (n = 6 per group). Behavioral data were analyzed using two-way repeated-measures ANOVA followed by appropriate post hoc multiple-comparison testing.

Effects of CMPP on nociceptive and depressive-like behaviors in reserpine-treated mice.

(A) Electronic von Frey test showing paw withdrawal threshold on days 4 and 6. Reserpine significantly reduced withdrawal threshold relative to the vehicle control group, indicating mechanical allodynia. CMPP at 10 mg/kg and 20 mg/kg significantly increased withdrawal threshold compared with the reserpine group, whereas 5 mg/kg showed limited efficacy. (B) Pressure application measurement (PAM) test showing mechanical withdrawal threshold on days 4 and 6. Reserpine significantly decreased withdrawal threshold, consistent with mechanical hyperalgesia. CMPP improved this response in a dose-dependent manner, with 10 mg/kg and 20 mg/kg producing the most consistent effects. (C) Forced swim test showing immobility time on days 4 and 6. Reserpine significantly increased immobility time compared with the vehicle control group, indicating depressive-like behavior. CMPP reduced immobility duration, with 10 mg/kg and 20 mg/kg showing stronger effects than 5 mg/kg. Gabapentin (10 mg/kg, p.o.) served as the positive control. Data are expressed as mean ± SEM (n = 6 per group). Behavioral data were analyzed using two-way repeated-measures ANOVA followed by appropriate post hoc multiple-comparison testing.

Pressure Application Measurement test

Two-way repeated-measures ANOVA of PAM withdrawal thresholds demonstrated a significant main effect of treatment (F(5,30) = 65.90, p < 0.0001), a significant main effect of time (F(1,30) = 19.12, p = 0.0001), and a significant treatment × time interaction (F(5,30) = 5.43, p = 0.0011). Post hoc comparisons showed that, on day 4, reserpine-treated mice had significantly lower withdrawal thresholds than vehicle controls (p = 0.0222). Relative to the reserpine group, CMPP 10 mg/kg (p = 0.0426) and CMPP 20 mg/kg (p = 0.0007) significantly increased withdrawal threshold, whereas CMPP 5 mg/kg (p = 0.4241) and gabapentin (p = 0.1681) did not reach statistical significance. CMPP 10 mg/kg was significantly more effective than 5 mg/kg (p = 0.0003), with no significant difference between 10 mg/kg and 20 mg/kg (p = 0.6227). On day 6, reserpine markedly reduced withdrawal threshold compared with the control group (p < 0.0001). Gabapentin (p < 0.0001), CMPP 10 mg/kg (p < 0.0001), and CMPP 20 mg/kg (p < 0.0001) significantly reversed this reduction, whereas CMPP 5 mg/kg again did not (p = 0.6261). CMPP 10 mg/kg was significantly more effective than 5 mg/kg (p < 0.0001) and did not differ significantly from gabapentin (p = 0.4337) or 20 mg/kg (p = 0.4811) (Fig. 2B).

Effects of CMPP on depressive-like behavior and spatial memory deficit in reserpine-treated mice

Forced swim test

Two-way repeated-measures ANOVA of immobility time revealed a significant main effect of treatment (F(5,30) = 42.03, p < 0.0001), a significant main effect of time (F(1,30) = 16.55, p = 0.0003), and a significant treatment × time interaction (F(5,30) = 4.62, p = 0.0031). Post hoc analysis showed that, on day 4, reserpine-treated mice displayed significantly longer immobility times than vehicle-treated controls (p < 0.0001). Compared with the reserpine group, CMPP 10 mg/kg significantly reduced immobility (p < 0.0001), and CMPP 20 mg/kg also produced a significant reduction (p = 0.0014), whereas CMPP 5 mg/kg (p = 0.2733) and gabapentin (p = 0.1312) did not reach significance on day 4. CMPP 10 mg/kg was significantly more effective than 5 mg/kg (p = 0.0167), while no significant difference was observed between 10 mg/kg and 20 mg/kg (p = 0.8159). On day 6, reserpine again significantly increased immobility time relative to controls (p < 0.0001). Gabapentin (p < 0.0001) and all three CMPP doses significantly reduced immobility compared with the reserpine group (5 mg/kg: p < 0.0001; 10 mg/kg: p < 0.0001; 20 mg/kg: p < 0.0001). However, CMPP 10 mg/kg remained significantly more effective than 5 mg/kg (p = 0.0003) and did not differ significantly from gabapentin (p = 0.9985) or from 20 mg/kg (p = 0.9958) (Fig. 2C).

Morris water maze test

Escape latency in the Morris water maze was analyzed using two-way repeated-measures ANOVA, which revealed a significant main effect of treatment (F(5,30) = 201.78, p < 0.0001), whereas neither the main effect of time (F(1,30) = 0.33, p = 0.5677) nor the treatment × time interaction (F(5,30) = 1.85, p = 0.1330) reached statistical significance. Post hoc analysis showed that, on day 4, reserpine-treated mice exhibited significantly longer escape latencies than vehicle-treated controls (p < 0.0001), indicating impaired spatial learning and memory. Compared with the reserpine group, gabapentin significantly reduced escape latency (p < 0.0001). CMPP at 5 mg/kg, 10 mg/kg, and 20 mg/kg also significantly reduced escape latency relative to the reserpine-only group (all p < 0.0001). In addition, the effect of 10 mg/kg was significantly greater than that of 5 mg/kg (p = 0.0369), whereas no significant difference was observed between 10 mg/kg and 20 mg/kg (p = 0.5613). The escape latency in the 10 mg/kg group did not differ significantly from that in the vehicle control group (p = 0.7844), suggesting near-complete behavioral normalization.

On day 6, reserpine again significantly increased escape latency compared with the control group (p < 0.0001). Gabapentin significantly reversed this impairment (p < 0.0001 vs. reserpine). Likewise, CMPP at 5 mg/kg, 10 mg/kg, and 20 mg/kg significantly reduced escape latency relative to the reserpine-treated group (all p < 0.0001). The 10 mg/kg dose remained significantly more effective than 5 mg/kg (p = 0.0026), while no significant difference was detected between 10 mg/kg and 20 mg/kg (p = 0.3564). Furthermore, the 10 mg/kg group did not differ significantly from either the control group (p = 0.7567) or the gabapentin group (p = 0.0609). These findings indicate that CMPP ameliorated reserpine-induced cognitive impairment, with 10 mg/kg providing robust efficacy comparable to the higher dose (Fig. 3).Fig. 3Effects of CMPP on spatial learning and memory in the Morris water maze. Escape latency to reach the hidden platform was measured on days 4 and 6. Reserpine-treated mice showed significantly prolonged escape latency compared with the vehicle control group, indicating impaired spatial learning and memory. CMPP at 5, 10, and 20 mg/kg significantly reduced escape latency relative to the reserpine group, with 10 mg/kg showing greater efficacy than 5 mg/kg and an effect broadly comparable to that of 20 mg/kg. Gabapentin (10 mg/kg, p.o.) also significantly improved performance. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using two-way repeated-measures ANOVA followed by appropriate post hoc multiple-comparison testing.

Effects of CMPP on spatial learning and memory in the Morris water maze. Escape latency to reach the hidden platform was measured on days 4 and 6. Reserpine-treated mice showed significantly prolonged escape latency compared with the vehicle control group, indicating impaired spatial learning and memory. CMPP at 5, 10, and 20 mg/kg significantly reduced escape latency relative to the reserpine group, with 10 mg/kg showing greater efficacy than 5 mg/kg and an effect broadly comparable to that of 20 mg/kg. Gabapentin (10 mg/kg, p.o.) also significantly improved performance. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using two-way repeated-measures ANOVA followed by appropriate post hoc multiple-comparison testing.

Effect of CMPP on markers of oxidative stress

Reduced glutathione (GSH)

Brain GSH levels differed significantly among groups, as shown by one-way ANOVA (F(3,20) = 99.63, p < 0.0001). Post hoc analysis demonstrated that reserpine-treated mice had significantly lower GSH levels than vehicle-treated controls (p < 0.0001). Relative to the reserpine-only group, both gabapentin (p < 0.0001) and CMPP (p < 0.0001) significantly increased brain GSH levels. In addition, GSH levels in the CMPP-treated group were significantly higher than those in the gabapentin-treated group (p = 0.036). These findings indicate that CMPP markedly attenuated the reserpine-induced depletion of brain GSH but did not completely restore it to control values (Fig. 4A).Fig. 4Effects of CMPP on oxidative stress markers and whole-brain MAO-A activity in reserpine-treated mice. (A) Brain reduced glutathione (GSH, μmol/mg protein). Reserpine significantly decreased GSH levels relative to the vehicle control group, whereas both gabapentin and CMPP significantly increased GSH relative to the reserpine-only group. CMPP produced a greater increase than gabapentin, although GSH levels remained below control values. (B) Brain thiobarbituric acid reactive substances (TBARS, nmol/mg protein). Reserpine significantly increased TBARS levels compared with the vehicle control group, indicating enhanced lipid peroxidation. Both gabapentin and CMPP significantly reduced TBARS relative to the reserpine-only group, and CMPP restored TBARS to values not significantly different from control. (C) Whole-brain monoamine oxidase-A (MAO-A) activity (U/L). Reserpine significantly increased MAO-A activity relative to the vehicle control group. Both gabapentin and CMPP significantly reduced MAO-A activity compared with the reserpine-only group, with CMPP producing a stronger reduction than gabapentin, although activity remained above control levels. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Effects of CMPP on oxidative stress markers and whole-brain MAO-A activity in reserpine-treated mice. (A) Brain reduced glutathione (GSH, μmol/mg protein). Reserpine significantly decreased GSH levels relative to the vehicle control group, whereas both gabapentin and CMPP significantly increased GSH relative to the reserpine-only group. CMPP produced a greater increase than gabapentin, although GSH levels remained below control values. (B) Brain thiobarbituric acid reactive substances (TBARS, nmol/mg protein). Reserpine significantly increased TBARS levels compared with the vehicle control group, indicating enhanced lipid peroxidation. Both gabapentin and CMPP significantly reduced TBARS relative to the reserpine-only group, and CMPP restored TBARS to values not significantly different from control. (C) Whole-brain monoamine oxidase-A (MAO-A) activity (U/L). Reserpine significantly increased MAO-A activity relative to the vehicle control group. Both gabapentin and CMPP significantly reduced MAO-A activity compared with the reserpine-only group, with CMPP producing a stronger reduction than gabapentin, although activity remained above control levels. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Lipid peroxidation index (TBARS)

Brain TBARS levels differed significantly among groups (one-way ANOVA: F(3,20) = 92.30, p < 0.0001). Tukey's post hoc test showed that reserpine-treated mice had significantly higher TBARS levels than vehicle-treated controls (p < 0.0001), indicating increased lipid peroxidation. Compared with the reserpine-only group, both gabapentin (p < 0.0001) and CMPP (p < 0.0001) significantly reduced TBARS levels. No significant difference was observed between the CMPP and gabapentin groups (p = 0.9027), and TBARS levels in the CMPP-treated group did not differ significantly from those in the control group (p = 0.8843). These findings indicate that CMPP effectively attenuated reserpine-induced lipid peroxidation and restored TBARS values to near-control levels (Fig. 4B).

The effect of CMPP on whole-brain MAO-A activity

Brain MAO-A activity differed significantly among groups (one-way ANOVA: F(3,20) = 172, p < 0.0001). Post hoc analysis showed that reserpine treatment significantly increased MAO-A activity relative to the vehicle control group (p < 0.0001). Both gabapentin (p < 0.0001) and CMPP (p < 0.0001) significantly reduced MAO-A activity compared with the reserpine-only group. Moreover, MAO-A activity in the CMPP-treated group was significantly lower than that in the gabapentin-treated group (p < 0.0001), although it remained significantly higher than that in the control group (p = 0.0237). These findings indicate that CMPP markedly suppressed the reserpine-induced elevation in MAO-A activity, with a greater effect than gabapentin, but did not fully normalize the enzyme activity to baseline control levels (Fig. 4C).

Effect of CMPP on serum cytokine expression levels

Serum TNF-α levels differed significantly among groups, as shown by one-way ANOVA (F(3,20) = 51.89, p < 0.0001). Tukey's post hoc analysis demonstrated that reserpine-treated mice had significantly higher TNF-α levels than vehicle-treated controls (p < 0.0001). Relative to the reserpine-only group, both gabapentin (p < 0.0001) and CMPP (p < 0.0001) significantly reduced serum TNF-α levels. No significant difference was observed between the CMPP and gabapentin groups (p = 0.3528), and TNF-α levels in the CMPP-treated group did not differ significantly from those in the control group (p = 0.8685). These findings indicate that CMPP effectively attenuated the reserpine-induced increase in systemic TNF-α and restored values toward control levels (Fig. 5A).Fig. 5Effects of CMPP on serum pro-inflammatory cytokines in reserpine-treated mice.(A) Serum tumor necrosis factor-α (TNF-α, pg/mL). Reserpine significantly increased TNF-α levels relative to the vehicle control group. Both gabapentin and CMPP significantly reduced TNF-α levels compared with the reserpine-only group, and TNF-α levels in the CMPP-treated group were not significantly different from those in the control or gabapentin groups. (B) Serum interleukin-1β (IL-1β, pg/mL). Reserpine significantly increased IL-1β levels relative to the vehicle control group. Both gabapentin and CMPP significantly reduced IL-1β levels compared with the reserpine-only group. However, IL-1β levels differed significantly between the CMPP and gabapentin groups, indicating an endpoint-dependent difference in the anti-inflammatory effects of the two treatments. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Effects of CMPP on serum pro-inflammatory cytokines in reserpine-treated mice.

(A) Serum tumor necrosis factor-α (TNF-α, pg/mL). Reserpine significantly increased TNF-α levels relative to the vehicle control group. Both gabapentin and CMPP significantly reduced TNF-α levels compared with the reserpine-only group, and TNF-α levels in the CMPP-treated group were not significantly different from those in the control or gabapentin groups. (B) Serum interleukin-1β (IL-1β, pg/mL). Reserpine significantly increased IL-1β levels relative to the vehicle control group. Both gabapentin and CMPP significantly reduced IL-1β levels compared with the reserpine-only group. However, IL-1β levels differed significantly between the CMPP and gabapentin groups, indicating an endpoint-dependent difference in the anti-inflammatory effects of the two treatments. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Serum IL-1β levels also differed significantly among groups (one-way ANOVA: F(3,20) = 61.89, p < 0.0001). Post hoc analysis showed that reserpine-treated mice exhibited significantly higher IL-1β levels than vehicle-treated controls (p < 0.0001). Compared with the reserpine-only group, gabapentin (p < 0.05) and CMPP (p < 0.0001) both significantly reduced serum IL-1β levels. A significant difference was detected between the CMPP and gabapentin groups (p < 0.0001). These data indicate that CMPP significantly suppressed the reserpine-induced elevation in systemic IL-1β and restored this inflammatory marker to near-control levels (Fig. 5B).

Impact of CMPP on the levels of neurotransmitters

As shown in Fig. 6, brain levels of serotonin, norepinephrine, and glutamate differed significantly among groups, as shown by one-way ANOVA for serotonin (F(3,20) = 181.2, p < 0.0001), norepinephrine (F(3,20) = 121.8, p < 0.0001), and glutamate (F(3,20) = 146.6, p < 0.0001). Post hoc analysis revealed that reserpine-treated mice had significantly lower serotonin and norepinephrine levels, and significantly higher glutamate levels, than vehicle-treated controls (all p < 0.0001), confirming marked disruption of monoaminergic and excitatory neurotransmission.Fig. 6Effects of CMPP on brain neurotransmitter levels in reserpine-treated mice. (A) Brain serotonin (5-HT, ng/mg tissue). Reserpine significantly reduced serotonin levels relative to the vehicle control group. Both gabapentin and CMPP significantly increased serotonin levels compared with the reserpine-only group; however, serotonin levels in the CMPP-treated group remained lower than those in the gabapentin and control groups. (B) Brain norepinephrine (NE, ng/mg tissue). Reserpine significantly reduced norepinephrine levels relative to the vehicle control group. Both gabapentin and CMPP significantly increased norepinephrine levels compared with the reserpine-only group, with CMPP producing a greater increase than gabapentin, although values remained below control levels.(C) Brain glutamate (Glu, μmol/mg tissue). Reserpine significantly increased glutamate levels relative to the vehicle control group. Both gabapentin and CMPP significantly reduced glutamate levels compared with the reserpine-only group, with CMPP producing a greater reduction than gabapentin and restoring glutamate to values not significantly different from those of the control group. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Effects of CMPP on brain neurotransmitter levels in reserpine-treated mice. (A) Brain serotonin (5-HT, ng/mg tissue). Reserpine significantly reduced serotonin levels relative to the vehicle control group. Both gabapentin and CMPP significantly increased serotonin levels compared with the reserpine-only group; however, serotonin levels in the CMPP-treated group remained lower than those in the gabapentin and control groups. (B) Brain norepinephrine (NE, ng/mg tissue). Reserpine significantly reduced norepinephrine levels relative to the vehicle control group. Both gabapentin and CMPP significantly increased norepinephrine levels compared with the reserpine-only group, with CMPP producing a greater increase than gabapentin, although values remained below control levels.

(C) Brain glutamate (Glu, μmol/mg tissue). Reserpine significantly increased glutamate levels relative to the vehicle control group. Both gabapentin and CMPP significantly reduced glutamate levels compared with the reserpine-only group, with CMPP producing a greater reduction than gabapentin and restoring glutamate to values not significantly different from those of the control group. Data are expressed as mean ± SEM (n = 6 per group). Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Relative to the reserpine-only group, both gabapentin and CMPP significantly increased brain serotonin levels (gabapentin: p < 0.0001; CMPP: p = 0.0225). However, serotonin levels in the CMPP-treated group remained significantly lower than those in the gabapentin-treated group (p = 0.0048) and the control group (p < 0.0001), indicating partial restoration. Similarly, both gabapentin (p = 0.0217) and CMPP (p < 0.0001) significantly increased norepinephrine levels compared with the reserpine group. Norepinephrine levels in the CMPP-treated group were significantly higher than those in the gabapentin-treated group (p = 0.0272), although they remained significantly lower than those in the control group (p < 0.0001), again indicating partial recovery.

For glutamate, both gabapentin and CMPP significantly reduced the reserpine-induced elevation relative to the reserpine-only group (both p < 0.0001). Moreover, glutamate levels in the CMPP-treated group were significantly lower than those in the gabapentin-treated group (p < 0.0001) and did not differ significantly from those in the control group (p = 0.4382). Collectively, these findings indicate that CMPP significantly attenuated the neurotransmitter disturbances induced by reserpine by partially restoring serotonin and norepinephrine levels and normalizing glutamate levels toward those of the control group.

Discussion

This study provides convincing evidence that CMPP, a novel heterocyclic pyrrole derivative, exerts anti-reserpine-induced pain depression-like effects in mice. CMPP treatment significantly alleviated nociceptive hyperalgesia, depressive-like behaviour, and cognitive dysfunction. Biochemically, CMPP reestablished antioxidant potential in the brain, partially restored monoaminergic balance and normalized glutamatergic disturbance, and inhibited monoamine oxidase-A (MAO-A) activity. In addition, it showed the ability to inhibit systemic pro-inflammatory cytokine release. Taken together, these results reveal CMPP as a promising multi-target compound and therapeutic candidate for the treatment of FM-like syndromes characterized by the coexistence of pain, depression, and cognitive impairment. An important feature of the present study is that the behavioral phase included three doses of CMPP (5, 10, and 20 mg/kg), allowing a preliminary evaluation of dose dependence. Across the behavioral assays, the 10 mg/kg dose produced clearer efficacy than 5 mg/kg, while its effects were largely comparable to those observed with 20 mg/kg. On this basis, 10 mg/kg was selected as the working dose for biochemical characterization. This strategy allowed us to identify an effective dose that was pharmacologically robust without advancing an apparently non-superior higher dose to all downstream analyses.

According to previous reports [[31], [32], [33]], reserpine-treated mice were significantly sensitive to mechanical stimuli, as reflected by a decrease in withdrawal responses in the electronic von Frey and pressure application tests. CMPP pre-treatment remarkably reversed the above responses, indicating that CMPP exerted antinociceptive effects that were comparable to those of gabapentin, a commonly used agent for fibromyalgia and neuropathic pain [34].

Reserpine also increased immobility in the forced swim test, a measure of depression-like behavior [18]. CMPP treatment significantly reduced this reserpine-induced increase in immobility time, indicating an antidepressant-like effect. Reserpine increased escape latency in the Morris water maze, indicating impaired spatial learning and memory, whereas CMPP treatment reduced escape latency, suggesting an improvement in cognitive performance. These results are consistent with clinical reports of pain-depression comorbidity, typically characterized by affective and cognitive disturbances [35,36].

Our observations presented in this study showed that reserpine significantly increased the activation of TNF-α and IL-1β, providing evidence of an association between inflammation, pain sensitization, and depression [5,37]. These cytokines reduce the pain threshold via increased excitability of Aδ and C fibers [38] and induce depression-like symptoms via HPA axis activation and disturbance in the monoamine system [17,39]. Proinflammatory mediators also stimulate the release of prostaglandins and substance P, thus facilitating hyperalgesia [40].

CMPP significantly decreased TNF-α and IL-1β levels, indicating its potent anti-inflammatory properties. This effect is consistent with the results of cytokine-targeted interventions, such as TNF-α blockers (e.g., infliximab and etanercept), which reduce depressive symptoms in clinical samples [41]. Therefore, the anti-inflammatory effect of CMPP may contribute to its dual action against nociceptive and affective responses.

A key point in interpreting the present findings is that inflammation, oxidative stress, and monoaminergic dysfunction are not independent processes but form a mutually reinforcing network. Pro-inflammatory cytokines such as TNF-α and IL-1β can reduce monoaminergic signaling through several mechanisms, including activation of indoleamine 2,3-dioxygenase and diversion of tryptophan metabolism toward the kynurenine pathway, which may reduce serotonin availability while increasing neuroactive metabolites that promote glutamatergic dysregulation. In parallel, inflammatory signaling can impair monoamine synthesis by reducing the availability of tetrahydrobiopterin, a critical cofactor for serotonin and catecholamine biosynthesis, and may also alter monoamine transporter function. Within this framework, the reduction in TNF-α and IL-1β observed after CMPP treatment may have contributed indirectly to the partial restoration of serotonin and norepinephrine and to the normalization of glutamate levels [[42], [43], [44], [45]].

The relationship between oxidative stress and MAO-A activity is also likely to be mechanistically important. MAO-A, located on the outer mitochondrial membrane, catalyzes monoamine degradation and generates hydrogen peroxide and reactive aldehydes as by-products, thereby linking monoamine depletion to oxidative injury. Excessive MAO-A activity can therefore amplify reactive oxygen species formation, promote lipid peroxidation, and further destabilize neuronal function. Conversely, oxidative and inflammatory states can aggravate monoaminergic impairment, creating a feed-forward cycle in which monoamine degradation, oxidative stress, and neuroinflammation perpetuate one another. From this perspective, the reduction in MAO-A activity after CMPP treatment may not only preserve monoamine availability but also lessen oxidative burden, which is consistent with the observed increase in GSH and decrease in TBARS. Taken together, our findings support a model in which CMPP acts at multiple interconnected levels by suppressing inflammatory signaling, improving antioxidant defenses, reducing MAO-A-associated monoamine degradation, and thereby weakening the reciprocal reinforcement among neuroinflammation, oxidative stress, excitatory imbalance, and monoamine depletion that sustains nociceptive hypersensitivity, depressive-like behavior, and cognitive dysfunction in the reserpine model. Although the present study does not establish direct causality among these pathways, it strongly supports an integrated multi-target mechanism rather than separate parallel effects [46,47].

Monoaminergic dysfunction is a characteristic feature of the reserpine model, where depletion of 5-HT and NE by inhibiting vesicular monoamine transporters and elevating MAO-A activity has been reported [48]. Our findings support these abnormalities, and we found significantly lower serotonin and norepinephrine levels and increased glutamate levels in reserpine-treated animals. CMPP treatment partially restored those levels, including the reduction in monoamine levels and decreased glutamatergic overactivity. Although the present findings support a role for monoaminergic modulation in the effects of CMPP, the neurochemical analysis was limited to serotonin and norepinephrine and did not include dopamine, which should be addressed in future mechanistic studies.

Descending pain inhibition and cognitive processing are governed by monoamines [49], whereas central sensitization and persistent excitatory transmission are driven by glutamate release [50]. Our positive control, gabapentin, has been reported to decrease glutamate levels by inhibiting branched-chain amino acid aminotransferase and increasing glutamate dehydrogenase activity [51]. In addition to increasing monoamines, the capacity of CMPP to partially restore monoaminergic levels and normalize glutamatergic imbalance supports its broader mechanistic profile.

Reserpine-mediated upregulation of MAO-A shifts the additional burden of monoamine degradation, leading to elevated reactive oxygen species (ROS) and oxidative damage [52]. This was confirmed in our study, in which reserpine increased TBARS levels and decreased GSH levels. CMPP markedly reduced the activity of MAO-A and opposed the alteration of antioxidant equilibrium, which implies that it cannot only resist neurotransmitter consumption but also shield against ROS-mediated neuronal injury. This dual activity of CMPP classifies it as a compound exhibiting both psychotropic and neuroprotective activities.

Cytokines have wide-ranging influences on neurotransmitters, including the activation of the kynurenine pathway (which causes serotonin depletion and glutamatergic metabolite generation) and the depletion of tetrahydrobiopterin (BH4), a cofactor required for monoamine synthesis [43,53]. By lowering cytokine levels, CMPP may indirectly stabilize monoamine levels and reduce excitatory glutamatergic signaling. Therefore, our results suggest that CMPP acts as a therapeutic agent by dual regulation of inflammatory, oxidative, and monoaminergic functions.

Although gabapentin served as an appropriate positive control in the present model, the differences between gabapentin and CMPP are likely rooted in their fundamentally different mechanisms of action. Gabapentin primarily binds to the α2δ-1 auxiliary subunit of voltage-gated calcium channels, reducing calcium-dependent presynaptic neurotransmitter release and thereby dampening neuronal hyperexcitability and central sensitization. Its established analgesic benefit is therefore mainly linked to modulation of excitatory transmission rather than to direct regulation of inflammatory cytokines, monoamine catabolism, or oxidative balance [54,55]. By contrast, the present findings suggest that CMPP has a broader pharmacological profile, simultaneously affecting TNF-α, IL-1β, GSH, TBARS, MAO-A activity, and monoaminergic/glutamatergic markers. This wider scope may explain why CMPP outperformed gabapentin in selected biochemical endpoints, particularly those more directly linked to oxidative stress and monoamine degradation. In this regard, MAO-A is not only a catabolic enzyme for serotonin and norepinephrine but also a source of hydrogen peroxide and reactive aldehydes, such that stronger MAO-A suppression by CMPP would be expected to preserve monoamine availability while also reducing oxidative burden [47,56]. A similar logic may apply to the greater effects of CMPP on GSH, norepinephrine, and glutamate, because these endpoints lie at the intersection of inflammation, redox imbalance, and monoaminergic dysfunction. At the same time, gabapentin showed a stronger effect on serotonin, indicating that the superiority of CMPP was not universal but rather endpoint-dependent. Thus, the present data support the interpretation that gabapentin mainly attenuates the excitatory component of the model, whereas CMPP appears to interrupt several mutually reinforcing pathological pathways simultaneously, which may account for its broader biochemical efficacy. However, because direct target-binding and pathway-interference experiments were not performed in the present study, these mechanistic distinctions should be interpreted as pharmacologically plausible rather than definitively established.

It will also be necessary to properly position the current results relative to clinically available agents for pain–depression comorbidity. Duloxetine, which is a serotonin–norepinephrine reuptake inhibitor and one of the well-established pharmacological treatments for fibromyalgia and chronic pain conditions, has its therapeutic effect attributed solely to increased descending monoaminergic inhibition through blockade of serotonin and norepinephrine uptake [57]. Rather, the present study proposes that CMPP might regulate a wider network of pathological processes affecting important inflammatory cytokines, oxidative stress markers, MAO-A activity, and depletion of monoamines (serotonin and norepinephrine), as well as glutamatergic imbalance. This broader profile could suggest multi-target potential, but the current data did not show an advantage in comparison to duloxetine or with existing therapies since head-to-head comparisons were never accomplished. For this reason, the main scientific contribution of these findings should be interpreted as the first description of CMPP as a multi-target bioactive compound in a model of pain–depression comorbidity induced by reserpine. Further studies should be undertaken to directly compare CMPP with clinically relevant agents (for example duloxetine), ideally in matched experimental settings, to better establish its relative efficacy, shared mechanisms and possible therapeutic advantages [58].

From a medicinal chemistry point of view, the multi-target profile of CMPP may more likely emerge from the combination of several pharmacologically productive structural motifs as opposed to one major attribute. Maleimide/cyclic imide core is a well-known bioactive scaffold and compounds containing maleimide- or cyclic imide have been associated with anti-inflammatory activity in medicinal chemistry studies, rendering plausibility of this fragment being the contributor for the anti-inflammatory and redox related effects seen here [59,60]. The other notable element is the aminoacetylene/propargyl-like moiety, as analogous motifs had previously featured multiple times in neuroprotective- and monoamine oxidase-directed ligands, such as compounds whose activities have been associated with enhancing monoaminergic control and neuronal survivability signaling. [61,62]. At the same time, the morpholine ring is a privileged motif in medicinal chemistry that has been used across CNS-relevant scaffolds to modulate polarity, solubility and target recognition [63,64]. Similarly, piperazine (especially when conjugated with a heteroaryl group such as pyridine) is an established fragment of bioactive and CNS-targeted ligands since it modifies basicity, molecular orientation, and therefore receptor or enzyme interaction [65,66]. In this regard, the analgesic, antidepressant-like, antioxidant and anti-inflammatory actions of CMPP might be indicative of a well-coordinated structural arrangement with (i) the maleimide moiety being responsible for anti-inflammatory and redox sensitive activities; (ii) the aminoacetylene portion providing neuroprotective and monoaminergic effects; and (iii) areas associated with improved physicochemical properties of behavior relevant to CNS-targets in relation to mechanism--driven therapeutic dimensions. This interpretation is furthermore also supported by the original chemical report that describes CMPP as a member of a larger class of aminoacetylene–maleimide hybrids. However, these assignments should be regarded as hypothesis-generating rather than definitive, because the present study did not compare CMPP with close analogs lacking individual structural features. Therefore, future analog-based studies that systematically vary the chlorine substituent, morpholine ring, and pyridyl-piperazine region will be needed to establish a true SAR and identify which elements are most responsible for the observed behavioral and biochemical effects.

This study has some limitations that must be acknowledged. First, although behavioral dose screening was performed using three doses of CMPP (5, 10, and 20 mg/kg), the biochemical and mechanistic analyses were conducted only at the selected 10 mg/kg dose. Therefore, a full multi-dose biochemical characterization is still needed to define the therapeutic window more comprehensively. Second, only adult male mice were included in the present study. Male animals were selected for this initial proof-of-concept evaluation to reduce hormonal variability and allow a controlled first assessment of the therapeutic potential of CMPP in the reserpine-induced pain–depression dyad model. However, this design limits the translational generalizability of the findings, particularly because fibromyalgia is more prevalent in women. Future studies should therefore investigate CMPP in female mice and directly compare both sexes to determine whether sex-dependent differences exist in behavioral responsiveness, biochemical effects, optimal dose range, and underlying mechanisms. Third, upstream molecular signaling pathways and direct target interactions were not examined. Future studies should therefore investigate pathways such as NF-κB, MAPK, and Nrf2/ARE, together with receptor-binding, enzyme-interaction, and target-validation approaches, to define the molecular targets responsible for the multi-target pharmacological profile of CMPP. In addition, MAO-A activity was assessed in whole-brain homogenates rather than in discrete brain regions. Although this approach was suitable for an initial global neurochemical assessment, it does not allow region-specific interpretation. Future studies should therefore examine MAO-A activity in anatomically defined regions implicated in pain–depression comorbidity, including the hippocampus, prefrontal cortex, and amygdala. Another limitation is that dopamine levels were not measured. Although serotonin and norepinephrine were assessed as key monoaminergic mediators and glutamate was evaluated as an index of excitatory imbalance, the lack of dopamine analysis limits the completeness of the neurochemical characterization, particularly because dopamine is also implicated in pain modulation, motivation, and mood regulation and is known to be affected in reserpine-based models. Consequently, future studies should include a dopamine profiling to evaluate monoaminergic involvement in the effects of CMPP more comprehensively. A further limitation is that a formal a priori power analysis to determine sample size was not conducted. Statistically significant effects were identified on several endpoints, but future confirmatory studies can include prospective power calculations and potentially larger group sizes where indicated to further strengthen the robustness of the results. Moreover, The present study has an additional limitation: the brief 6-day experimental period. While this time-point was suitable for initial efficacy testing in the reserpine model, it does not reflect the chronic nature of pain–depression comorbidity and allows no assessments on long-term tolerability and safety. Therefore, it is recommended that future studies utilize longer treatment paradigms and post-treatment observation periods, together with dedicated toxicological and organ-safety assessments for CMPP, to better define drug persistence and safety profiles under more chronic conditions. A further limitation of the current study is that serotonin, norepinephrine, and glutamate were quantified using commercial ELISA-based assays. Although these assays are suitable for preliminary neurochemical profiling, more advanced analytical methods such as HPLC-ECD or LC-MS/MS provide greater selectivity, sensitivity, and confidence in analyte identification. Therefore, future studies should validate the present monoamine findings using HPLC-ECD or LC-MS/MS and expand the neurochemical analysis to include dopamine and relevant metabolites for a more comprehensive characterization of CMPP's effects on monoaminergic signaling [67,68].

Although the present study demonstrates that CMPP improves behavioral outcomes and favorably modulates inflammatory, oxidative, and neurochemical markers, the upstream molecular targets responsible for these effects remain to be defined. In particular, signaling pathways such as NF-κB, MAPK, and Nrf2/ARE are plausible candidates because of their well-established roles in coordinating inflammatory activation, oxidative stress responses, and neuronal survival in pain- and depression-related pathology. Likewise, dedicated receptor-binding, enzyme-interaction, and target-engagement studies will be necessary to determine whether CMPP acts directly on monoamine-related enzymes, inflammatory signaling components, ion channels, or other CNS-relevant targets. Accordingly, future studies should combine region-specific molecular analyses, protein-expression assays, and pharmacological target-validation approaches to define the primary signaling pathways and direct molecular targets underlying the multi-target effects of CMPP.

In summary, CMPP significantly attenuated reserpine-induced pain hypersensitivity, depressive-like behavior, and cognitive impairment in mice. These effects were associated with suppression of pro-inflammatory cytokines, improvement of antioxidant defenses, partial restoration of monoaminergic balance, reduction of glutamatergic disturbance, and inhibition of MAO-A activity. Taken together, these findings identify CMPP as a promising multi-target compound in a reserpine-induced preclinical model of pain–depression comorbidity. However, further studies are required to confirm its efficacy in additional animal models relevant to fibromyalgia and chronic pain, including sex-comparative and mechanistic investigations, before broader translational conclusions can be drawn.

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Institutional review board statement

Animal experimental procedures were approved by the animal ethics committee at Hashemite University (22/2/2024/2025), and were in accordance with the guidelines of the US National Institutes of Health on the use and care of laboratory animals, and with the Animal Research: Reporting of in Vivo Experiments (ARRIVE) guidelines.

Declaration of using AI tool

During the preparation of this manuscript, the author(s) used Paperpal for language improvement, sentence structure enhancement, and grammar checking. After using this tool/service, the author(s) reviewed and edited the content as needed and take(s) full responsibility for the content of the published article.

Funding

The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at the Hashemite University, Zarqa, Jordan, for funding this research. (Grant number: 54/2020, 49/2020). The Deanship of Scientific Research at Yarmouk University , Irbid Jordan for funding the research ( Grant number 1/2024).

CRediT authorship contribution statement

Lorca Alzoubi: Conceptualization, Funding acquisition, Methodology, Validation. Yousra Bsieso: Investigation, Methodology. Omar Gammoh: Data curation, Formal analysis. Alaa A.A. Aljabali: Validation, Writing – review & editing. Bader A. Salameh: Resources, Software. Abdelrahim Alqudah: Conceptualization, Funding acquisition, Project administration. Esam Qnais: Supervision, Writing – review & editing. Ahmad M. Al-malkawi: Data curation, Methodology, Resources. Kayed Abu-Safieh: Methodology, Resources, Visualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.