A New Tick on the Clock: Indole-Based Optimization of Melatonin Receptor Modulators

Emine Erdag

doi:10.33069/cim.2025.0014

Chronobiol Med > Volume 7(2); 2025 > Article

Erdag: A New Tick on the Clock: Indole-Based Optimization of Melatonin Receptor Modulators

Original Article

Chronobiology in Medicine 2025;7(2):88-94.

Published online: June 27, 2025

DOI: https://doi.org/10.33069/cim.2025.0014

A New Tick on the Clock: Indole-Based Optimization of Melatonin Receptor Modulators

Emine Erdag^1,²

¹Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Near East University, Nicosia, Cyprus

²DESAM Research Institute, Near East University, Nicosia, Cyprus

Corresponding author: Emine Erdag, PhD, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Near East University, Near East Boulevard, Nicosia 99138, Cyprus.
Tel: 90-5338898921, E-mail: emine.erdag@neu.edu.tr

Received March 15, 2025 Revised April 9, 2025 Accepted April 21, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

Melatonin is a key regulator of circadian rhythms and sleep-wake cycles, exerting its effects through MT1 and MT2 receptors. Despite the clinical use of selective MT1/MT2 agonists, their short half-lives, low bioavailabilities, and rapid first-pass metabolism limit their efficacy in sleep and circadian rhythm disorders. This study aimed to identify and evaluate novel piperazine-substituted indole derivatives with enhanced receptor binding, prolonged systemic circulation, and improved pharmacokinetic properties as potential next-generation melatonin receptor modulators.

Methods

Seven piperazine-substituted indole derivatives were selected based on structure-activity relationship studies and structural similarity to melatonin. Molecular docking was conducted using AutoDock 4.2, followed by molecular dynamics (MD) simulations in GROMACS 2023.1 to assess receptor-ligand stability. Binding free energy calculations (molecular mechanics/Poisson–Boltzmann surface area [MM/PBSA] method) were performed to quantify ligand-receptor interactions. Pharmacokinetic properties were predicted using ADMETlab 2.0.

Results

Among the tested compounds, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole, 3-{[4-(2,5-Dimethylphenyl) piperazin-1-yl]methyl}-1H-indole, and 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole exhibited the highest binding affinities for MT1 and MT2 receptors, with MM/PBSA free energies significantly lower than those of reference compound, ramelteon. MD simulations confirmed stable ligand-receptor interactions, with low root-mean-square deviation values over 100 ns, indicating minimal structural fluctuations. The tested derivatives demonstrated enhanced pharmacokinetic profile and minimal toxicity risks, suggesting greater therapeutic potential for circadian rhythm modulation.

Conclusion

The identified piperazine-substituted indole derivatives demonstrated strong receptor binding, enhanced bioavailability, and prolonged half-life. These findings suggested that these derivatives hold promise for circadian rhythm-related disorders and sleep disturbances.

Keywords: Melatonin receptor; Circadian rhythm; Indole; Molecular docking; Pharmacokinetics

INTRODUCTION

Melatonin is an endogenous hormone synthesized primarily in the pineal gland, playing a crucial role in the regulation of circadian rhythms, sleep-wake cycles, and various physiological processes [1]. Structurally, melatonin contains an indole ring, which facilitates its interaction with MT1 and MT2 receptors, both of which are G-protein coupled receptors involved in the modulation of circadian rhythms and sleep regulation [2].

MT1 receptors are predominantly expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus and contribute to sleep induction by promoting the onset of sleep. Conversely, MT2 receptors regulate circadian phase shifting, aiding in the adaptation to environmental light-dark cycles [3]. Dysregulation of these receptors is associated with sleep disorders, depression, and neurodegenerative diseases [4]. In addition to their well-established role in sleep-wake regulation, melatonin receptor agonists have shown promising therapeutic effects in various neurodegenerative and psychiatric disorders. Recent studies highlighted their neuroprotective, anti-inflammatory, and circadian-restorative properties, particularly in conditions such as Alzheimer’s disease, Parkinson’s disease, and major depressive disorder. These findings underscore the broader therapeutic potential of MT1 and MT2 modulation beyond sleep disorders, extending to complex central nervous system (CNS) pathologies characterized by circadian dysfunction and oxidative stress [5,6]. Given the significance of melatonin receptors, several synthetic agonists were developed to mimic the effects of endogenous melatonin. One of the most notable examples is ramelteon, a high-affinity MT1/MT2 agonist used in the treatment of insomnia and circadian rhythm sleep disorders, such as non-24-hour sleep-wake disorder [7]. Ramelteon demonstrates advantages over traditional hypnotics, such as benzodiazepines, due to its non-sedative mechanism and lack of dependence potential [8].

Despite the success of ramelteon, its clinical application is limited by its relatively short half-life (approximately 1–2 hours), which may reduce its sustained efficacy in regulating sleep-wake cycles [8,9]. This short duration of action may not be sufficient for patients requiring prolonged sleep maintenance, necessitating additional dosing. Additionally, ramelteon has a low oral bioavailability, meaning that a significant portion of the administered dose does not reach systemic circulation due to extensive hepatic first-pass metabolism [10]. This may lead to inconsistent therapeutic effects among individuals. Furthermore, ramelteon’s primary active metabolite, M-II, has a longer half-life compared to the parent compound but possesses significantly lower receptor affinity, which may limit its pharmacological efficacy [9,11]. Additionally, interindividual variability in metabolism and first-pass hepatic degradation can affect its bioavailability and therapeutic outcomes [12]. Therefore, there remains a need for novel agonists with improved pharmacokinetic properties, greater receptor selectivity, enhanced bioavailability, and prolonged half-life. Current melatonin agonists exhibit limitations in efficacy, metabolism, or patient variability [13]. Therefore, the search for new melatonin receptor modulators continues, particularly for conditions involving circadian rhythm disruptions, such as jet lag, shift work disorder, and mood disorders.

In recent years, piperazine has emerged as a widely explored scaffold due to its versatile pharmacological properties, including its role in modulating receptor activity and enhancing drug-like characteristics [14]. Several studies have demonstrated that the inclusion of a piperazine moiety in ligands can enhance receptor interactions by optimizing hydrogen bonding and hydrophobic interactions [15]. Given these advantages, the incorporation of piperazine into indole derivatives may contribute to improved receptor modulation and drug-like properties.

In this study, the binding affinities, structural stability, and pharmacokinetic profiles of seven piperazine-substituted indole derivatives were investigated as potential melatonin receptor modulators. In silico evaluations were utilized to analyze the interaction profiles of these compounds with MT1 and MT2 receptors, providing insights into their potential as therapeutic agents for circadian rhythm-related disorders.

METHODS

Ligand selection and preparation

The synthesis of seven selected piperazine-substituted indole derivatives and their structural characterization were reported in the previously published work. In this study, piperazine-substituted indole derivatives were selected based on their high percentage yields in their synthesis [16]. These compounds were chosen due to their structural resemblance to melatonin and their potential interactions with MT1 and MT2 receptors. The two-dimensional (2D) structures of the selected ligands were initially sketched using ChemDraw 20.0 (Revvity Signals Software, Inc.; https://revvitysignals.com/products/research/chemdraw) and subsequently converted to three-dimensional (3D) structures using Open Babel 3.1.1 (https://openbabel.org/). Energy minimization was performed, ensuring that the ligands were in their lowest energy conformations before docking. The optimized ligand structures were then saved in Protein Data Bank (PDB) format for further molecular docking studies.

Molecular docking

Molecular docking simulations were conducted to predict the binding conformations and interaction profiles of the selected piperazine-substituted indole derivatives within the active sites of MT1 and MT2 receptors. The crystallographic structures of MT1 and MT2 receptors were retrieved from the PDB (https://www.wwpdb.org/; PDB ID: 7DB6 for MT1, PDB ID: 6ME9 for MT2). Before docking, the receptor structures were preprocessed using AutoDockTools 1.5.7 (https://autodocksuite.scripps.edu/), where water molecules were removed, polar hydrogens were added, and Gasteiger charges were assigned to ensure proper electrostatic interactions during docking.

The grid box dimensions were defined to encompass the orthosteric binding site of each receptor, centering around the binding residues previously identified in co-crystallized ligand structures. The grid spacing was set to 0.375 Å, ensuring high-resolution sampling of the binding site. The docking simulations were performed using AutoDock 4.2, employing the Lamarckian Genetic Algorithm (LGA) with 100 independent runs per ligand-receptor complex. The binding poses were ranked based on binding energy, with the most stable pose (lowest energy) selected for further post-docking analysis.

Molecular dynamics simulations

The stability and dynamic behavior of the ligand-receptor complexes were assessed by molecular dynamics (MD) simulations, which were performed using GROMACS 2023.1 (https://manual.gromacs.org/) with the CHARMM36 force field. The receptor-ligand complexes were solvated in an explicit TIP3P water model, ensuring a 10 Å buffer zone around the system. The system was neutralized by adding counterions (Na⁺ or Cl^–) to maintain electrostatic stability, using the same procedure as reported before. Additionally, a physiological salt concentration of 0.15 M NaCl was used to ensure electrostatic stability and to closely simulate in vivo ionic conditions [17,18].

After system preparation, energy minimization was conducted using the steepest descent algorithm to remove steric clashes and optimize the initial conformation. The system was then equilibrated in a stepwise manner. First, the NVT (number of particles, volume, and temperature) ensemble was applied, where the system was gradually heated from 0 K to 310 K over 100 ps using the Berendsen thermostat, allowing it to reach thermal equilibrium [19]. The production MD phase was carried out for 100 ns at 310 K and 1 atm pressure, with a 2 fs timestep. The Particle Mesh Ewald (PME) method was used for long-range electrostatics, and the LINear Constraint Solver (LINCS) algorithm was applied to constrain bond lengths, ensuring simulation stability [20]. System trajectory data were extracted every 10 ps for post-simulation analyses.

The root-mean-square deviation (RMSD) was calculated to evaluate the overall stability of the ligand-receptor complexes over time. RMSD values were computed for the Cα backbone of the receptor and the ligand within the binding site, using the initial docked structure as a reference. Lower RMSD values were associated with more stable binding interactions, whereas significant fluctuations suggested possible ligand repositioning or weaker binding affinity [21].

Molecular mechanics/Poisson–Boltzmann surface area calculations

Binding free energy calculations were conducted using the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) approach to quantitatively evaluate the binding affinity of each ligand-receptor complex. The binding energies of each complex were calculated using a previously published equation [22]. The calculations were performed using g_mmpbsa, a GROMACS-compatible MM/PBSA tool, applied to 500 evenly extracted snapshots from the last 20 ns of the MD simulation to ensure convergence and reliable energy estimations.

Pharmacokinetic predictions

The pharmacokinetic properties of the selected piperazine-substituted indole derivatives were evaluated using ADMETlab 2.0, a machine learning-based tool for predicting absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters [23]. These computational predictions provide insights into the potential oral bioavailability, blood-brain barrier (BBB) penetration, metabolic stability, clearance, and toxicity of the compounds.

The absorption profile of each ligand was assessed by predicting human intestinal absorption (HIA) and Caco-2 permeability, which are key determinants of oral drug uptake. The predicted pKa and LogD values at physiological pH (7.4) provided additional information regarding the ionization state and lipophilicity of the compounds, which directly influence their passive diffusion across biological membranes [23,24].

In terms of distribution, BBB penetration was predicted to determine the ability of the compounds to reach the CNS and effectively modulate melatonin receptors. Given that MT1 and MT2 receptors are expressed in the CNS, a high BBB permeability score was considered favorable. Plasma protein binding (PPB) was also evaluated to estimate the extent to which the compounds bind to albumin and α1-acid glycoprotein, which can influence their free (active) concentration in circulation. Compounds with lower PPB are expected to have greater systemic availability, while those with excessive PPB may have reduced pharmacological effects [23,24].

Metabolic stability was analyzed by predicting interactions with major cytochrome P450 (CYP) enzymes, including CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4. These enzymes play a crucial role in hepatic metabolism and drug clearance. The compounds were classified based on whether they act as substrates or inhibitors of these enzymes. Inhibition of CYP isoforms may lead to drug-drug interactions and altered metabolic clearance, affecting the pharmacokinetic profile. Additionally, intrinsic clearance (CLint) values were predicted to estimate the rate of hepatic elimination, providing insights into metabolic half-life and systemic exposure [23,24].

The excretion properties of the ligands were predicted by assessing renal clearance and elimination half-life (T1/2). A longer half-life suggests prolonged activity, which is beneficial for maintaining sustained receptor engagement, whereas compounds with rapid clearance may require frequent dosing [24,25].

Potential toxicity risks were evaluated by predicting hERG (the human ether-à-go-go-related gene) potassium channel inhibition, which is associated with cardiotoxicity and QT interval prolongation. Hepatotoxicity risk was assessed to determine the likelihood of liver damage, an essential consideration for long-term administration. Furthermore, mutagenicity and carcinogenicity predictions were performed using computational models trained on Ames test and rodent carcinogenicity data [24].

RESULTS

The MM/PBSA calculations provided critical insights into the binding affinities of the piperazine-substituted indole derivatives towards MT1 and MT2 receptors (Table 1). Among the seven ligands tested, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole, 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole, and 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole exhibited the strongest interactions, with MM/PBSA binding free energies significantly surpassing those of the other ligands tested. Specifically, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole demonstrated the highest affinity for MT1 (-51.34±1.56 kJ/mol) and MT2 (-41.44±1.53 kJ/mol), followed by 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole (-48.46±1.82 kJ/mol for MT1, -45.94±2.04 kJ/mol for MT2). The third most potent ligand, 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole, exhibited binding free energies of -46.90±1.68 kJ/mol for MT1 and -39.58±1.38 kJ/mol for MT2.

MD simulations further supported these findings, revealing that the three most active ligands maintained stable interactions with both receptors over the 100 ns simulation period. Their RMSD values remained relatively low, indicating minimal fluctuations in receptor-ligand complexes. Among them, 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole exhibited the most stable RMSD pattern, with values of 1.35 ± 0.52 Å for MT1 and 1.76±0.34 Å for MT2, suggesting optimal binding stability. In contrast, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole and 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole showed slightly higher fluctuations, but still within the range of stable interactions.

Other ligands, such as 3-{[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-methyl}-1H-indole and 3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole, exhibited weaker binding affinities and higher RMSD fluctuations, suggesting lower receptor compatibility. For example, the binding free energy of 3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole was the weakest among all derivatives (-37.45±1.69 kJ/mol for MT1 and -28.95±1.62 kJ/mol for MT2), which was consistent with its less stable binding during MD simulations.

The ADMET properties of all ligands were analyzed to assess their suitability for oral administration and CNS penetration (Table 2). The top three ligands once again demonstrated superior pharmacokinetic properties, exhibiting enhanced oral absorption, prolonged half-life, and favorable BBB penetration, which are essential characteristics for effective melatonin receptor modulation.

Regarding absorption and bioavailability, the most active ligands showed significantly higher intestinal absorption rates compared to the other compounds. Notably, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole exhibited the highest HIA value of 98.4%, ensuring optimal systemic uptake. In addition, Caco-2 permeability values suggested effective passive diffusion across intestinal membranes. The top three ligands consistently demonstrated permeability scores classified as “high” to “very high,” which are indicative of their strong potential for oral bioavailability and systemic circulation.

BBB penetration emerged as another crucial determinant of the compounds’ therapeutic potential, given that MT1 and MT2 receptors are primarily localized in the CNS. Among the most active ligands, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole exhibited high BBB permeability, while 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole showed moderate-to-high penetration, both of which were superior to melatonin and existing MT1/MT2 agonists. In contrast, 3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole and N-[2-(1,6,7,8-Tetramethyl-2,3-dihydro-1H-inden-5-yl)ethyl]propionamide (ramelteon) exhibited poorer BBB permeability, making them less suitable for CNS-targeted therapies and limiting their effectiveness as melatonin receptor modulators.

Regarding clearance and elimination half-life, the top three ligands exhibited prolonged systemic circulation, a key advantage for maintaining receptor engagement over an extended period. In particular, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole exhibited a half-life of 5 hours, while 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole had a half-life of 4 hours. In contrast, 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole exhibited a shorter half-life of 3 hours, likely due to its CYP1A2-mediated metabolism. These values remain significantly higher than ramelteon, which has a half-life of only 1–2 hours.

The toxicity predictions further supported the safety profile of the most active derivatives. None of these compounds exhibited hERG potassium channel inhibition, indicating a low risk of cardiotoxicity. Additionally, hepatotoxicity risks were predicted to be low for 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole and 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole, reinforcing their potential suitability for long-term administration without significant hepatic concerns. These findings collectively highlighted the therapeutic promise of these newly designed melatonin receptor modulators, which not only exhibit stronger receptor binding but also possess favorable pharmacokinetic and toxicity profiles, making them excellent candidates for further drug development.

DISCUSSION

The superior pharmacokinetic and binding properties observed for the top three ligands can be attributed to their distinct chemical structures, which significantly influence receptor interactions, metabolic stability, and systemic circulation. The strong MM/PBSA binding affinities of 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole, 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole, and 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole suggest that these compounds establish highly stable interactions within the MT1 and MT2 receptor binding sites. The extended conjugated aromatic rings in their structures likely enhance π-π stacking interactions with key residues, stabilizing their binding conformations. Moreover, the presence of piperazine substitution appears to be crucial for receptor affinity, as it facilitates additional hydrophobic and electrostatic interactions, which have been reported in previous structure-activity relationship studies [15,26,27].

A major advantage of these ligands over existing melatonin analogs, including ramelteon, lies in their improved oral absorption and bioavailability. While ramelteon exhibits low oral bioavailability (approximately 1.8%) due to extensive first-pass metabolism [8,10], the top three ligands in this study demonstrated high HIA values, with 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole reaching 98.4%. This suggested enhanced systemic availability, which may translate into more consistent pharmacological effects. Additionally, high Caco-2 permeability scores indicated that these ligands may efficiently diffuse across intestinal membranes, further supporting their potential as orally administered therapeutic agents.

For CNS-targeted drugs, BBB penetration is another critical factor. The most effective derivatives tested in this study exhibited high-to-moderate BBB permeability, which is essential for effective engagement with MT1 and MT2 receptors located in the SCN [28,29]. This property distinguishes them from less effective derivatives, such as 3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole and N-[2-(1,6,7,8-Tetramethyl-2,3-dihydro-1H-inden-5-yl) ethyl]propionamide (ramelteon), both of which exhibited poor BBB penetration and, consequently, lower therapeutic potential for circadian rhythm modulation. The naphthyl and dimethylphenyl groups presented in the most effective derivatives likely enhance lipophilicity and facilitate passive diffusion across the BBB, an essential feature that correlates well with previous reports on melatonin receptor-targeted ligands [30,31].

Another key finding of this study was the metabolic stability of the most active ligands. Unlike ramelteon, which is rapidly metabolized via CYP1A2, CYP2C9, and CYP3A4 [32], two of the top three ligands did not significantly inhibit major CYP isoforms, indicating a low risk of drug-drug interactions. However, 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole exhibited moderate CYP1A2 inhibition, suggesting potential metabolic interactions that should be investigated further. This was particularly relevant given that CYP1A2 metabolism plays a major role in melatonin and ramelteon clearance [33]. In contrast, 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole and 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole were not identified as major CYP inhibitors, reducing the potential risk of drug-drug interactions, which is a common concern for sleep disorder medications [34].

Elimination half-life is another major parameter influencing the sustained activity of melatonin receptor agonists [35]. The top three derivatives exhibited prolonged half-lives, with 3-{[4-(1-Naphthylmethyl)piperazin-1-yl]methyl}-1H-indole showing the longest half-life of 5 hours, followed by 3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole (4 hours) and 3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole (3 hours). This represents a marked improvement over ramelteon, which has a half-life of only 1–2 hours [35]. A longer half-life suggests extended receptor engagement and sustained therapeutic effects, potentially reducing the need for multiple daily doses and improving patient compliance [36,37]. The presence of electron-donating groups in the top ligands’ structures may contribute to their metabolic stability, delaying rapid hepatic clearance and prolonging systemic exposure.

From a toxicity perspective, hERG inhibition was not detected in the most active ligands, suggesting a low risk of cardiotoxicity. However, 3-{[4-(1-Piperonyl)piperazin-1-yl]methyl}-1H-indole and 3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole exhibited significant hERG inhibition, highlighting potential cardiotoxic concerns. Additionally, hepatotoxicity risk was predicted to be low for two of the three most active ligands, supporting their suitability for long-term administration. In contrast, compounds with halogen-substituted phenyl groups exhibited higher predicted hepatotoxicity, which correlates with previous studies indicating that halogenated aromatic rings can contribute to metabolic instability and liver toxicity [38,39].

Compared to other approved melatoninergic agents such as tasimelteon and agomelatine, the tested indole-based derivatives in this study demonstrated several notable advantages. Tasimelteon, while effective in treating non-24-hour sleep-wake disorder, has shown moderate bioavailability (approximately 38%) and significant interindividual variability in hepatic metabolism via CYP1A2 and CYP3A4, often resulting in inconsistent therapeutic outcomes. Similarly, agomelatine, although known for its antidepressant effects via MT1/MT2 agonism and 5-HT2C antagonism, has limited receptor selectivity and hepatotoxicity concerns that require regular liver function monitoring during treatment. In contrast, the top three indole-piperazine derivatives exhibited higher predicted BBB penetration, lower CYP-related interactions, and more favorable toxicity profiles, indicating superior pharmacokinetic behavior and reduced risk of metabolic complications [40,41]. These findings align well with recent advancements in melatonin receptor research, where efforts were directed toward developing longer-acting and more bioavailable agonists to overcome the limitations of current therapeutics such as ramelteon and tasimelteon [42,43]. The superior performance of the top three most effective derivatives in this study suggested that structural modifications, such as extended aromatic moieties and optimized lipophilicity, were possible key strategies for improving melatonin receptor modulators. The predicted therapeutic mechanisms of the tested derivatives, including sleep onset induction via MT1, phase shift regulation via MT2, and combined circadian alignment through dual receptor affinity, were visually summarized in Figure 1.

This study highlights the therapeutic potential of piperazine-substituted indole derivatives as next-generation melatonin receptor modulators with enhanced pharmacokinetics, superior receptor binding, and a favorable safety profile. These properties suggest that the identified ligands could offer significant advantages in the treatment of circadian rhythm-related disorders, sleep disturbances, and mood disorders. Future research should focus on in vitro and in vivo validation to confirm efficacy and safety, particularly through animal models of sleep regulation and circadian dysfunction. Additionally, further structure-based optimization may enhance MT1/MT2 selectivity while minimizing off-target interactions, ultimately improving therapeutic outcomes. Refining the structure-activity relationships and optimizing the pharmacokinetic profile will be essential to advancing these compounds toward clinical development as potential melatonin receptor-targeted therapeutics.

NOTES

Conflicts of Interest

The author has no potential conflicts of interest to disclose.

Availability of Data and Material

The data generated or analyzed during the study are available from the corresponding author upon reasonable request.

Funding Statement

None

Acknowledgments

None

Figure 1.

Schematic illustration of the expected role of indole-based derivatives on MT1/MT2 receptors, which may improve suprachiasmatic nucleus (SCN)-driven circadian alignment.

Table 1.

The MM/PBSA binding free energy and RMSD values for various ligands docked to MT1 and MT2 receptors

Name of ligands	MM/PBSA (kJ/mol)		RMSD (Å)
Name of ligands	MT1 receptor complex	MT2 receptor complex	MT1 receptor complex	MT2 receptor complex
3-{[4-(4-Acetylphenyl)piperazin-1-yl]methyl}-1H-indole	-46.90±1.68	-39.58±1.38	2.47±0.22	2.37±0.25
3-{[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-methyl}-1H-indole	-42.66±1.46	-31.22±2.27	2.01±0.61	2.40±0.43
3-{[4-(1-Napthylmethyl)piperazin-1-yl]methyl}-1H-indole	-51.34±1.56	-41.44±1.53	1.43±0.72	2.50±0.68
3-{[4-(1-Piperonyl)piperazin-1-yl]methyl}-1H-indole	-40.42±2.04	-37.47±2.16	2.26±0.51	2.15±0.73
3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole	-37.45±1.69	-28.95±1.62	2.24±0.73	2.04±0.48
3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl]methyl}-1H-indole	-48.46±1.82	-45.94±2.04	1.35±0.52	1.76±0.34
N-[2-(1,6,7,8-Tetramethyl-2,3-dihydro-1H-inden-5-yl)ethyl]propionamide	-45.46±1.82	-38.35±1.73	1.49±0.36	1.25±0.47

MM/PBSA, molecular mechanics/Poisson–Boltzmann surface area; RMSD, root-mean-square deviation

Table 2.

The predicted pharmacokinetic and toxicity properties of a series of indole-based ligands, along with ramelteon as a reference compound

Ligand name	HIA (%)	Caco-2 permeability	BBB penetration	PPB (%)	CYP inhibition (major isoforms)	CLint (mL/min/kg)	Half-life (T1/2) (h)	hERG inhibition	Hepatotoxicity risk
3-{[4-(1-Naphthylmethyl)piperazin-1-yl] methyl}-1H-indole	98.4	Very high	High	67.8	No major CYP inhibition	6.2	5	Low	Low
3-{[4-(2,5-Dimethylphenyl)piperazin-1-yl] methyl}-1H-indole	96.8	High	Moderate to high	72.1	No major CYP inhibition	8.4	4	Low	Low
3-{[4-(4-Acetylphenyl)piperazin-1-yl] methyl}-1H-indole	94.5	High	Moderate	74.2	CYP1A2 inhibition	9.1	3	Low	Moderate
3-{[4-(4-Trifluoromethylphenyl)piperazin-1-yl]-methyl}-1H-indole	85.7	Moderate	Moderate	80.1	CYP2C9, CYP3A4	14.3	3	Moderate	Low
3-{[4-(1-Piperonyl)piperazin-1-yl]methyl}-1H-indole	88.3	Moderate	Low	79.5	CYP2C19, CYP2D6	15.2	2.8	High	Moderate
3-{[4-(4-Bromophenyl)piperazin-1-yl]methyl}-1H-indole	76.2	Low	Low	85.6	CYP3A4	17.1	2	High	High
N-[2-(1,6,7,8-Tetramethyl-2,3-dihydro-1H-inden-5-yl)ethyl]propionamide (Ramelteon)	85–90	Low	Low to moderate	82–90	CYP1A2, CYP2C9, CYP3A4	15–20 mL/min/kg	1–2 h	Low	Moderate

HIA, human intestinal absorption; BBB, blood–brain barrier; PPB, plasma pretein binding; CYP, cytochrome P450; CLint, intrinsic clearance

REFERENCES

1. Samanta S. Physiological and pharmacological perspectives of melatonin. Arch Physiol Biochem 2022;128:1346–1367.

2. Stauch B, Johansson LC, Cherezov V. Structural insights into melatonin receptors. FEBS J 2020;287:1496–1510.

3. Ruan W, Yuan X, Eltzschig HK. Circadian rhythm as a therapeutic target. Nat Rev Drug Discov 2021;20:287–307.

4. Xiao X, Rui Y, Jin Y, Chen M. Relationship of sleep disorder with neurodegenerative and psychiatric diseases: an updated review. Neurochem Res 2024;49:568–582.

5. Talbot NC, Luther PM, Spillers NJ, Ragland AR, Kidder EJ, Kelkar RA, et al. Neuroprotective potential of melatonin: evaluating therapeutic efficacy in Alzheimer’s and Parkinson’s diseases. Cureus 2023;15:e50948.

6. Shin JW. Neuroprotective effects of melatonin in neurodegenerative and autoimmune central nervous system diseases. Encephalitis 2023;3:44–53.

7. Kim HK, Yang KI. Melatonin and melatonergic drugs in sleep disorders. Transl Clin Pharmacol 2022;30:163–171.

8. Zammit GK. Ramelteon: a novel hypnotic indicated for the treatment of insomnia. Psychiatry (Edgmont) 2007;4:36–42.

9. Miyamoto M. Pharmacology of ramelteon, a selective MT1/MT2 receptor agonist: a novel therapeutic drug for sleep disorders. CNS Neurosci Ther 2009;15:32–51.

10. Pandi-Perumal SR, Spence DW, Verster JC, Srinivasan V, Brown GM, Cardinali DP, et al. Pharmacotherapy of insomnia with ramelteon: safety, efficacy and clinical applications. J Cent Nerv Syst Dis 2011;3:51–65.

11. Erman M, Seiden D, Zammit G, Sainati S, Zhang J. An efficacy, safety, and dose-response study of ramelteon in patients with chronic primary insomnia. Sleep Med 2006;7:17–24.

12. Stielow M, Witczyńska A, Kubryń N, Fijałkowski Ł, Nowaczyk J, Nowaczyk A. The bioavailability of drugs-the current state of knowledge. Molecules 2023;28:8038.

13. Hardeland R. Melatonin: signaling mechanisms of a pleiotropic agent. Biofactors 2009;35:183–192.

14. Romanelli MN, Manetti D, Braconi L, Dei S, Gabellini A, Teodori E. The piperazine scaffold for novel drug discovery efforts: the evidence to date. Expert Opin Drug Discov 2022;17:969–984.

15. Szczepańska K, Podlewska S, Dichiara M, Gentile D, Patamia V, Rosier N, et al. Structural and molecular insight into piperazine and piperidine derivatives as histamine H3 and sigma-1 receptor antagonists with promising antinociceptive properties. ACS Chem Neurosci 2022;13:1–15.

16. Erdag E. Synthesis and characterization of 3-substituted indole derivatives as novel Mannich bases. Asian J Chem 2021;33:781–784.

17. Haskologlu IC, Erdag E, Uludag O, Abacioglu N. A chronobiological approach: the potential of photoswitchable drug derivatives in the treatment of Alzheimer’s disease. Chronobiol Med 2024;6:194–204.

18. Haskologlu IC, Erdag E, Sehirli AO, Uludag O, Abacioglu N. Exploring the therapeutic potential of benzoxazolone derivatives on the circadian clock: an in silico and hypothetical approach. Chronobiol Med 2024;6:87–99.

19. Brooks BR, Brooks CL 3rd, Mackerell AD Jr, Nilsson L, Petrella RJ, Roux B, et al. CHARMM: the biomolecular simulation program. J Comput Chem 2009;30:1545–1614.

20. Harvey MJ, De Fabritiis G. An implementation of the smooth particle mesh Ewald method on GPU hardware. J Chem Theory Comput 2009;5:2371–2377.

21. Fusani L, Palmer DS, Somers DO, Wall ID. Exploring ligand stability in protein crystal structures using binding pose metadynamics. J Chem Inf Model 2020;60:1528–1539.

22. Ozverel CS, Erdag E. Enhancing quality of life in multiple sclerosis patients through coadministration of FDA-approved immunomodulators and melatonin. Chronobiol Med 2024;6:135–142.

23. Xiong G, Wu Z, Yi J, Fu L, Yang Z, Hsieh C, et al. ADMETlab 2.0: an integrated online platform for accurate and comprehensive predictions of ADMET properties. Nucleic Acids Res 2021;49:W5–W14.

24. Dong J, Wang NN, Yao ZJ, Zhang L, Cheng Y, Ouyang D, et al. ADMETlab: a platform for systematic ADMET evaluation based on a comprehensively collected ADMET database. J Cheminform 2018;10:29.

25. Horde GW, Gupta V. Drug clearance. In: StatPearls [Internet]. Treasure Island: StatPearls Publishing; 2023. Available at: https://www.ncbi.nlm.nih. gov/books/NBK557758. Accessed March 12, 2025.

26. Zhang RH, Guo HY, Deng H, Li J, Quan ZS. Piperazine skeleton in the structural modification of natural products: a review. J Enzyme Inhib Med Chem 2021;36:1165–1197.

27. Ikwu FA, Shallangwa GA, Mamza PA, Uzairu A. In silico studies of piperazine derivatives as potent anti-proliferative agents against PC-3 prostate cancer cell lines. Heliyon 2020;6:e03273.

28. Neumaier F, Zlatopolskiy BD, Neumaier B. Drug penetration into the central nervous system: pharmacokinetic concepts and in vitro model systems. Pharmaceutics 2021;13:1542.

29. Liu J, Clough SJ, Hutchinson AJ, Adamah-Biassi EB, Popovska-Gorevski M, Dubocovich ML. MT1 and MT2 melatonin receptors: a therapeutic perspective. Annu Rev Pharmacol Toxicol 2016;56:361–383.

30. Ferreira MA Jr, Azevedo H, Mascarello A, Segretti ND, Russo E, Russo V, et al. Discovery of ACH-000143: a novel potent and peripherally preferred melatonin receptor agonist that reduces liver triglycerides and steatosis in diet-induced obese rats. J Med Chem 2021;64:1904–1929.

31. Mineiro R, Rodrigues Cardoso M, Catarina Duarte A, Santos C, Cipolla-Neto J, Gaspar do Amaral F, et al. Melatonin and brain barriers: the protection conferred by melatonin to the blood-brain barrier and blood-cerebrospinal fluid barrier. Front Neuroendocrinol 2024;75:101158.

32. Obach RS, Ryder TF. Metabolism of ramelteon in human liver microsomes and correlation with the effect of fluvoxamine on ramelteon pharmacokinetics. Drug Metab Dispos 2010;38:1381–1391.

33. Klomp F, Wenzel C, Drozdzik M, Oswald S. Drug-drug interactions involving intestinal and hepatic CYP1A enzymes. Pharmaceutics 2020;12:1201.

34. Pagel JF, Parnes BL. Medications for the treatment of sleep disorders: an overview. Prim Care Companion J Clin Psychiatry 2001;3:118–125.

35. Laudon M, Frydman-Marom A. Therapeutic effects of melatonin receptor agonists on sleep and comorbid disorders. Int J Mol Sci 2014;15:15924–15950.

36. Binder U, Skerra A. Strategies for extending the half-life of biotherapeutics: successes and complications. Expert Opin Biol Ther 2025;25:93–118.

37. Baryakova TH, Pogostin BH, Langer R, McHugh KJ. Overcoming barriers to patient adherence: the case for developing innovative drug delivery systems. Nat Rev Drug Discov 2023;22:387–409.

38. Vickers AE, Sloop TC, Lucier GW. Mechanism of action of toxic halogenated aromatics. Environ Health Perspect 1985;59:121–128.

39. Hansen LG. Halogenated aromatic compounds (1st ed). In: Cockerham LG, Shane BS, editors. Basic environmental toxicology. Boca Raton: CRC Press, 2019, p.199-230.

40. Johnsa JD, Neville MW. Tasimelteon: a melatonin receptor agonist for non-24-hour sleep-wake disorder. Ann Pharmacother 2014;48:1636–1641.

41. de Bodinat C, Guardiola-Lemaitre B, Mocaër E, Renard P, Muñoz C, Millan MJ. Agomelatine, the first melatonergic antidepressant: discovery, characterization and development. Nat Rev Drug Discov 2010;9:628–642.

42. Williams WP 3rd, McLin DE 3rd, Dressman MA, Neubauer DN. Comparative review of approved melatonin agonists for the treatment of circadian rhythm sleep-wake disorders. Pharmacotherapy 2016;36:1028–1041.

43. Comai S, Gobbi G. Melatonin, melatonin receptors and sleep: moving beyond traditional views. J Pineal Res 2024;76:e13011.