Can Piperazine-Bearing Fluoro-Benzoxazolone Derivatives Be a Chronotherapeutic Solution for Pain Management?

Ismail Celil Haskologlu; Emine Erdag; Orhan Uludag

doi:10.33069/cim.2025.0044

Chronobiol Med > Volume 7(3); 2025 > Article

Haskologlu, Erdag, and Uludag: Can Piperazine-Bearing Fluoro-Benzoxazolone Derivatives Be a Chronotherapeutic Solution for Pain Management?

Original Article

Chronobiology in Medicine 2025;7(3):154-161.

Published online: September 30, 2025

DOI: https://doi.org/10.33069/cim.2025.0044

Can Piperazine-Bearing Fluoro-Benzoxazolone Derivatives Be a Chronotherapeutic Solution for Pain Management?

Ismail Celil Haskologlu¹

, Emine Erdag²

, Orhan Uludag³

¹Department of Pharmacology, Faculty of Pharmacy, Near East University, Nicosia, Cyprus

²Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Near East University, Nicosia, Cyprus

³Department of Pharmacology, Faculty of Pharmacy, Gazi University, Ankara, Türkiye

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 July 13, 2025 Revised August 30, 2025 Accepted September 1, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://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

Chronic pain management remains limited due to the restricted efficacy and adverse effects of conventional analgesics. A chronotherapeutic approach targeting melatonin MT1 and MT2 receptors offers an alternative solution, as these receptors play a role in the modulation of circadian rhythms and pain. This study aimed to develop piperazine-bearing fluoro-benzoxazolone derivatives with improved binding affinity and pharmacokinetic properties compared to melatonin for MT1 and MT2 receptors.

Methods

Four piperazine-bearing fluoro-benzoxazolone derivatives (Compound 1–4) were synthesized via the Mannich reaction. Binding affinities were assessed using molecular docking and molecular mechanics/Poisson–Boltzmann surface area calculations, with melatonin as the reference. Key pharmacokinetic properties were predicted using ADMETLab 3.0.

Results

Compound 2 demonstrated the highest binding affinity and stability among the four synthesized compounds. The high affinity and stability indicated enhanced interactions with target receptors compared to melatonin. All synthesized compounds exhibited improved pharmacokinetic profiles relative to melatonin.

Conclusion

The synthesized 5-fluoro-benzoxazolone derivatives exhibited promising binding affinities and pharmacokinetic profiles, highlighting their potential as alternative analgesic agents for MT1 and MT2 receptors in chronotherapy applications.

Keywords: Melatonin receptors; Melatonin; Chronotherapy; Analgesic activity; 5-fluoro benzoxazolone; Piperazine

INTRODUCTION

Conventional analgesics represent an important therapeutic class for the alleviation of pain and inflammation associated with several pathological conditions. Effective pain management, especially in cases of chronic and inflammatory pain, remains a significant challenge due to the limited efficacy and side effects of conventional analgesics [1]. Chronotherapy, a field that explores how drugs interact with biological rhythms, aims to improve therapeutic outcomes by synchronizing pain treatment with the body’s natural cycles. At the core of these biological rhythms lies the circadian clock, which plays a pivotal role in regulating fundamental physiological processes such as pain perception, immune responses, and sleep patterns [2,3]. The suprachiasmatic nucleus in the hypothalamus functions as the body’s primary biological clock, aligning internal rhythms with environmental cues to regulate biological rhythms [4]. This intrinsic timing mechanism not only controls sleep-wake cycles but also modulates pain sensitivity and immune responses [4,5], suggesting that a targeted chronotherapeutic approach could enhance analgesic effects while minimizing adverse outcomes.

Melatonin, secreted by the pineal gland in response to darkness, acts as a key regulator of circadian rhythms [6]. By acting on MT1 and MT2 receptors in the hypothalamus, melatonin coordinates physiological processes according to the day-night cycle, influencing everything from sleep initiation to immune system regulation. Furthermore, these receptors are integral components of the neuroanatomical pain modulation network, suggesting their role in regulating pain perception and response through interactions with key brain regions involved in nociception and analgesia [7]. While MT1 receptors are primarily involved in promoting sleep onset, MT2 receptors are essential for adjusting the circadian phase, responding to light signals, and modulating pain sensitivity. Activation of MT2 receptors plays a notable role in pain modulation, reducing pain transmission in both the central and peripheral nervous systems by suppressing pro-inflammatory cytokine release and enhancing inhibitory neurotransmission via the GABAergic pathway [8].

Utilizing compounds that selectively target MT1 and MT2 receptors in harmony with circadian rhythms could lead to more effective and sustained analgesic effects, particularly during periods of increased pain sensitivity [9]. To investigate this potential, our study focused on benzoxazolone and piperazine derivatives, nitrogen-containing pharmacophores with well-documented analgesic properties and broad pharmacological activity [10,11]. Benzoxazolone derivatives containing piperazine structures may offer a promising foundation for developing new analgesic agents, as they are known to modulate immune responses and inhibit pain signaling [11,12]. As the most electronegative element, fluorine can enhance molecular polarity by altering electron distribution, allowing for stronger electrostatic interactions with biological targets, particularly receptors [13,14]. Therefore, the incorporation of fluorine at the 5-position of the benzoxazolone ring may offer advantages in biological interactions. In this study, fluorine-substituted benzoxazolone derivatives were synthesized and evaluated for their potential efficacy as potential analgesic agents targeting MT1 and MT2 receptors, using an in silico approach. This approach aimed to enhance analgesic efficacy and align with chronopharmacological principles, providing a targeted chronotherapeutic treatment strategy for chronic pain management.

METHODS

Chemicals

All chemicals were purchased from Merck (Darmstadt). The FT-IR spectra were recorded on a Spectrum Two FT-IR Spectrometer (PerkinElmer, Inc.). The nuclear magnetic resonance (NMR) spectra of the synthesized derivatives were obtained on a Jeol 400 MHz spectrometer (JEOL USA, Inc.), operating at 400 MHz for ¹H and 100 MHz for ¹³C, with fully decoupled ¹³C spectra reported. Chemical shifts were expressed in parts per million (ppm) compared to tetramethylsilane (TMS, 0.00 ppm) as the reference. Elemental analysis was performed using an EA1110 Elemental Analyzer (Fison Instruments Ltd, Farnborough). Mass spectra were recorded with an Agilent 6200 Series TOF/ESI-MS instrument (Agilent Technologies).

Synthesis procedure

The 5-fluoro-benzoxazolone derivatives were synthesized by incorporating piperazine derivatives with different aromatic groups via the Mannich reaction (Figure 1). 5-Fluoro-2(3H)-benzoxazolone (15 mmol) and equimolar quantities of selected piperazine derivatives were dissolved in 20 mL of methanol, following a previously published procedure [15]. Formalin (37%) was subsequently added to the reaction mixture and refluxed for 2 hours. After the reaction, the mixture was cooled in an ice bath to facilitate precipitation. The precipitate was then collected through vacuum filtration, and the resulting solid product was further purified by recrystallization using an appropriate solvent.

Ligand preparation

In this study, synthesized 5-fluoro-benzoxazolone derivatives were utilized as ligands, and their binding affinities toward MT1 and MT2 receptors were analyzed. The chemical structures of each ligand were initially created using ChemDraw (version 12.0, CambridgeSoft Corporation), and the Simplified Molecular Input Line Entry System (SMILES) codes generated were then utilized to convert their two-dimensional representations into mol2 files via LigandScout (version 4.0, Inte GmbH). During ligand preparation, all water molecules and heteroatoms were removed, and polar hydrogen atoms were added. Subsequently, Gasteiger charges were calculated to achieve an accurate distribution of partial charges across each atom.

Molecular docking

Molecular docking studies were conducted using AutoDock Vina to determine the binding affinities of the ligands toward human MT1 and MT2 receptors. The 3D structures of these receptors were obtained from the Protein Data Bank (PDB entries 7DB6 and 6ME6, respectively) in PDB format. For the molecular docking simulations, a grid box was set to fully encompass the receptor’s active site. A grid box with dimensions of 30×30×30 Å and a grid spacing of 0.375 Å was used to cover all potential interaction points within the binding region, ensuring that ligands could access optimal binding positions within the active site. The binding poses were visualized using Biovia Discovery Studio Visualizer 2021.

MD simulations and MM/PBSA

Molecular dynamics (MD) simulations were conducted for ligands exhibiting the highest binding affinity in molecular docking studies to evaluate the stability of ligand-receptor complexes. These simulations were performed using GROMACS 2020.4 software with a previously published procedure [16]. The final 20 ns of the 100 ns MD simulations were used to calculate binding free energies of ligand-receptor complexes via the molecular mechanics/Poisson–Boltzmann surface area (MM/PBSA) method, as per the published procedure, with mean and standard deviation (SD) values reported in kJ/mol for each complex [17].

Statistical analysis

Statistical analyses were performed to evaluate the binding energies of the MT1 and MT2 receptor-ligand complexes for synthesized compounds. Binding energy values were obtained using the MM/PBSA method, and the binding energies were analyzed for each ligand. The results were presented as mean±SD. To compare the binding energies of each synthesized compound (Compounds 1–4) with melatonin, which served as the control, a one-way analysis of variance (ANOVA) followed by post-hoc Dunnett’s multiple comparison tests was performed. Significance was determined at p<0.05. Statistical analyses were performed using GraphPad Prism (version 8.0, GraphPad Software).

Pharmacokinetic analysis

The key pharmacokinetic properties of melatonin and the synthesized 5-fluoro-benzoxazolone derivatives (Compounds 1–4) were predicted using ADMETLab 3.0 (https://admetmesh.scbdd.com/service/evaluation/index/). Parameters analyzed included lipophilicity (logP), blood–brain barrier (BBB) penetration, plasma protein binding (PPB), and half-life (t_1/2).

RESULTS

The spectral analysis data of the synthesized molecules provided conclusive evidence supporting their chemical structures. Detailed results are summarized in Table 1. The interaction profiles of the synthesized molecules (ligands) with the MT1 and MT2 receptors were assessed through binding affinity (Table 2) and MM/PBSA binding energy calculations, with melatonin serving as a reference compound (Table 3).

In the MT1 receptor-ligand complexes (PDB entry: 7DB6), Compound 2 emerged as the ligand with the most pronounced binding affinity (−9.1 kcal/mol), followed by Compound 4 (−8.5 kcal/mol), Compound 3 (−7.8 kcal/mol), and Compound 1 (−7.3 kcal/mol). Melatonin, by comparison, showed a binding affinity of −6.4 kcal/mol. The MM/PBSA binding energies mirrored these trends, with Compound 2 exhibiting the most favorable binding energy (−262.85±1.38 kJ/mol) in the series, followed closely by Compound 4 (−241.63±1.83 kJ/mol). The remaining compounds also demonstrated stronger binding interactions with the MT1 receptor compared to melatonin (−152.46±1.37 kJ/mol).

Similarly, for the MT2 receptor-ligand complexes (PDB entry: 6ME6), Compound 2 displayed superior binding affinity (−8.5 kcal/mol), followed by Compound 4 (−8.0 kcal/mol), with Compounds 1 and 3 each showing a binding affinity of −7.6 kcal/mol. In contrast, melatonin exhibited a significantly weaker binding affinity of −5.8 kcal/mol. Consistent with these findings, the MM/PBSA binding energy for Compound 2 (−218.12±1.62 kJ/mol) was markedly more favorable than that of the other compounds, indicating a robust interaction with the MT2 receptor. Compound 4 also demonstrated relatively strong binding energy (−203.57± 1.42 kJ/mol), whereas the binding energy of melatonin was substantially weaker (−118.26±1.32 kJ/mol). The statistical analysis of both MT1 and MT2 receptor-ligand complexes using Dunnett’s multiple comparison tests indicated that all compounds exhibited significantly higher negative binding energies (more favorable) relative to melatonin (p<0.0001) (Figures 2 and 3).

The interaction profiles of Compounds 2 and 4, as well as melatonin, with the MT2 receptor were analyzed to identify common and distinct binding interactions. Across all ligands, several consistent interactions were observed, including hydrogen bonding and hydrophobic interactions with key residues in the MT2 binding pocket (Table 4). Notably, Phe206 and Trp264 engaged in π-π stacking interactions with the aromatic systems of both Compound 2 and Compound 4, as well as melatonin.

Among all ligands, Compound 2 demonstrated unique π-π stacking interactions due to its naphthalene ring, which allowed for additional stabilizing interactions with Tyr282 in the MT2 receptor, a feature not observed with melatonin or Compound 4. In contrast, Compound 4, with its 2-methylbenzyl group, showed enhanced hydrophobic interactions with Leu229 and Val208, increasing binding stability through van der Waals forces, albeit less prominently than the naphthalene ring of Compound 2. Melatonin was observed to form key hydrogen bonds with Ser138 and Asn162, two residues known for facilitating ligand entry and stabilization within the MT2 binding site. However, Compounds 2 and 4 formed additional hydrogen bonds with Asp320, enhancing their binding affinity relative to melatonin.

All synthesized compounds exhibited higher lipophilicity (logP) values compared to melatonin, which has a logP of 1.169. The synthesized compounds showed logP values ranging from 2.517 to 3.532, suggesting enhanced membrane permeability. All synthesized compounds demonstrated a high probability of BBB penetration, contrasting with melatonin’s low probability (0.012). Furthermore, Compounds 1, 2, and 4 displayed significantly higher plasma protein binding rates (97.113%, 97.273%, and 98.332%, respectively) than melatonin (51.164%). When evaluating the half-life, the t₁/₂ of melatonin was determined to be 0.839 hours, while Compound 1 exhibited a half-life of 1.767 hours, Compound 2 demonstrated the longest half-life at 2.056 hours, Compound 3 showed a half-life of 1.819 hours, and Compound 4 displayed a half-life of 1.713 hours.

DISCUSSION

Melatonin is a naturally occurring compound known for its pain modulation and circadian rhythm regulation properties through MT1 and MT2 receptors [18]. However, melatonin’s binding affinity and bioavailability are limited, constraining its clinical efficacy [19]. In this study, the stronger binding of piperazine-bearing fluoro-benzoxazolone derivatives to the melatonin receptors suggested that these derivatives could serve as potential alternatives to melatonin for pain management and circadian rhythm regulation.

The benzoxazolone core structure present in the synthesized compounds contributed to the higher affinity for MT1 and MT2 receptors compared to the indole nucleus found in melatonin. Benzoxazolone, as a bicyclic system containing both aromatic and lactam functionalities, provides a rigid scaffold that promotes stable binding interactions within the receptor binding site [20]. This structure allows for favorable π-π interactions with aromatic residues in the receptor, similar to the indole ring of melatonin. Enhanced rigidity and electron density can facilitate stronger non-covalent interactions [21–23]. Fluorine’s high electronegativity could create a polar region that engages in dipole-dipole interactions or hydrogen bonding with amino acid residues in the receptor’s binding site, thereby stabilizing the ligand-receptor complex. Furthermore, the small size of fluorine might preserve the overall steric compatibility of the ligand within the binding pocket, allowing the molecule to retain an optimal conformation without steric hindrance [24,25]. These chemical features of the benzoxazolone ring and 5-fluoro substitution likely account for the increased binding affinity observed for the synthesized compounds, positioning them as promising candidates for further development over melatonin in melatonin receptor modulation. Additionally, interaction profiles revealed key π-π stacking interactions with aromatic residues such as Phe206 and Trp264, which played a crucial role in stabilizing the binding of all compounds to the MT2 receptor. Specifically, the unique naphthalene-2-yl group of Compound 2 engaged in additional π-π stacking interactions with Tyr282, further stabilizing the receptor-ligand complex. This finding aligns with the literature indicating that large, planar aromatic systems can enhance receptor-ligand stability through such interactions [26,27].

Specifically, Compounds 2 and 4 demonstrated the highest binding affinity and stability in molecular docking and MM/PBSA binding energy analyses, suggesting their potential as potent agonists for melatonin receptors. Modifications at the 3-position of the benzoxazolone ring with piperazine derivatives have resulted in compounds that interact with the receptor with greater affinity. The consistently higher binding affinities and binding energies observed for Compound 2 across both MT1 and MT2 receptors can be attributed to its unique naphthalene-2-yl methyl group. This structural feature likely facilitates extensive π-π stacking interactions with aromatic amino acid residues within the receptor binding pocket. The rigid structure and substantial surface area of naphthalene moiety could allow Compound 2 to establish stronger and more stable interactions compared to other compounds, similar to previous reports [28,29]. Although Compound 4, containing a 2-methylbenzyl group, exhibited lower binding affinity compared to Compound 2, it still demonstrated higher binding affinity than melatonin. The 2-methylbenzyl group provided favorable π-π interactions and additional hydrophobic stabilization due to the methyl substituent, which enhanced receptor binding in hydrophobic pockets for Compound 4. However, these interactions were weaker compared to those of Compound 2. The electron-rich naphthalene ring allowed for more significant van der Waals and electrostatic interactions within the binding site, thereby strengthening Compound 2’s binding profile.

The lower binding affinity observed for Compounds 1 and 3 compared to others could be attributed to the structural and electronic characteristics of their substituents, which limited optimal interactions with the MT1 and MT2 receptor binding sites. In Compound 1, the 2,5-dimethylphenyl group, while providing some degree of hydrophobic interaction with the receptor’s binding pocket, lacked the extensive π-π stacking potential seen in the larger naphthalene ring of Compound 2. This group has two methyl substitutions, which may add bulk but do not significantly enhance binding through π-π or dipole interactions. Moreover, the methyl groups potentially increased steric hindrance without contributing additional polar or electrostatic interactions, resulting in a weaker interaction with the binding site compared to the more planar and electron-dense naphthalene and 2-methylbenzyl groups in Compounds 2 and 4, respectively.

In Compound 3, the cyclohexyl moiety introduced substantial hydrophobic bulk, but as a saturated ring, it lacks the aromatic character needed for π-π interactions with aromatic amino acid residues in the receptor. The cyclohexyl ring might provide limited specificity in binding interactions. Additionally, the cyclohexyl group is conformationally flexible [30], which could reduce binding stability by allowing multiple orientations within the binding pocket, potentially weakening its overall affinity relative to the more rigid aromatic systems in Compounds 2 and 4.

The pharmacokinetic analysis of the piperazine-bearing fluoro-benzoxazolone derivatives highlights their potential as effective alternatives to melatonin for targeting MT1 and MT2 receptors. The higher lipophilicity values observed for Compounds 1–4 suggested improved permeability across cellular membranes, a critical factor for CNS drugs [31]. The ability to penetrate the BBB more effectively than melatonin is particularly valuable, as MT1 and MT2 receptors are predominantly located within the brain [32]. This improved BBB permeability may facilitate stronger and more consistent interactions with these receptors, potentially leading to enhanced efficacy in modulating circadian rhythm and alleviating pain.

Compounds 1, 2, and 4 exhibited significantly higher PPB compared to melatonin. This necessitates careful consideration in dose adjustment. On the other hand, the longer half-life of piperazine-bearing fluoro-benzoxazolone derivatives compared to melatonin may allow them to exert therapeutic effects over an extended period. This pharmacokinetic characteristic aligns well with the therapeutic needs of circadian rhythm modulation, as it supports consistent receptor interaction throughout the day and night cycle [33,34].

The piperazine-bearing fluoro-benzoxazolone derivatives, with their improved pharmacokinetic profiles, may address these limitations by potentially offering enhanced efficacy and longer-lasting effects. The higher affinity for CNS receptors, combined with prolonged half-life and improved BBB penetration, could position these compounds as promising candidates for further development in therapies targeting circadian rhythm disorders and chronic pain.

This study has certain limitations. The absence of in vitro absorption evaluations and in vivo studies prevents definitive conclusions regarding the efficacy of these compounds in biological systems. Future experimental studies could support these findings by examining the pharmacokinetic profiles, bioavailability, and potential toxicity of these compounds. Despite the limitations, the in silico findings supported the potential use of piperazine-bearing fluoro-benzoxazolone derivatives as melatonin receptor agonists in treating chronic pain.

NOTES

Conflicts of Interest

The authors have 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.

Author Contributions

Conceptualization: Ismail Celil Haskologlu, Emine Erdag. Data curation: all authors. Formal analysis: all authors. Investigation: all authors. Methodology: Ismail Celil Haskologlu, Emine Erdag. Software: Emine Erdag. Supervision: Orhan Uludag. Visualization: all authors. Writing—original draft: all authors. Writing—review & editing: all authors.

Funding Statement

None

Acknowledgments

None

Figure 1

Synthesis of 5-fluoro-benzoxazolone derivatives with various piperazine substituents. The reaction scheme illustrates the Mannich reaction of 5-fluoro-2(3H)-benzoxazolone with different piperazine derivatives (R-substituted) to form four distinct compounds.

Figure 2

Comparison of MM/PBSA binding energies of MT1 receptor-ligand complexes for melatonin and synthesized 5-fluoro-benzoxazolone derivatives (Compounds 1–4). Statistical significance is denoted by *p<0.0001, compared to melatonin. MM/PBSA, molecular mechanics/Poisson–Boltzmann surface area.

Figure 3

Comparison of MM/PBSA binding energies of MT2 receptor-ligand complexes for melatonin and synthesized 5-fluoro-benzoxazolone derivatives (Compounds 1–4). Statistical significance is denoted by *p<0.0001, compared to melatonin. MM/PBSA, molecular mechanics/Poisson–Boltzmann surface area.

Table 1

The IUPAC names and detailed characterization analysis of synthesized compounds

Name of ligands	Characterization analysis
5-Fluoro-3-[4-(2,5-dimethylphenyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 1)	¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.3–6.7 (m, 6H, Ar-CH, J=7 Hz), 4.7 (s, 2H, CH₂), 2.9 (t, 4H, pip-CH₂ H₂, H₆, J=5 Hz), 2.8 (t, 4H, pip-CH₂ H₃, H₅, J=5 Hz), 2.3 (s, 3H, CH₃), 2.2 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 155.1, 150.9, 141.1, 136.1, 132.8, 130.9, 129.4, 124.1, 122.6, 119.8, 110.8, 109.8 (Ar-C), 65.1 (CH₂), 51.5, 51.1 (pip-C), 21.2, 17.4. ESI-MS m/z: [M+H]⁺=384.2, [M+Na]⁺=406.2. Calculated for C₁₉H₂₁FN₃O₂: C, 67.85%; H, 6.29%; N, 12.49%; Found: C, 67.70%; H, 6.31%; N, 12.45%.
5-Fluoro-3-[4-naphthalen-2-ylmethylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 2)	¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.1–6.8 (m, 10H, Ar-CH, J=7 Hz), 4.7 (s, 2H, CH₂), 3.9 (s, 2H, CH₂), 2.9 (t, 4H, pip-CH₂ H₃, H₆, J=5 Hz), 2.8 (t, 4H, pip-CH₂ H₃, H₅, J=5 Hz). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 155.1, 150.9, 142.5, 141.1, 136.1, 133.8, 132.8, 130.9, 129.4, 127.3, 125.7, 124.1, 122.6, 119.8, 110.8, 109.8 (Ar-C), 65.1 (CH₂), 60.9 (CH₂), 53.0, 50.5 (pip-C). ESI-MS m/z: [M+H]⁺=434.2, [M+Na]⁺=456.2. Calculated for C₂₃H₂₁FN₃O₂: C, 71.84%; H, 5.50%; N, 10.92%; Found: C, 71.65%; H, 5.53%; N, 10.89%.
5-Fluoro-3-[4-(4-cyclohexylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 3)	¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.3–7.0 (m, 3H, Ar-CH, J=7 Hz), 4.7 (s, 2H, CH₂), 2.7 (t, 4H, pip-CH₂ H₂, H₆, J=5 Hz), 2.5 (t, 4H, pip-CH₂ H₃, H₅, J=5 Hz), 2.2 (m, 1H, cyclohexyl-CH H₁), 1.8 (q, 4H, cyclohexyl-CH₂ H₂, H₆, J=6 Hz), 1.2 (m, 4H, cyclohexyl-CH₂ H₃, H₅), 1.1 (m, 1H, cyclohexyl-CH H₃).¹³C NMR (100 MHz, CDCl₃), δ (ppm): 155.3, 142.5, 132.0, 123.7, 122.5, 109.9 (Ar-C), 64.4 (CH₂), 63.3 (cyclohexyl-CH), 50.9, 48.6 (pip-C), 28.9, 26.2, 25.8 (cyclohexyl-CH₂). ESI-MS m/z: [M+H]⁺=401.3, [M+Na]⁺=423.3. Calculated for C₂₀H₂₅FN₃O₂: C, 68.55%; H, 7.19%; N, 11.99%; Found: C, 68.35%; H, 7.15%; N, 11.96%.
5-Fluoro-3-[4-(2-methylbenzyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 4)	¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.3–6.9 (m, 7H, Ar-CH, J=7 Hz), 4.7 (s, 2H, CH₂), 3.5 (s, 2H, CH₂), 2.9 (t, 4H, pip-CH₂ H₂, H₆, J=5 Hz), 2.8 (t, 4H, pip-CH₂ H₃, H₅, J=5 Hz), 2.3 (s, 3H, CH₃). ¹³C NMR (100 MHz, CDCl₃), δ (ppm): 155.0, 141.0, 137.4, 136.1, 132.9, 130.2, 129.3, 127.1, 125.4, 122.5, 110.7, 109.3 (Ar-C), 64.8 (CH₂), 60.6 (CH₂), 52.7, 50.6 (pip-C), 19.1 (CH₃). ESI-MS m/z: [M+H]⁺=370.2, [M+Na]⁺=392.2. Calculated for C₁₉H₂₁FN₃O₂: C, 67.85%; H, 6.29%; N, 12.49%; Found: C, 67.60%; H, 6.32%; N, 12.45%.

IUPAC, International Union of Pure and Applied Chemistry.

Table 2

The negative docking score (binding affinity) and MM/PBSA binding energies of tested ligands in MT1 receptor

Name of ligands	Ligand-MT1 receptor complex (PDB entry: 7DB6)

	Binding affinity (kcal/mol)	MM/PBSA binding energy (kJ/mol)
5-Fluoro-3-[4-(2,5-dimethylphenyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 1)	−7.3	−179.36±1.24
5-Fluoro-3-[4-naphthalen-2-ylmethylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 2)	−9.1	−262.85±1.38
5-Fluoro-3-[4-(4-cyclohexylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 3)	−7.8	−201.36±1.23
5-Fluoro-3-[4-(2-methylbenzyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 4)	−8.5	−241.63±1.83
N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide (Melatonin)	−6.4	−152.46±1.37

MM/PBSA, molecular mechanics/Poisson–Boltzmann surface area.

Table 3

The negative docking score (binding affinity) and MM/PBSA binding energies of tested ligands in MT2 receptor

Name of ligands	Ligand-MT2 receptor complex (PDB entry: 6ME6)

	Binding affinity (kcal/mol)	MM/PBSA binding energy (kJ/mol)
5-Fluoro-3-[4-(2,5-dimethylphenyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 1)	−7.6	−183.36±1.57
5-Fluoro-3-[4-naphthalen-2-ylmethylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 2)	−8.5	−218.12±1.62
5-Fluoro-3-[4-(4-cyclohexylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 3)	−7.6	−185.25±1.25
5-Fluoro-3-[4-(2-methylbenzyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 4)	−8.0	−203.57±1.42
N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide (Melatonin)	−5.8	−118.26±1.32

MM/PBSA, molecular mechanics/Poisson–Boltzmann surface area.

Table 4

The main interactions between ligands and amino acid residues in the binding sites of MT1 and MT2 receptors

Name of ligand	Receptor	Key residues	Interaction type	Specific Interaction details
5-Fluoro-3-[4-(2,5-dimethylphenyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 1)	MT2	Phe206, Trp264	π-π stacking, hydrophobic	Limited π-π interactions with Phe206 and Trp264
	MT1	Leu229, Val208	Hydrophobic	Hydrophobic interaction without significant π-π stacking
5-Fluoro-3-[4-naphthalen-2-ylmethylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 2)	MT2	Phe206, Trp264, Tyr282	π-π stacking, hydrophobic	Strong π-π stacking with Phe206, Trp264, and Tyr282 due to naphthalene ring
	MT1	Phe179, Tyr282, Trp264	π-π stacking, hydrogen bonding	Additional π-π stacking with Tyr282; strong hydrophobic interaction
5-Fluoro-3-[4-(4-cyclohexylpiperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 3)	MT2	Ser138, Asn162	Hydrogen bonding	Less pronounced hydrophobic interaction due to cyclohexyl group
	MT1	Leu229	Hydrophobic	Limited interaction due to lack of aromatic system
5-Fluoro-3-[4-(2-methylbenzyl)piperazin-1-ylmethyl]-3H-benzoxazol-2-one (Compound 4)	MT2	Phe206, Trp264, Leu229, Val208	π-π stacking, hydrophobic	2-Methylbenzyl group enhances hydrophobic stability, π-π stacking with Phe206, Trp264
	MT1	Phe179, Trp264	π-π stacking, hydrophobic	π-π stacking similar to MT2 interactions but less stable
Melatonin	MT2	Ser138, Asn162	Hydrogen bonding	Key hydrogen bonds with Ser138 and Asn162; limited π-π stacking
	MT1	Leu229, Val208	Hydrophobic	Minimal hydrophobic interaction with receptor residues

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