Anti-inflammatory and analgesic potential of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives: Insights from molecular simulation and in vivo studies

Muhammad Ibrar Khan; Muzaffar Abbas; Humaira Nadeem; Ashraf Ullah Khan; Sana Zafar; Iqra Zulfiqar; Siraj Khan; Muhammad Mustaqeem; Reham M Alahmadi; Ashraf Atef Hatamleh; Eun Kyoung Seo; Salman Khan

doi:10.25259/AJC_163_2025

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Original Article

:19;

1632025

doi:

10.25259/AJC_163_2025

Anti-inflammatory and analgesic potential of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives: Insights from molecular simulation and in vivo studies

Muhammad Ibrar Khan^a,b,c,, Muzaffar Abbas^c, Humaira Nadeem^d, Ashraf Ullah Khan^e,, Sana Zafar^a,b, Iqra Zulfiqar^d, Siraj Khan^f,, Muhammad Mustaqeem^g, Reham M Alahmadi^h, Ashraf Atef Hatamleh^h, Eun Kyoung Seoⁱ, Salman Khan^a,b,*

aPharmacological Sciences Research Lab, Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan

bDepartment of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan

cDepartment of Pharmacology, Faculty of Pharmacy, Capital University of Science & Technology, Islamabad Expressway, Islamabad, Pakistan

dDepartment of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Riphah International University, Islamabad, Pakistan

eDepartment of Pharmacy, City University of Science and Information Technology (CUSIT), Peshawar, KPK, Pakistan.

fDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Capital University of Science & Technology, Islamabad Expressway, Islamabad, Pakistan

gInstitute of Chemistry University of Sargodha, Sargodha, Pakistan

hDepartment of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia.

iCollege of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul, South Korea

*Corresponding author: E-mail address: skhan@qau.edu.pk (S. Khan)

Received: 2025-02-18, Accepted: 2025-10-21, Epub ahead of print: 2026-01-08, Published: 2026-02-02

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Abstract

Inflammation is the central pathway of various pathological condition occurring as a result of injury or infection and severely affects quality of life. It is well documented that cyclooxygenase 2 (COX-2) signaling plays a significant role in potentiating inflammation. In the current study, two 2,3-disubstituted thiazolidine-4-one derivatives, (E)-3-benzyl-2-((Z)-(1-(4-bromophenyl)ethylidene)hydrazono)thiazolidin-4-one (BEHT) and (E)-2-((Z)-(1-(4-chlorophenyl)ethylidene)hydrazono)-3-(4-nitrobenzoyl)thiazolidin-4-one (CEHNT) were synthesized and their anti-inflammatory, anti-oxidant and analgesic potential were investigated. The synthesized compounds were spectroscopically analyzed by infrared (IR) spectroscopy, nuclear magnetic resonance (¹H NMR, and ¹³C NMR). Preliminary total anti-oxidant capacity (TAC), total reducing power (TRP), and free radical scavenging potential were evaluated. In vitro COX-2 inhibitory effect of both compounds were also investigated. Subsequently, acute and sub-acute toxicity of BEHT and CEHNT were investigated. Next, acute analgesic and anti-inflammatory activities which include hot plate test, acetic acid induced writhing behavior, formalin induced paw licking test, and carrageenan induced inflammation model were established. Additionally, molecular docking and simulation were carried out to interpret the variable inhibitory activity of BEHT and CEHNT against COX-2 signaling. The in vitro findings demonstrate promising anti-oxidant, free radical scavenging and COX-2 inhibitory potential of synthesized derivatives. Both compounds show no signs of acute (1000 mg/kg) or subacute toxicity (500 mg/kg) in mice based on histopathology, body weight, and serum biomarkers. Moreover, the compounds exhibit strong anti-inflammatory and analgesic potential in acetic acid-induced writhing behavior, heat-induced hypersensitivity, formalin-induced paw licking test, and carrageenan-induced inflammation model. The molecular docking (MD) and simulation results claimed that BEHT and CEHNT possess strong binding affinities and interaction towards COX-2, demonstrating the mechanism for their anti-inflammatory and analgesic activities. The current findings indicate that BEHT or CEHNT exert marked anti-inflammatory and analgesic activities.

Keywords

COX-2

Docking

Inflammation

Thiazolidin-4-one

Toxicity

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1. Introduction

Acute inflammation is the body’s immune response to injury or infection. It is generally accompanied by pain and swelling and is often vital to the healing process. However, persistent inflammation is associated with increased hypersensitivity, resulting in the initiation of chronic pain. Chronic inflammation and pain are connected to anxiety and depression, and may cause disturbance of daily activities, profoundly affecting the quality of life of an individual [1]. Following injury, acute inflammatory pain occurs due to the release of various cytokines, chemokines, and infiltration of immune cells such as neutrophils and macrophages. Acute inflammatory pain last for a few days and is accompanied by chronic inflammation and pain when unresolved [2-4]. Several studies propose that the pathophysiology of chronic pain implicates a multifaceted interaction between the immune and nervous systems. Chronic pain is a neuroinflammatory ailment facilitated comparably by neuronal and/or non-neuronal cells. The circulating immune cells, which include monocytes, neutrophils, and T-cells, are recruited to the area of tissue injury and/or inflammatory site and frequently intrude into the peripheral and central nervous systems (CNS). The expression of several inflammatory mediators (cytokines/chemokines) is enhanced as a consequence of stimulation of these cells, causing direct sensitization of peripheral or central neurons and act indirectly on immune cells that regulate the chronic pain [5].

Numerous studies have described that inflammation is linked with enhanced production of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and Interleukin-1 beta (IL-1β). Subsequently, TNF-α and IL-1β causes sensitization of several nociceptors, resulting in pain hypersensitivity in response to mechanical and heat stimuli [6,7]. Similarly, exposure to TNF-α and IL-1β triggers the enhanced expression of cyclooxygenase-2 (COX-2) during an inflammatory condition and further exacerbates the acute inflammatory pain response. Additionally, COX-2 is associated with the release of prostaglandins, further worsening the inflammatory condition. Non-steroidal anti-inflammatory drugs (NSAIDS) are commonly used medication that exerts their anti-inflammatory effects by inhibiting the COX-2 enzyme [8-10].

Analgesics are the primary therapeutic medication used to treat acute and chronic inflammatory pain, which includes opioids and NSAIDS. However, the adverse effects of these drugs contribute to considerable declines in health and quality of life. Opioids are associated with life-threatening adverse effects, which include tolerance, addiction, dependence, and respiratory depression. The misuse and abuse of prescription opioids has become highly ubiquitous and concerning [11,12]. In the US, approximately 70,000 deaths occurred due to seizures and respiratory depression caused by opioids [13]. Similarly, NSAIDS are associated with gastrointestinal irritation, constipation, cardiovascular problems, and nephrotoxicity. These challenges potentiate the need for novel, safe, and effective treatment options that promise to suppress the acute and chronic inflammation and inflammatory pain. Thiazolidin-4-one is one of the privileged pharmacophores in medicinal chemistry and is associated with various biological activities, including anti-oxidant, anti-inflammatory, and anti-microbial activities [14]. The current study aims to design and synthesize novel 2,3-disubstituted thiazolidine-4-one derivatives and to investigate their anti-inflammatory and analgesic potential.

2. Materials and Methods

2.1. Chemical and reagents

Thiosemicarbazide, chloroacetophenone, bromoacetophenone, ethyl chloroacetate, benzoyl chloride, 4-Nitrobezoyl chloride, acetic acid, anhydrous acetone, anhydrous potassium carbonate, ethanol, methanol, n-hexane, ethyl acetate, petroleum ether, dimethyl sulfoxide (DMSO), ascorbic acid, etc., were used during the recent study. All the chemicals used in the present study were obtained from Sigma-Aldrich and used without additional purification. Gallenkamp melting point (m.p) apparatus was used to estimate the m.p of compounds. The Thermo Scientific NICOLET IS10 spectrophotometer was used to analyze the IR spectra. A Bruker AM-300 spectrophotometer was used to record nuclear magnetic spectra (¹H NMR) spectra at 300 and 100 MHz, respectively, in deuterated DMSO/CDCl₃ as a solvent and TMS (tetramethylsilane) as an internal standard at Quaid-i-Azam University, Islamabad.

2.2. General procedure for the synthesis of 2,3-disubstituted thiazolidine-4-one derivatives (a-b)

2,3-disubstituted thiazolidine-4-one derivatives (a-b) were synthesized according to the procedure as reported with certain modifications [15]. The synthesis of target compounds has been shown in Schemes 1 & 2. Briefly, the reaction was initiated between thiosemicarbazide and selected carbonyl compounds using ethanol as a reaction medium and acetic acid as a catalyst. As a result, thiosemicarbazone was formed, which reacted with ethyl chloroacetate. The reaction takes place in the presence of sodium acetate using methanol as a reaction medium, leading to the formation of 1,3-thiazolidin-4-one derivatives. Subsequently, thiazolidinones reacted with benzoyl chloride or 4-nitrobenzoyl chloride in the presence of anhydrous acetone and anhydrous potassium carbonate, resulting in the formation of 2,3-disubstituted thiazolidine-4-one derivatives (a-b).

2.2.1. Synthesis of BEHT

A solution of 4-Bromoacetophenone (5 mmol) and thiosemicarbazide (5 mmol) was dissolved in 25 mL ethanol and catalytic amounts (1 or 2 drops) of acetic acid was added to it. The reaction mixture was magnetically stirred for 8 to 24 h at room temperature (RT). The formation of the thiosemicarbazone product was monitored by thin-layer chromatography (TLC) (petroleum ether and ethyl acetate, 2:1) and dried under vacuum. The intermediate thiosemicarbazone (5 mmol) was then reacted with ethyl 2-chloroacetate (5 mmol) and sodium acetate (5 mmol) in methanol (25 mL) at RT for 24 h. The subsequent intermediate product was further reacted with benzyl chloride to obtain the final product (E)-3-benzyl-2-(((Z)-1-(4-bromophenyl)ethylidene)hydrazineylidene)thiazolidin-4-one (BEHT). The final product was purified by column chromatography. C₁₈H₁₆BrN₃OS; yield 74%; R_f = 0.4, m.wt.;402.31, Fourier-transform infrared (FTIR) cm^-1(C=N) 1604.77, (C=C) 1570.05, (C=O) 1705.07 (Figure S1), ¹H NMR (300 MHz, DMSO-d₆) δ ppm 2.47 (s, 3H, methyl),4.0 (s,2H, thiazolidine), 4.8 (s, 2H, CH2), 7.23-7.40 (m, 5H, Ar-H) 7.60-7.65 (d, 2H, Ar-H) 7.75-7.80 (d,2H, Ar-H) (Figure S2),¹³C NMR (DMSO-d₆, 100 MHz, δ ppm): 13.86 (CH₃), 32.09, 46.77 (CH₂),128.41, 133.88, 136.50 (Ar–C), 146.52 (C=N), 178.97 (C=O) (Figure S3).

Figure S1

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Figure S2

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Figure S3

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2.2.2. Synthesis of CEHNT

A solution of 4-Chloroacetophenone (5 mmol) and thiosemicarbazide (5 mmol) was dissolved in 25 mL of ethanol, and catalytic amounts (1 or 2 drops) of acetic acid were added to it. The reaction mixture was magnetically stirred for 8 to 24 h at RT. The formation of the thiosemicarbazone product was monitored by TLC (petroleum ether and ethyl acetate, 2:1) and dried under vacuum. The intermediate thiosemicarbazone (5 mmol) was then reacted with ethyl 2-chloroacetate (5 mmol) and sodium acetate (5 mmol) in methanol (25 mL) at RT for 24 h. The subsequent intermediate product ((Z)-2-(((Z)-1-(4-chlorophenyl)ethylidene)hydrazineylidene)thiazolidin-4-one) was further reacted with benzoyl chloride to obtain the final product BEHT. The final product was purified by column chromatography. C₁₈H₁₃ClN₄O₄S; yield 70%; R_f = 0.5, m.wt.;416.84, FTIR cm^-1(NO) asymmetric stretching 1523.68, symmetric stretching 1338.59 (C=C) 1579.70, (C=O) 1703.14, (C=N) 1602.05 (Figure S4), ¹H NMR (300 MHz, CDCl₃) δ ppm 2.47 (s, 3H, methyl), 3.88, (s, 2H, thiazolidine), 7.4-7.6, (t, 2H, Ar-H) 7.98, (m, 2H, Ar-H) 7.6, (m, 2H, Ar-H) 7.9, (m, 2H, Ar-H) 8.44 (m, 2H, Ar-H) (Figure S5), ¹³C NMR (CDCl₃, 100 MHz, δ ppm): 14.93, 32.38, 123.78, 127.99, 128.58, 129.53, 136.18, 142.68, 147.64, 161.86, 162.16, 171.82 (Figure S6).

Figure S4

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Figure S5

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Figure S6

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2.3. In vitro anti-oxidant potential of BEHT and CEHNT

2.3.1. Total antioxidant capacity and total repression power

The total antioxidant capacity (TAC) and total reducing power (TRP) of both compounds (BEHT and CEHNT) were estimated as reported previously [16-18]. Briefly, TAC was determined by mixing the sample solution (DMSO 4 mg/mL) with ammonium molybdate, sulfuric acid, and sodium phosphate, followed by incubation (90°C for 95 min). Later, the absorbance was measured at 645 nm by a spectrophotometer. Similarly, TRP was evaluated by incubating the sample solution with phosphate buffer (pH 6.6) and potassium ferricyanide (1%), followed by trichloroacetic acid (10%). Next, ferric chloride was added, and absorbance was determined at 700 nm. In both assays, DMSO was used as a negative control, while ascorbic acid was used as a positive control. The results were presented as μg ascorbic acid equivalent per mg (μg AAE/mg) of sample.

2.3.2. Free radical scavenging assay

In order to investigate the free radical scavenging capacity of BEHT and CEHNT, the DPPH (1, 1-diphenyl-2-picryl-hydrazyl) assay was performed [19,20]. In short, the sample solution (DMSO 4 mg/mL) was mixed with DPPH agent and added to the 96-well plate. The samples were then subjected to incubation at 37°C for 1 h, and absorbance was calculated at 517 nm. The reference standard used in the current assay was ascorbic acid, while DMSO was used as a negative standard. The fraction of the sample’s radical scavenging potential is determined by the following formula (Eq. 1):

(1)

% scavenging activity = (1 - A_{s} {/A}_{n}) \times 1 00.

A_s = DPPH solution absorbance with the sample.

A_n = negative control absorbance (having the reagent solution with no sample).

2.3.3. COX-2 enzyme inhibitory assay

The COX-2 enzyme inhibitory assay was performed following established protocols [21]. The method is based on the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), resulting in a measurable colorimetric change. BEHT and CEHNT were tested in three different concentrations (10, 25, and 50 µM) in triplicate using a 96-well plate, with celecoxib (10 µM) used as the reference inhibitor. Test samples were pre-incubated with the enzyme in Tris-HCl buffer (100 mM, pH 8.0, containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 1 µM hematin) for 5 min at 30°C. The reaction was started by the addition of arachidonic acid (150 µM) and permitted to continue for 5 min. Afterwards, the absorbance was recorded at 590 nm using a microplate reader. Percent (%) inhibition was calculated comparative to the vehicle control (1% DMSO).

2.4. Analgesic and anti-inflammatory potential of BEHT and CEHNT

2.4.1. Animals

BALB/c mice, 5-6 weeks old of either sex, weighing 25-30 g, were used in the present study. The animals were preserved in a controlled environment (seven animals per cage) at RT (24 ± 1°C) and control humidity (37.5 ± 5%) with open access to food and water. The animal experiments in the recent study were accomplished in the facility of the faculty of pharmacy, Capital University of Science & Technology (CUST), Islamabad, under the guidelines of the bio-ethical committee (REC-CUST-PH-01-23).

2.4.2. Hot plate test

Eddy’s hot plate was used to investigate the anti-nociceptive effect of synthesized compounds [22,23]. The male BALB/c mice were allocated to the following groups (n=5 mice/group). Group 1 received only normal saline (10 mL/kg, i.p., negative control). Group 2 was administered tramadol (1 mg/kg, i.p., positive control). Groups 3, 4, and 5 were administered BEHT or CEHNT at different doses (1, 5, 10 mg/kg, i.p.). All the treatments were dissolved in 2% DMSO and were administered prior to testing. The temperature was set at 54 ± 0.5°C on the hot plate, and the pain threshold was noted at different time intervals. To avoid tissue damage, 30 seconds was considered the cut-off time.

2.4.3. Writhing test

The present study explored the analgesic potential of synthesized compounds (BEHT & CEHNT) in the acetic acid-induced writhing test. The animals were allocated into five groups (n=5 mice/group). The control group was administered normal saline (10 mL/kg, i.p.), the positive control received tramadol (1 mg/kg, i.p.), treatment groups received BEHT or CEHNT at various doses (1, 5, 10 mg/kg, i.p.). Acetic acid (1%, 10 mL/kg, i.p.) was injected into each animal after 30 min of treatment, and were observed for writhing behavior (abdominal constrictions) according to previous protocols [24,25].

2.4.4. Formalin-induced paw licking test

Formalin test was accomplished to assess the effect of BEHT or CEHNT on formalin-induced pain response as reported [26,27]. Briefly, 2.5% formalin (20 µL) was administered via the intraplanter (i.pl.) route to each animal’s right hind paw. Diclofenac sodium (5 mg/kg, i.p.) and BEHT or CEHNT (1, 5, 10 mg/kg, i.p.) were given 30 min before formalin administration. All the mice were observed for paw licking, flinching, or biting as indicative of pain response in two different phases. In the early phase, the pain response was measured for the initial 5 min, while in the late phase, the readings were recorded from 20-40 min.

2.4.5. Carrageenan-induced inflammation model in mice

In order to explore the anti-inflammatory potential of BEHT or CEHNT, an acute inflammation pain model induced by carrageenan was established in mice with slight modification [28,29]. Female mice weighing 25-30 g and 5-6 weeks old were acclimatized 1 week before the experiment, provided with normal food and water. The animals were then subjected to various groups (n=7 mice/group) as discussed below.

Group I: normal saline (10 mL/kg, i.p.), normal control

Group II: normal saline + 1% carrageenan (20 µL, intraplanter, i.pl.), negative control

Group III: diclofenac sodium (5 mg/kg, i.p.) + carrageenan, positive control.

Group IV: BEHT (1 mg/kg, i.p.) + carrageenan

Group V: BEHT (5 mg/kg, i.p.) + carrageenan

Group VI: BEHT (10 mg/kg, i.p.) + carrageenan

Group VII: CEHNT (1 mg/kg, i.p.) + carrageenan

Group VIII: CEHNT (5 mg/kg, i.p.) + carrageenan

Group IX: CEHNT (10 mg/kg, i.p.) + carrageenan

All the treatments were dissolved in normal saline and 2% DMSO and given 30 min prior to carrageenan induction. The paw edema and nociceptive pain behavior (mechanical allodynia and heat hyperalgesia) were investigated before treatment (0 hr) and after carrageenan administration (0.5, 1, 2,4, and 6 h).

2.4.5.1. Measurement of carrageenan-induced pain hypersensitivity

In the current study, mechanical allodynia and the hot plate test were executed to examine the effect of BEHT or CEHNT on carrageenan-induced pain hypersensitivity at different time intervals. Briefly, the animals were placed in a transparent chamber and habituated for 30 min prior to testing. Subsequently, von-frey filaments were perpendicularly applied to the right hind paw, and the paw withdrawal threshold (PWT) was recorded as reported previously. Afterwards, the Eddy hot plate was used to evaluate carrageenan-induced heat hyperalgesia. Animals were positioned individually in the hot plate, and paw withdrawal latency (PWL) or jumping response was observed. To protect tissue from any damage, 30 s was selected as the cut-off time as reported [29-31].

2.4.5.2. Paw edema test

The paw edema was evaluated in the carrageenan-induced inflammation in all the mice according to established procedures [30,32]. The paw edema was assessed in millimeter (mm) using a paw thickness meter (No. 2046 F, Mitutoyo, Kawasaki, Japan). To determine the anti-inflammatory potential of the newly synthesized derivative, the mice were given Diclofenac (5 mg/kg i.p.) and BEHT or CEHNT at various doses (1, 5, 10 mg/kg i.p.). The paw edema was recorded at various intervals of time (0, 30 min, 1,2, 4, & 6 h) following carrageenan induction.

2.5. Acute toxicity

The acute toxicity was performed according to the established OECD guidelines No. 425 [33]. Briefly, female mice (n = 5), 5-6 weeks old, were randomly selected for the acute toxicity analysis in three different groups. The control group was administered normal saline (300 mL/kg, i.p.). The toxicity group was administered a single dose of BEHT or CEHNT (1000 mg/kg, i.p.). The animals were frequently and independently observed for mortality or any clinical or behavioral signs of toxicity following dosing for the initial 24 h. The first 6 h were given special attention after administration of a high dose. Afterward, the animals were continuously assessed for the consecutive 7 days.

2.6. Subacute toxicity

The subacute toxicity study of BEHT and CEHNT was carried out in compliance to the OECD Guidelines No. 407 [34]. Female mice (n = 5) and 5-6 weeks’ old (weight 20-25 g) were randomly assigned into three diverse groups. Normal saline (300 ml/kg, i.p.) was administered to the control group. The toxicity group was administered BEHT or CEHNT (500 mg/kg, i.p.). To investigate any signs of general toxicity or behavioral changes, all the treatments were injected for a duration of 15 days. During the study, the animals were observed for body weight changes at various time intervals. Afterward, the animals were sacrificed on day 15, and the toxic effect, if any, of BEHT or CEHNT was evaluated on major organs (liver, kidney, and lungs) using hematoxylin & eosin (H & E) staining. Moreover, the blood samples were collected and investigated for serum biochemical markers, including the liver enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as renal function markers (Creatinine, Urea, and Uric acid).

2.7. Haematoxylin & Eosin staining

In order to explore the subacute toxic effect of BEHT or CEHNT (500 mg/kg, i.p.) on various organs, H & E staining was performed. Briefly, the animals were administered BEHT or CEHNT for 15 consecutive days. At the end of the experiment, all the organs (liver, kidney, and lungs) were collected, followed by formalin (10%) fixation. Subsequently, the organs were subjected to dehydration and paraffin embedding. The paraffin-embedded block were then cut into thin slices (4 µm) using microtome. The thin slices were fixed on microscopic slides and stained with H & E as reported [35].

2.8. Molecular docking studies

Molecular docking investigation of the BEHT and CEHNT was carried out against the protein target, i.e., COX-2. The protein was downloaded from the research collaboratory for structural bioinformatics-protein data bank (RCSB-PDB) site (https://www.rcsb.org) as a PDB file, and the PDB ID of the protein was 1CX2. The protein preparation was carried out by deleting the water molecules, co-crystalized ligand, and adding polar hydrogens, and the undesired chains were removed using UCSF Chimera Software. Both the Compounds were prepared in the ChemDraw software_16 and saved as PDB files. The energies of both compounds were minimized, following the addition of polar hydrogens. The configuration file from the co-crystalized ligand was retrieved and used during the docking studies. The AutoDock v1.1.2 software was used for the molecular docking analysis as reported previously [36-38].

2.9. Molecular dynamic (MD) simulation

The MD simulation was carried out using the GROMACS 2021.4-2 software to measure the dynamic stability of the protein-ligand system. During the MD simulation, the topologies of protein and ligand were produced. The CHARMM27 force field was used, and for solvation TIP3 box was used. The energy of the system was minimized using the steepest descent model. The system was equilibrated in different phases, i.e., moles, volume, temperature (NVT) and moles, pressure, temperature (NPT). The NVT equilibration was performed for 1 ns, while the NPT equilibration was performed for 2 ns. The final MD production run will be performed for 50 ns. The various components that were estimated during MD simulation include root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (RoG), solvent accessible surface area (SASA), and H-bonding as reported previously [39-42].

2.10. MM-PBSA, MM-GBSA, and per-residue decomposition

In this study, molecular mechanics poisson-boltzmann surface area (MMPBSA) and molecular mechanics generalized born surface area (MMGBSA) methods were employed to calculate the binding free energy of the ligand-protein complex after molecular dynamics (MD) simulation. The calculations were performed using the GROMACS-MMPBSA tool and decomposition analysis was conducted to assess the contribution of individual residues to the binding affinity. The MD trajectory was first processed by removing solvent and ions, followed by generating snapshots for MMPBSA/MMGBSA analysis. For each snapshot, binding free energy was determined by considering the van der Waals, electrostatic, polar, and non-polar solvation energy components. A total of 500 snapshots, evenly spaced throughout the equilibrated part of the simulation, were selected to ensure statistically robust energy estimates [40,41,43].

2.11. Methodology and results integrated narrative

In the present study, MMPBSA and MMGBSA analysis was performed to assess the bending free energy after MD simulation and determine the dynamic stability of both complexes. For the binding free energy calculations, the input file was generated and for this 1-500 snapshots were selected. The different parameters that were calculated for both the MM-PBSA and MM-GBSA analysis includes electrostatic energy (EEL), polar solvation energy (ENPOLAR), non-polar solvation energy (ENSURF), Poisson-Boltzmann/Generalized-Born Solvation Energy (EPB/EGB), Gas phase energy (GGSAS), solvation energy (GSOLV), van der Waals (VDWAALS), and total energy. Similarly, the per-residue decomposition analysis was executed to conclude the involvement of specific amino acid to the ligand and protein binding. The results were visualized using the gmx_mmpbsa ana function as reported.

2.12. Principle component analysis (PCA) and Gibbs free energy landscape

In the present study, the PCA analysis was performed for both the complexes to assess the ligand-protein complex molecular dynamic trajectories. The PCA analysis is employed to reduce the dimensionality for the assessment of the collective motions within the system by converting the trajectory data into orthogonal principle components. The initial few principal components were extracted to assess the key variations in the atomic position throughout the course of simulation. During this analysis, the C-alpha atoms are focused to identify the ligand binding associated with key conformational changes. After the PCA analysis, the 2D projection was used for the visualization of the resulting principal components, this visualization corresponds to the ligand-protein complex distinct cluster of conformations. This visualization reflects the stability and flexibility of the protein in complex with ligand, identifying the potential signaling of the conformational changes that can hinder or facilitate the binding. Furthermore, the Gibbs free energy landscape (FEL) was used to assess the ligand binding thermodynamics. The 3D graph of the FEL was generated by plotting the two key principal components obtained from the PCA against the free energy. The FEL showed the ridges and valleys that determine the unstable and stable conformation of the complexes. The favorable energy of the binding states is represented by the depth of the valleys, while energy barriers of the conformational states are represented by the height [44-46].

2.13. Statistical analysis

In the present study, GraphPad Prism (Version 8.0.2) was used for assessing statistical significance. The normality of distribution was evaluated by the Shapiro-Wilk test, the Kolmogorov-Smirnov test, & D’Agostino test. Afterwards, to determine statistical significance among various groups, one-way analysis of variance (ANOVA) or two-way repeated measure (RM) ANOVA was used, followed by post hoc Tukey’s multiple comparisons test. The data were plotted as mean ± standard error of mean (SEM) or mean ± standard deviation (SD). *P<0.05, **P<0.01, ***P<0.001 demonstrate a marked difference between the treatment and the negative control group. ^#P<0.05, ^##P<0.01, ^###P<0.001 show marked difference between normal and negative control group.

3. Results and Discussion

3.1. In vitro antioxidant activities of BEHT and CEHNT

3.1.1. Total antioxidant capacity and reducing power evaluation of BEHT and CEHNT

The present study investigates the TAC and TPR of newly synthesized BEHT and CEHNT by TAC and TRP assay. The results demonstrated that both compounds show promising antioxidant and reducing power activities. CEHNT was noted to have slightly better TAC (251.2) and TRP (186.2) activities as compared to BEHT. : The TAC and TRP potential of BEHT were noted as 174.4 and 117.5, respectively. The results were stated as µg AAE/mg of sample, as indicated in Figure 1.

In vitro TAC and TRP activities of BEHT and CEHNT. The results indicated that BEHT and CEHNT show marked TAC and TRP activities.

3.1.2. Radical scavenging activity (DPPH) of BEHT and CEHNT

The free radical scavenging potential of newly synthesized BEHT and CEHNT was determined by DPPH assay with slight modification in the protocols. The results of the DPPH assay indicated that both compounds exhibit marked anti-oxidant activities, which is reflected by the decolorization of DPPH to yellow colored diphenyl picrylhydrazine. The percent (%) free radical scavenging potntial of BEHT and CEHNT were noted as 27.6±3.05 and 39.1±1.72, respectively. The results were indicated as percentage ± SD.

3.1.3. Inhibitory effect of BEHT and CEHNT on COX-2 enzyme

The results of the COX-2 inhibitory assay showed that the synthesized compounds promisingly inhibit the COX-2 enzyme activity. The inhibitory effects of both compounds were observed to be concentration-dependent. The highest percent inhibition for BEHT and CEHNT at 50 µM was noted as 63 and 70%, respectively. The reference drug, elecoxib (10 µM) showed 87% inhibition capacity. These-findings signify the inhibitory effect of BEHT and CEHNT on the COX-2 enzyme. The results have been demonstrated in Figure 2.

In vitro COX-2 inhibitory activity of BEHT and CEHNT at 10, 25, and 50 µM compared with the reference inhibitor celecoxib (10 µM). Data are expressed as mean ± SD (n = 3). Both compounds revealed a concentration-dependent COX-2 inhibition, with maximum activity at 50 µM approaching that of celecoxib.

3.2. Effect of BEHT and CEHNT on heat-induced hyperalgesia

The inhibitory effects of BEHT and CEHNT were investigated against heat-induced pain hypersensitivity. Mice that showed pain hypersensitivity <15 s at 54 ± 0.5°C were designated for the study. The results indicated that tramadol (1 mg/kg, i.p.) substantially attenuates heat-induced paw withdrawal threshold (PWT). Similarly, the heat-induced pain hypersensitivity was markedly suppressed by BEHT and CEHNT (5, 10 mg/kg, i.p.), and the PWL was significantly enhanced in a dose-dependent manner as compared to the control group. Moreover, the maximum inhibitory response was observed after 60 min of drug administration. However, BEHT or CEHNT (1 mg/kg, i.p.) shows no significant inhibitory effects on heat-induced hyperalgesia. The results of BEHT or CEHNT have been indicated in Figure 3 and Figure 4, respectively.

Effects of BEHT (1, 5, & 10 mg/kg) in (a) heat-induced hyperalgesia, (b) acetic acid-induced writhing behavior, and (c) formalin (2.5%)-induced paw licking in mice. Data were presented as mean ± SE (n = 5 mice/group). Statistical analyses were performed using one-way or two-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001 showed statistical significance among treatment and control group.

Effects of CEHNT (1, 5, & 10 mg/kg) in (a) heat-induced hyperalgesia, (b) acetic acid-induced writhing behavior, and (c) formalin (2.5%)-induced paw licking in mice. Data were presented as mean ± SE (n = 5 mice/group). Statistical analyses were performed using one-way or two-way ANOVA followed by Tukey’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.001 showed statistical significance among treatment and control group.

3.3. Analgesic effect of BEHT and CEHNT on acetic acid-induced writhing pain response

In the current study, administration of acetic acid (1%, 10 mL/kg, i.p.) induced the writhing pain behavior indicated as abdominal constriction in all the saline or drug-treated groups. The saline-treated (10 mL/kg, i.p.) control group was observed with the maximum number of writhing responses. However, pre-treatment with BEHT or CEHNT (1, 5, 10 mg/kg, i.p.) potentially suppresses acetic acid-induced writhing pain behavior as compared to the saline-treated group. Furthermore, the pain-inhibitory effects were observed in a dose-dependent manner. Similarly, prior administration of tramadol (1 mg/kg, i.p.) markedly suppressed the acetic acid-induced writhing response in comparison to the control group. The results of BEHT or CEHNT have been indicated in Figures 3(b) and 4(b), respectively.

3.4. Effect of BEHT and CEHNT on formalin-induced paw licking response

The current study assessed the inhibitory effect of BEHT or CEHNT on formalin-induced nociception. Administration of formalin (2.5%, 20 µL i.pl.) to the right hind paw of each animal pre-treated with normal saline (10 mL/kg, i.p.) causes a significant increase in paw licking, flinching, or biting as indicative of pain nociception in both phases of the formalin test. Similar results were observed in the initial phase (0-5 min) of the formalin test in all treatment groups. However, the formalin-induced nociception was promisingly reduced in the late phase (20-40 min) of the formalin test when pre-treated with diclofenac (2, 5 mg/kg, i.p.) or BEHT (1, 5, 10 mg/kg, i.p.) or CEHNT (5, 10 mg/kg, i.p.) when compared to the control group. Additionally, the inhibitory effect of BEHT or CEHNT was dose-dependent. Also, the current results revealed that CEHNT (1 mg/kg, i.p.) shows no significant inhibitory effect on formalin-induced pain behavior in the late phase as well. The results of BEHT or CEHNT have been indicated in Figures 3(c) and 4(c), respectively.

3.5. Anti-nociceptive effect of BEHT and CEHNT on carrageenan-induced pain hypersensitivity

In the current study, we examined the anti-nociceptive effects of BEHT or CEHNT in the carrageenan-induced mechanical allodynia and heat hyperalgesia. Carrageenan (20 µL, i.pl.) administration into the right hind paw significantly increases the nociceptive response (PWT or PWL) to mechanical or heat stimuli as compared to the normal group. The current results revealed that pre-treatment with diclofenac (5 mg/kg, i.p.) profoundly ameliorates the carrageenan-induced pain hypersensitivity in response to mechanical or heat stimuli as compared to the saline-treated (10 mL/kg, i.p.) carrageenan group. Similar effects were observed in BEHT or CEHNT treated groups at doses of 5 or 10 mg/kg administered via the i.p route. However, BEHT or CEHNT (1 mg/kg, i.p.) showed no significant anti-nociceptive effects as compared to the carrageenan group. All the results have been presented in Tables 1 and 2.

Table 1. Effects of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives, BEHT or CEHNT (1, 5, & 10 mg/kg, i.p.) on carrageenan-induced mechanical allodynia in mice.

Treatment	Baseline	30 min	1 h	2 h	4 h	6 h
Normal	2.11±0.88	1.83±0.29	2.11±0.88	2.11±0.88	2.20±0.82	2.11±0.88
Carrageenan	2.20±0.82	0.27±0.20^###	0.24±0.19^##	0.27±0.20^##	0.21±0.14^##	0.48±0.17^#
Diclofenac (5 mg/kg)	2.11±0.88	1.14±0.44*	1.17±0.21***	1.11±0.30***	1.31±0.66*	1.26±0.44*
BEHT (1 mg/kg)	2.03±0.92	0.42±0.34	0.69±0.38	0.62±0.30	0.60±0.41	0.66±0.25
BEHT (5 mg/kg)	2.20±0.82	0.71±0.28	1.03±0.41*	1.00±0.55	0.80±0.35*	0.94±0.28*
BEHT (10 mg/kg)	2.29±0.88	1.06±0.37**	1.17±0.47***	1.00±0.33**	1.14±0.50*	1.06±0.28**
CEHNT (1 mg/kg)	2.40±1.13	0.51±0.38	0.77±0.37	0.57±0.21	0.51±0.40	0.69±0.23
CEHNT (5 mg/kg)	2.29±0.76	0.74±0.25*	1.09±0.54*	0.86±0.34*	0.91±0.40*	0.97±0.50
CEHNT (10 mg/kg)	2.14±0.90	0.97±0.37*	1.46±0.47**	1.09±0.54*	1.29±0.54*	1.20±0.45*

Assessment of mechanical allodynia using von-frey test. The values are expressed in grams, reflecting the force required to elicit a PWT. Each value represents the mean PWT in the respective experimental groups. Higher filament weights correspond to higher mechanical thresholds, signifying reduced pain hypersensitivity to mechanical stimuli. Data were presented as mean ± SD, (n = 7 mice/group). Statistical analyses were performed using 2-way RM ANOVA followed by Tukey’s multiple comparisons test. *P<0.05, **P<0.01, ***P<0.01 indicate significant difference between treatment and carrageenan group. #P<0.05, ##P<0.01, ###P<0.001 show marked difference between normal and carrageenan group.

Table 2. Effects of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives, BEHT or CEHNT (1, 5, & 10 mg/kg, i.p.) on carrageenan-induced heat hyperalgesia in mice.

Treatment	Baseline	30 min	1 h	2 h	4 h	6 h
Normal	27.1±1.57	27.6±2.30	26.6±1.62	27.0±1.73	26.4±1.81	27.0±0.82
Carrageenan	26.1±1.46	13.6±1.90^###	13.0±2.16^###	11.4±2.07^###	12.0±1.91^###	13.0±2.16^###
Diclofenac (5 mg/kg)	26.4±1.81	17.7±1.80*	17.6±2.37*	16.6±1.72**	17.4±2.23**	19.1±2.54**
BEHT (1 mg/kg)	25.6±1.27	13.1±1.77	13.9±2.34	14.1±1.35	15.3±1.80	15.4±1.90
BEHT (5 mg/kg)	25.7±1.89	14.0±1.41	15.6±1.40	15.7±2.06*	17.9±2.54**	18.4±1.27**
BEHT (10 mg/kg)	25.1±1.57	17.0±1.41*	18.4±2.51**	19.1±2.48***	19.6±1.62***	19.1±2.67**
CEHNT (1 mg/kg)	26.0±1.63	14.3±1.38	15.1±2.12	14.6±1.51	14.9±2.12	14.3±2.14
CEHNT (5 mg/kg)	27.0±2.00	15.3±1.80	16.7±1.80*	16.9±1.86**	18.4±2.37**	17.0±1.29*
CEHNT (10 mg/kg)	25.9±1.57	18.0±2.31*	19.0±2.16**	18.3±1.11***	19.7±3.04**	20.0±2.00***

Assessment of heat-induced pain hypersensitivity using the hot plate test. The values are expressed in seconds (s), reflecting the time required to elicit a PWL. Each value represents the mean PWL in the respective experimental groups. Increased PWL corresponds to higher thermal thresholds, signifying reduced pain hypersensitivity to heat stimuli. Data were presented as mean ± SD (n = 7 mice/group). Statistical analyses were performed using 2-way RM ANOVA followed by Tukey’s multiple comparisons test *P<0.05, **P<0.01, ***P<0.01 indicate significant difference between treatment and carrageenan group. ###P<0.001 shows a marked difference between the normal and carrageenan group.

3.6. Anti-inflammatory effect of BEHT and CEHNT on carrageenan-induced paw edema

Carrageenan-induced inflammatory pain model was established to explore the inhibitory effect of BEHT or CEHNT on carrageenan-induced paw edema. It was noted that 1% carrageenan (20 µL, i.pl.) administration into the right hind paw markedly enhanced paw edema in saline-treated (10 mL/kg, i.p.) carrageenan group when compared to the normal group. However, the carrageenan-induced paw edema was significantly reversed by diclofenac (5 mg/kg, i.p.) or BEHT or CEHNT (5, 10 mg/kg i.p.) dose dependently as compared to the carrageenan group. Moreover, BEHT or CEHNT (1 mg/kg, i.p.) produced no profound effects on carrageenan-induced paw edema (Table 3). Additionally, representative images of each group were captured from the carrageenan-induced paw during the acute inflammation model. Figures 5(a-c) show the representative images of different groups captured at various time intervals. The images clearly indicate the anti-inflammatory effects of BEHT or CEHNT dose dependently as evidence from reduction in paw swelling and edema. The supplementary images of each group (n =3) is provided in supplementary file (Figure S8 a-i).

Figure S8

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Table 3. Effects of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives, BEHT or CEHNT (1, 5 & 10 mg/kg. i.p.) on carrageenan induced paw edema in mice.

Treatment	Baseline	30 min	1 h	2 h	4 h	6 h
Normal	1.94±0.13	1.96±0.14	1.96±0.13	1.93±0.11	1.96±0.10	1.94±0.10
Carrageenan	2.01±0.12	2.67±0.20^####	2.74±0.19^###	2.64±0.15^###	2.64±0.11^###	2.59±0.15^###
Diclofenac (5 mg/kg)	2.03±0.88	2.21±0.44**	2.23±0.21**	2.20±0.30**	2.14±0.66***	2.13±0.44**
BEHT (1 mg/kg)	1.99±0.09	2.56±0.10	2.63±0.11	2.53±0.11	2.56±0.10	2.51±0.12
BEHT (5 mg/kg)	1.99±0.11	2.36±0.10	2.44±0.10*	2.41±0.09	2.36±0.08**	2.41±0.12
BEHT (10 mg/kg)	1.97±0.08	2.37±0.10	2.33±0.11**	2.36±0.10*	2.34±0.10**	2.26±0.11**
CEHNT (1 mg/kg)	2.01±0.13	2.54±0.15	2.57±0.8	2.57±0.8	2.50±0.12*	2.50±0.12
CEHNT (5 mg/kg)	2.00±0.08	2.37±0.05*	2.43±0.08*	2.40±0.06*	2.41±0.07**	2.36±0.08**
CEHNT (10 mg/kg)	1.94±0.08	2.29±0.07*	2.31±0.07**	2.30±0.10**	2.30±0.12***	2.26±0.10***

Paw edema test investigating inflammation after carrageenan administration in different experimental groups. Paw thickness was measured in millimeters (mm) at various time points. An increase in paw thickness indicates enhanced inflammatory response. Data were presented as mean ± SD, (n = 7 mice/group). 2-way RM ANOVA followed by Tukey’s multiple comparisons test were used to analyze the data statistical. *P<0.05, **P<0.01, ***P<0.01 indicate significant difference between treatment and carrageenan group. ###P<0.001 shows a marked difference between the normal and carrageenan group.

Anti-inflammatory effect of BEHT or CEHNT on carrageenan-induced paw inflammation. (a-c) Representative images of carrageenan-induced right hind paw of different groups were captured at various time intervals (0, 1, 2, 4, & 6 h). The images indicated that both BEHT & CEHNT (1, 5, 10 mg/kg, i.p.) dose dependently reverse carrageenan-induced inflammation in paw tissue, reflecting the effectiveness of the synthesized compounds.

3.7. Acute and sub-acute toxicity analysis of BEHT and CEHNT in mice

In the recent study, a high dose of BEHT or CEHNT (1000 mg/kg, i.p.) was administered to all the mice to observe any signs of mortality or general toxicity. After administration of an acute high dose, the mice were investigated for 7 days. The results indicated that during the observation, none showed any signs of clinical toxicity or death. Similarly, no visible signs of mortality and clinical toxicity were noted during the sub-acute toxicity analysis of BEHT or CEHNT (500 mg/kg, i.p.) in the entire 15-day treatment. The animals treated with both compounds show no marked changes in body weight (Table 4). At the end of 15 days, the animals were sacrificed, and the major organs were processed for histopathological examination. However, during the sub-acute toxicity studies, no histopathological changes were observed in the kidney, liver, and lungs, signifying the safety of both BEHT and CEHNT (Figure 6). The supplementary H & E images of each group (n =3) have been provided in the supplementary file (Figure S7a-c). Similarly, BEHT and CEHNT treatment demonstrated no signification alteration in serum biochemical parameters (ALT, AST, urea, creatinine, and acetic acid) as compared to the normal group, which further confirms the safety of synthesized compounds (Table 5).

Figure S7

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Table 4. Effect of BEHT and CEHNT (500 mg/kg, i.p) on the body weight of mice during subacute toxicity.

Day	Normal	BEHT (500 mg/kg)	CEHNT (500 mg/kg)
0	22.0 ± 1.2	22.8 ± 1.6	21.4 ± 1.1
1	22.2 ± 1.1	23.0 ± 1.7	21.8 ± 0.8
3	22.6 ± 0.9	24.2 ± 1.5	22.8 ± 1.1
5	23.0 ± 1.2	25.0 ± 1.7	23.8 ± 1.1
7	24.8 ± 1.5	26.0 ± 1.6	25.2 ± 0.8
9	25.2 ± 1.5	26.6 ± 1.7	25.6 ± 1.3
12	26.2 ± 0.8	26.4 ± 1.5	25.8 ± 1.3
15	27.6 ± 1.1	27.2 ± 1.3	26.8 ± 1.3

The values are expressed as mean±SD (n = 5 mice/group). The body weight was measured in grams (g). Statistical significance was performed using two-way RM ANOVA followed by the Tukey multiple comparison test. The results showed that treatment with BEHT or CEHNT cause no substantial changes in the mice’s body weight, as compared to the normal group demonstrated no apparent toxicity.

H & E staining sections of the kidney (a-c) from mice (n = 3). G: glomeruli; RT: renal tubules. H & E staining sections of liver (d-f) from mice (n = 3). CV: central vein; green arrow: hepatocytes; black arrow: sinusoids. H & E staining sections of lung (g-i) from mice (n = 3). B: bronchioles; A: alveoli. All the group shows normal histological architecture.

Table 5. Effect of BEHT and CEHNT (500 mg/kg) on serum biochemical markers in sub-acute toxicity.

Parameters	Normal	BEHT (500 mg/kg)	CEHNT (500 mg/kg)
Serum ALT (SGPT, U/L)	24.7 ± 3.5	24.0 ± 6.2	26.7 ± 4.0
Serum AST (SGOT, U/L)	19.3 ± 4.0	26.0 ± 6.0	21.7 ± 5.7
Serum Urea (mg/dL)	16.0 ± 3.1	15.0 ± 2.6	18.0 ± 3.0
Serum Creatinine (mg/dL)	0.6 ± 0.12	0.8 ± 0.15	0.7 ± 0.06
Serum Uric acid (mg/dL)	3.2 ± 0.31	3.4 ± 0.35	3.2 ± 0.42

The values are presented as mean±SD. Statistical significance was performed using one-way ANOVA followed by Tukey multiple comparison test. The results revealed no significant changes in serum biochemical parameters as compared to normal group demonstrated no potential toxicity.

3.8. Molecular docking

The molecular docking of BEHT and CEHNT was investigated against the COX-2 (PDB ID: 1CX2) using the AutoDock vina. The molecular docking analysis showed the binding energy of the BEHT with COX-2 as -9.0 kcal∙mol^-1, while the CEHNT exhibited the binding energy of -7.3 kcal∙mol^-1 with the COX-2 protein. The molecular docking is visualized using Discover Studio Visualizer, and the results showed that both compounds interacted with the target protein via numerous hydrophilic and hydrophobic bonds, as shown in Figure 7. CEHNT shows the formation of the conventional hydrogen bond with GLN A:350, TYR A:355, SER A:579, and PHE A:580. Moreover, CEHNT shows non-conventional hydrogen and hydrophobic binding affinities with HIS A:351 and HIS A:356, respectively. Similarly, BEHT formed a single conventional hydrogen bond against TYR A:355 while many hydrophobic interactions were noted against various amino acids, including ARG A:120, ARG A:120, LEU A:531, LEU A:359, VAL A:116, TYR A:348, LEU A:352, VAL A:349, VAL A:349, VAL A:349, VAL A:349, TRP A:387, TRP A:387, MET A:522, MET A:522, LEU A:384, VAL A:523, and ALA A:527.

Computational (docking) analysis of BEHT and CEHNT against COX-2. The binding affinities of (a, b) BEHT and (c, d) CEHNT BEHT and CEHNT were investigated using AutoDockVina. The results were visualized as 3D and 2D images using Discovery Studio Visualizer 2021. The green color in 2D images represents conventional hydrogen bonds while other colors represent hydrophobic interactions.

3.9. MD simulation analysis

The MD simulation was performed to establish the dynamic stability of the ligand-protein complex. The numerous parameters that were estimated during MD simulation include RMSD, RMSF, RoG, SASA, and H-bonds. The RMSD assesses the mean distance of the ligand-protein atoms over time compared to the reference structure. Lower the value of RMSD indicates a more stable complex, while a higher value of the RMSD indicates a less stable complex. The results of Figure 8 indicate that the BEHT-COX-2 complex is more stable having a lower RMSD value, and the RMSD value fluctuates between 0.2 to 0.4 nm, while the CEHNT-protein complex is less stable as compared to the BEHT-COX-2 complex. In the case of the CEHNT-COX-2 complex, initially the value of RMSD is lower, but a sharp increase was observed after 10 ns. The RMSD value fluctuates between 0.3 to 0.8 nm in the case of CEHNT-COX-2 complex and shows less stability over time in contrast to BEHT-COX-2 complex. The RMSF indicates the fluctuation of each residue within the complex over time. The lower RMSF indicates less flexibility and vice versa. The results of Figure 9 indicate that both complexes show a similar pattern; however, the BEHT showed more fluctuation at 3000, 4000, and 6000 (number of residue) as compared to the CEHNT. This fluctuation indicates that within certain region of the protein when BEHT shows more fluctuation and proteins are more flexible at certain region after binding with the BEHT. The RoG indicates the overall compactness of the protein after binding with the ligand. The lower RoG suggest that protein overall maintain the compactness throughout the simulation period following binding with the ligand. The results of the Figure 10 indicate that RoG value of both complexes remain stable and no significant deviation or fluctuation was observed. The protein maintained its structure and compactness throughout the simulation. The SASA gives insight into the exposure of the protein or ligand-protein complex to solvent. The stable SASA indicates that ligand-protein complex remains stable during exposure to the solvent. The results of the present study indicate that both complexes remain stable during exposure to the solvent and no significant deviation have been observed, as shown in the Figure 11. During MD simulation, the number of hydrogens bond between the ligand and COX-2 over the course of time were evaluated. The present study showed that the CEHNT form maximum of five hydrogen bonds, while the BEHT form maximum of 4 bonds with the COX-2 protein as shown in the Figure 12.

RMSD plot of BEHT–COX-2 and CEHNT–COX-2 complexes following MD simulation for 50 ns. BEHT–COX-2 complex shows greater stability with RMSD values ranging from 0.2 to 0.4 nm, while CEHNT–COX-2 exhibits higher fluctuations (0.3–0.8 nm), indicating lower stability.

The RMSF analysis of the BEHT-COX2 and CEHNT-COX2 complexes for 50 ns and changes in the complexes over 50 ns course of time. RMSF plot of BEHT–COX-2 and CEHNT–COX-2 complexes showing similar overall patterns. Advanced fluctuations were observed in the BEHT complex around residues 3000, 4000, and 6000, specifying improved flexibility in those regions upon binding.

The RoG analysis of the BEHT and CEHNT with the COX2 protein over the course of 50 ns. The RoG plot of BEHT–COX-2 and CEHNT–COX-2 complexes indicates stable values throughout the simulation, showing maintained structural compactness with no substantial deviations.

SASA plot of both compound complexes with COX-2 over the simulation period (50 ns), displaying stable solvent exposure with no substantial deviations, showing structural stability.

Hydrogen bond analysis of CEHNT and BEHT with COX-2 protein over the course of 50 ns, showing a maximum of five and four hydrogen bonds, respectively.

3.10. MM-PBSA, MM-GBSA, and per-residue decomposition

Before generating the snapshots for MM-PBSA, MM-GBSA, and per-residue calculations, the trajectory of the MD simulation was prepared by removing the ions and solvents. The binding free energy for each snapshot was measured by assessing the parameters, such as electrostatic, van der Waals, non-polar solvation, and solvation energy components. During the binding free energy calculations, 500 snapshots were selected through the simulation and evenly spaced for statistically robust energy calculations. The results of the MM-PBSA and MM-GBSA for both complexes revealed that total binding energy was negative and contributed to favorable binding energy, with significant contributions from the electrostatic, van der Waals, GGAS, and non-polar solvation energy components, as shown in Figures 13-16. The per-residue decomposition was performed to assess the contribution of the key amino acids or residues towards ligand binding interaction. The results showed various key residues involved in the interaction with both ligands, and their corresponding favorable energy contributions are shown in Figures 17 and 18. The per-residue decomposition analysis showed that some of the parameters exhibited a similar pattern with the MM-PBSA and MM-GBSA but different energies.

(a-h), MM-PBSA analysis of BEHT-COX-2 complex. The results demonstrate MM-PBSA binding free energy analysis of BEHT–COX-2 complex, showing negative total binding energies with major contributions from van der Waals, electrostatic, and non-polar solvation components, indicating favorable ligand–protein interactions. (a) The EEL shows negative binding energy, (b) represent ENPOLAR energy is also negative, (c) shows the EPB energy, which is positive and unfavorable, (d) shows the GGAS which is favorable, (e) shows the GSOLV energy, which is positive and unfavorable, (f) represents VDWAALS energy, which is negative and favorable, (g) represents the total energy which is negative and indicates favorable binding interaction between the ligand-protein system. However, the (h) represent the GGAS (EEL and VDWAALS) and GSOLV (EPB and ENPOLAR) and total energy of the system.

MM-GBSA binding free energy analysis of the BEHT–COX-2 complex. The results indicate favorable binding with negative total free energy. Marked contributions were detected from electrostatic, van der Waals, and non-polar solvation energy components. (a) The EEL shows negative binding energy, (b) represent ENPOLAR energy is also negative, (c) shows the EPB energy, which is positive and unfavorable, (d) shows the GGAS which is favorable, (e) shows the GSOLV energy, which is positive and unfavorable, (f) represents VDWAALS energy, which is negative and favorable, (g) represents the total energy which is negative and indicates favorable binding interaction between the ligand-protein system. However, the (h) represent the GGAS (EEL and VDWAALS) and GSOLV (EPB and ENPOLAR) and total energy of the system.

MM-PBSA analysis of the CEHNT-COX-2 protein complex for 500 frames. MM-PBSA binding free energy analysis of CEHNT–COX-2 complex, indicating negative total binding energies with significant contributions from van der Waals, electrostatic, and non-polar solvation components, signifying favorable ligand–protein interactions. (a) The EEL shows negative binding energy, (b) represent ENPOLAR energy is also negative, (c) shows the EPB energy, which is positive and unfavorable, (d) shows the GGAS which is favorable, (e) shows the GSOLV energy, which is positive and unfavorable, (f) represents VDWAALS energy, which is negative and favorable, (g) represents the total energy which is negative and indicates favorable binding interaction between the ligand-protein system. However, the (h) represent the GGAS (EEL and VDWAALS) and GSOLV (EPB and ENPOLAR) and total energy of the system.

MM-GBSA analysis of the CEHNT-COX-2 protein complex for 500 frames. The results show favorable binding with negative total free energy. Electrostatic, van der Waals, and non-polar solvation energies contributed significantly to the overall binding. (a) The EEL shows negative binding energy, (b) represent ENPOLAR energy is also negative, (c) shows the EPB energy, which is positive and unfavorable, (d) shows the GGAS which is favorable, (e) shows the GSOLV energy, which is positive and unfavorable, (f) represents VDWAALS energy, which is negative and favorable, (g) represents the total energy which is negative and indicates favorable binding interaction between the ligand-protein system. However, the (h) represent the GGAS (EEL and VDWAALS) and GSOLV (EPB and ENPOLAR) and total energy of the system.

Per-residue energy decomposition analysis of the BEHT-COX-2 protein complex. The results highlighting important amino acid residues contributing favorably to BEHT binding through significant van der Waals and electrostatic interactions. (a) represents the total energy of the per-residue decomposition analysis using the Poisson-Boltzmann equation, (b) represents the total decomposition analysis using the Generalized-Born surface area equation, (c) shows the heatmap analysis of each frame and each amino acid involved in the ligand-protein interaction and their respective energy (kcal/mol) using Poisson-Boltzmann equation, (d) shows the total energy of each amino acid and ligand energy (kcal/mol) using Poisson-Boltzmann equation, (e) shows the heatmap analysis of each frame and each amino acid involved in the ligand-protein interaction and their respective energy (kcal/mol) using Generalized-Born surface area equation, (f) shows the total energy of each amino acid and ligand energy (kcal/mol) using Generalized-Born surface area equation.

Pre-residue decomposition analysis of the CEHNT-COX-2 protein complex identifying key residues with favorable energy contributions to CEHNT binding, primarily through electrostatic and van der Waals interactions. (a) represents the total energy of the per-residue decomposition analysis using the Poisson-Boltzmann equation, (b) represents the total decomposition analysis using the Generalized-Born surface area equation, (c) shows the heatmap analysis of each frame and each amino acid involved in the ligand-protein interaction and their respective energy (kcal/mol) using Poisson-Boltzmann equation, (d) shows the total energy of each amino acid and ligand energy (kcal/mol) using Poisson-Boltzmann equation, (e) shows the heatmap analysis of each frame and each amino acid involved in the ligand-protein interaction and their respective energy (kcal/mol) using Generalized-Born surface area equation, (f) shows the total energy of each amino acid and ligand energy (kcal/mol) using Generalized-Born surface area equation.

3.11. Principle component analysis and Gibbs free energy landscape

The 2D plot of the BEHT-COX-2 complex showed FEL with the PC1 on the X-axis and PC2 on the Y-axis. The PCA analysis color gradient ranges from blue to red, indicating the free energy scale. The red color shows the regions with high energy and less thermodynamically stable regions, while the blue color indicates low energy regions with high thermodynamic stability. The BEHT complex with the COX-2 revealed well-defined blue regions, i.e., PC1=0.25, and PC2=-0.25, which is a highly stable conformation within this region. This indicates that this area is highly thermodynamically favorable and stable for this component, and the system exists in this state more often. The 3-D visualization of the BEHT with the COX-2 reveals clear imaging of the energy basin, and the downwards depression of the surface gives clear indications of the existence of the free energy minimum. The smooth slope moving towards the basin shows the transition state between different states. Similarly, the 2D visualization of the CEHNT compared to the BEHT reveals multiple basins of low energy as evident from the purple and blue colors, which shows that the system is under several different stable conformational states compared to BEHT when projected onto these components. These sub-states with different conformations suggest that during the MD simulation, the system adopts. Furthermore, the 3D plot of the CEHNT exhibited a more significant difference in height in contrast to BEHT, which shows that the energy barriers towards the stable state are higher than those of BEHT, and more energy is required for the system to transition between these states, as shown in Figure 19.

PCA and Gibbs FEL of (a, b) BEHT–COX-2 and (c, d) CEHNT–COX-2 complexes. BEHT exhibits a well-defined, thermodynamically stable energy basin, while CEHNT displays multiple low-energy conformational states with higher energy barriers, indicating greater structural variability. The free energy landscape unit is kj/mol, while the images are system generated and the 2D images on the Y-axis only represent the scale of energy i.e., kj/mol only. While the 3D images on x-axis and y-axis only shows the principal components only i.e., PC1 and PC2.

3.12. Discussion

Injury or infection to tissues is accompanied by an acute inflammatory response, whereas chronic inflammation is maladaptive and provokes a substantial amount of adverse pain. The inflammatory mediators, such as proinflammatory cytokines and chemokines, that are released during an inflammatory condition, act on nociceptive nerve endings, causing a marked increase in pain hypersensitivity, potentiating allodynia and hyperalgesia [47]. Multiple studies reported the role of COX-2 enzyme, TNF-α, and IL-1β in developing the inflammatory insult in response to tissue injury or infection. Subsequently, an inflammatory response leads to the production of reactive oxygen species (ROS) and a decrease in the antioxidant level, resulting in oxidative stress. It is well documented that oxidative stress is significantly correlated with inflammation [48]. In the current study, we designed and synthesized two novel 2,3-disubstitued thiazolidine-4-one derivatives and investigated their antioxidant and anti-inflammatory potential. Moreover, the compounds were also evaluated for their toxicological effects.

Preliminary in vitro activities were performed to investigate the antioxidant potential of BEHT and CEHNT. TAC and TRP were evaluated as measures of antioxidant potential as reported in previous studies [49]. The findings revealed that both compounds demonstrated promising total anti-oxidant capacity and reducing power, proposing significant anti-oxidant potential of BEHT and CEHNT. Similarly, percent radical scavenging potential was also investigated using the DPPH assay [20], and it was noted that both compounds show substantial radical scavenging capacity, suggesting their antioxidant potential. Numerous studies reported that inflammatory response is associated with ROS generation, accompanied by oxidative damage of tissues due to compromise antioxidant defense system. Antioxidants play a major role in overcoming the enhanced production of ROS during inflammation and improve the antioxidant defense system [50-52]. The current investigation revealed consistent findings that BEHT and CEHNT possess promising anti-oxidant potential, suggesting their effectiveness in resolving the inflammatory processes by suppressing the oxidative stress induced by ROS. generation.

Furthermore, the in vitro COX-2 inhibitory potential of BEHT and CEHNT was assessed at concentrations of 10, 25, and 50 µM. Both compounds demonstrated a concentration-dependent rise in inhibition when compared with the vehicle control (1% DMSO). Celecoxib (10 µM), used as the reference inhibitor, produced strong inhibition as expected. Notably, BEHT and CEHNT at 50 µM exhibited inhibition levels approaching those of celecoxib, showing marked COX-2 suppressive potential. These findings suggest that both compounds interact promisingly with the COX-2 enzyme, evidenced by the decrease oxidation of tetramethylphenylenediamine (TMPD) and corresponding reduction in absorbance at 590 nm. The observed inhibitory activity of BEHT and CEHNT further supports their potential role as anti-inflammatory agents, consistent with the results obtained in vivo. The current investigation provides preliminary but convincing evidence that BEHT and CEHNT possess significant COX-2 inhibitory activity, justifying further mechanistic and structural studies [53].

Inflammatory response is associated with improved mechanical and heat-induced pain hypersensitivity. It is well documented that pro-inflammatory cytokines sensitize nociceptors, causing pathological pain such as hyperalgesia and allodynia [54,55]. The recent research further demonstrates the acute anti-inflammatory effects of BEHT and CEHNT in in vivo studies. Initially, writhing behavior induced by acetic acid, heat hyperalgesia, and paw licking behavior (formalin-induced) were evaluated. The newly synthesized BEHT and CEHNT treatment ameliorated writhing response, paw licking or flinching, and heat-induced pain hypersensitivity. Additionally, the response of both compounds (BEHT & CEHNT) was noted in dose dose-dependent manner, and the maximum effect was observed at 10 mg/kg. These findings strongly suggest the analgesic and anti-inflammatory activities of BEHT and CEHNT, signifying their pharmacological effectiveness in alleviating pain and inflammation. Subsequently, an acute inflammation pain model induced by carrageenan was established to further confirm the anti-inflammatory potential of BEHT and CEHNT. Several studies described that carrageenan application into the plantar area of mice significantly induced paw edema and nociception, accompanied by pain hypersensitivity [56]. The same findings were observed in the current studies. However, prior administration of newly synthesized BEHT and CEHNT promisingly reverses carrageenan-induced paw edema and mechanical or heat-induced pain hypersensitivity. The anti-inflammatory response of both BEHT and CEHNT observed was dose-dependent. The findings of the present experiment revealed that BEHT or CEHNT at 1 mg/kg show no significant effect. However, BEHT or CEHNT at 5 or 10 mg/kg produced promising anti-inflammatory effects and suppressed the pain hypersensitivity and paw edema significantly. These results further emphasize the anti-inflammatory and anti-nociceptive potential of BEHT and CEHNT, suggesting their effectiveness in attenuating inflammatory conditions.

It is well documented that inflammation occurs in response to tissue damage or injury, resulting in the consequent production of pro-inflammatory cytokines such as TNF-α and IL-1β. These cytokines result in stimulation and increased expression of COX-2. As a result, release of prostaglandins occurs, which is associated with the initiation of inflammation and pain [57,58]. Numerous studies reported the induction of inflammation by carrageenan administration into the plantar area of mice. Similar studies demonstrate the role of COX-2 in triggering the inflammatory condition accompanied by pain hypersensitivity in response to carrageenan [59]. In the recent study, BEHT and CEHNT were targeted against carrageenan-induced inflammation. To understand the possible mechanism of BEHT and CEHNT against inflammation and inflammation-induced pain, molecular docking and MD simulation were performed against COX-2. These studies revealed promising binding affinities of BEHT and CEHNT, demonstrated by hydrogen and hydrophobic interaction with COX-2. Likewise, both compounds show stable complex formation with COX-2, which is illustrated by hydrogen bonding and significant residue formation with COX-2 over the course of 50 ns. The PCA and Gibbs FEL also revealed thermodynamically stable and favorable complex formation between the synthesized compounds and COX-2. These findings signify the anti-inflammatory role of synthesized compounds via targeting the COX-2 signaling.

Acute and subacute toxicity were performed in order to evaluate the toxic effects of BEHT and CEHNT on general animal behavior and histological changes according to OECD Guidelines No. 407. The potential histopathological changes of BEHT and CEHNT on major organs kidney, liver, and lungs, were investigated using H & E staining after sub-acute dosing to mice. The current findings demonstrated that administering a single acute high dose of BEHT or CEHNT (1000 mg/kg, i.p.) shows no significant alteration in mice’s general behavioral responses and appearance. Additionally, no signs of toxicity or mortality were observed during the 7-day time period. Similarly, during subacute toxicity studies, the animals were kept under observation for 15 15-day period after administering BEHT or CEHNT (500 mg/kg/day, i.p.). The findings revealed no signs of toxicity or behavior changes. Moreover, the animals were sacrificed on day 15, and the major organs kidney, liver. and the lungs were collected to investigate any signs of histological toxicity. It was noted that BEHT or CEHNT shows no signs of histological toxicity in the kidney, liver, or lungs. The current conclusions propose that both BEHT and CEHNT show a wide range of therapeutic safety during in vivo study.

4. Conclusions

The present study investigates the anti-inflammatory and analgesic activities of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives (BEHT & CEHNT). The recent findings demonstrate that both compounds show antioxidant potential in vitro. The compounds possess promising acute analgesic and anti-inflammatory effects in vitro and in vivo. Moreover, no evidence of acute or subacute toxicity was found. Additionally, BEHT and CEHNT show strong binding affinities and interaction against COX-2, and this is confirmed by in vitro COX-2 enzyme inhibition, which demonstrates a possible molecular mechanism for their anti-inflammatory or analgesic potential. However, a more in-depth investigation should be performed to strengthen the current findings.

Acknowledgment

The authors extend their appreciation to the Ongoing Research Funding program (ORF - 2025 - 543) King Saud University, Riyadh, Saudi Arabia. This research was funded by the Researchers Supporting Project (No. 14609), National Research Programme for Universities (NRPU), Higher Education Commission (HEC), Pakistan.

CRediT authorship contribution statement

Muhammad Ibrar Khan: Conceptualization, Investigation, Methodology, Software, Visualization, and Writing-original draft. Muzaffar Abbas: Conceptualization, Project administration, Software, Co-supervision, Validation, and Visualization. Humaira Nadeem: Conceptualization, Methodology. Ashrafullah Khan: Software, Visualization, and Writing-review-editing. Sana Zafar: Investigation, Methodology, and Writing-review-editing. Iqra Zulfiqar: Methodology, Investigation. Siraj Khan: Visualization, Writing-review-editing. Muhammad Mustaqeem: Software, Visualization. Reham M Alahmadi: Visualization, Funding acquisition. Ashraf Atef Hatamleh: Visualization, Funding acquisition. Salman Khan: Conceptualization, Data curation, Formal analysis, Project administration, Software, Validation, Visualization, and Supervision.

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.

Declaration of generative AI and AI-assisted technologies in the writing process

The authors confirm that there was no use of artificial intelligence (AI)-assisted technology for assisting in the writing or editing of the manuscript and no images were manipulated using AI.

Supplementary data

Supplementary material to this article can be found online at https://dx.doi.org/10.25259/AJC_163_2025.

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1. Introduction

2. Materials and Methods

2.1. Chemical and reagents
2.2. General procedure for the synthesis of 2,3-disubstituted thiazolidine-4-one derivatives (a-b)
2.3. In vitro anti-oxidant potential of BEHT and CEHNT
2.4. Analgesic and anti-inflammatory potential of BEHT and CEHNT
2.5. Acute toxicity
2.6. Subacute toxicity
2.7. Haematoxylin & Eosin staining
2.8. Molecular docking studies
2.9. Molecular dynamic (MD) simulation
2.10. MM-PBSA, MM-GBSA, and per-residue decomposition
2.11. Methodology and results integrated narrative
2.12. Principle component analysis (PCA) and Gibbs free energy landscape
2.13. Statistical analysis

3. Results and Discussion

3.1. In vitro antioxidant activities of BEHT and CEHNT
3.2. Effect of BEHT and CEHNT on heat-induced hyperalgesia
3.3. Analgesic effect of BEHT and CEHNT on acetic acid-induced writhing pain response
3.4. Effect of BEHT and CEHNT on formalin-induced paw licking response
3.5. Anti-nociceptive effect of BEHT and CEHNT on carrageenan-induced pain hypersensitivity
3.6. Anti-inflammatory effect of BEHT and CEHNT on carrageenan-induced paw edema
3.7. Acute and sub-acute toxicity analysis of BEHT and CEHNT in mice
3.8. Molecular docking
3.9. MD simulation analysis
3.10. MM-PBSA, MM-GBSA, and per-residue decomposition
3.11. Principle component analysis and Gibbs free energy landscape
3.12. Discussion

4. Conclusions

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Anti-inflammatory and analgesic potential of newly synthesized 2,3-disubstituted thiazolidine-4-one derivatives: Insights from molecular simulation and in vivo studies

Abstract

Keywords

COX-2

Docking

Inflammation

Thiazolidin-4-one

Toxicity

1. Introduction

2. Materials and Methods

2.1. Chemical and reagents

2.2. General procedure for the synthesis of 2,3-disubstituted thiazolidine-4-one derivatives (a-b)

2.2.1. Synthesis of BEHT

2.2.2. Synthesis of CEHNT

2.3. In vitro anti-oxidant potential of BEHT and CEHNT

2.3.1. Total antioxidant capacity and total repression power

2.3.2. Free radical scavenging assay

2.3.3. COX-2 enzyme inhibitory assay

2.4. Analgesic and anti-inflammatory potential of BEHT and CEHNT

2.4.1. Animals

2.4.2. Hot plate test

2.4.3. Writhing test

2.4.4. Formalin-induced paw licking test

2.4.5. Carrageenan-induced inflammation model in mice

2.4.5.1. Measurement of carrageenan-induced pain hypersensitivity

2.4.5.2. Paw edema test

2.5. Acute toxicity

2.6. Subacute toxicity

2.7. Haematoxylin & Eosin staining

2.8. Molecular docking studies

2.9. Molecular dynamic (MD) simulation

2.10. MM-PBSA, MM-GBSA, and per-residue decomposition

2.11. Methodology and results integrated narrative

2.12. Principle component analysis (PCA) and Gibbs free energy landscape

2.13. Statistical analysis

3. Results and Discussion

3.1. In vitro antioxidant activities of BEHT and CEHNT

3.1.1. Total antioxidant capacity and reducing power evaluation of BEHT and CEHNT

3.1.2. Radical scavenging activity (DPPH) of BEHT and CEHNT

3.1.3. Inhibitory effect of BEHT and CEHNT on COX-2 enzyme

3.2. Effect of BEHT and CEHNT on heat-induced hyperalgesia

3.3. Analgesic effect of BEHT and CEHNT on acetic acid-induced writhing pain response

3.4. Effect of BEHT and CEHNT on formalin-induced paw licking response

3.5. Anti-nociceptive effect of BEHT and CEHNT on carrageenan-induced pain hypersensitivity

3.6. Anti-inflammatory effect of BEHT and CEHNT on carrageenan-induced paw edema

3.7. Acute and sub-acute toxicity analysis of BEHT and CEHNT in mice

3.8. Molecular docking

3.9. MD simulation analysis

3.10. MM-PBSA, MM-GBSA, and per-residue decomposition

3.11. Principle component analysis and Gibbs free energy landscape

3.12. Discussion

4. Conclusions

Acknowledgment

CRediT authorship contribution statement

Declaration of competing interest

Declaration of generative AI and AI-assisted technologies in the writing process

Supplementary data

References

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