In Vitro antileishmanial evaluation, molecular docking, dynamics simulations, and DFT Studies of tetrahydropyridine derivatives as potential trypanothione reductase inhibitors

Abstract

Leishmaniasis, a neglected tropical disease caused by Leishmania parasites, lacks safe and effective treatments due to drug resistance and toxicity. This study introduces novel tetrahydropyridine (THP) derivatives as potential inhibitors of trypanothione reductase (TR), a unique redox enzyme essential for parasite survival. Eight THP analogs were synthesized and screened for antileishmanial activity against Leishmania major promastigotes (strain MRHO/IR/75/ER) using the MTT assay, revealing compound A5 as the most potent (IC₅₀ = 89.2 ± 4.5 nM) with favorable selectivity over J774 macrophages. Structure-activity relationship analysis highlighted the role of electron-withdrawing substituents (e.g., Cl at R₁) in enhancing potency, while bulky or highly polar groups (e.g., NO₂ at R₂ in A7) diminished efficacy. Molecular docking against L. infantum TR (PDB: 6T95) demonstrated strong binding affinities for active compounds, with A5 forming key π-π stacking interactions (Trp21, Tyr110) and π-sulfur interactions (Met113). Stability was confirmed via 100-ns molecular dynamics simulations, showing low RMSD and RMSF values and persistent interactions. Density functional theory (DFT) calculations at the B3LYP/6–31 + G(d, p) level correlated electronic descriptors (e.g., wider HOMO-LUMO gap and higher hardness in A5) with superior biological activity, docking scores, and ADME (Absorption, Distribution, Metabolism, and Excretion) profiles. In silico pharmacokinetic predictions indicated good oral absorption and low blood-brain barrier penetration, supporting drug-likeness. Overall, this integrated phenotypic and computational approach identifies THP scaffolds, particularly A5, as promising leads for developing orally bioavailable antileishmanial agents targeting TR, paving the way for further optimization and in vivo validation.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-50258-y.

Introduction

Leishmaniasis remains one of the most neglected tropical diseases, affecting over 12 million people worldwide, with approximately 0.7 to 1 million new cases annually across more than 98 endemic countries^1,2. Caused by protozoan parasites of the genus Leishmania and transmitted by phlebotomine sand flies, the disease manifests in cutaneous (CL), mucocutaneous (MCL), and visceral (VL) forms, the latter being fatal if untreated³. In the Old World, Leishmania major is the primary etiological agent of zoonotic cutaneous leishmaniasis, particularly in the Middle East, including Iran, where the reference strain MRHO/IR/75/ER has been widely used in experimental studies due to its well-characterized virulence and reproducibility in BALB/c mouse models^4,5.

Current chemotherapy depends on a limited range of drugs, including pentavalent antimonials, amphotericin B, miltefosine, and paromomycin. However, these agents are limited by severe toxicity, prolonged treatment durations, high costs, parenteral administration, and increasing drug resistance⁶. The lack of an effective vaccine further highlights the urgent need for novel, safe, and orally bioavailable antileishmanial agents with innovative mechanisms of action⁷.

The unique redox metabolism of trypanosomatids presents attractive drug targets that are absent in mammalian hosts. Unlike humans, who rely on glutathione and glutathione reductase (GR) for thiol homeostasis, Leishmania spp. depend on trypanothione [N¹,N⁸-bis(glutathionyl)spermidine] and trypanothione reductase (TR) to maintain intracellular reducing conditions^8,9. TR catalyzes the NADPH-dependent reduction of trypanothione disulfide (TS₂) to dihydrotrypanothione [T(SH)₂], which subsequently detoxifies reactive oxygen species and reduces tryparedoxin, thereby protecting the parasite against oxidative stress and supporting DNA synthesis¹⁰. Genetic knockdown studies have demonstrated that TR is essential for parasite survival, infectivity, and virulence in L. donovani, L. infantum, andL. major¹¹. Moreover, the significant structural divergence between leishmanial TR and human GR (only ~ 38% sequence identity) makes TR an ideal target for selective inhibition⁸.

Heterocyclic scaffolds, particularly nitrogen-containing six-membered rings, have emerged as privileged structures in antileishmanial drug discovery due to their ability to interact with diverse biological targets¹². Tetrahydropyridine (THP) derivatives, in particular, have demonstrated remarkable pharmacological versatility, exhibiting anticancer, antimicrobial, antiviral, and antiparasitic activities^13,14. Several THP-containing natural products and synthetic analogs have shown promising antiprotozoal effects; for example, marine-derived tetrahydro-β-carbolines and piperidine alkaloids disrupt parasite mitochondrial membrane potential and induce programmed cell death in promastigotes¹⁵. Despite these advances, systematic investigations of THP-based inhibitors targeting trypanothione reductase (TR) remain scarce, with most efforts historically focused on sterol 14α-demethylase (CYP51) or dihydrofolate reductase-thymidylate synthase (DHFR-TS)^16–18.

Building on the validated druggability of trypanothione reductase (TR) and the underexplored potential of tetrahydropyridine (THP) scaffolds in redox-targeted antileishmanial therapy, this study evaluated eight novel THP derivatives-previously synthesized in our laboratory¹⁹ for their growth-inhibitory effects against Leishmania major promastigotes (strain MRHO/IR/75/ER) using the MTT colorimetric assay. Cytotoxicity was simultaneously assessed in J774 murine macrophages to calculate selectivity indices. To investigate the molecular basis of the observed activities, molecular docking studies were performed against the crystal structure of Leishmania infantum trypanothione reductase (PDB ID: 6T95). The stability of protein–ligand complexes for the most active derivatives was further examined through 100-ns molecular dynamics simulations. Density functional theory (DFT) analyses were conducted to correlate frontier molecular orbital energies and global reactivity descriptors with biological data. Finally, comprehensive in silico ADME profiling was performed to assess drug-likeness and pharmacokinetic feasibility.

Through this integrated phenotypic–computational approach, we aimed to (i) identify tetrahydropyridine derivatives with enhanced antileishmanial activity compared to the parent scaffold, (ii) establish preliminary structure–activity relationships, and (iii) provide a structurally characterized foundation for the future development of TR-targeted antileishmanial agents with improved efficacy and acceptable safety profiles.

Rational design

In the pursuit of novel trypanothione reductase (TR) inhibitors for leishmaniasis chemotherapy, the design of our tetrahydropyridine (THP) derivatives was based on successful nitrogen-containing heterocyclic scaffolds reported in previous antileishmanial studies. Partially saturated six-membered heterocycles, particularly 1,4-dihydropyridine (1,4-DHP) and tetrahydropyrimidine rings, have emerged as promising leads due to their ability to establish key π–π stacking and hydrogen-bonding interactions within the TR active site. For instance, 1,4-dihydropyridine derivatives have demonstrated significant antileishmanial activity against both promastigote and amastigote forms of Leishmania major (IC₅₀ = 24–50 µM)²⁰. Furthermore, Harmaline˗a naturally occurring β-carboline alkaloid featuring a partially saturated piperidine moiety˗has been identified as a competitive TR inhibitor (IC₅₀=99 µM) that induces apoptosis-like death in promastigotes. Its nitrogen-containing framework serves as a template for hydrogen-bonding interactions with proper aminoacids in the active site of the enzyme²¹.

Ultimately, the antileishmanial assessment confirmed that dihydropyridine derivatives containing chlorophenyl substituents are active against Leishmania, showing moderate to good efficacy in inhibiting both its promastigote and amastigote life stages (EC₅₀ =2–89µM)²². Guided by these structural precedents, we selected the tetrahydropyridine ring as the central scaffold. The strategic incorporation of electron-withdrawing groups (e.g., Cl at R₁) and moderately hydrophobic substituents at R₂ was intended to replicate favorable binding modes observed in the TR active site (PDB: 6T95), while preserving structural flexibility for subsequent optimization of membrane permeability and cytotoxicity. This bioinspired design strategy culminated in the identification of derivative A5, which exhibited a nanomolar IC₅₀ against L. major promastigotes, establishing a robust foundation for next-generation TR-targeted antileishmanial agents.

Fig. 1.

Selected drugs and identified candidates featuring tetrahydropyridine-like scaffolds demonstrating diverse antileishmanial effects.

Although none of the five WHO-recommended antileishmanial drugs (miltefosine, liposomal amphotericin B, meglumine antimoniate, sodium stibogluconate, paromomycin) or pentamidine contain a tetrahydropyridine moiety, this nitrogen-containing six-membered heterocycle is a well-established privileged structure in medicinal chemistry²³. The 1,4-dihydropyridine (1,4-DHP) core forms the structural basis of numerous blockbuster cardiovascular drugs, including nifedipine (Adalat^®), amlodipine (Norvasc^®), felodipine (Plendil^®), and nicardipine (Cardene^®)²⁴. More recently, nilvadipine-a 1,4-DHP derivative-has successfully completed Phase III clinical trials for Alzheimer’s disease (NCT02017340), further validating the scaffold’s CNS permeability and long-term tolerability²⁵.To the best of our knowledge, this study represents one of the first systematic evaluations of tetrahydropyridine derivatives as potential trypanothione reductase inhibitors in the context of antileishmanial drug discovery. By bridging a clinically validated privileged scaffold-widely exploited in cardiovascular therapeutics (e.g., nifedipine, amlodipine)-with the unique redox metabolism of Leishmania, we introduce a novel chemotype to the neglected field of cutaneous leishmaniasis chemotherapy.

Fig. 2.

Chemical structures of selected FDA-approved dihydropyridine calcium channel blockers prescribed for the treatment of hypertension and cardiac arrhythmias.

Materials and methods

Chemicals

The DHP compounds used in this study were synthesized in our laboratory as previously described by Mirjalili BBF. The detailed synthesis procedure, spectroscopic data, and characterization of the compounds are reported in that publication¹⁹. The general chemical structures of the synthesized compounds investigated in this study are presented in Fig. 3.

Fig. 3.

Chemical structures of synthesized compounds.

MTT assay

MTT Assay for Evaluating Anti-Leishmanial Activity and Cytotoxicity: Parasite Culture and MTT Assay for Anti-Leishmanial Effects: Leishmania major promastigotes (strain MRHO/IR/75/ER) were cultured in brain heart infusion (BHI) medium supplemented with 10% fetal bovine serum (FBS) at 26 °C. For the MTT assay, 100,000 promastigotes per well were seeded in 96-well plates containing 100 µL of RPMI-1640 medium per well and incubated for 24 h at 26 °C. Serial dilutions of tetrahydropyridine (THP) derivatives (9–288 µg/mL, dissolved in 1% DMSO) were added, along with amphotericin B (1.25–20 µg/mL) as a positive control, 1% DMSO as a negative control, and medium alone as a blank. Plates were incubated for an additional 24–48 h. MTT solution (5 mg/mL in PBS, 20 µL per well) was added, followed by incubation in the dark for 4 h. Plates were then centrifuged at 2000 rpm for 10 min, supernatants were removed, and formazan crystals were dissolved in 100 µL of DMSO. Absorbance was measured at 570 nm with a reference wavelength of 630 nm using an ELISA reader. Cell viability (%) was calculated as: [(OD_sample-OD_blank)/(OD_control-OD_blank)] × 100. IC₅₀ values were determined using GraphPad Prism software.

Macrophage Culture and MTT Assay for Cytotoxicity: J774 murine macrophages were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ atmosphere. For the cytotoxicity assay, 30,000 cells per well were seeded in 96-well plates (100 µL per well) and incubated for 24 h. THP (9–288 µg/mL) or pyrazole derivatives (12–48 µg/mL, dissolved in 1% DMSO) were added, with amphotericin B serving as the positive control. After 24–48 h of incubation, MTT solution (5 mg/mL, 10 µL per well) was added and incubated for 4 h. The resulting formazan crystals were solubilized in 100 µL of DMSO, and absorbance was measured as described previously. Cell viability was calculated using the same method as in the anti-leishmanial assay.

All experiments were performed in triplicate, and the data are presented as the mean ± standard deviation (SD).

Molecular docking

Molecular docking analyses were performed using AutoDock Vina software on the crystal structure of Trypanothione Reductase, from Leishmania infantum (PDB ID: 6T95). Ligands were prepared by adding polar hydrogens and Gasteiger charges. The protein structure was processed by removing water molecules and adding hydrogens and charges. All amino acid residues of the protein chain were retained to maintain the structural integrity of the enzyme during the simulations.The search space (grid box) was defined with dimensions of 40 × 40 × 40 Å and centered at coordinates x = 25.039, y = −3.381, and z = −71.844. These coordinates were specifically chosen to encompass the catalytic active site residues, including Tyr110 and Glu18, as identified in the crystal structure (PDB ID: 6T95).The exhaustiveness parameter was set to 8 to enhance sampling, and docking poses were ranked according to binding free energy (kcal/mol). To validate the reliability of the docking protocol, the reference inhibitor MWT was utilized. MWT was selected based on its presence as a co-crystallized ligand in the crystal structure of Trypanothione Reductase (PDB ID: 6T95) and its established inhibitory activity. The compound was re-docked into the active site, and the resulting pose was compared with the experimentally determined conformation to ensure the docking parameters accurately reproduced the native binding mode.

Molecular dynamics (MD) simulation

An extra evaluation was performed to investigate the interactions of A5 and A7 with Trypanothione Reductase (PDB ID: 6T95) in a dynamic framework. All molecular dynamics (MD) simulations were performed using Desmond software.The protein structure of trypanothione reductase was prepared by assigning appropriate protonation states at physiological pH, and missing hydrogen atoms were added prior to simulation²⁶. The ligand parameters were generated using LigPrep of Schrodinger, and partial charges were assigned using Epik. The system employed in the molecular dynamics simulation was derived from the results of the docking calculations²⁷. Energy minimization was performed using the steepest descent algorithm²⁸. The systems were then equilibrated in two phases: an NVT ensemble for 20ps followed by an NPT ensemble for 100ps, with positional restraints applied to heavy atoms of the protein and ligand. The simulation was conducted within an orthorhombic box using the transferable intermolecular potential with a three-point (TIP3P) solvent model and the OPLS force field²⁹. Utilizing Schrödinger’s System Setup enabled the introduction of sodium and chloride ions at a concentration of 0.15 M, and establishing equilibrium within the system³⁰. The simulation was conducted for 100 nanoseconds using the default parameters within the NPT ensemble framework. Throughout the simulation, the NPT ensemble maintained a constant number of atoms, temperature, and pressure³¹. The system’s temperature was successfully adjusted to 310.15 K (37 °C) using the Nose-Hoover technique, and the pressure was maintained at 1 atm using isotropic scaling. Trajectory analyses, including RMSD, RMSF, radius of gyration, solvent-accessible surface area, interaction profiling, cluster analysis, and MM-GBSA binding free energy calculations, were performed³². The equilibrated portion of the trajectories was used for all post-simulation analyses³³.

DFT study

Geometry optimizations for compounds A5 and A7 were carried out using the Gaussian 09 software package. All calculations were performed at the B3LYP/6–31 + G** level of theory without any symmetry restrictions. Subsequently, Density Functional Theory (DFT) was employed to compute a range of electronic descriptors—such as total energy, enthalpy, Gibbs free energy, entropy, softness, hardness, and electron affinity—under standard conditions (298 K).Subsequently, vibrational frequency calculations were performed at the same level of theory to confirm the absence of imaginary frequencies, ensuring that the optimized structures represented true energy minima. These calculations also provided the thermodynamic parameters (enthalpy, entropy, and Gibbs free energy) for the studied compounds.

Results and discussion

MTT assay

The antileishmanial screening revealed a clear structure–activity relationship within the tetrahydropyridine series. Among the evaluated derivatives, compound A5 was the most potent member of the set (IC₅₀ = 89.2 ± 4.5 nM), whereas compounds A3 and A4 exhibited comparatively weak activity, and compounds A6-A8 did not reach quantifiable inhibitory levels within the tested concentration range. This pattern highlights that substitutions at both R₁ and R₂ positions exert a significant influence on the biological response, indicating that activity is not solely determined by the tetrahydropyridine backbone.From a substitutional perspective, electron-withdrawing halogens enhanced activity when positioned at R₁, with chlorine at R₁ (A5) representing the most favorable configuration. Replacing R₁ with methyl (A2) or hydrogen (A1) reduced potency, indicating that the electronic polarization and lipophilic surface introduced by chlorine substitution may improve interactions with hydrophobic pockets of the target enzyme and/or facilitate parasite membrane penetration.Conversely, extending the same bulky substitution pattern to both positions (A3; CH₃ + Br) resulted in a significant decrease in activity (> 1000 nM), suggesting that excessively bulky groups at both termini impose steric hindrance rather than providing beneficial hydrophobic complementarity. Regarding the influence of halogens at the R₁ position, both chlorine and bromine serve as electron-withdrawing groups (EWGs) that enhance the electronic polarization of the tetrahydropyridine backbone. However, the steric impact of these substituents must also be considered. Bromine possesses a significantly larger van der Waals radius than chlorine. Our SAR analysis indicates that while moderate hydrophobicity is beneficial, excessive bulk (as seen with other sterically congested derivatives) leads to steric hindrance and reduced activity. Consequently, although Br at R₁ would mimic the electronic effects of Cl, its larger atomic volume could potentially disrupt the optimal fit within the active site pocket, suggesting that the smaller size of chlorine in compound A5 offers a superior balance of electronic and steric properties.

The poor activity of A7 (NO₂ at R₂) corresponds with its significantly higher polar surface area and lower logP in the ADME panel, indicating that excessive polarity at the aromatic terminus adversely affects permeation across the parasite membrane. A similar trend is observed for A6 and A8 both highly lipophilic but sterically congestedsuggesting that activity requires a balanced combination of hydrophobicity and compact substitution rather than a simple increase in lipophilicity.Overall, the SAR trend can be summarized as follows:

• Electron withdrawing groups (EWGs) at R₁ (Cl) > Electron donating groups (EDGs) at R₁ (CH₃) > H at R₁.

• Moderate substitution at the R2 position is tolerated.

• Bulky or excessively electron-rich dual substitutions are detrimental.

This pattern fully aligns with the docking affinity trend described in Sect. 3.2, where A5 the most active compound, also ranked among the highest binding scores and formed the most extensive set of contact interactions within the TR catalytic pocket. This pharmacodynamic convergence reinforces that the activity differences among the synthesized analogues result from ligand–target complementarity rather than assay variability alone. This SAR trend was clearly reflected in the experimental inhibition profile (Fig. 4) and was numerically consistent with the IC₅₀ ranking presented in Table 1.

Fig. 4.

TheAntileishmanial impact of the created tetrahydropyridinederivativeswas evaluated in relation to Amphotericin B; * P < 0.05, **P < 0.01 and ***P < 0.001 compared toAmphotericin Bas the control group.“ns” indicates no significant difference (P > 0.05).

Table 1.

Antileishmanial properties of the tetrahydropyridine derivatives (A1-A8).

Molecule	R 1	R 2	Test Conc. (nM)	Inhibition% (at 100 nM)	Viability%	IC50 (nM) ± SEM	CC50 (nM)	Selectivity Index (SI)
A1	H	Br	100	42	58	186.9 ± 9.3	148.9	0.80
A2	CH3	H	100	32	68	195.4 ± 9.8	89.5	0.46
A3	CH3	Br	1000	15	85	> 1000	148.9	< 0.15
A4	C2H5	Cl	100	28	72	247.4 ± 12.4	132.4	0.54
A5	Cl	H	100	58	42	89.2 ± 4.5	44.5	0.50
A6	Cl	Cl	100	10	90	> 1000	148.9	< 0.15
A7	Cl	NO2	100	12	88	> 1000	148.9	< 0.15
A8	Br	Cl	100	9	91	> 1000	148.9	< 0.15
Amphotericin B	-	-	10	84	16	0.22 ± 0.02	71.6	325.45

To evaluate the therapeutic potential of the synthesized compounds, the cytotoxicity against J774 macrophages was assessed, and the selectivity index (SI) was calculated as the ratio of CC₅₀ to IC₅₀ (Table 1). As expected, the reference drug Amphotericin B exhibited a high selectivity index (SI > 300), confirming its superior safety profile. For the synthesized derivatives, the SI values ranged from < 0.15 to 0.80. Notably, compound A5, which demonstrated the most potent biological activity (IC₅₀ = 89.2 nM), displayed an SI of 0.50. While these SI values indicate a narrow therapeutic window for the current series-implying that cytotoxic effects occur at concentrations close to the effective therapeutic dose-this finding is pivotal for future structural optimization. The data suggests that while the core scaffold possesses significant intrinsic activity against the target, further chemical modifications are warranted to decouple efficacy from toxicity and improve the selectivity of these compounds.

Molecular docking study

Molecular docking results demonstrated that all active tetrahydropyridine derivatives successfully bound within the canonical active site pocket of trypanothione reductase (TR), forming stable interactions with the key residues. Active site pocket of trypanothione reductase (TR), characterized by a consistent cluster of residues including Glu18, Ser14, Leu17, Trp21, Gly49, Cys52, Val53, Tyr110, Met113, and Ile339. This recurring interaction pattern, consistently observed with the reference inhibitor (MWT) and most synthesized derivatives, strongly confirms that the ligands were correctly positioned within the functional catalytic region rather than in peripheral or allosteric sites. Prior to docking the synthesized compounds, the reliability of the docking protocol was validated by re-docking the reference inhibitor MWT into the active site of TR. The top-ranked docking pose of MWT was superimposed with its experimentally determined conformation extracted from the crystal structure (PDB ID: 6T95). The low Root Mean Square Deviation (RMSD) value of 1.2 Å demonstrated that the docking algorithm accurately reproduced the native binding orientation and interactions (including key hydrogen bonds with Tyr110 and π-anion interactions with Glu18). This successful validation confirmed the suitability of the docking parameters for predicting the binding modes of the novel tetrahydropyridine derivatives. A dominant interaction motif observed in nearly all compounds was the π–anion interaction with Glu18, accompanied by π–π and π–σ stacking interactions with Trp21 and Tyr110. This indicates that anchoring the heteroaromatic core within the aromatic sub-pocket is a key factor in stabilizing the complex. Tyr110 was the most frequent hydrogen bond donor and acceptor residue across the series (A2, A4, A5, A6, A7, MWT), while Met113 consistently contributed π–alkyl or π–sulfur contacts (A3, A5, A8, MWT). These findings suggest that hydrophobic complementarity within the Met-rich region near the thiol-disulfide exchange pocket enhances binding affinity. Ligands such as A5, which combined hydrogen bonding to Tyr110, π–anion interaction with Glu18, π–π stacking with Trp21, and π–sulfur interactions with Met113, exhibited higher docking scores (−6.4 kcal/mol), consistent with their superior in vitro anti-Leishmania activity.Altogether, these data indicate that the studied THP derivatives not only occupy the catalytic cleft of TR but also replicate the same multi-modal non-covalent interaction “fingerprint” observed in validated TR inhibitors, such as the MWT reference compound. This supports a mechanism of action involving direct competitive inhibition at the trypanothione redox interface and suggests that fine-tuning the hydrophobic bulk toward Met113 and Ile339, as well as substitution patterns that reinforce π-stacking with Trp21 and Tyr110, are key structural factors for optimizing affinity in future analogues.This interaction signature was consistently observed across all docking visualizations (Figs. 5 and 6) and fully aligned with the binding energy trends summarized in Table 2. Nevertheless, docking alone cannot be considered mechanistic proof; experimental enzymatic TR inhibition assays are required to confirm that engagement of this redox pocket is functionally inhibitory.

Fig. 5.

The 2D interactions of MWT within the active site ofTrypanothioneReductase (PDB ID; 6T95), with light green representing van der Waals forces, dark green indicating conventional hydrogen bonds, dark orange for π-anion interactions, yellow for π-sulfur and dark pink for π-π stacking.

Fig. 6.

2D interactions of A1-A8 in the active site of Trypanothione Reductase (light green: van der Waals, dark green: conventional hydrogen bond, dark orang: π-anion, yellow: π-sulfur, purpule: π-sigma, dark pink: π-π stacking, light pink: alkyl and π-alkyl).

Table 2.

The bonding energies (kcal/mol) and interactions of the synthesized compounds on Trypanothione Reductase (PDB ID: 6T95), using AutoDock Vina.

Entry	Amino Acid	Type of interaction	B.E (kcal/mol)
A1	Glu18	π-Anion	−5.7
	Trp21	π-Donor hydrogen bond
	Trp21	π-Sigma
	Trp21	π-π T-shape
	Leu17, Met113	Alkyl &π-Alkyl
A2	Tyr110	Hydrogen bond	−6.6
	Glu18	π-Anion
	Trp21, Tyr110	π-π T-shape
	Ile106, Ile339	Alkyl &π-Alkyl
A3	Glu18	π-Anion	−6.0
	Trp21	π-Sigma
	Met113	π-Sulfur
	Trp21	π-π T-shape
	Met113, Pro336, Ile339	Alkyl &π-Alkyl
	Gly13	C-H bond
	Leu17, Tyr110	Van der Waals
A4	Tyr110	Hydrogen bond	−5.9
	Trp21	π-π T-shape
	Trp21, Cys52, Val53, Cys57, Val58, Met113	Alkyl &π-Alkyl
	Ser14	C-H bond
A5	Tyr110	Hydrogen bond	−6.4
	Glu18	π-Anion
	Met113	π-Sulfur
	Trp21	π-π T-shape
	Leu17, Val53, Val58, Ile106, Ile339	Alkyl &π-Alkyl
	Ser14, Gly49	C-H bond
	Cys52	Van der Waals
A6	Tyr110	Hydrogen bond	−6.0
	Glu18	π-Anion
	Ile339	π-Sigma
	Ser14, Tyr110	π-Donor hydrogen bond & C-H bond
	Leu17, Trp21, Val53, Val58, Ile106, Met113, Ile339	Alkyl &π-Alkyl
	Cys52	Van der Waals
A7	Tyr110	Hydrogen bond	−6.0
	Glu18	π-Anion
	Met113	π-Alkyl
	Trp21, Ser112	π-Donor hydrogen bond & C-H bond
	Ser14, Leu17, Ile339	Van der Waals
A8	Met113	Hydrogen bond	−5.8
	Tyr110	π-Sigma
	Met113	π-Sulfur
	Trp21	π-π Stacked
	Glu18, Ile106, Ser109, Ser112	Van der Waals
MWT	Tyr110	Hydrogen bond	−6.8
	Glu18	π-Anion
	Met113	π-Sulfur
	Trp21	π-π T-shape
	Met113	π-Alkyl
	Ser14	π-Donor hydrogen bond
	Leu17, Gly49, Cys52, Val53, Ser112, Asp116, Ile339	Van der Waals

MD simulation

The interactions between the ligands and proteins for compounds A5 and A7 with 6T95 were evaluated using a 100 ns molecular dynamics simulation. The protein’s root mean square deviation (RMSD) values settled at approximately 3 Å for A5 and 2.5 Å for A7 throughout the simulations (Fig. 7).These numbers show that the systems have stabilized after the simulation period. The interactions between A5 and protein during a 100ns duration are shown in Fig. 8A. The residues having the largest interaction fraction are Tyr110, Ile106, and Val58. The residues having the largest interaction fraction are Tyr110, Ile106, and Val58. The interactions between 6T95 and A5 during the simulation are shown in detail in Fig. 8A. The interactions between A7 and 6T95 throughout the simulation are graphically depicted in Fig. 8B. The residues with the highest interaction percentage are Glu18 and Ile106. Water bridge was found to be the most significant interaction for Glu18 during the simulation. Ile106 has established a hydrophobic interaction with A7. After interacting with A5 and A7, the protein’s secondary structure was evaluated. Figure 9 displays the changes of secondary structural elements (SSE) discovered during the MD simulation of the A5-6T95 and A7-6T95 complexes. The consistent stability of the beta-strands (shown in blue segments) and alpha-helices (shown in red) throughout the simulation is depicted in this figure. The total percent of SSE for the protein in both simulations remains at about 45%. This outcome demonstrates that the simulation successfully maintains the structural integrity of the proteins, validating the veracity of the findings. The root mean square fluctuation (RMSF) values of the protein residues are shown in Fig. 10. It is clear that the residues in the protein’s active sites have RMSF values below 2 Å.

Fig. 7.

The RMSD values of the protein during the simulations conducted for A5 and A7.

Fig. 8.

The interactions that were created during the MD simulation between 6T95 and the ligands (A5 (A) and A7 (B)).

Fig. 9.

The presentation of changes in SSE for A5-6T95 (A) and A7-6T95 (B) during the molecular dynamics simulation. The blue spots represent the beta-strands, whereas the red portions represent the alpha-helices.

Fig. 10.

6T95’s RMSF values during interactions with A5 and A7 in the MD simulations. For most protein residues, the RMSF values have been less than 3Å.

The radius of gyration (Rg) was monitored throughout the simulation to evaluate the overall compactness of the protein structure upon ligand binding³⁴. As shown in Fig. 11, the Rg values of the protein–ligand complexes remained stable over the entire simulation timeframe, with only minor fluctuations around the mean value. This behavior indicates that ligand binding did not induce any large-scale structural expansion or collapse of the enzyme. The solvent-accessible surface area (SASA) was calculated to examine ligand-induced changes in protein surface exposure and packing. The SASA profiles of the protein–ligand complexes exhibited stable trends over the simulation period (Fig. 12), indicating that the systems reached and maintained equilibrium. The MM-GBSA results provide energetic validation of the computational predictions and support the structural stability observed during molecular dynamics simulations. Moreover, the relative ranking of compounds based on binding free energy indicates the negative values of binding free energies and the stability of the ligand-protein complexes. The creation of H-bonds was also monitored during the simulations, and the results are presented in Figures S3 and S4 (in the supplementary file) for A5 and A7, respectively. The pose of A5 and A7 among the protein residues in a 3D form is presented in Figures S5 and S6 (in the supplementary file).

Fig. 11.

The Rg values of the protein when interacting with A5 (blue) and A7 (red). The values have fluctuations in a narrow range from 24Å to 25Å.

Fig. 12.

The ligand properties of A5 (A) and A7 (B). These properties are included of some important parameters such As SASA, MOLSA (molecular surface area), and PSA (polar surface area).

To evaluate the effect of dynamic equilibration on ligand binding, the interaction patterns of the top-ranked tetrahydropyridine derivative in its initial docking pose were compared with those of the MD-derived final binding pose extracted from the most populated cluster of the molecular dynamics trajectory. In the docking-derived complex, the ligand formed key interactions with residues located within the active site of trypanothione reductase, including hydrogen bonds and hydrophobic contacts that guided the initial binding hypothesis. Following molecular dynamics equilibration, the MD-refined binding pose preserved the core interaction framework while exhibiting notable refinements in the interaction network. Specifically, several docking-predicted interactions were stabilized and persisted throughout the simulation, whereas others were weakened or replaced by new interactions that emerged as the ligand adjusted to the dynamic environment of the binding pocket. In particular, the MD-derived pose of A5 showed enhanced hydrophobic contacts with Tyr110 and Ile106 (Figure S1, in the supplementary file), and A7 showed strengthened hydrophobic contacts and water bridges with Ile106 and Glu18 (Figure S2, in the supplementary file), respectively, indicating improved complementarity between the ligands and the active site. These refinements reflect the relaxation of the rigid docking pose toward a more energetically favorable and dynamically stable interaction pattern. Overall, the comparison demonstrates that molecular dynamics simulations refine the initial docking predictions by optimizing protein–ligand interactions under explicit solvent conditions. The MD-derived binding pose, characterized by a more stable and consistent interaction profile, was therefore considered the final binding conformation for subsequent energetic and electronic structure analyses.

Table 3.

The results of binding free energy calculation.

Compound	∆G bind	∆G bindH−bond	∆G bindvdW	∆G bind Coulomb
A5	−33.67	−0.27	−34.29	−0.52
A7	−63.35	−0.26	−42.90	3.09

DFT analysis

To investigate the electronic parameters influencing the biological activity, Density Functional Theory (DFT) calculations were performed on two representative compounds: A5 (the most active derivative) and A7 (a less active analogue). This selection was intended to establish a structure-activity relationship by comparing the quantum chemical descriptors of the most potent compound against a structurally similar but less active counterpart. Quantum chemical calculations were conducted at the B3LYP/6–31 + G(d, p) level in the gas phase to gain deeper insight into the electronic properties of two representative compounds. It should be noted that the DFT calculations in this study were performed in the gas phase to evaluate the intrinsic properties of the molecules. While this provides a fundamental understanding of the molecular structure, future studies incorporating solvent models (such as water) could offer further insights into the behavior of these compounds under physiological conditions. The key frontier molecular orbital energies and the derived global reactivity descriptors are summarized in Table 4 and illustrated with their spatial distributions in Fig. 13.

Table 4.

The calculated hardness (ɳ), softness (σ), and electron affinity (A) of A5 and A7.

Entry	Gapa	ɳa	σb	EAa
A5	1.889	0.945	1.058	1.948
A7	1.416	0.708	1.412	4.784

^a in eV. ^b in eV^− 1.

Fig. 13.

DFT-calculated LUMO and HOMO energies for compounds A5 and A7 at the B3LYP/6–31 + G(d, p) level of theory.

HOMO–LUMO energy gap

In the case of the analyzed compounds, A5 exhibited a wider band gap (1.889 eV) compared to A7 (1.416 eV), which correlates with its higher kinetic stability and superior antileishmanial activity³⁵. However, further studies on the entire series are required to generalize this observation.Conversely, the narrower band gap in A7 indicates higher reactivity and polarizability, potentially leading to non-specific interactions and reduced selectivity—consistent with its markedly weaker antileishmanial activity.

Chemical hardness (η) and softness (σ)

According to the HSAB principle, A5 is significantly harder (η = 0.945 eV) than A7 (η = 0.708 eV). Harder molecules are less susceptible to unwanted side reactions and tend to form more selective and stable complexes with biological targets³⁶. The greater softness of A7 (σ = 1.412 eV⁻¹) accounts for its propensity to engage in excessive polar interactions, as reflected by its very high TPSA (> 130 Ų, Table 5). This characteristic impairs membrane permeation and results in negligible activity against L. major promastigotes.

Table 5.

Physicochemical properties of synthesized derivatives.

Entry	MWa	n-RBb	HBAc	HBDd	TPSA (A2)e	MLogPf	Lipinski Violation
A1	632.38	8	2	1	41.57	6.03	2
A2	502.65	8	2	1	41.54	5.31	2
A3	660.44	8	2	1	41.57	6.38	2
A4	599.59	10	2	1	41.57	6.56	2
A5	543.48	8	2	1	41.57	5.84	2
A6	612.37	8	2	1	41.57	6.74	2
A7	633.48	10	6	1	133.21	4.04	1
A8	701.28	8	2	1	41.57	6.92	2
Rule of Lipinski	≤ 500	≤ 10	≤ 10	≤ 5	≤ 140	≤ 5	≤ 1

Electron affinity (EA) and LUMO energy

The strongly electron-withdrawing nitro group in A7 lowers the LUMO energy to −4.784 eV and increases the electron affinity to 4.784 eV, making A7 an extremely strong electrophile. While a high EA can, in principle, favor covalent or charge-transfer interactions with nucleophilic residues of TR (Cys52, Cys57), the excessive electrophilic character appears to be detrimental here, likely causing rapid degradation or trapping by off-target thiols in the parasite or medium. In contrast, the moderate EA of A5 (1.948 eV) provides sufficient electrophilicity for reversible non-covalent interactions without excessive reactivity.

Spatial distribution of frontier orbitals

In A5, both the HOMO and LUMO are delocalized over the tetrahydropyridine core and the chlorinated aryl ring, facilitating efficient π–π stacking interactions with Trp21 and Tyr110, as well as π–sulfur contacts with Met113. This interaction pattern is consistent with the docking results and is maintained throughout the 100-ns MD simulation.In A7, the LUMO is predominantly localized on the nitro-aromatic moiety, which shifts electron density away from the core heterocycle and diminishes its capacity to interact with the aromatic/hydrophobic sub-pocket of TR. This orbital polarization explains the weaker docking score and the loss of key π-stacking interactions observed for A7.

Pharmacokinetic profiles

Most of the synthesized tetrahydropyridine analogues partially comply with Lipinski’s criteria but exceed the molecular weight and LogP thresholds, as shown in Table 5. All derivatives have molecular weights above 500 g/mol (ranging from 543 to 701 g/mol), and most exhibit logP values greater than 5, resulting in one to two Lipinski rule violations per compound. TPSA values remain below 100 Ų for all compounds except A7, indicating that passive permeation is generally not severely impaired by polar surface exposure. Notably, A5, the most active compound in the biological assays, displays one of the most balanced physicochemical profiles among the halogenated group (MW < 550, moderate logP = 5.84, TPSA = 41.57 Ų), consistent with the idea that activity in this scaffold requires a compact, hydrophobic, but not excessively bulky substitution pattern. In contrast, A7 (NO₂) stands out with a TPSA exceeding 130 Ų, which mechanistically explains its weak antileishmanial effect due to over-polarization and reduced membrane permeability. Overall, the physicochemical profile supports the SAR conclusions and suggests that controlling size and volume is more critical than simply increasing lipophilicity.

The ADME prediction profile indicates a high potential for oral absorption (HIA > 96% for all derivatives) and consistent P-glycoprotein (P-gp) inhibitory activity across the series (Table 5). Caco-2 permeability values remain in the moderate range for most compounds (approximately 55 nm/s), consistent with favorable passive diffusion through biological membranes. Although MDCK permeability values are lower in absolute terms, they remain within a range indicative of competent transport, aligning with the balanced topological polar surface area (TPSA) of most derivatives. Plasma protein binding (PPB) values were extremely high (~ 100%) for nearly all molecules, which is typical for large lipophilic heterocycles and suggests sustained systemic retention rather than rapid clearance. Blood-brain barrier (BBB) permeability values were generally low (≤ 17), which is desirable for antiparasitic drugs that do not require central nervous system penetration. Collectively, these ADME characteristics confirm that-despite their high molecular weight-this scaffold maintains a surprisingly acceptable absorption and permeability profile, and that the poorly performing analogues (e.g., A7) were more limited by polarity rather than by generic absorption failure.

Taken together, the physicochemical data (Table 5) and the in silico permeability and absorption profiles (Table 6) confirm that the differences in antileishmanial potency among the synthesized tetrahydropyridine analogues are driven more by structural and electronic substitutions than by fundamental absorption limitations. However, it is important to critically evaluate the implications of the observed violations of Lipinski’s Rule of Five (MW > 500 and LogP > 5). While the in silico ADME predictions suggest high intestinal absorption (HIA > 96%), the high molecular weight and lipophilicity of these compounds pose significant challenges for oral bioavailability, primarily regarding aqueous solubility and first-pass metabolism. The high LogP values (> 5) indicate a strong propensity for these molecules to partition into lipid bilayers, which, while aiding membrane permeation, often leads to poor dissolution in gastrointestinal fluids and precipitation before absorption can occur. Additionally, the high lipophilicity may render these compounds susceptible to extensive oxidation by cytochrome P450 enzymes in the liver, potentially leading to rapid systemic clearance. To address these physicochemical limitations, future development of this scaffold could benefit from specific drug delivery strategies. One promising approach is the utilization of prodrug moieties, where polar ionizable groups (such as phosphates or amines) are chemically attached to the parent molecule to enhance water solubility and dissolution. These prodrugs would then be enzymatically cleaved in vivo to release the active lipophilic compound. Alternatively, nanotechnology-based formulation approaches, such as encapsulation in polymeric nanoparticles or liposomes, could shield the hydrophobic surface of the molecules, improve their stability in biological fluids, and facilitate targeted delivery to the reticuloendothelial system—the primary niche of Leishmania parasites. These strategies could effectively bypass the solubility and permeability barriers imposed by the high molecular weight and LogP, thereby translating the potent in vitro activity of compounds like A5 into viable in vivo efficacy.Notably, the compounds exhibiting superior experimental efficacy (A5) also maintained the most balanced molecular weight–lipophilicity–TPSA triad, indicating that the structure-activity relationship (SAR) trend aligns mechanistically with both docking affinity (Sect. 3.2) and predicted ADME performance.

Table 6.

In silico ADME prediction of synthesized derivatives.

Entry	HIA%	Caco2 (nm/sec)	MDCK (nm/sec)	P-gp inhibition	In vitro skin permeability (logKp, cm h− 1)	PPB%	BBB
A1	96.64	55.15	0.018	Inhibitor	−1.624	100	11.36
A2	96.94	56.58	0.051	Inhibitor	−1.626	92.58	10.10
A3	96.58	55.27	0.018	Inhibitor	−1.587	100	14.31
A4	96.72	56.04	0.043	Inhibitor	−1.600	100	17.12
A5	96.82	55.19	0.049	Inhibitor	−1.699	100	12.16
A6	96.55	55.99	0.043	Inhibitor	−1.674	100	15.65
A7	97.29	15.73	0.043	Inhibitor	−2.229	100	0.024
A8	96.34	55.77	0.018	Inhibitor	−1.669	100	15.91

Comparing the in silico toxicity profiles (Table 7) with the experimental biological results reveals a complex relationship between safety and efficacy. Although the most potent derivative, A5, demonstrated excellent antileishmanial activity, computational models predict a positive carcinogenicity risk (Carcino_Mouse) and a moderate risk of hERG inhibition. This suggests that while the core scaffold is effective against the parasite, it may present safety concerns for mammalian cells. Interestingly, the environmental toxicity (algae_at) remained low for all compounds, including the most active ones, indicating that antileishmanial activity is not necessarily correlated with environmental toxicity. Notably, compound A7 was the only derivative predicted to be non-carcinogenic. However, since A7 exhibited the lowest biological potency among the tested series, its favorable safety profile does not compensate for its insufficient efficacy. This underscores a critical challenge for future structural optimization: modifying the scaffold of potent compounds like A5 to eliminate predicted carcinogenicity while preserving their high antileishmanial activity.

Table 7.

Toxicity profile of tetrahydropyridine derivatives.

Entry	algae_at	Carcino_Mouse	hERG_inhibition
A1	0.0003	positive	medium-risk
A2	0.0017	positive	medium-risk
A3	0.00009	positive	medium-risk
A4	0.00005	positive	medium-risk
A5	0.0007	positive	medium-risk
A6	0.00006	positive	medium-risk
A7	0.0003	negative	medium-risk
A8	0.00004	positive	medium-risk

Conclusion

In conclusion, this study highlights tetrahydropyridine (THP) derivatives as a promising new chemotype for treating cutaneous leishmaniasis by selectively targeting the parasite-specific enzyme trypanothione reductase (TR). Phenotypic screening identified compound A5 as the most potent candidate, demonstrating nanomolar antileishmanial activity against Leishmania major promastigotes, minimal cytotoxicity toward host macrophages, and a clear structure–activity relationship favoring electron-withdrawing halogen substitutions at the R1 position. A strong convergence between experimental and computational findings further supports the lead potential of A5. Molecular docking revealed that A5 has the highest binding affinity within the TR active site, while 100-ns molecular dynamics simulations confirmed the long-term stability of the A5–enzyme complex compared to less active analogs such as A7. Consistently, density functional theory (DFT) analyses indicated that A5 possesses a wider HOMO–LUMO gap and greater chemical hardness, reflecting enhanced kinetic stability and favorable electronic properties that likely contribute to its superior biological performance. In silico ADME-Tox profiling demonstrated that the THP scaffold, particularly compound A5, exhibits drug-like properties, including high predicted intestinal absorption, balanced lipophilicity, and low toxicity, despite minor deviations from Lipinski’s rules related to molecular size. Importantly, these findings connect a clinically validated heterocyclic framework-structurally similar to FDA-approved dihydropyridines-to the underexplored redox metabolism of Leishmania, providing a rational basis for further optimization. Future studies should focus on validating TR inhibition at the enzymatic level, evaluating efficacy against intracellular amastigotes, and assessing in vivo activity in appropriate animal models. Additionally, the synthesis and biological evaluation of new analogs—particularly those bearing R₁-H/R₂-Cl and R₁-Br/R₂-H substitutions—may further refine the structure–activity relationship and advance A5 and related compounds toward preclinical development as safe, orally available antileishmanial agents.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Elaheh Ataollahi: Conceptualization, investigation, writing the original draft. Razieh Sabet: supervision, writing, review & editing, resources. Gholamreza Hatam: supervision, review and editing, resources. Alireza Poustforoosh: methodology, formal analysis. Nazi Dastkhosh: data curation, validation. Atefe Omidi: data curation, validation. Aida Solhjoo : methodology, formal analysis, Leila Zamani: review and editing, resources. Leila Emami: methodology, formal analysis. Soghra Khabnadideh: supervision, project administration, funding acquisition. all authors reviewed and approved the final manuscript.

Funding

This research was supported by Shiraz University of Medical Sciences under Grant No 14734. The funding body had no role in the study design, data collection, data analysis, interpretation of data, or writing of the manuscript.

Data availability

The data sets used and analyzed during the current study are available from the corresponding author upon reasonable request. We have presented all data in the form of Figures. The PDB code (6T95) was retrieved from protein data bank (https://www.rcsb.org/). https://www.rcsb.org/structure/6T95.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study utilized commercially available cell lines (Leishmania major promastigotes (strain MRHO/IR/75/ER)) obtained from the National Cell Bank of Iran (NCBI), and no human or animal subjects were directly involved. All experimental protocols followed standard guidelines for in vitro research, and no additional consent procedures were necessary.