Synthesis and biological evaluation of pyridine-based derivatives as potential inhibitors of Leishmania major propagation

Abstract

Leishmaniasis is a neglected tropical disease for which current treatment options remain severely limited and associated with considerable toxicity, underscoring the urgent need for novel therapeutic agents with innovative chemical structures. To address this gap, we designed, synthesized, and tested a series of fourteen hybrid molecules (3a–g and 5a–g) that combine a pyridine core with either thiosemicarbazone or thiazolidinone functionalities, and evaluated their activity against Leishmania major in vitro. Among these, the pyridine–thiazolidinone hybrids (5a–g) demonstrated numerically better efficacy compared to their pyridine–thiosemicarbazone counterparts (3a–g). Significantly, compound 5e emerged as a promising dual-stage agent, demonstrated to be effective against both promastigote forms (IC₅₀ = 8.42 ± 0.14 µM), which is comparable to the reference drug miltefosine (IC₅₀ = 7.83 µM), and axenic amastigotes forms (IC₅₀ = 8.88 ± 0.16 µM), similarly aligned with the activity of miltefosine (IC₅₀ = 8.07 µM). Mechanistic exploration via a folate-reversal assay pointed to an antifolate mode of action, proposed, modeling-supported target enzymes DHFR-TS and PTR1. Parasite viability was restored in a dose–responsive manner when co-administered with folic or folinic acid, with recovery rates reaching up to 74% at 20 µM and 83% at 100 µM folic acid, depending on the compound assessed. Additionally, 5e exhibited a favorable in vitro cytotoxicity profile in VERO cells (CC₅₀ = 393.04 µM; selectivity index = 46.63). To gain further insight, molecular docking and 100 ns molecular dynamics (MD) simulations were carried out to explore the possible binding interactions and dynamic stability of 5e within the PTR1 active site. These computational analyses support the compound's proposed inhibitory capacity against Lm-PTR1. Collectively, our results introduce a promising new class of antileishmanial agents that act through selective disruption of the folate biosynthesis pathway, highlighting their considerable therapeutic promise in the fight against leishmaniasis.

Fourteen hybrid molecules (3a–g, 5a–g) combining a pyridine core with thiosemicarbazone or thiazolidinone moieties were designed, synthesized, and evaluated in vitro against Leishmania major.

1. Introduction

Neglected Tropical Diseases (NTDs) constitute a diverse group of twenty-one infectious diseases that impose a disproportionate impact on over one billion people living in resource-constrained areas worldwide.¹ Among these, leishmaniasis represents a group of NTDs caused by infection with over twenty distinct protozoan species belonging to the genus Leishmania. The clinical manifestations are primarily categorized into three main phenotypes: the cutaneous form, which is characterized by ulcerative skin lesions; the mucocutaneous form, involving destructive lesions of the mucous membranes; and the visceral form, the most severe variant, marked by involvement of parenchymal organs.^2–4 The transmission cycle is chiefly maintained through the hematophagous activity of infected female phlebotomine sandflies, which act as essential biological vectors during blood-feeding.⁵ The clinical presentation of leishmaniasis ranges from severe cutaneous ulcerations to visceral disease, which, if left untreated, can result in systemic organ failure and pose a substantial threat to patient survival.^6,7

Therapeutic interventions for leishmaniasis remain notably limited.⁸ Pentavalent antimonials, specifically sodium stibogluconate and meglumine antimoniate, along with amphotericin B deoxycholate, are associated with significant systemic toxicity and require prolonged intravenous administration, thereby limiting their feasibility in resource-constrained settings.⁹ Paromomycin demonstrates inconsistent clinical efficacy,¹⁰ while miltefosine, the only widely recommended oral treatment for cutaneous leishmaniasis in endemic areas, faces increasing challenges due to emerging drug resistance, considerable teratogenic risks, and high costs.^11,12 These challenges are further exacerbated by the persistent absence of an effective vaccine and the concerning rise of multidrug-resistant Leishmania strains, collectively constituting a critical therapeutic crisis. Consequently, the advancement of novel antileishmanial agents with improved efficacy, reduced adverse effects, and shorter treatment regimens has emerged as an urgent scientific priority.¹³

Previous studies have indicated that inhibition of dihydrofolate reductase-thymidylate synthase (DHFR-TS) elicits a compensatory mechanism mediated by pteridine reductase 1 (PTR1), which ensures sufficient folate availability to maintain parasite survival.¹⁴ This observation suggests that effective antileishmanial interventions should concurrently proposed, modeling-supported targets both DHFR-TS and PTR1. However, this perspective remains contentious, as deletion of the ptr1 gene has been demonstrated to be lethal to the parasite, presumably because PTR1 serves as the sole enzyme responsible for pterin reduction, a process essential for parasite development and metacyclogenesis.^15,16 Consequently, PTR1 is regarded by some researchers as a validated drug target, prompting efforts to inhibit its enzymatic activity.^17,18 Despite progress in this field, a notable limitation remains, as existing studies have not adequately explored the potential development of drug-resistant parasite strains. In response to this challenge, medicinal chemistry has made considerable advancements in the rational design and optimization of novel chemical scaffolds aimed at producing candidate compounds with improved safety profiles and therapeutic effectiveness.^19,20

Nitrogen-containing heterocycles are widely recognized for their significant biological activity^21–23 and are essential to contemporary drug design and discovery initiatives.²⁴ Among these, the six-membered pyridine ring stands out due to its frequent occurrence in natural products, including alkaloids like nicotine, vitamins such as niacin, and various coenzymes.²⁵ Consequently, compounds featuring the pyridine scaffold have attracted broad interdisciplinary interest, largely owing to their role as pharmacophores in medicinal chemistry.²⁶ This structural versatility has facilitated the development of a wide array of broad-spectrum therapeutic agents with diverse biological effects, including antibacterial, antiviral, anticancer, and antiparasitic activities.^27–30 Furthermore, numerous studies have underscored the promise of pyridine derivatives as antileishmanial agents.^31,32 Bekhit and co-workers prepared a series of pyridine-2,6-dicarboxylic acid derivatives and evaluated their antileishmanial activity against Leishmania major. All synthesized compounds exhibited biological activity, with compound (I, Fig. 1) demonstrating the highest potency against both primary developmental stages of the parasite, promastigotes and amastigotes. Notably, this compound showed greater efficacy compared to the reference drug miltefosine.³³ Additionally, other pyridine derivatives, exemplified by structures (II and III, Fig. 1), have been reported to possess significant antileishmanial properties.^34,35

Fig. 1. Overview of established antileishmanial agents, classified according to their principal pharmacophoric frameworks: pyridine-based derivatives (I–III), carbothioamide compounds (IV–VI), and thiazolidinone-containing structures (VII–IX). The figure depicts the newly synthesized target molecules developed in the present study, designated as series 3a–g and 5a–g.

Other fragments that have attracted considerable attention owing to their chemical and pharmacological characteristics are the thiosemicarbazones, which are notable for their broad spectrum of biological activities,^36–38 particularly their leishmanicidal effects.^39,40 In a recent study, Paula Roberta da Silva and colleagues developed a novel series of thiosemicarbazone derivatives incorporating an indole framework, designed as antileishmanial agents. Their findings revealed that the majority of the synthesized compounds exhibited significant activity, with compound (IV, Fig. 1) emerging as the most potent, demonstrating high effectiveness against both promastigote and amastigote forms of Leishmania.⁴¹ Additionally, structures (V & VI, Fig. 1) demonstrated efficacy against two distinct forms of Leishmania.^42,43 Furthermore, cyclization of thiosemicarbazones to thiazolidinone derivatives has been explored as a possible approach for developing antileishmanial agents.^44,45 These compounds form an important class of heterocycles known for a broad spectrum of biological activities, including antitoxoplasma, antibacterial, antitumor, and anti-inflammatory properties,^46–48 and have recently demonstrated promising activity against leishmaniasis.^49,50 Although the thiazolidinone scaffold holds considerable promise as a viable antileishmanial pharmacophore, only a limited number of studies have reported such derivatives with antileishmanial potential (structures VII–IX, Fig. 1).^51–53 This scarcity underscores the need to broaden the chemical diversity in antileishmanial research, particularly through the conjugation of thiazolidinone moieties with the biologically active pyridine framework.

Expanding upon these findings and our prior research in this domain 2–4, 13, we employed a rational design approach for the target compounds, utilizing FDA-approved drugs containing pyridine moieties as structural templates.⁵⁴ This core scaffold was then combined with biologically active carbothioamide and thiazolidinone moieties known for their antileishmanial properties (Fig. 1). This integrative approach led to the synthesis of compounds 3a–g and 5a–g (Fig. 1), which represent a new chemical series conceived to harness the complementary advantages of each structural component. The novel framework was systematically constructed to assess its effectiveness against leishmaniasis, with the goal of uncovering promising lead candidates that could support the development of improved therapies for this overlooked tropical illness. Additionally, we carried out in silico studies and molecular dynamics simulations to examine how these lead compounds interact with the target binding site, providing valuable insights into the molecular basis of their potential antileishmanial action.

2. Results and discussion

2.1. Chemistry

The synthetic approaches utilized for the preparation of the target hybrid compounds are comprehensively outlined in Scheme 1. The precursor, N-(pyridin-2-yl)hydrazinecarbothioamide (1), was synthesized following a previously reported procedure. The target compounds 3a–g were then obtained via a condensation reaction between aldehyde derivatives 2a–g and compound 1, carried out in absolute ethanol with a few drops of acetic acid (Scheme 1). Subsequently, these intermediates 3a–g were subjected to cyclization by refluxing with ethyl bromoacetate (4) in ethanol in the presence of anhydrous sodium acetate, yielding the corresponding thiazolidinone derivatives 5a–g (Scheme 1).

Scheme 1. Synthesis of the target compounds 3a–g and 5a–g.

The newly created hybrids were analyzed for their structures using various spectral techniques, such as proton nuclear magnetic resonance (¹H-NMR), carbon-13 nuclear magnetic resonance (¹³C-NMR).

2.2. Biological assessments

2.2.1. In vitro antileishmanial activities

To assess the antileishmanial activity, all synthesized compounds (3a–g and 5a–g) were subjected to in vitro evaluation against both the extracellular promastigote and axenic amastigotes forms of Leishmania major. The half-maximal inhibitory concentration (IC₅₀) values were determined concurrently with the established antileishmanial agent miltefosine, providing a quantitative basis for comparative potency analysis (Table 1).

Table 1. In vitro activity of the investigated target compounds and standard reference drugs, expressed in micromolar concentrations (µM), against various developmental stages of Leishmania major.

Compound	R	IC50 (µg µM−1)a
		Anti-promastigote		Anti-amastigote
		µg ml−1	µM	µg ml−1	µM
3a	H	6.91 ± 0.08	26.98 ± 0.32	7.46 ± 0.09	29.11 ± 0.36
3b	CH3	7.63 ± 0.09	28.24 ± 0.34	8.60 ± 0.11	31.82 ± 0.42
3c	OCH3	9.94 ± 0.13	34.73 ± 0.46	10.62 ± 0.12	37.10 ± 0.42
3d	F	9.00 ± 0.13	32.82 ± 0.48	8.77 ± 0.12	31.98 ± 0.46
3e	Cl	12.45 ± 0.12	42.82 ± 0.42	12.50 ± 0.11	43.00 ± 0.38
3f	Br	8.92 ± 0.11	26.62 ± 0.34	9.36 ± 0.10	27.92 ± 0.32
3g	NO2	10.10 ± 0.13	33.53 ± 0.46	9.94 ± 0.10	32.99 ± 0.36
5a	H	8.12 ± 0.12	28.58 ± 0.44	8.24 ± 0.11	28.98 ± 0.42
5b	CH3	5.62 ± 0.06	18.86 ± 0.22	6.33 ± 0.05	21.24 ± 0.18
5c	OCH3	5.30 ± 0.08	16.88 ± 0.28	5.56 ± 0.10	17.68 ± 0.32
5d	F	4.45 ± 0.07	14.74 ± 0.26	4.80 ± 0.06	15.89 ± 0.22
5e	Cl	2.68 ± 0.04	8.42 ± 0.14	2.83 ± 0.05	8.88 ± 0.16
5f	Br	4.71 ± 0.11	12.98 ± 0.32	5.92 ± 0.08	16.31 ± 0.24
5g	NO2	6.80 ± 0.11	20.64 ± 0.36	7.64 ± 0.14	23.22 ± 0.44
Miltefosine	—	3.19 ± 0.13	7.83 ± 0.34	3.28 ± 0.09	8.07 ± 0.24
Amphotericin B	—	0.03 ± 0.00	0.04 ± 0.001	0.13 ± 0.00	0.15 ± 0.01

^aIC₅₀ values are the mean ± S.D. for three experiments. The concentration range is 0.04–40 µg mL⁻¹.

The in vitro biological assessment of the synthesized compound library demonstrated moderate to notable antileishmanial efficacy against both the extracellular promastigote and the obligate axenic amastigotes stages of L. major. Specifically, the anti-promastigote activity screening revealed significant inhibitory effects across the compound series. The 3a–g series exhibited IC₅₀ values within the micromolar range, spanning from 26.62 ± 0.34 to 42.82 ± 0.42 µM, whereas the 5a–g series showed numerically improved potency, with IC₅₀ values ranging from 8.42 ± 0.14 to 28.58 ± 0.44 µM. 5e exhibited IC₅₀ values within a similar micromolar range to miltefosine under the assay conditions used.

Among the inhibitors targeting the promastigote stage, compound 5e emerged as a prominent lead candidate, exhibiting notable micromolar potency with an IC₅₀ value of 8.42 ± 0.14 µM. This results of inhibitory concentration is comparable to that of miltefosine, thereby establishing compound 5e as effective anti-promastigote agent. Additionally, several other derivatives within the 5a–g series demonstrated micromolar inhibition within a low-to-moderate range, underscoring the favorable structural characteristics inherent to this series. In contrast, compound 3e showed significantly reduced promastigote inhibition, with an IC₅₀ of 42.82 ± 0.42 µM, reflecting approximately a 5.5-fold decrease in activity relative to miltefosine.

The structure–activity relationships (SARs) between the thiosemicarbazone and thiazolidinone series, as derived from the antileishmanial activity data presented in Table 1, can be summarized as follows. Concerning the inhibitory effects on promastigotes exhibited by compounds 3a–g and 5a–g, the thiazolidinone core generally demonstrated greater potency compared to the thiosemicarbazone scaffold. Specifically, substitution at the R position within the thiazolidinone series typically enhanced activity relative to the unsubstituted hydrogen analogue. For instance, compound 5e, bearing a chloro substituent at R, exhibited the highest potency among both series, with an IC₅₀ value of 8.42 ± 0.14 µM, representing an approximate 3.4-fold increase in activity compared to the unsubstituted compound 5a (IC₅₀ = 28.58 ± 0.44 µM). Conversely, the introduction of a chloro substituent at the R position in the thiosemicarbazone series led to a marked decrease in activity, as exemplified by compound 3e (R Cl), which was the least potent derivative (IC₅₀ = 42.82 ± 0.42 µM). This finding suggests that substitution on the thiazolidinone core is highly favorable for promastigote inhibition, whereas similar modifications on the thiosemicarbazone core are detrimental. Furthermore, the presence of electron-withdrawing groups such as bromine and fluorine at the R position influenced activity within each series; however, no consistent trend was observed, indicating that their effects are secondary. Notably, halogen substitutions (Br and F) generally enhanced activity more effectively than other substituents such as methyl (CH₃), methoxy (OCH₃), nitro (NO₂), or unsubstituted hydrogen.

Furthermore, assessment against the clinically pertinent axenic amastigotes stage revealed comparably moderate inhibitory effects across the synthesized compound library. The 3a–g series exhibited IC₅₀ values ranging from 27.92 ± 0.32 to 37.10 ± 0.42 µM, whereas the 5a–g series demonstrated numerically better micromolar inhibition, with IC₅₀ values spanning 8.88 ± 0.16 to 28.98 ± 0.42 µM. Collectively, these activities compare favorably with miltefosine (IC₅₀ = 8.08 ± 0.22 µM), indicating considerable efficacy against the obligate intracellular parasite stage. While axenic amastigotes were employed as a model for initial anti-leishmanial evaluation, it is acknowledged that this does not fully simulate the intracellular environment encountered in the mammalian host, where amastigotes reside within the host cell.

Notably, compound 5e achieved effective amastigote inhibition, yielding an IC₅₀ value of 8.88 ± 0.16 µM, which approximates the activity of miltefosine and underscores the exceptional dual-stage inhibitory potential of this molecule. In contrast, compound 3e exhibited numerically diminished amastigote inhibition (IC₅₀ = 43.00 ± 0.38 µM), corresponding to approximately 5.6-fold lower activity relative to miltefosine.

Structure–activity relationship (SAR) analysis of the inhibitory effects on axenic amastigotes revealed that the thiazolidinone series (compounds 5a–g) exhibited greater potency compared to the thiosemicarbazone series (compounds 3a–g), consistent with observations in promastigote assays. Within the thiazolidinone derivatives, halogen substitutions at the R position were associated with enhanced potency relative to other substituents such as methyl (CH₃), methoxy (OCH₃), or nitro (NO₂) groups which improve the capacity of hydrogen-bonding. Notably, the chloro-substituted derivative 5e demonstrated the more activity, with an IC₅₀ value of 8.88 ± 0.16 µM followed by the fluoro-substituted 5d and bromo-substituted 5f, which exhibited IC₅₀ values of 15.89 ± 0.22 µM and 16.31 ± 0.24 µM, respectively. Furthermore, Halogen-specific phenomena encompass factors such as polarizability, halogen bonding interactions, and the unique characteristics exhibited by fluorine—namely its small atomic radius and high electronegativity—in contrast to chlorine and bromine, which are comparatively larger and exhibit greater polarizability. In contrast, within the thiosemicarbazone series, the chloro-substituted compound 3e showed the lowest potency (IC₅₀ = 43.00 ± 0.38 µM), indicating that chloro substitution at the R position alone may negatively impact activity in the absence of other favorable substituents. Furthermore, variations at the R position involving hydrogen or bromine substitutions had only marginal effects on amastigote activity among related analogues, suggesting a secondary role in modulating intracellular efficacy.

In summary, the results demonstrate that substitution at the R position within the thiazolidinone series serves as the primary determinant of antileishmanial efficacy in compounds 5a–g, whereas analogous substitutions at the R position in the thiosemicarbazone series produce only a modest modulatory impact. Collectively, the structure–activity relationship (SAR) analysis suggests that a rational simplification of the core scaffold through a ring-cyclization strategy, coupled with selective chlorine substitution, successfully generated the more active compounds in the present series (5e). This compound exhibits a good dual-stage antileishmanial activity, comparable to that of miltefosine against both promastigote and amastigote forms, thereby positioning it as a promising candidate for subsequent preclinical and clinical development.

2.2.2. The antileishmanial activity of hybrid 5e, when opposed by antagonistic factors, can be counteracted through the addition of folic acid and folinic acid

Following the confirmation of the in vitro activity of compound 5e (Table 1), we proceeded to investigate the biochemical mechanism underlying its effect through a targeted folate-reversal assay. This assay assesses whether supplementation with exogenous folate can competitively counteract the proposed inhibition of dihydrofolate reductase (DHFR) and pteridine reductase 1 (PTR1) induced by the compound, thereby providing preliminary evidence for the involvement of folate-dependent pathways in its antileishmanial activity.

To further elucidate the potential role of the folate pathway, a folate-reversal assay was conducted on the most effective analog, 5e, using trimethoprim as a reference DHFR inhibitor. Parasites were treated with 5e at a concentration of 20 µM, exceeding their IC₅₀ values, followed by exposure to either folic acid or folinic acid at concentrations of 20 and 100 µM. The addition of folic acid at 100 µM nearly completely reversed the inhibitory effect of trimethoprim, restoring parasite viability to 99%, thereby supporting the assay's sensitivity and specificity. Similarly, the antileishmanial activity of compound 5e was reversed in a dose-dependent manner by both forms of folate. Parasite survival increased from 28% in untreated controls to 83% and 89% upon treatment with 100 µM folic acid and folinic acid, respectively. The extent and kinetics of this reversal were quantitatively comparable to those observed with trimethoprim, with partial reversal evident at 20 µM folate concentrations, indicating a concentration-dependent protective effect.

Collectively, these findings provide supportive evidence that compound 5e exerts its antileishmanial effects potentially through interference with folate-dependent enzymes such as DHFR and/or PTR1. Folate-mediated restoration of parasite viability is consistent with a possible antifolate mechanism but does not definitively establish target engagement or enzyme inhibition (Table 2).

Table 2. The effectiveness of folate pathway inhibition was assessed in vitro and expressed as a percentage of viable cells^a.

Compound	No competitor added	Folic acid		Folinic acid
		20 µM	100 µM	20 µM	100 µM
5e	28%	74%	83%	86%	89%
Trimethoprim (100 µM)	72%	—	99%	—	—

^aPercentage of viable cells = 100 − % AP; where % AP is the percentage growth inhibition.

2.2.3. Assessment of cytotoxicity in vitro

To evaluate the favorable in vitro cytotoxicity profile in VERO cells of the most active compound 5e, its cytotoxicity was examined using African green monkey kidney (VERO) cells in accordance with a previously established protocol.⁵⁵ The cells were exposed to a series of concentrations of the test compounds for 72 hours, and the CC₅₀ values were determined as the concentrations that resulted in a 50% reduction in cell viability. The selectivity index (SI) was calculated by dividing the CC₅₀ by the corresponding IC₅₀ values against axenic amastigotes. Compound 5e demonstrated a CC₅₀ value of 393.04 µM, indicating substantial potency in its antileishmanial activity (IC₅₀ = 8.42 µM), which corresponded to an SI value of 46.63. These results indicate a marked selectivity of compound 5e for the parasite relative to mammalian cells (Table 3).

Table 3. The cytotoxicity (CC₅₀) of hybrid compound 5e was evaluated using normal VERO cells, and the selectivity indices against Leishmania amastigotes were subsequently determined.

Comp.	CC50a µg ml−1 (µM)	Antileishmanial
		IC50 µM	SIb
5e	125 (393.04)	8.88	44.26

^aCC₅₀ refers to the concentration at which half of the cells survive.

^bSI = CC₅₀/IC₅₀.

VERO cells represent a standard and well-validated mammalian cell line for preliminary cytotoxicity assessment, yet evaluation of selectivity against intracellular amastigotes in the presence of the host cell would provide a more clinically relevant interpretation of compound 5e activity that may be integrated into future studies.

2.3. Molecular modeling

2.3.1. ADME profile

A compound can only be considered a promising therapeutic candidate if it demonstrates both potent biological activity and favorable ADMET properties. In the present study, the ADME profile of compound 5e was evaluated using ADMETlab 3.0, a fully updated and commercially accessible web-based platform.

As shown in Fig. 2, the predicted physicochemical properties of compound 5e were evaluated using ADMETlab 3.0 and compared with those of the reference drug Miltefosine. Compound 5e exhibited a molecular weight (MW) of 318.03 g mol⁻¹, log P of 3.06, TPSA of 57.59 Ų, one hydrogen-bond donors (nHD), and five hydrogen-bond acceptors (nHA). In comparison, the corresponding values for Miltefosine were 407.32 g mol⁻¹, 1.29, 58.59 Ų, zero, and five, respectively. Most descriptors of 5e were located within the recommended ranges of the predictive model and were broadly comparable to those of Miltefosine. Although the predicted log P value of 5e was slightly above the optimal range, suggesting increased lipophilicity, the remaining physicochemical parameters were consistent with accepted drug-likeness criteria. The predicted ADME properties of 5e were generally favorable within the computational model and provide preliminary support for further investigation. However, these data represent computational estimates generated by ADMETlab 3.0 and should not be considered experimentally validated pharmacokinetic properties. Experimental pharmacokinetic and toxicological studies will be required to further confirm the ADME characteristics of 5e.

Fig. 2. ADMETlab 3.0's pharmacokinetic prediction for compound 5e compared with Miltefosine.

2.3.2. Molecular docking of compound 5e to LmPTR1 and LmDHFR

According to the evaluated anti-Leishmania activities, compound 5e was selected for molecular docking studies followed by molecular dynamics (MD) simulations. Redocking of the folate reference ligand was used to validate the docking parameters, and the overlay and low RMSD values verified its validity (Fig. S39). After initial visual inspection, the binding conformations of compound 5e within the active site of LmPTR1 and LmDHFR were selected based on their lowest docking scores (−7.9 and −7.7 kcal mol⁻¹, respectively) and shown in (Fig. 3). Molecular docking analysis revealed that, within LmPTR1, compound 5e forms one hydrogen bond with Val230, π–cation interaction with Arg17, π–sulfur interaction with His241, π–π stacking with Phe113, and hydrophobic contacts with Leu188, Met183, and NDP302 (nicotinamide adenine dinucleotide phosphate cofactor, reduced form). On the other hand, in the LmDHFR active site, compound 5e exhibits a hydrogen bond with Val156, π–π stacking with Phe56, π–sulfur interaction with Met53, and multiple hydrophobic interactions involving Ala32, Val87, and Leu94.

Fig. 3. Docking interaction profile for 5e inside the active site of (A) LmPTR1 and (B) LmDHFR.

To evaluate the stability of the binding of compound 5e within the active sites of LmPTR1 and LmDHFR, the docking poses generated using AutoDock Vina were employed for subsequent 100 ns MD simulations.

2.3.3. Molecular MD simulations of 5e in complex with LmPTR1 and LmDHFR

To evaluate the conformational stability of the 5e–LmPTR1 and 5e–LmDHFR complexes obtained from molecular docking, a 100 ns molecular dynamics (MD) simulation was conducted (Fig. 4 and 5). The root mean square deviation (RMSD) of both the ligand (5e) and the backbone atoms of LmPTR1/LmDHFR was subsequently calculated. The RMSD trajectories reached a stable plateau, indicating that the 5e–LmPTR1 complex achieved proper equilibration during the simulation. Furthermore, the stability of ligand 5e within the active site was supported by consistent solvent-accessible surface area (SASA) and radius of gyration (R_g) profiles. The center of mass of 5e remained near that of LmPTR1 (2.5 ± 0.5 nm) and LmDHFR (3.0 ± 0.5 nm), suggesting stable binding throughout the simulation. In addition, root mean square fluctuation (RMSF) analysis revealed similar fluctuation patterns between the apo and ligand-bound forms of both proteins, further supporting the stable accommodation of 5e within the active sites of the two targets.

Fig. 4. MD simulation analysis for 5e–LmPTR1 complex over a 100 ns simulation.

Fig. 5. MD simulation analysis for 5e–LmDHFR complex over a 100 ns simulation.

A limitation of the MD analysis is that only a single 100-ns trajectory was performed for each complex. Although the simulations provided valuable insights into the stability of the predicted protein–ligand interactions, additional independent replicate simulations and longer sampling times would further strengthen the robustness and reproducibility of the observed behavior.

In addition, while the biological assays and molecular docking results support target engagement, no enzyme kinetic or mechanistic studies were performed to determine whether the inhibition is reversible, irreversible, competitive, noncompetitive, or allosteric. Therefore, the precise mode of inhibition requires further investigation through dedicated biochemical and biophysical studies.

3. Conclusions

In summary, we prepared a library of fourteen hybrid molecules (3a–g and 5a–g) featuring a pyridine scaffold linked to either thiosemicarbazone or thiazolidinone moieties, and assessed their in vitro antileishmanial activity. The thiazolidinone derivatives (5a–g) generally showed lower numerical IC₅₀ values than the corresponding thiosemicarbazone derivatives under the conditions tested. Compound 5e was particularly noteworthy, displaying effective dual-stage efficacy against promastigotes (IC₅₀ = 8.42 µM) and axenic amastigotes (IC₅₀ = 8.88 µM), comparable to miltefosine. Mechanistic studies using a folate-reversal assay indicated an antifolate mode of action, proposed, modeling-supported target of the enzymes PTR1/DHFR-TS; co-administration with folic or folinic acid restored parasite growth in a dose-dependent fashion (up to 83% recovery). Furthermore, 5e exhibited a favorable in vitro cytotoxicity profile in VERO cells in mammalian (CC₅₀ = 393.04 µM, selectivity index = 44.26). To rationalize these findings, we performed molecular docking and 100-ns MD simulations, which provided computational support for the stable accommodation of 5e within the Lm-PTR1 binding pocket and its would-be as effective anti-leishmanial agent.

4. Materials and methods

4.1. Chemistry

Melting points were determined using the Electrothermal IA 9000 apparatus, with no subsequent corrections applied to the recorded values. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data for some synthesized compounds were obtained using a JEOL JMS-700 instrument, manufactured in Tokyo, Japan. Nuclear magnetic resonance (NMR) spectroscopy, including both ¹H and ¹³C NMR analyses, was performed on Bruker 500 MHz spectrometers housed at the Faculty of Pharmaceutical Science, Tokushima Bunri University, Japan. Chemical shifts are reported in parts per million (δ, ppm), and coupling constants are presented in hertz (Hz). Elemental analyses (FLASH 2000 CHNS/O analyzer, Thermo Scientific (USA)) was carried out at the Regional Center for Mycology and Biotechnology (RCMB), Al-Azhar University, Nasr City, Cairo. Thin-layer chromatography (TLC) was employed to monitor the reactions, utilizing silica gel on aluminum sheets (60 F254, Merck) with a chloroform/methanol (9.8 : 0.2 v/v) eluent. Compound 1 was synthesized according to previously established methods.⁵⁶

4.1.1. General procedure for the target compound 3a–g

A mixture of N-(pyridin-2-yl)hydrazinecarbothioamide (1) (0.01 mol) and an equimolar amount of various aldehyde derivatives (2a–g), specifically phenyl, 4-methylphenyl, 4-methoxyphenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, and 4-nitrophenyl (0.01 mol each), was refluxed in 15 mL of absolute ethanol in the presence of 0.5 mL from glacial acetic acid as a catalyst for approximately 6–8 hours. After completion of the reaction, the mixture was allowed to cool, and the resulting precipitate was isolated by filtration, washed with ethanol, and dried. The obtained solid was then; the substance was recrystallized from 95% ethanol by heating the solution to near boiling using a recrystallization apparatus consisting of a conical flask, a beaker, and filter paper to afford the desired compounds 3a–g.

4.1.1.1. 2-Benzylidene-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3a)

Yellow powder in 92% yield, m.p 158–160 °C; ¹H NMR (500 MHz, CDCl₃): δ = 7.11–7.13 (1H, m, H–Ar), 7.41–7.42 (3H, m, H–Ar), 7.72–7.78 (3H, m, H–Ar), 7.96 (1H, s, H–C N), 8.38 (1H, d, J = 4.0 Hz, H–Ar), 8.90 (1H, d, J = 8.0 Hz, H–Ar), 9.87 (1H, s, NH), 14.95 (s, 1H, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 115.39, 120.53, 127.75, 128.90, 130.91, 132.78, 137.72, 143.47, 148.12, 151.49 (C N), 174.06 (C S). LREIMS: 256; HREIMS: 256.0787 (calcd for C₁₃H₁₂N₄S; 256.0783). Elemental analysis: calcd: % C, 60.92; H, 4.72; N, 21.86. Found: % C, 61.02; H, 4.83; N, 21.99.

4.1.1.2. 2-(4-Methylbenzylidene)-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3b)

Yellow powder in 86% yield, m.p 185–186 °C; ¹H NMR (500 MHz, CDCl₃): δ = 2.11(3H, s, CH₃), 6.92 (2H, d, J = 9.0 Hz, H–Ar), 7.10–7.13 (1H, m, H–Ar), 7.69 (2H, d, J = 7.5 Hz, H–Ar), 7.68 (1H, s, H–C N), 8.37 (1H, d, J = 4.0 Hz, H–Ar), 8.88 (1H, d, J = 8.0 Hz, H–Ar), 9.92 (1H, s, NH), 14.86 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 20.75 (CH₃), 114.39, 115.63, 120.45, 125.47, 129.48, 129.77, 137.83, 143.62, 147.87, 151.52, 161.91 (C N), 176.04 (C S). Analysis for C₁₄H₁₄N₄S (270.35), calcd: % C, 62.20; H, 5.22; N, 20.72. Found: % C, 62.32; H, 5.31; N, 20.81.

4.1.1.3. 2-(4-Methoxybenzylidene)-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3c)

Buff powder in 78% yield, m.p 138–139 °C; ¹H NMR (500 MHz, CDCl₃): δ = 3.86 (3H, s, OCH₃), 6.92 (2H, d, J = 9.0 Hz, H–Ar), 7.11–7.13 (1H, m, H–Ar), 7.69 (2H, d, J = 8.5 Hz, H–Ar), 7.75–7.79 (1H, m, H–Ar), 7.89 (1H, s, H–C N), 8.37 (1H, d, J = 4.0 Hz, H–Ar), 8.88 (1H, d, J = 8.0 Hz, H–Ar), 10.00 (1H, s, NH), 14.87 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 55.44 (OCH₃), 114.38, 115.68, 120.45, 125.49, 129.48, 137.84, 143.68, 147.85, 151.50 (C N), 161.90 (C–OCH₃), 176.03(C S). LREIMS: 286; HREIMS: 286.0887 (calcd For C₁₄H₁₄N₄OS; 286.0888). Elemental analysis: calcd: % C, 58.72; H, 4.93; N, 19.57. Found: % C, 58.83; H, 5.03; N, 19.69.

4.1.1.4. 2-(4-Fluorobenzylidene)-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3d)

Buff powder in 88% yield, m.p 152–153 °C; ¹H NMR (500 MHz, CDCl₃): δ = 7.09–7.14 (3H, m, H–Ar), 7.72–7.78 (3H, m, H–Ar), 7.93 (1H, s, H–C N), 8.38 (1H, s, H–Ar), 8.89 (1H, d, J = 8.0 Hz, H–Ar), 9.95 (1H, s, NH), 14.94 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 115.40, 116.09, 120.59, 129.71, 137.77, 142.23, 148.10, 151.44, 163.32 (C N), 165.32 (C–F), 174.05 (C S). LREIMS: 274; HREIMS: 274.0686 (calcd for C₁₃H₁₁FN₄S; 274.0688). Elemental analysis: calcd: % C, 56.92; H, 4.04; N, 20.42. Found: % C, 57.02; H, 4.12; N, 20.53.

4.1.1.5. 2-(4-Chlorobenzylidene)-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3e)

Yellow powder in 78%, m.p 195–196 °C; ¹H NMR (500 MHz, CDCl₃): δ = 6.93 (2H, s, H–Ar), 7.01–7.07 (2H, m, H–Ar), 7.11–7.14 (1H, m, H–Ar), 7.55–7.79 (1H, m, H–Ar), 7.86 (1H, s, H–C N), 8.37 (1H, d, J = 4.0 Hz, H–Ar), 8.84 (1H, d, J = 8.0 Hz, H–Ar), 9.93 (1H, s, NH), 14.93 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 115.84, 120.61, 128.12, 137.67, 139.22, 140.72, 143.68, 148.12, 151.49, 153.63 (C N), 173.99 (C S). Analysis for C₁₃H₁₁ClN₄S (290.76), calcd: % C, 53.70; H, 3.81; N, 19.27. Found: % C, 53.81; H, 3.92; N, 19.29.

4.1.1.6. 2-(4-Bromobenzylidene)-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3f)

Yellow powder in 89% yield, m.p 182–183 °C; ¹H NMR (500 MHz, CDCl₃): δ = 7.54–7.55 (3H, m, H–Ar), 7.60 (2H, d, J = 8.5 Hz, H–Ar), 7.76 (1H, t, J = 7.5 Hz, H–Ar), 7.89 (1H, s, H–C N), 8.38 (1H, d, J = 4.0 Hz, H–Ar), 8.87 (1H, d, J = 8.0 Hz, H–Ar), 10.05 (1H, s, NH), 14.98 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 115.46, 120.67, 125.27, 129.06, 131.76, 132.18, 137.84, 142.21, 148.03, 151.35 (C N), 174.13 (C S). LREIMS: 334; HREIMS: 333.9886 (calcd for C₁₃H₁₁BrN₄S; 333.9888). Elemental analysis: calcd: % C, 46.58; H, 3.31; N, 16.71. Found: % C, 46.71; H, 3.40; N, 16.783.

4.1.1.7. 2-(4-Nitrobenzylidene)-N-(pyridin-2-yl)hydrazine-1-carbothioamide (3g)

Yellow powder in 76% yield, m.p 177–179 °C; ¹H NMR (500 MHz, CDCl₃): δ = 7.17 (1H, brs, H–Ar), 7.76–7.79 (1H, m, H–Ar), 7.91–8.00 (3H, m, H–Ar), 8.27 (2H, d, J = 8.5 Hz, H–Ar), 8.39 (1H, s, H–C N), 8.87 (1H, d, J = 4.0 Hz, H–Ar), 9.77 (1H, s, NH), 15.10 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 115.47, 120.95, 124.24, 128.22, 137.99, 139.93, 148.10, 148.82, 151.16 (C N), 152.69 (C–NO₂), 174.66 (C S). Analysis for C₁₃H₁₁N₅O₂S (301.32), calcd: % C, 51.82; H, 3.68; N, 23.24. Found: % C, 51.93; H, 3.80; N, 23.35.

4.1.2. Synthesis of the target compound 5a–g

A mixture of the key intermediate pyridinehydrazinocarbothioamids (3a–g) (1 mmol) and ethyl bromoacetate (0.01 mol) was prepared in 15 mL of absolute ethanol, to which anhydrous sodium acetate was added. This reaction mixture was subjected to reflux conditions for 6–8 h. The reaction was monitored with TLC. After completion of the reaction, the formed solid was collected, washed with ethanol. The compound was recrystallized from a solvent mixture of ethanol and dimethylformamide (9 : 1) by heating the solution to near boiling using a recrystallization apparatus consisting of a conical flask, a beaker, and filter paper to obtain the target compounds 5a–g.

4.1.2.1. 2-(2-Phenylhydrazono)-3-(pyridin-2-yl)thiazolidin-4-one (5a)

Yellow powder in 74% yield, m.p 159–160 °C; ¹H NMR (500 MHz, CDCl₃): δ = 3.97 (2H, s, CH₂), 7.37–7.44 (5H, m, H–Ar), 7.71–7.73 (2H, m, H–Ar), 7.90–7.93 (1H, m, H–Ar), 8.70–8.71 (1H, m, H–Ar), 11.31 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 32.75 (CH₂), 123.50, 124.45, 128.17, 128.67, 130.88, 134.07, 138.76, 143.41, 148.08, 150.02, 171.57 (C O). Analysis for C₁₄H₁₂N₄OS (284.33), calcd: % C, 59.14; H, 4.25; N, 19.70; found: % C, 59.25; H, 4.37; N, 19.80.

4.1.2.2. 3-(Pyridin-2-yl)-2-(2-(p-tolyl)hydrazono)thiazolidin-4-one (5b)

Yellow powder in 68% yield, m.p 189–191 °C; ¹H NMR (500 MHz, CDCl₃): δ = 2.24 (3H, s, CH₃), 3.98 (2H, s, CH₂), 7.06 (2H, t, J = 8.5 Hz, H–Ar), 7.40–7.44 (2H, m, H–Ar), 7.70–7.72 (2H, m, H–Ar), 7.90–7.93 (1H, m, H–Ar), 8.70–8.71 (1H, m, H–Ar), 11.06 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 23.05 (CH₃), 32.74 (CH₂), 115.79, 115.97, 123.47, 124.46, 130.04, 130.11, 130.36, 138.75, 148.07, 150.04, 171.51 (C O). Analysis for C₁₅H₁₄N₄OS (298.36), calcd: % C, 60.38; H, 4.73; N, 18.78. Found: % C, 60.51; H, 4.83; N, 18.90.

4.1.2.3. 2-(2-(4-Methoxyphenyl)hydrazono)-3-(pyridin-2-yl)thiazolidin-4-one (5c)

Yellow powder in 82% yield, m.p 191–192 °C; ¹H NMR (500 MHz, CDCl₃): δ = 3.83 (3H, s, OCH₃), 3.96 (2H, s, CH₂), 6.89 (2H, d, J = 8.5 Hz, H–Ar), 7.40–7.43 (2H, m, H–Ar), 7.65 (2H, d, J = 8.5 Hz, H–Ar), 7.89–7.92 (1H, m, H–Ar), 8.69–8.70 (1H, m, H–Ar), 11.20 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 32.75 (CH₂), 55.37 (OCH₃), 114.15, 123.51, 124.40, 126.85, 129.81, 138.73, 143.30, 148.14, 149.99, 155.35 (C–OCH₃), 171.60 (C O). Analysis for C₁₅H₁₄N₄O₂S (314.36), calcd: % C, 57.31; H, 4.49; N, 17.82; found: % C, 57.42; H, 4.60; N, 17.93.

4.1.2.4. 2-(2-(4-Fluorophenyl)hydrazono)-3-(pyridin-2-yl)thiazolidin-4-one (5d)

Yellow powder in 79% yield, m.p 188–190 °C; ¹H NMR (500 MHz, CDCl₃): δ = 3.96 (2H, s, CH₂), 6.89 (2H, d, J = 8.5 Hz, H–Ar), 7.40–7.43 (2H, m, H–Ar), 7.85 (2H, d, J = 8.5 Hz, H–Ar), 7.89–7.92 (1H, m, H–Ar), 8.69–8.71 (1H, m, H–Ar), 11.12 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 32.75 (CH₂), 114.15, 123.50, 124.39, 126.86, 129.81, 135.51, 138.71, 143.41, 148.16, 150.01, 171.59 (C O). Analysis for C₁₄H₁₁FN₄OS (302.32), calcd: % C, 55.62; H, 3.67; N, 18.53. Found: % C, 55.74; H, 3.77; N, 18.65.

4.1.2.5. 2-(2-(4-Chlorophenyl)hydrazono)-3-(pyridin-2-yl)thiazolidin-4-one (5e)

Yellow powder in 69% yield, m.p 200–202 °C; ¹H NMR (500 MHz, CDCl₃): δ = 3.98 (2H, s, CH₂), 7.35 (2H, d, J = 8.5 Hz, H–Ar), 7.39–7.44 (2H, m, H–Ar), 7.64 (2H, d, J = 8.5 Hz, H–Ar), 7.90–7.93 (1H, m, H–Ar), 8.70–8.71 (1H, m, H–Ar), 11.37 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 32.75 (CH₂), 123.47, 124.61, 129.00, 129.15, 129.29, 129.75, 132.57, 136.82, 138.76, 148.03, 150.05 (C N), 171.49 (C O). Analysis for C₁₄H₁₁ClN₄OS (318.77), calcd: % C, 52.75; H, 3.48; N, 17.58; found: % C, 52.88; H, 3.59; N, 17.70.

4.1.2.6. 2-(2-(4-Bromophenyl)hydrazono)-3-(pyridin-2-yl)thiazolidin-4-one (5f)

Yellow powder in 84% yield, m.p 189–190 °C; ¹H NMR (500 MHz, CDCl₃): δ = 3.98 (s, 2H, CH₂), 7.39–7.45 (m, 2H, H–Ar), 7.51 (d, 2H, J = 8.5 Hz, H–Ar), 7.57 (d, 2H, J = 8.5 Hz, H–Ar), 7.90–7.94 (m, 1H, H–Ar), 8.70–8.71 (m, 1H, H–Ar), 11.23 (s, 1H, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 32.75 (CH₂), 123.47, 124.49, 125.27, 129.49, 129.94, 131.96, 132.12, 132.99, 138.77, 148.02, 150.05, 171.49 (C O). Analysis for C₁₄H₁₁BrN₄OS (363.23), calcd: % C, 46.29; H, 3.05; N, 15.42; found: % C, 46.40; H, 3.14; N, 15.55.

4.1.2.7. 2-(2-(4-Nitrophenyl)hydrazono)-3-(pyridin-2-yl)thiazolidin-4-one (5g)

Red powder in 74% yield, m.p 199–201 °C; ¹H NMR (500 MHz, CDCl₃): δ = 4.01 (2H, s, CH₂), 7.40 (1H, d, J = 8.0 Hz, H–Ar), 7.44–7.46 (1H, m, H–Ar), 7.86 (2H, d, J = 8.5 Hz, H–Ar), 7.92–7.95 (1H, m, H–Ar), 8.23 (2H, d, J = 8.5 Hz, H–Ar), 8.70–8.71 (1H, m, H–Ar), 11.09 (1H, s, NH). ¹³C NMR (126 MHz, CDCl₃) δ = 32.77(CH₂), 123.43, 123.97, 124.61, 128.66, 131.86, 133.64, 138.83, 139.94, 147.88, 148.91, 150.09, 171.37(C O). Analysis for C₁₄H₁₁N₅O₃S (329.33), calcd: % C, 51.06; H, 3.37; N, 21.27; found: % C, 51.17; H, 3.47; N, 21.39.

4.2. Biological evaluations

4.2.1. In vitro evaluation of antileishmanial efficacy

4.2.1.1. In vitro antileishmanial activity on L. major promastigotes

The antileishmanial activity of the compounds was assessed using a colorimetric assay. First, each compound was dissolved in DMSO and diluted to a working concentration of 1 mg mL⁻¹, ensuring the final DMSO concentration in the wells remained at or below 0.1% to prevent interference with parasite growth. For the assay, both the test compounds and the positive controls (amphotericin B and miltefosine) were subjected to three-fold serial dilutions in fresh culture medium, producing a concentration series from 40 down to 0.04 µg mL⁻¹. Into each well of a flat-bottom 96-well plate, 100 µL of culture medium containing 3 × 10⁶ promastigotes of L. major was dispensed. The serially diluted compounds were then added to the parasite cultures in duplicate. Negative control wells were set up with parasites, medium, and DMSO only. The plates were incubated at ambient temperature (21 ± 1 °C) for 24 hours. Subsequently, 10 µL of Alamar blue indicator (prepared by dissolving 12.5 mg of resazurin in 100 mL of distilled water) was added to every well. After further 48-hour incubation, the absorbance was read at dual wavelengths (540 nm and 630 nm). The assay relies on the metabolic reduction of the blue, non-fluorescent resazurin dye to pink, highly fluorescent resorufin, with the signal intensity directly correlating with the number of living cells.^57–59

4.2.1.2. In vitro antileishmanial activity on L. major amastigotes

To assess antileishmanial activity, a serial dilution of each test compound was prepared in a 96-well microtiter plate, yielding final concentrations between 0.04 and 40 µg mL⁻¹ in 50 µL of culture medium. This was followed by the addition of 50 µL of a culture containing axenic amastigotes at a density of 2 × 10⁷ cells per mL to each well. The plates were then maintained in a humidified incubator at 31 °C with 5% CO₂ for a total of 72 hours. After 68 hours of incubation, a 10-µL aliquot of a resazurin fluorochrome solution (prepared by dissolving 12.5 µg in 100 mL PBS, pH 7.2) was introduced into every well. The resultant fluorescence, indicative of cell viability, was quantified after the full 72-hour period using a Victor³ Multilabel Counter, set to an excitation of 530 nm and an emission of 590 nm. The half-maximal effective concentration (IC₅₀) for each compound was derived by fitting the data to sigmoidal dose–response curves using GraphPad Prism 3.0 software. All results are expressed as the mean ± standard deviation of triplicate biological runs, with each concentration tested in duplicate wells. Standard reference drugs and negative controls (culture medium alone and 1% DMSO) were run concurrently, and background fluorescence readings were subtracted from all experimental values.^60,61

4.2.2. Reversal of antileishmanial activity by folic acid and folinic acid

To evaluate whether folic and folinic acids inhibit folate activity, each test compound was preincubated with either 20 or 100 µM of folic or folinic acid in the presence of 10⁶ log-phase Leishmania parasites. Following incubation, the parasites were washed, resuspended in phosphate-buffered saline (PBS), and kept at room temperature for 1 hour. The cells were then pelleted by centrifugation, the supernatant discarded, and the pellet resuspended in fresh culture medium before being seeded into 24-well plates. Test compounds or trimethoprim were subsequently added at the specified concentrations, and the plates were incubated for 48 hours. Parasite viability was assessed by calculating the percentage of growth inhibition using the equation: % inhibition = 100 × (C − T)/C, where C represents the parasite count per mL in untreated control wells, and T denotes the average number of motile parasites per mL in treated samples.⁶²

4.2.3. In vitro cytotoxicity evaluation

Compounds that showed the highest antileishmanial activity were subsequently evaluated for in vitro cytotoxicity using established and validated procedures. Briefly, Vero cells were seeded in 96-well plates at a density of 1 × 10⁵ cells per well and exposed to varying concentrations (0–100 µM) of the test compounds for 72 hours. Incubation was carried out at 37 °C in a humidified atmosphere containing 5% CO₂. The cells were maintained in Eagle's minimal essential medium (MEM; Gibco) supplemented with 5% heat-inactivated fetal bovine serum. Cytotoxicity was assessed using the crystal violet staining method, and the half-maximal cytotoxic concentration (CC₅₀) values, defined as the compound concentration required to reduce cell growth by 50%, were calculated using Excel and GraphPad Prism. At least three independent experiments were performed for each compound, with each condition tested in duplicate.^55,59

4.3. Molecular modeling

4.3.1. Molecular docking

The crystal structure of the L. major PTR1 enzyme (PDB ID: 7PXX⁶³) was obtained from the Protein Data Bank in PDB format. For the L. major DHFR enzyme, the AlphaFold model (code AF-P07382-F1 (ref. 66)) was utilized.^64,65 This model was aligned with the Escherichia coli DHFR structure (PDB ID: 4QLF⁶⁷) using PyMOL software,⁶⁸ after which NAD and folate molecules were incorporated into the model. In the docking study, three-dimensional structures of folate and selected ligands were generated and optimized using MarvinSketch. Molecular docking simulations were performed with AutoDock Vina.⁶⁹ The active site coordinates were defined as x = −54.1, y = 20.1, and z = 1.4, with a grid box size of 19.3 × 17.7 × 19.8 Å. Visualization of the docking results was conducted using Discovery Studio 2025, which facilitated both three-dimensional rendering and two-dimensional schematic representations.

4.3.2. Molecular dynamic simulation

To investigate the interaction stability of compound 5e within the active site, molecular dynamics (MD) simulations were performed based on the optimal docking conformation. The CHARMM force field parameters for tyrosinase were utilized to generate the input files for MD calculations using CHARMM-GUI solution builder.^70,71 The topologies of the selected ligands were generated using the CGenFF server and CHARMM General Forcefield. The CHARMM-GUI solution builder consists of five steps. In the initial step, the tool reads the protein–ligand combination's coordinates. The size and structure of the system are determined during the second phase, which involves solvating the protein–ligand combination. In this phase, Na⁺ and Cl⁻ ions are added to neutralize the system.

In order to mimic a large system using a unit cell that is then repeated in all directions, the third phase creates Periodic Boundary Conditions (PBC). The simulation is limited to the atoms that are part of the PBC box. Na⁺ ions have been added to neutralize complexes with dimensions of 77 Å, 77 Å, and 77 Å for the PBC box in x, y, and z, respectively.⁷² At this point, faulty connections are removed through short minimization. The fourth and fifth steps are system and production equilibration. Equilibration is done in two stages: NPT ensemble and NVT ensemble, to ensure the system has reached the proper temperature and pressure.

The desired changes, such as the number of MD run steps, the frequency of trajectory saving, the energy calculation, etc., are made after downloading the equilibration and production input files. The Verlet cutoff scheme was used to buffer the neighbor-searching list, and a 12 Å cutoff distance was used for non-bonded interactions, whilst the particle mesh Ewald (PME) technique was used for long-range electrostatic interactions. Before the production simulation, the steepest descent algorithm (5000 steps) was used to minimize the system's energy.

The complex was then equilibrated to stabilize its temperature and pressure by exposing it to NVT and NPT ensembles and simulating for 125 ps at 310.15 K using 400 kJ mol⁻¹ nm⁻² and 40 kJ mol⁻¹ nm⁻² positional restrictions on the backbone and side chains, respectively. After that, the complex is subjected to a 100 ns production simulation run at 310.15 K and 1 bar in the NPT ensemble. The temperature was controlled by Nose–Hoover thermostats, while the pressure was controlled by Parrinello–Rahman barostats. The LINCS method was used to limit H-bonds using CHARMM-GUI inputs. The V-rescale thermostat was used at 300 K with a coupling constant of 1 ps. The trajectories were saved every two ps. NPT assembly simulations lasting 100 ns were conducted for the production phase. The GROMACS 2024.4 program was used to simulate the complexes for 100 ns. GROMACS tools were used to examine the trajectories of the MD simulations. The VMD molecular graphics application was used for contact frequency analysis, detailed hydrogen-bonding data, and trajectory visualization.⁷⁴ MM/PBSA (molecular mechanics/Poisson–Boltzmann surface area) calculations were performed using g_mmpbsa, a GROMACS tool for determining binding affinity.⁷³

Author contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, and acquisition of data, analysis, or interpretation. Also, they took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agreed to be accountable for all aspects of the work.

Conflicts of interest

The authors declare no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Data availability

ALL required data inserted in manuscript and supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra07150c.