Thiolactomycin-based morpholino and piperidino conjugates: synthesis, antimalarial evaluation, and PfFAS-II inhibition

Abstract

Background and purpose:

Malaria remains a formidable global healthcare challenge due to emerging drug-resistant Plasmodium spp., underscoring the urgent need for novel antimalarial scaffolds. Thiolactomycin, a FAS-II inhibitor, is a promising lead for structural optimization. This study aims to design, synthesize, and evaluate a series of thiomorpholino and piperidino conjugates bearing a thiolactone moiety as target-specific antimalarial agents.

Experimental approach:

A series of thiolactomycin-based conjugates was synthesized, including intermediate 7b and conjugate 4g. Antimalarial activity was evaluated in vitro against the Plasmodium falciparum D10 strain. To elucidate the mechanism, multi-target docking studies of 7b and 4g were performed against key FAS-II enzymes, followed by 200 ns explicit-solvent MD simulations and MM-PBSA binding free energy calculations targeting PfFabI. In silico pharmacokinetics and drug-likeness profiling of potent molecules were also carried out.

Findings/Results:

Biological evaluation identified 7b and 4g as potent compounds with IC₅₀ values of 0.2 and 1.81 μM, respectively. However, the higher phenotypic potency of 7b was accompanied by mutagenic liability (Ames positive) revealed by toxicological profiling. Conversely, 4g exhibited superior binding affinity (-7.2 kcal/mol) against PfFabI. MD simulations revealed an induced-fit stabilization of the 4g-PfFabI complex and the complex achieved superior binding free energy (ΔG_Total = -22.14 kcal/mol) compared to thiolactomycin.

Conclusion and implications:

Conjugate 4g was identified as a safer potent compound from the series. MD simulation of the 4g-PfFabI complex revealed an induced fit stabilization involving persistent H-bonds that were absent in docking. These findings suggest 4g as a promising molecule for further optimization.

INTRODUCTION

Malaria can be a potentially fatal disease transmitted through the bites of mosquitoes, which introduce the disease-causing Plasmodium parasite into humans (1). It remains a significant challenge in the sub-Saharan Africa region and parts of Asia owing to various environmental, biological, and socio-economic factors (2). A recent global WHO report suggested that nearly a quarter of a billion malaria cases occurred, with over half a million deaths only in the year 2022 (3). Quinine, from the bark of the cinchona tree, was the first established drug used in the treatment of malaria, and now several drugs are clinically used, such as chloroquine, mefloquine, artemisinin, artesunate, amodiaquine, etc. (Fig. 1) (4,5). The most widely used drugs are artemisinin-based combination therapies (ACTs) as per the recommendation of WHO as the first-line treatment (6,7). Unfortunately, multidrug-resistant strains of Plasmodium falciparum have emerged that are also resistant to artemisinin-based combination therapies (8,9). Further exacerbating the scenario, the vector mosquitoes are increasingly developing resistance to insecticides that are used to control them (10,11). Therefore, there is a pressing need to develop new antimalarial agents, underscored by the surge in recent scientific literature (12,13). One of the promising lead molecules is thiolactomycin 1, which exhibits significant inhibition of beta-ketoacyl-ACP synthase III (FabH), β-ketoacyl-acyl carrier protein synthases (Kas) A and B enzymes, which are essential in the fatty acid synthase II pathway of the bacterial cellular system (14,15). Interestingly, this fatty acid synthesis II (FAS-II) pathway is also present in the Plasmodium parasite and is distinct from the human FAS-I system, making it a potential therapeutic target (16,17). This approach is also exploited in the development of thiolactomycin-based agents for treating tuberculosis, a fatal mycobacterial infection exacerbated by evolving drug resistance (18).

Fig. 1.

Drugs used in malarial treatment.

In the 2000s, only limited investigations were undertaken toward the development of newer thiolactomycin-based antimalarial compounds. One study reported that the modification at the C3 and C5 positions of the thiolactone ring resulted in increased activity (19) . Further studies from the same research group demonstrated that the attachment of alkyl moieties on the hydroxyl group at the C4 position led to a remarkable increase in potency (20) . Based on these reports, we selected the C4 position as a suitable site for structural diversification that could lead to enhanced effect. Furthermore, in the literature, 3D QSAR models were employed to identify the key structural elements that affect the antimalarial potency of thiolactone-based compounds (21).

The models suggested that hydrophobic side chains with steric bulk around the core structure could be useful in the design of new thiolactone-based antimalarial agents. Based on these findings, we intended to incorporate moderately bulky groups at the C4 position to enhance hydrophobic interactions while maintaining the potency of the thiolactomycin pharmacophore. In recent decades, the antitubercular potential of thiolactomycin-based compounds was extensively explored, and we have reported newer quinoline-thiolactone conjugates (2 and 3) as antitubercular and antibacterial with encouraging potency (22,23). However, the promising antimalarial potential of the thiolactomycin-based compounds remains largely unexplored that warrant further investigation.

Based on the promising potential of modifications at the C4 position, we hypothesized that incorporating basic heterocyclic motifs would be advantageous. Heterocyclic groups are known to enhance solubility, improve cellular uptake into the parasite, and facilitate different electronic or hydrogen-bonding interactions with the receptor proteins at the molecular level. Piperidine was selected as it has a well-established presence in antimalarial drugs like mefloquine and exhibits a balance between basicity and lipophilicity (24). On the other hand, thiomorpholine, with the presence of sulfur, offers distinct characteristics that may affect the polarity and interaction pattern differently than that of morpholine. We envisaged that conjugating piperidine and thiomorpholine motifs at the C4 position of a thiolactomycin core would lead to a potential antimalarial lead (Fig. 2). Therefore, a series of these conjugates was synthesized and investigated for antimalarial and antitubercular activity. The promising compounds were taken forward for extensive molecular docking studies against a set of selected enzymes involved in the FAS-II pathway of P. falciparum, followed by in silico pharmacokinetics and lead-likeness profiling. Following the application of the Ames filter for toxicity screening, 200 ns explicit-solvent molecular dynamics (MD) simulations and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations were performed to understand the plausible mechanism of action of the prioritized lead.

Fig. 2.

Designing new thiolactone-piperidine/morpholine conjugates.

MATERIALS AND METHODS

Chemistry

Synthesis of thiolactomycin-based thiomorpholine and piperidine conjugates

The target thiolactomycin-based conjugates were synthesized through a two-step diversification strategy starting from the previously reported 4-hydroxy thiolactone intermediate (6). In the first step, O-alkylation of intermediate 6 was achieved using a series of dibromoalkanes containing different carbon chain lengths (C2-C12) in the presence of anhydrous potassium carbonate as a base. The reactions were carried out under reflux in dry acetone to afford the corresponding monobromoalkyl thiolactone intermediates (7a-i). These intermediates served as precursors for subsequent nucleophilic substitution reactions. The detailed synthesis and spectral data of monobromoalkyl thiolactone derivatives with longer carbon chains, i.e., 7f-i, are mentioned in our earlier report (23).

In the second step, the monobromoalkyl intermediates (7a-i) were reacted with either thiomorpholine or piperidine to generate two distinct series of thiolactomycin derivatives. To a solution of monobromoalkyl thiolactone derivatives 7a-i in dry acetone, potassium carbonate and either thiomorpholine or piperidine were added and refluxed for 24-48 h. The reaction proceeded via nucleophilic displacement of the terminal bromide group, furnishing the corresponding thiomorpholine conjugates (4a-i) and piperidine conjugates (5a-i). Upon completion, the reaction mixtures were filtered through Celite, concentrated under reduced pressure, and extracted with ethyl acetate. The organic layers were washed with brine, dried over anhydrous sodium sulfate, and concentrated. Final purification was achieved by silica gel column chromatography to afford the desired products in good to excellent yields.

The synthesized compounds were characterized by infrared (IR) spectroscopy, nuclear magnetic resonance spectroscopy (¹H and ¹³C NMR), electron ionization mass spectrometry (EIMS), and elemental analysis, which collectively confirmed the proposed chemical structures and purity of the target molecules.

In vitro P. falciparum assay

The chloroquine-sensitive strain of P. falciparum (D10) was continuously cultured at 5% haematocrit following a method adapted from Trager and Jensen (25). The parasites were grown in O-positive human red blood cells, and the growth medium contained Albumax II (GIBCO/Invitrogen, USA), RPMI 1640 (Biowhittaker, USA), 25 mM HEPES (Sigma, USA), 1% sodium bicarbonate, hypoxanthine (44 mg/L), and gentamicin (40 mg/mL). The cultures were maintained at 37 °C in a gas mixture of 3% oxygen, 4% carbon dioxide, and 93% nitrogen. To synchronize the parasites at the ring stage, 5% D-sorbitol (Sigma, USA) was used, following the Lambros and Vandenberg protocol. The drug activity was assessed in vitro using a parasitic lactate dehydrogenase assay, modified from Makler et al. (26). For the assay, parasites were cultured at 1% haematocrit and 2% parasitaemia for 48 h, both with and without the test compound in 96-well microtiter plates. After incubation, Malstat reagent (Flow Inc., USA) was added as a colorimetric indicator to measure parasite viability. IC50 values were calculated from the resulting data using a sigmoidal dose-response curve analysis.

Molecular modeling studies

The 3D structures of the ligands 4g, 7b, control (thiolactomycin), and co-crystallized native ligands were generated utilizing Simplified Molecular Input Line Entry System (SMILES) employing NovaPro online tools. The crystal structures of the four selected target FAS-II pathway proteins were retrieved from the RCSB Protein Data Bank (27) which includes Escherichia coli FabB co-crystallized with thiolactomycin (PDB ID: 1FJ4) (28), Plasmodium falciparum FabI co-crystallized with triclosan (PDB ID: 1NHG) (29), P falciparum enoyl-ACP-reductase (PfFabI) co-crystallized with NAD and inhibitor 7-(4-chloro-2-hydroxyphenoxy)-4-methyl-2H-chromen-2-one (PDB ID: 4IGE) (30) and PfFabZ co-crystallized with 8-(benzyloxy)-5-chloroquinoline (PDB ID: 3AZA) (31). All 3D structures were visualized using Biovia Discovery visualization software. Water molecules, co-crystallized ligands, and HETATM entities were removed from the active site, and the binding pockets were analyzed based on the native ligand positions in the crystal structures.

The docking grid was centered on the thiolactomycin binding site for FabB (1FJ4), on the native inhibitor-binding pocket for FabI (1NHG and 4IGE), and on the inhibitor-bound catalytic tunnel for FabZ (3AZA) to fully cover the active-site residues and permit ligand flexibility. The docking grid was centered on the active-site pocket of each receptor using the following coordinates: FabB (1FJ4), x = 4.79, y = -20.35, z = 0.53; FabI (1NHG), x = 51.55, y = 93.27, z = 34.34; FabI (4IGE), x = 51.83, y = 90.98, z = 34.45; and FabZ (3AZA), x = -3.59, y = 30.89, z = 16.25. The docking grid box dimensions were set to 60 × 60 × 60 Å with a grid spacing of 0.375 Å to fully encompass the catalytic residues and accommodate the ligands’ conformational flexibility. Additionally, energy minimization was performed using CHARMM force field implementation modules incorporated within the software. The MGL AutoDock 4.2 tools protocol was employed to evaluate the binding energies between the selected ligands and the receptor molecules (32). Docking was carried out using AutoDock 4.2 with the Lamarckian genetic algorithm. For each ligand, 10 independent docking runs were performed with a population size of 150, a maximum of 2.5 × 10^-6 energy evaluations, and 27,000 generations, while the mutation and crossover rates were kept at the default values. The resulting poses were clustered using a root mean square deviation (RMSD) tolerance of 2.0 Å, and the lowest-energy pose from the most populated cluster was selected as the representative binding mode. AutoDock was applied to calculate the interaction affinity in kcal/mol. The docking and molecular simulation methods followed previously reported protocols (23).

MD studies

The stability and dynamic behavior of the docked complexes of the lead compound 4g and thiolactomycin (control) with PfFabI (PDB: 1NHG) were investigated using 200 ns MD simulations performed with the GROMACS package (33). Protein parameters were defined employing the CHARMM force field, while the topology files for the ligand were generated using the SwissParam server to ensure compatibility (34). Each protein-ligand complex was prepared by removing crystallographic water molecules and non-essential heteroatoms, followed by solvation in an appropriate periodic boundary condition box using the selected water model. The systems were neutralized by adding counter ions and then energy-minimized to remove steric clashes and unfavorable contacts via the steepest-descent algorithm (50,000 steps or F_max < 10.0 kJ/mol.nm). The system was equilibrated under positional restraints on solute heavy atoms in two 100 ps stages: an NVT ensemble (300 K, velocity-rescaling thermostat, τ = 0.1 ps) followed by an NPT ensemble (1 bar, Parrinello-Rahman barostat, τ = 2.0 ps, compressibility = 4.5 × 10^-5 bar^-1) to stabilize system density. Production MD runs were executed for 200 ns, and the post-simulation analysis to calculate the RMSD, root mean square fluctuation (RMSF), radius of gyration (Rg), and hydrogen bond analysis was performed using GROMACS built-in utilities, and the XMGRACE tool was used to generate the plots.

To estimate the relative binding free energy of the complexes, the gmx_MMPBSA workflow was applied to representative frames extracted from the equilibrated trajectories (35). The MM-PBSA method was used to compute the total binding free energy based on the molecular mechanics, polar solvation, and nonpolar solvation contributions. The protein, ligand, and complex groups were defined for each receptor system, and the solvent and ions were excluded from the final energy decomposition. The binding free energy values were reported as the average over the selected trajectory frames.

Absorption, distribution, metabolism, excretion, toxicity, and drug-likeness

Pharmacological (absorption, distribution, metabolism, excretion, toxicity (ADMET)) and physicochemical properties of the synthesized molecules were evaluated using Molinspiration and SwissADME online web tools (36). The lipophilicity (logP), topological polar surface area (TPSA), and drug likeliness were determined for all the synthesized compounds.

RESULTS

Chemistry

The 4-hydroxy thiolactone intermediate (6), serving as the core scaffold for the design of thiolactone-piperidine and thiolactone-thiomorpholine conjugates, was synthesized via a previously reported four-step synthetic route (23). The thiolactone intermediate 6 was subsequently alkylated with various dibromoalkanes containing different methylene carbon spacers, resulting in the formation of corresponding monobromoalkyl derivatives 7a-i (Scheme 1). The reactions were carried out in the presence of anhydrous potassium carbonate used as a base and refluxed in dry acetone (~ 56 °C) to afford the derivatives in 61-65% yield. The formation of these derivatives was confirmed by spectroscopic analysis. In the ¹H NMR spectra, the disappearance of the hydroxyl proton signal and the appearance of characteristic methylenes (-CH2) signals in the range of δ 1.26-4.10 ppm, including the methylene groups attached to oxygen and bromine atoms, confirm the successful alkylation step. The desired new thiolactone-morpholine conjugates (4a-i) were synthesized by a straightforward condensation reaction involving nucleophilic substitution of monobromoalkyl derivatives (7a-i) with thiomorpholine in dry acetone using anhydrous potassium carbonate as base. Similarly, thiolactone-piperidine conjugates (5a-i) were synthesized by the reaction of the same derivatives with piperidine under analogous conditions. The reaction yields were satisfactory, ranging from 64% to 84%. The formation of the desired conjugates was unequivocally confirmed by spectroscopic analysis. In the ¹H NMR spectra, the appearance of additional characteristic methylene signals (δ 1.81-2.82 ppm), attributable to the introduced heterocyclic moieties, provided clear evidence of successful conjugation. The methyl protons on the thiolactone core were observed at δ 1.58 and 1.83 ppm, corresponding to the CH3 groups at the C5 and C4 positions, respectively. The remaining proton of the C4 appeared as a distinct signal in the δ 4.32-4.49 ppm range. Further structural confirmation was obtained through IR spectroscopy and elemental analysis, which were consistent with the proposed structures.

Scheme 1.

Reagents and conditions: i) Br-(CH₂)_n-Br, anh. K₂CO₃, dry acetone, reflux; ii) thiomorpholine, anh. K₂CO₃, dry acetone, reflux; iii) piperidine, anh. K₂CO₃, dry acetone, reflux.

3,5-Dimethyl-4-(2-bromoethoxy)-5H-thiophen-2-one (7a)

Prepared from 4-hydroxy thiolactone 6 (1.44 g, 10 mmol), anhydrous potassium carbonate (5.53 g, 40 mmol) and 1,2-dibromoethane (3.58 mL, 40 mmol) to afford 1.53 g of 7a as a colorless liquid. Yield: 61%; IR (neat): 2929, 2856, 1678, 1627 cm^-1; ¹H NMR (200 MHz; CDCb) δ 1.59 (3H, d, J = 7.0 Hz, CH₃ linked to thiolactone (TL) ring), 1.86 (3H, s, =CCH₃ attached to alkenyl carbon in TL ring), 3.57 (2H, t, J = 7.0 Hz, -CH₂Br), 4.39-4.65 (3H, m, ring CH and -OCH₂); EIMS (m/z): 253 [M+2]⁺; Anal. calcd for C₈H₁₁BrO₂S: C, 38.26; H, 4.41%. Found: C, 38.20; H, 4.36%.

3,5-Dimethyl-4-(3-bromopropoxy)-5H-thiophen-2-one (7b)

Prepared from 4-hydroxy thiolactone 6 (1.44 g, 10 mmol), anhydrous potassium carbonate (5.53 g, 40 mmol) and 1,3-dibromopropane (4.2 mL, 40 mmol) following the general procedure to afford 1.7 g of 7b as a colorless liquid. Yield: 64%; IR (neat): 2976, 2929, 1678, 1633 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.54 (3H, d, J = 7.03 Hz, -CH₃ linked to TL ring), 1.9 (3H, s, =CCH₃ attached to alkenyl carbon in TL ring), 2.24-2.36 (2H, m, -CH2-alkane spacer), 3.51 (2H, t, J = 7.03 Hz, -CH₂Br), 4.10-4.48 (3H, m, TL ring -CH- and -OCH2-); ¹³C NMR (100MHz; CDCl₃): δ 8.78, 19.57, 28.96, 32.24, 41.63, 68.07, 114.19, 177.39, 194.98; EIMS (m/z): 265 [M]⁺; Anal. calcd for C₉H₁₃BrO₂S: C, 40.77; H, 4.94%. Found: C, 40.71; H, 4.88%.

3,5-Dimethyl 4-(4-bromobutoxy)-5H-thiophen-2-one (7c)

Prepared from 4-hydroxy thiolactone 6 (1.44 g, 10 mmol), anhydrous potassium carbonate (5.53 g, 40 mmol) and 1,4-dibromobutane (4.8 mL, 40 mmol) by following the general procedure to afford 1.8 g of 7c as a colorless liquid. Yield: 65%; IR (neat): 2984, 2931, 1674, 1629 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.59 (3H, d, J = 7.0 Hz, -CH₃ linked to TL ring), 1.9 (3H, s, =CCH₃ attached to alkenyl carbon in TL ring), 1.95-2.1 (4H, m, -CH₂-CH₂- alkane spacer), 3.5 (2H, t, J = 7.0 Hz, -CH₂Br), 4.10-4.45 (3H, m, TL ring -CH- and -OCH₂-); FABMS (m/z): 281 [M+2]⁺; Anal. calcd for C₁₀H₁₅BrO₂S: C, 43.02; H, 5.42%. Found: C, 42.98; H, 5.35%.

3,5-Dimethyl-4-[(5-bromopentyl)oxy]-5H-thiophen-2-one (7d)

Prepared from 4-hydroxy thiolactone 6 (1.44 g, 10 mmol), anhydrous potassium carbonate (5.53 g, 40 mmol) and 1,5-dibromopentane (5.5 mL, 40 mmol) by following the general procedure to afford 1.90 g of 7d as a colorless liquid. Yield: 65%; IR (neat): 2995, 2944, 1671, 1630 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.54 (3H, d, J = 7.0 Hz, -CH3 linked to TL ring), 1.66-1.86 (4H, m, -CH₂-CH₂- alkane spacer), 1.94 (3H, s, =CCH3 attached to alkenyl carbon in TL ring), 1.96-2.10 (2H, m, Br-C-CH₂-), 3.5 (2H, t, J = 7.03 Hz, -CH₂Br), 4.1-4.4 (3H, m, TL ring -CH- and -OCH₂-); EIMS (m/z): 293 [M]⁺; Anal. calcd for C₁₁H₁₇BrO₂S: C, 45.06; H, 5.84%. Found: C, 44.98; H, 5.78%.

3,5-Dimethyl-4-[(6-bromohexyl)oxy]-5H-thiophen-2-one (7e)

Prepared from 4-hydroxy thiolactone 6 (1.44 g, 10 mmol), anhydrous potassium carbonate (5.53 g, 40 mmol) and 1,6-dibromohexane (6.2 mL, 40 mmol) by following the general procedure to afford 2.02 g of 7e as a colorless liquid. Yield: 66%; IR (neat): 2985, 2953, 1667, 1623 cm^-1; ¹H NMR (300 MHz; CDCl₃) δ 1.4-1.52 (4H, m, -CH₂-CH₂- alkane spacer), 1.54 (3H, d, J = 7.0 Hz, -CH3 linked to TL ring), I. 65-1.8 (2H, m, -CH₂-), 1.94 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 1.96-2.0 (2H, m, Br-C-CH₂-), 3.51 (2H, t, J = 7.0 Hz, - CH₂Br), 4.1-4.4 (3H, m, TL ring -CH- and - OCH₂-); EIMS (m/z): 307 [M]⁺; Anal. calcd for C₁₂H₁₉BrO₂S: C, 46.91; H, 6.23%. Found: C, 46.87; H, 6.15%

3,5-Dimethyl 4-[2-(1,4-thiazinan-4-yl)ethoxy]-5H-thiophen-2-one (4a)

Prepared from 7a (251 m, 1 mmol), anhydrous potassium carbonate (553 m, 4 mmol) and thiomorpholine (0.14 mL, 1.3 mmol) by following the general procedure to afford 175 mg of 4a as a pale-yellow oily liquid. Yield: 64%; IR (neat): 2636, 2862, 1677, 1627 cm^-1; ¹H NMR (200 MHz; CDCb) 5 1.58 (3H, d, J = 6.7 Hz, -CH3 linked to TL ring), 1.82 (3H, s, =CCH3 attached to alkenyl carbon of TL ring), 2.56-2.66 (4H, m, -CH₂-S-CH₂- thiomorpholine ring), 2.66-2.73 (2H, t, J = 6.7 Hz, -N-CH₂- alkane spacer linked to thiomorpholine), 2.74-2.82 (4H, m, -CH₂-N-CH₂-thiomorpholine ring), 4.05-4.43 (3H, m, TL ring -CH- and -OCH₂-); FABMS (m/z): 273 [M]⁺; Anal. calcd for C₁₂H₁₉NO₂S₂: C, 52.71; H, 7.00; N, 5.12%. Found: C, 52.69; H, 6.95; N, 5.08%.

3,5-Dimethyl-4-[3-(1,4-thiazinan-4-yl) propoxy]-5H-thiophen-2-one (4b)

Prepared from 7b (265 m, 1 mmol), anhydrous potassium carbonate (553 m, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 189 mg of 4b as a pale-yellow oily liquid. Yield: 66%; IR (neat): 2935, 2862, 1678, 1629 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), 1.82 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 1.85-1.98 (2H, m, -C-CH₂-C-alkane spacer), 2.46 (2H, t, J = 6.7 Hz, -N-CH₂- alkane spacer linked to thiomorpholine), 2.60-2.71 (8H, m, -CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 4.07-4.44 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 288 [M+1]⁺; Anal. calcd for C₁₃H₂₁NO₂S₂: C, 54.32; H, 7.36; N, 4.87%. Found: C, 54.29; H, 7.31; N, 4.82%.

3.5-Dimethyl-4-[3-(1,4-thiazinan-4-yl)butoxy]-5H-thiophen-2-one (4c)

Prepared from 7c (279 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 201 mg of 4c as a pale-yellow oily liquid. Yield: 67%; IR (neat): 2937, 2859, 1680, 1631 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), 1.82 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 1.97-2.07 (4H, m, -C-CH₂-CH₂-C- alkane spacer), 2.46 (2H, t, J = 6.7 Hz, -N-CH₂- alkane spacer linked to thiomorpholine), 2.60-2.71 (8H, m, -CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 4.07-4.44 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 302 [M+1]⁺; Anal. calcd for C₁₄H₂₃NO₂S₂: C, 55.78; H, 7.69; N, 4.65%. Found: C, 55.75; H, 7.71; N, 4.62%.

3,5-Dimethyl-4-([5-(1,4-thiazinan-4-yl) pentyl] oxy)-5H-thiophen-2-one (4d)

Prepared from 7d (293 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 214 mg of 4d as a pale-yellow oily liquid. Yield: 68%; IR (neat): 2934, 2861, 1677, 1628 cm^-1; ¹H NMR (300 MHz; CDCl₃) δ 1.4-1.53 (4H, m, -C-CH₂-CH₂-C- alkane spacer), 1.58 (3H, d, J = 6.7 Hz, -CH3 linkded to TL ring), 1.62-1.74 (2H, m, -C-CH₂-C-O-alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.34 (2H, t, J = 6.7 Hz, -N-CH2-alkane spacer linked to thiomorpholine), 2.55-2.75 (8H, m, -CH₂-S-CH₂- and -CH₂-N- CH₂- of thiomorpholine ring), 4.03-4.36 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 316 [M+1]⁺; Anal. calcd for C₁₅H₂₅NO₂S₂: C, 57.11; H, 7.99; N, 4.44%. Found: C, 57.08; H, 7.88; N, 4.35%.

3,5-Dimethyl-4-([6-(1,4-thiazinan-4-yl)hexyl] oxy)-5H-thiophen-2-one (4e)

Prepared from 7e (307 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 227 mg of 4e as a pale-yellow oily liquid. Yield: 69%; IR (neat): 2932, 2860, 1677, 1628 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.30-1.47 (6H, m, -C-CH₂-CH₂-CH₂-C-alkane spacer), 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), 1.67- 1.74 (2H, m, -C-CH₂-C-O-alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.32 (2H, t, J = 6.7 Hz, -N-CH2-alkane spacer linked to thiomorpholine), 2.62-2.71 (8H, m, -CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 3.92-4.49 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 330 [M+1]⁺; Anal. calcd for C₁₆H₂₇NO₂S₂: C, 58.32; H, 8.26; N, 4.25%. Found: C, 58.26; H, 8.23; N, 4.20%.

3,5-Dimethyl-4-([8-(1,4-thiazinan-4-yl)octyl] oxy)-5H-thiophen-2-one (4f)

Prepared from 7f (335 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the above general procedure to afford 278 mg of 4f as a pale-yellow oily liquid. Yield: 78%; IR (neat): 2930, 2858, 1678, 1629 cm^-1; ¹H NMR (300 MHz; CDCl₃) δ 1.24-1.51 (10H, m, -C-(CH₂)₅-C- alkane spacer), 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), 1.67-1.73 (2H, m, -C-CH₂-C-O- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.32 (2H, t, J = 6.7 Hz, -N-CH₂- alkane spacer linked to thiomorpholine), 2.56-2.78 (8H, m, -CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 4.0-4.39 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 358 [M+1]⁺; Anal. calcd for C₁₈H₃₁NO₂S₂: C, 60.46; H, 8.74; N, 3.92%. Found: C, 60.43; H, 8.70; N, 3.88%.

3,5- Dimethyl-4-([9-(1,4-thiazinan-4-yl)nonyl] oxy)-5H-thiophen-2-one (4g)

Prepared from 7g (349 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 293 mg of 4g as a pale-yellow oily liquid. Yield: 79%; IR (neat): 2928, 2857, 1678, 1630 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.14-1.5 (12H, m, -C-(CH₂)₆-C-alkane spacer), 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), I. 66-1.72 (2H, m, -C-CH₂-C-O- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.32 (2H, t, J = 6.7 Hz, -N-CH2- alkane spacer linked to thiomorpholine), 2.62-2.68 (8H, m, -CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 3.9-4.40 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 372 [M+1]⁺; Anal. calcd for C₁₉H₃₃NO₂S₂: C, 61.41; H, 8.95; N, 3.77%. Found: C, 61.38; H, 8.92; N, 3.73%.

3,5- Dimethyl-4-([10-(1,4-thiazinan-4-yl)decyl] oxy)-5H-thiophen-2-one (4h)

Prepared from 7h (363 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 317 mg of 4h as a pale-yellow oily liquid. Yield: 82%; IR (neat): 2927, 2854, 1679, 1629 cm^-1; ¹H NMR (200 MHz; CDCl₃) 5 1.25-1.44 (14H, m, -C-(CH₂)₇-C- alkane spacer), 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), 1.65-1.73 (2H, m, -C-CH₂-C-O- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.31 (2H, t, J = 6.7 Hz, -N-CH₂- alkane spacer linked to thiomorpholine), 2.61-2.68 (8H, m, -CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 4.06-4.32 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 386 [M+1]⁺; Anal. calcd for C₂₀H₃₅NO₂S₂: C, 62.29; H, 9.15; N, 3.63%. Found: C, 62.22; H, 9.12; N, 3.56%.

3.5- Dimethyl-4-([12-(1,4-thiazinan-4-yl)dodecyl] oxy)-5H-thiophen-2-one (4i)

Prepared from 7i (391 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and thiomorpholine (0.14 mL, 1.3 mmol) following the general procedure to afford 347 mg of 4i as a pale-yellow oily liquid. Yield: 84%; IR (neat): 2926, 2852, 1680, 1628 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.07-1.56 (18H, m, -C-(CH₂)₉-C- alkane spacer), 1.58 (3H, d, J = 6.7 Hz, -CH₃ linked to TL ring), 1.66-1.72 (2H, m, -C-CH₂- C-O- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.33 (2H, t, J = 6.7 Hz, -N-CH₂- alkane spacer linked to thiomorpholine), 2.63-2.69 (8H, m, - CH₂-S-CH₂- and -CH₂-N-CH₂- of thiomorpholine ring), 4.03-4.36 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 414 [M+1]⁺; Anal. calcd for C₂₂H₃₉NO₂S₂: C, 63.87; H, 9.50; N, 3.39%. Found: C, 63.84; H, 9.44; N, 3.36%.

3,5- Dimethyl-4-(2-piperidinoethoxy)-5H-thiophen-2-one (5a)

Prepared from 7a (251 mg, 1 mmol), potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 176 mg of 5a as a pale-yellow oily liquid. Yield: 69%; IR (neat): 2935, 2863, 1674, 1628 cm^-1; ¹H NMR (200 MHz; CDCl₃+DMSO-d₆) δ 1.59 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.79 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.26 (2H, t, J = 7.1 Hz, γ-CH₂- of piperidine), 2.44-2.74 (10H, m, α-(CH₂)₂, β- (CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.14-4.49 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 256 [M+1]⁺; Anal. calcd for C₁₃H₂₁NO₂S: C, 61.14; H, 8.29; N, 5.48%. Found: C, 61.04; H, 8.21; N, 5.42%.

3,5-Dimethyl-4-(3-piperidinopropoxy)-5H-thiophen-2-one (5b)

Prepared from 7b (265 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 194 mg of 5b as a pale-yellow oily liquid. Yield: 72%; IR (neat): 2933, 2861, 1676, 1629 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.58 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.82 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 1.83- 1.97 (4H, m, γ-CH₂- of piperidine and - CH₂- alkane spacer), 2.29-2.51 (10H, m, α- (CH₂)₂, β-(CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.11-4.44 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 270 [M+1]⁺; Anal. Calcd for C₁₄H₂₃NO₂S: C, 62.42; H, 8.61; N, 5.20%. Found: C, 62.35; H, 8.56; N, 5.20%.

3,5- Dimethyl-4-(3-piperidinobutoxy)-5H-thiophen-2-one (5c)

Prepared from 7c (279 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 209 mg of 5c as a pale-yellow oily liquid. Yield: 74%; IR (neat): 2932, 2859, 1675, 1631 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.59 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.81 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 1.84- 1.99 (6H, m, γ-CH₂- of piperidine & - (CH₂)₂- alkane spacer), 2.27-2.54 (10H, m, α- (CH₂)₂, β-(CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.11-4.46 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 284 [M+1]⁺; Anal. calcd for C₁₅H₂₅NO₂S: C, 63.56; H, 8.89; N, 4.94%. Found: C, 63.51; H, 8.86; N, 4.92%.

3,5- Dimethyl-4-[(5-piperidinopentyl)oxy]-5H-thiophen-2-one (5d)

Prepared from 7d (293 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 220 mg of 5d as a pale-yellow oily liquid. Yield: 74%; IR (neat): 2932, 2860, 1676, 1630 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.30-1.53 (4H, m, -(CH₂)₂- alkane spacer), 1.58 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.6-1.82 (4H, m, γ-CH2- of piperidine and -(CH₂)- alkane spacer), 1.83 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.23-2.51 (10H, m, α- (CH₂)₂, β-(CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.02-4.39 (3H, m, TL ring -CH- and -OCH₂-); ESIMS (m/z): 298 [M+1]⁺; Anal. calcd for C₁₆H₂₇NO₂S: C, 64.61; H, 9.15; N, 4.71%. Found: C, 64.56; H, 9.11; N, 4.67%.

3,5- Dimethyl-4-[(5-piperidinohexyl)oxy]-5H-thiophen-2-one (5e)

Prepared from 7e (307 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 242 mg of 5e as a pale-yellow oily liquid. Yield: 78%; IR (neat): 2930, 2858, 1678, 1629 cm^-1; ¹H NMR (300 MHz; CDCl₃) δ 1.23-1.54 (6H, m, -(CH₂)₃- alkane spacer), 1.58 (3H, d, J = 6.9 Hz, -CH3 linked to TL ring), 1.61-1.81 (4H, m, γ-CH₂- of piperidine and -(CH₂)- alkane spacer), 1.83 (3H, s, =CCH3 attached to alkenyl carbon of TL ring), 2.2-2.49 (10H, m, α-(CH₂)₂, β- (CH2)2 of piperidine and -N-CH2- alkane spacer), 4.01-4.36 (3H, m, TL ring -CH- and - OCH2-); ESIMS (m/z): 312 [M+1]⁺; Anal. calcd for C₁₇H₂₉NO₂S: C, 65.55; H, 9.38; N, 4.50%. Found: C, 65.50; H, 9.31; N, 4.45%.

3,5- Dimethyl-4-[(5-piperidinooctyl)oxy]-5H- thiophen-2-one (5f)

Prepared from 7f (335 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 269 mg of 5f as a pale-yellow oily liquid. Yield: 79%; IR (neat): 2931, 2855, 1679, 1628 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.2-1.54 (10H, m, -(CH₂)₅- alkane spacer), 1.58 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.62-1.81 (4H, m, γ-CH₂- of piperidine and -(CH₂)- alkane spacer), 1.85 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.3-2.56 (10H, m, α-(CH₂)₂, β-(CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.07-4.38 (3H, m, TL ring -CH- and - OCH₂-); ESIMS (m/z): 340 [M+1]⁺; Anal. calcd for C₁₉H₃₃NO₂S: C, 67.21; H, 9.80; N, 4.13%. Found: C, 67.18; H, 9.74; N, 4.07%.

3,5- Dimethyl-4-[(5-piperidinononyl)oxy]-5H- thiophen-2-one (5g)

Prepared from 7g (349 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 282 mg of 5g as a pale-yellow oily liquid. Yield: 80%; IR (neat): 2929, 2856, 1679, 1630 cm^-1; ¹H NMR (200 MHz; CDCl₃) δ 1.1-1.57 (12H, m, -(CH₂)₆- alkane spacer), 1.58 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.61-1.81 (4H, m, Y-CH₂- of piperidine and -(CH₂)- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.23-2.5 (10H, m, α-(CH₂)₂, β-(CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.02-4.41 (3H, m, TL ring -CH- and - OCH₂-); ESIMS (m/z): 354 [M+1]⁺; Anal. calcd for C₂₀H₃₅NO₂S: C, 67.94; H, 9.98; N, 3.96%. Found: C, 67.90; H, 9.91; N, 3.90%.

3,5- Dimethyl-4-[(5 -piperidinodecyl)oxy]-5H- thiophen-2-one (5h)

Prepared from 7h (363 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 294 mg of 5h as a pale-yellow oily liquid. Yield: 80%; IR (neat): 2929, 2855, 1679, 1632 cm^-1; ¹H NMR (300 MHz; CDCl₃) 5 1.2-1.53 (14H, m, -(CH₂)₇- alkane spacer), 1.57 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.59-1.81 (4H, m, γ-CH₂- of piperidine and -(CH₂)- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.2-2.48 (10H, m, α-(CH₂)₂, β-(CH₂)₂ of piperidine and -N-CH₂- alkane spacer), 4.04-4.21 (3H, m, TL ring -CH- and - OCH₂-); ESIMS (m/z): 368 [M+1]⁺; Anal. calcd for C₂₁H₃₇NO₂S: C, 68.62; H, 10.15; N, 3.81%. Found: C, 68.66; H, 10.08; N, 3.76%.

3,5- Dimethyl-4-[(5 -piper idinododecyl)oxy]- 5H-thiophen-2-one (5i)

Prepared from 7i (391 mg, 1 mmol), anhydrous potassium carbonate (553 mg, 4 mmol), and piperidine (0.1 mL, 1.3 mmol) following the general procedure to afford 320 mg of 5i as a pale-yellow oily liquid. Yield: 81%; IR (neat): 2927, 2853, 1680, 1630 cm^-1; ¹H NMR (200 MHz; CDCl₃) 5 1.14-1.52 (18H, m, -(CH₂)₉- alkane spacer), 1.58 (3H, d, J = 6.9 Hz, -CH₃ linked to TL ring), 1.65-1.78 (4H, m, γ-CH₂- of piperidine and -(CH₂)- alkane spacer), 1.84 (3H, s, =CCH₃ attached to alkenyl carbon of TL ring), 2.24-2.44 (10H, m, α-(CH₂)₂, β-(CH₂)₂ of piperidine and-N-CH₂- alkane spacer), 4.02-4.38 (3H, m, TL ring -CH- and -OCH2-); ESIMS (m/z): 396 [M+1]+; Anal. calcd for C₂₃H₄₁NO₂S: C, 69.82; H, 10.44; N, 3.54%. Found: C, 69.78, H, 10.40, N, 3.49%.

In vitro antimalarial activity

The synthesized thiolactone-thiomorpholine (4a-i) and thiolactone-piperidine (5a-i) conjugates were screened for in vitro inhibitory activity against the P. falciparum D10 strain. The design of these analogues was predicated on literature precedents suggesting that increasing hydrophobicity at the C4 position can enhance the potency of thiolactomycin derivatives. In addition to these, the intermediates 7a-i were also evaluated to provide a starting point for comparison that could help to understand how much the bromo- alkyl chain contributes to the potency before heterocyclic rings are introduced. The antimalarial activity of these conjugates was carried out by calculating the IC₅₀ values (μM), and the results are provided (Table 1).

Table 1.

In vitro activity of thiolactomycin-based morpholino/piperidino conjugates (4a-f and 5a-f) and intermediates (7a-7i) against Plasmodium falciparum D10.

Compound	IC50 ± SD (μM)
7a	5.88 ± 0.61
7b	0.2 ± 0.02
7c	4.10 ± 0.35
7d	9.89 ± 1.18
7e	1.9 ± 0.20
7f	2.65 ± 0.23
7g	1.9 ± 0.19
7h	9.89 ± 1.10
7i	1.9 ± 0.21
4a	> 40
4b	11.76 ± 1.22
4c	> 40
4d	2.53 ± 0.27
4e	3.88 ± 0.31
4f	3.00 ± 0.31
4g	1.81 ± 0.19
4h	> 40
4i	6.88 ± 0.70
5a	40.84 ± 5.1
5b	> 40
5c	> 40
5d	> 40
5e	> 40
5f	> 40
5g	> 40
5h	> 40
5i	> 40
Chloroquine	0.018 ± 0.002

Experimental results indicated that the monobromoalkyl series generally showed superior phenotypic activity compared to the heterocyclic conjugates. Among the tested compounds, the bromoalkyl derivative 7b, containing a three-methylene spacer, was the most potent hit with an IC50 value of 0.2 μM. Other bromoalkyl derivatives such as 7e, 7g, and 7i also showed good activity (IC₅₀ value of 1.9 μM), though this was nearly tenfold lower than that of 7b. In the heterocyclic series, the thiomorpholine conjugate 4g, featuring a nine- carbon aliphatic spacer, exhibited the highest activity with an IC₅₀ value of 1.81 μM. In contrast, the piperidine series (5a-i) exhibited poor efficacy, with most compounds showing IC₅₀ values exceeding 40 μM.

Thiolactomycin serves as a lead molecule in the design of antitubercular agents, and many of its derivatives are reported to possess significant antitubercular properties (22). Therefore, all the synthesized thiolactomycin- based derivatives were also evaluated for their potency against the Mycobacterium tuberculosis H37Rv strain. Unfortunately, all the tested derivatives exhibited little activity with MIC values exceeding 16 μg/mL.

Molecular docking

The synthesized thiolactone-thiomorpholine/ piperidine conjugates and their monobromoalkyl thiolactone precursors were evaluated for antimalarial activity, and compounds 4g and 7b were identified to be the most potent. In this study, to understand the plausible mechanism of action, we selected a set of E. coli and P. falciparum FAS- II enzymes as molecular targets to evaluate the binding potential of thiolactone-based conjugates. The biological relevance and mechanistic diversity were taken into consideration in the selection of molecular targets. First, we selected E. coli FabB (PDB ID: 1FJ4) as a benchmark target since thiolactomycin is the key scaffold on which the new conjugates were designed. Moreover, thiolactomycin is a known inhibitor of β-ketoacyl- ACP synthase (FabB) in E. coli and serves as a standard control in KAS inhibition (28). After the initial assessment in the primary E. coli reference system of thiolactomycin, we extended our study to the P. falciparum FAS-II pathway to evaluate whether the thiolactone-based conjugates retained binding affinity towards P. falciparum-specific enzymes. FabI (an enoyl-ACP reductase enzyme) is a validated antimalarial target, as its inhibition stops the final reduction step in fatty acid elongation, essential for parasite survival (37). This will be critical to evaluate the conjugates' potential as targeted antimalarial agents. Therefore, we selected two different conformations of P. falciparum FabI enzymes (PDB IDs: 1NHG and 4IGE) in our study (29,30). By docking against both FabI structures (1NHG and 4IGE), we tested the consistency of binding across different inhibitor-bound conformations, ensuring robustness in this key molecular target. Finally, P. falciparum FabZ enzyme (PDB ID: 3AZA) was selected as a molecular target for our docking studies. FabZ is a β-hydroxyacyl-ACP dehydratase enzyme that catalyzes the dehydration step during elongation in the fatty acid biosynthesis cycle of P. falciparum (31). The inhibition of FabZ interferes with the formation of the apicoplast membrane, rendering it a valuable target for novel antimalarials. All four docking analyses provided significant results. In the first selected benchmark target, FabB (PDB ID: 1FJ4) with thiolactomycin as a control, the results indicated that both 4g and 7b showed binding affinity comparable to thiolactomycin (Fig. 3). Compounds 7b and 4g exhibited a binding affinity of -6.48 and -6.40 kcal/mol, respectively. These values aligned closely with that of thiolactomycin (-6.62 kcal/mol), suggesting that the active compounds maintain strong molecular interactions with the FabB binding site (Table 2). The subsequent molecular docking studies of the synthesized compounds 4g and 7b were conducted against three validated P. falciparum FAS-II enzymes (PDB ID: 1NHG, 4IGE, and 3AZA) and compared with thiolactomycin (control) and the respective co-crystallized native ligands (Fig. 4). In the docking with PfFabI protein (1NHG), compound 4g demonstrated the most favorable binding energy value of -7.2 kcal/mol better than the control, thiolactomycin (-6.7 kcal/mol) and compound 7b (-5.7 kcal/mol), though it is lower than the native ligand triclosan (-8.1 kcal/mol) (Table 3).

Fig. 3.

3D and 2D representation of molecular docking analysis of the active compounds with FabB, the target protein (PDB ID: 1FJ4). (A) 3D overlapped structures of compounds 4g and 7b along with thiolactomycin (control) in the protein active site. (B-D) 2D Binding interactions of 7b, 4g, or thiolactomycin (control)

Table 2.

Molecular docking analysis of the interactions of compounds 4g and 7b with FabB protein (1FJ4).

Compound	Final intermolecular energy (kcal/mol)	Hydrogen bonds	H-bond length (Ǻ)	Amino acid residues involved in van der Waals interaction	Amino acid residues involved in other non-covalent interactions
4g	-6.40	A:GLY394:HN - :UNL1:S2	2.24	ASP268, GLY393, ASP265, THR395, GLY391, ALA271, PHE392, VAL270, THR300, GLY305, THR302, GLY205	Alkyl/Pi-Alkyl: HIS298, VAL304, PRO303, PRO272, ALA206 Pi-Sulfur: GLY394
7b	-6.48	A:GLY393:HN - :UNL1:O1 A:GLY394:HN - :UNL1:O1	2.28 1.96	ALA271, VAL270, THR395, ASN396, GLY305, GLU309, HIS298, GLY391	Alkyl/Pi-Alkyl: PHE390, PRO272 Pi-Sulfur: PHE392
Thiolactomycin	-6.62	A:HIS298:HE2 - :UNL1:O1 A:HIS333:HE2 - :UNL1:O1 :UNL1:H14 - A:VAL270:O	2.02 2.00 2.74	CYS163, GLY305, GLY391, ASP268, GLY394, GLY393, ALA272, THR300, THR302	Alkyl/Pi-Alkyl: PHE229, PHE392, PRO272 Pi-Sulfur: PHE390

Fig. 4.

2D Representation of binding interactions of the active compounds 4g and 7b with three Plasmodium falciparum-specific enzymes. Thiolactomycin was used as the positive control along with native co-crystallized ligands. Native ligands: triclosan (1NHG), chromene derivative (4IGE), and quinoline derivative (3AZA).

Table 3.

Molecular docking analysis of the interactions of compounds 4g and 7b with Plasmodium falciparum-specific enzymes.

Compound	Final intermolecular energy (kcal/mol)	Hydrogen bonds	H-bond length (Å)	Amino acid residues involved in van der Waals interaction	Amino acid residues involved in other non-covalent interactions
PfFabI (PDB ID: 1NHG)
4g	-7.2	None	None	LYS285, THR266, ALA312, ALA217, EU265, LEU 315, ILE369, PRO314, GLY104, LY106, GLY110, GLY112, SER215, SER317, YR111, TYR277, PHE368, MET281	Alkyl/Pi-Alkyl: ALA319, ALA320 Pi-orbital: TYR265
7b	-5.7	ALA169: HN—UNL1:O GLY104: HA1—UNL1:O2	1.89 2.57	SER170, ASP168, ASN218, ALA217, ILE105, GLY106, LEU216, PHE167	Pi-Sigma: TRP131 Pi-Sulfur: TRP131 Alkyl/Pi-Alkyl: VAL134, TRP131
Thiolactomycin (control)	-6.7	ALA320: HN—UNL1:O1	2.84	ALA319, SER317, ILE323, LYS285, TYR277, LEU315, ALA312, LEU265, TYR111	Pi-Sigma: TYR267 Alkyl/Pi-Alkyl: ALA372, PRO314, ILE369, TYR267, PHE368
Triclosan (native ligand)	-8.1	SER215: O—TCL500:H7 GLY104: HA1—TCL500: O7 ARG318:HD2— TCL500:CL14	2.60 2.29 3.05	ASP107, ALA319, VAL134, GLY106, SN218, SER170, ASP168, GLY129, ALA103, ALA217, ILE105	Alkyl/Pi-Alkyl: ALA169, ARG318, LEU216, LYS240 Pi-orbital: TRP131, PHE167, ALA169, LEU216
PfFabI (PDB ID: 4IGE)
4g	-7.4	SER317: OG—UNL1:O1	3.08	THR266, LEU265, ALA312, ILE369, ALA219, ASN218, TYR277, PHE268, PRO314, LEU315, GLY313	Pi-Sulfur: TYR111 Alkyl/Pi-Alkyl: ALA217, VAL222, MET281, ALA319, ALA320, ILE323, TYR267
7b	-5.8	ALA169: N—UNL1:O1 TRP131:CD1—UNL1:O1	2.93 3.47	SER10, LYS240, ASN218, SER215, ALA217, ILE105, GLY106, GLY104,,PHE167, LEU216, ASP168	Pi-Sigma: TRP131 Alkyl/Pi-Alkyl: VAL134, TRP131
Thiolactomycin (control)	-6.6	None	None	SER317, ALA320, THR266, ALA312, ILE323, TYR277, GLY313, TYR111	Alkyl/Pi-Alkyl: ALA372, PRO314, ILE369, TYR267, PHE368
Chromene derivative (native ligand)	-9.8	ASN218: ND2— CHJ501:O10 ASN218: ND2—CHJ501:O11	3.07 2.98	ASP168, PHE167, ALA217, TYR111, SER317, GLY110, GLY106, ILE105, GLY104, LYS240	Pi-Alkyl: LEU216, ALA169, ALA319 Pi-Cation: TRP131 Pi-Pi Stacked: TRP131
PfFabI (PDB ID: 3AZA)
4g	-4.7	LYS181: N—UNL1:O1 HIS133:CD2—UNL1:N1	3.06 3.46	PRO182, ILE139, GLY142, MET140, GLN145, VAL183, LYS180	Alkyl/Pi-Alkyl: PRO141, VAL143, HIS133, TRP179
7b	-4.2	UNL1: C7—MET140:O	3.60	PHE134, GLY142, TRP179, LYS181, GLN145, VAL183, ILE139	Pi-Sulfur: HIS133 Alkyl:PRO182
Thiolactomycin (control)	-5.1	VAL143: N—UNL1:O2	3.15	PHE134, VAL183, LYS181, ILE139, GLN145, TRP179, GLY142, PRO141	Pi-Sulfur: HIS133 Alkyl:PRO182
Quinoline derivative (native ligand)	-6.6	GLY142: N—KM02:O8	2.99	PHE134, MET140, GLY145, VAL183, LYS181	Alkyl/Pi-Alkyl: ILE139, PRO182, PRO141, VAL143 Pi-Sigma: ILE139 Pi-Pi T-shaped: HIS133, TRP179

The binding pose of 4g showed a stable positioning within the active site through different intermolecular interactions with key residues involved in substrate recognition. Interestingly, a similar trend was observed in an alternate conformation of the P/FabI protein (4IGE) with a chromenone-based co-crystallized native ligand. Compound 4g again showed a superior binding energy value of -7.4 kcal/mol compared to thiolactomycin (-6.6 kcal/mol) and 7b (-5.8 kcal/mol). However, it bonded less strongly than the native chromenone-based ligand, which exhibited a binding energy value of -9.8 kcal/mol. Finally, docking with the PfFabZ protein (3AZA) showed that the overall binding affinities of the compounds were weaker compared to the other target proteins. Compounds 4g and 7b exhibited binding energies of -4.7 and -4.2 kcal/mol, respectively, which were lower compared to the control thiolactomycin (-5.1 kcal/mol) and the native quinoline ligand (-6.6 kcal/mol). This suggested limited compatibility of the synthesized compounds with the FabZ active site.

Overall, compound 4g consistently outperformed both thiolactomycin and compound 7b across all targets, indicating its potential as the prioritized candidate for further rigorous investigations.

ADMET and lead-likeness assessment

Before advanced experimental characterization, computational profiling provides a robust, cost-effective alternative for evaluating ADMET and lead-likeness (38). The in silico tools streamline the selection process and minimize the risks associated with downstream preclinical development. This is because of early identification of candidates with favorable pharmacokinetic properties in the discovery pipeline. Therefore, we employed in silico models to predict the pharmacokinetic and toxicological properties of compounds 7b, 4g, and thiolactomycin (Table 4). The model predicted both 7b and 4g to have high gastrointestinal absorption, with 7b demonstrating blood-brain barrier (BBB) permeability. However, a critical distinction in toxicological profiling was identified that flagged 7b as Ames-positive, indicating potential mutagenicity. In contrast, conjugate 4g and the parent thiolactomycin were predicted to be Ames-negative, with 4g displaying a bioavailability score of 0.55. Additionally, 4g followed major drug-likeness rules, although it displayed inhibition of several cytochrome P450 isoforms (CYP1A2, 2C19, and 2D6), unlike the smaller 7b and thiolactomycin molecules.

Table 4.

Key ADMET and drug-likeness calculations of compounds 4g and 7b.

Compound	ESOL	GIA	BBB-P	Inhibition of cytochrome enzymes					BA Score	Ames toxicity	Follow drug likeness filter
				CYP 1A2	CYP 2C19	CYP 2C9	CYP 2D6	CYP 3A4
4g	Soluble	High	No	Yes	Yes	No	Yes	No	0.55	No	4
7b	Moderately soluble	High	Yes	No	No	No	No	No	0.85	Yes	5
Thiolactomycin	Soluble	High	No	No	No	Yes	No	No	0.85	No	5

ESOL, Estimated solubility; GIA, gastrointestinal absorption; BBB-P, blood-brain barrier permeant; CYP, cytochrome P450 enzymes; BA, bioavailability.

MD simulation

MD simulations are a cornerstone of drug discovery, facilitating the elucidation of mechanistic interactions within enzyme- inhibitor complexes. In this study, MD was employed to evaluate the stability of the enzyme-inhibitor complex under physiological conditions, accounting for solvation effects and protein flexibility. Based on the integrated evaluation of biological potency, target affinity, and safety profile, 4g was prioritized over 7b for a high-resolution mechanistic study. Although intermediate 7b exhibited higher phenotypic activity, its lower binding affinity for PfFabI and mutagenic liability (Ames positive) precluded it from further study. Consequently, a 200 ns explicit-solvent MD simulation was performed on the 4g-PfFabI complex to assess its structural stability and conformational flexibility relative to the parent scaffold (thiolactomycin) and the Apo state.

Structural stability and flexibility (RMSD and RMSF)

RMSD was calculated to assess the time- dependent stability and equilibration of the PfFabI complexes. The results showed that the Apo protein stabilized near 0.4 nm after 20 ns, and in the final 30 ns of the simulation, a secondary drift toward 0.45 nm was observed (Fig. 5). On the other hand, both ligand-bound complexes demonstrated enhanced structural rigidity. Compound 4g equilibrated within the first 20 ns at around 0.3 nm with a slight upward shift at 165 ns to 0.38 nm. The control (thiolactomycin) followed a similar course to compound 4g, equilibrating at 0.28-0.3 nm. These results suggested that the binding of 4g effectively stabilized the PfFabI backbone, comparable to that of thiolactomycin, as inferred from a reduction in RMSD relative to the Apo state.

Fig. 5.

Molecular dynamics simulation profiles of PfFabI complexes (PBD ID: 1NHG) over 200 ns. (A) RMSD plot of PfFabI-lead compound 4g (light blue), PfFabI-Apo state (purple), and PfFabI-control TLM (blue) complexes' deviation during the 200 ns period. (B) RMSF plot with fluctuation per residue of the complexes. (C) Rg plot representing the compactness of the complexes. (D) Hydrogen bond plot depicting hydrogen bond formation of the complexes during the simulation. RMSD, Root mean square deviation; RMSF, root mean square fluctuation; Rg, radius of gyration.

RMSF analysis was carried out to depict the mobility at the local residue level. Significant flexibility was observed in the Apo form, with fluctuations peaking at 1.0 nm, predominantly in the loop regions surrounding residues 320-370. These fluctuations were significantly reduced to approximately 0.6 nm in the binding of compound 4g. Notably, in the control (thiolactomycin), the fluctuations were below 0.4 nm, which demonstrated the induction of rigidity in this specific loop. The lowering of RMSF values across the binding regions indicates that 4g successfully restricts the atomic displacement of the target residues upon complexation.

Structural compactness and hydrogen bond analysis

The Rg was studied to understand the compactness and folding integrity of the protein-ligand complexes. The Apo protein maintained an Rg value in the range of 1.92-1 . 9 5 nm, with decreased compactness toward the end of the run. The compound 4g-protein complex was almost identical to Apo compactness for the majority of the simulation, fluctuating around 1.93 nm. Interestingly, the control (thiolactomycin) exhibited an elevated level of compactness in the complex, with lower Rg values in the range of 1.88-1.90 nm after 20 ns.

The stability of the protein-ligand interface was further evaluated through hydrogen bond (H-bond) profiling. Compound ⁴g maintained a consistent interaction with the protein scaffold, forming 1 to 2 H- bonds throughout the 200 ns run. A periodic 3 H-bond interaction was observed, mainly during 15-35 ns and 135 ns duration. Similarly, the control (thiolactomycin) predominantly maintained 2 H-bonds, which underscores a stable and conserved binding mode for both ligands.

Thermodynamic binding free energy (MM- PBSA)

To quantify the binding affinity of compound 4g towards the PfFabI protein, MM- PBSA calculations were performed using 2001 frames from the stable trajectory (Fig. 6). In this, the components contributing to the total system energy of the entire complex and ligand- receptor free energy were calculated (Table 5). The results revealed that compound 4g achieved a total binding energy (ΔG_Total) of - 22.14 ± 6.49 kcal/mol, which was superior to the control that showed a value of 20.27 ± 2.05 kcal/mol. The analysis of the energetic components demonstrates that the van der Waals interactions (ΔV_dwaals) are the primary driving force for 4g, contributing -41.81 ± 4.94 kcal/mol, compared to -27.77 ± 3.97 kcal/mol for thiolactomycin.

Fig. 6.

Binding free energy trace plot for the lead 4g complex. Tracks the ΔG_Total over 2001 frames. The moving average (red dashed line) confirms thermodynamic convergence and a favorable binding trend.

Table 5.

Selected thermodynamic parameters and binding free energy components (kcal/mol) for PfFabI complexes. Data are expressed as mean ± SEM. Thiolactomycin served as the control.

Complex	ΔVdwaals	ΔE EL	ΔEPB	ΔGSoiv	ΔGTotal
System total (PB)
Compound 4g	-2000.52 ± 26.58	-18026.62 ± 132.95	-4209.06 ± 106.14	-4132.44 ± 105.25	-5676.17 ± 55.17
Thiolactomycin	-1962.77 ± 21.17	-18524.36 ± 104.46	-3674.84 ± 73.93	-3600.28 ± 73.56	-5763.61 ± 50.41
Binding affinity (MM-PBSA)
Compound 4g	-41.81 ± 4.94	53.28 ± 17.59	-29.22 ± 20.16	-33.62 ± 19.88	-22.14 ± 6.49
Thiolactomycin	-27.77 ± 3.97	-10.19 ± 2.30	20.63 ± 2.04	17.69 ± 2.03	-20.27 ± 2.05

Where system total (PB) is the Poisson-Boltzmann total system energy (entire complex); MM-PBSA, molecular mechanics Poisson-Boltzmann surface area (protein-ligand bond); ΔV_dwais, van der Waals enthalpy; ΔE_EL, electrostatic molecular energy; ΔE_PB, polar solvation energy / Poisson- Boltzmann; ΔG_solv, total solvation free energy; ΔG_Total, total binding free energy.

DISCUSSION

The SAR analysis of the designed thiolactomycin-based conjugates and their intermediates revealed distinct trends regarding the modification of the thiolactone scaffold. Based on literature precedent suggesting that increased hydrophobicity at the C4 position enhances potency, these conjugates were developed, and the resulting data supported this trend within both the thiomorpholine and monobromo alkyl series (Table 1). The high potency of the monobromo alkyl series highlights the bromo substituent as a key factor for target engagement, with derivative 7b exhibiting the highest activity among all tested compounds. However, the introduction of additional methylene groups in the monobromo alkyl chain resulted in a notable decrease in activity. Regarding the heterocyclic systems, the thiomorpholine ring was clearly superior to the piperidine ring. Specifically, the thiomorpholine conjugate 4g, featuring a nine-carbon spacer, exhibited significant antimalarial activity with an IC₅₀ value of 1.81 μM. This suggested that the thiomorpholine ring system was more favorable for enhancing antimalarial properties, as the additional sulfur atom may contribute to more effective interactions within the active site at the molecular level. In contrast, the thiolactone- piperidine conjugates (5a-i) exhibited considerably lower activity, with many compounds showing IC₅₀ values above 40 μM. The FAS-II pathway is crucial in the biosynthesis of the cell membrane in the Plasmodium parasite, and the enzymes in this pathway are validated targets in antimalarial drug development. Molecular docking studies were conducted on three different proteins (FabB, FabI, and FabZ) in the FAS-II pathway alongside an evaluation of alternate conformations of the most promising target, FabI. Thiolactomycin is a known inhibitor of β- ketoacyl-ACP synthase (FabB) in E. coli, and therefore, first docking was conducted against the FabB protein (PDB ID: 1FJ4). Subsequent docking studies were performed on three validated P. falciparum FAS-II enzymes, namely PfFabI (PDB ID: 1NHG and 4IGE) and PfFabZ (PDB ID: 3AZA), each representing a distinct catalytic step in the FAS-II pathway. Specifically, the FabB enzyme initiates fatty acid chain elongation, FabZ catalyzes the intermediary dehydration step, and FabI catalyzes the last reduction step. Integration of these targets allows for a systematic exploration of the inhibition potential of our thiolactone conjugates 4g and 7b across different mechanistic steps of fatty acid synthesis in the parasite. Overall, the docking results provide a plausible mechanism for the experimental antimalarial activity of the most potent compounds. These findings are further substantiated by detailed interaction analyses and binding mode comparisons.

Studies with the FabB protein (PDB ID: 1FJ4) revealed that the conjugates 4g and 7b showed stable binding in the active site of the protein through different molecular interactions (Fig. 3). The formation of a hydrogen bond is very impactful in ligand-target interactions. Compound 7b formed two hydrogen bonds with residues GLY393 and GLY394, with bond lengths of 2.98 Å and 1.96 Å, respectively (Table 2). On the other hand, compound 4g formed a single hydrogen bond with GLY394 with a bond length of 2.24 Å. Thiolactomycin, the control, formed three hydrogen bonds, notably with HIS298 and HIS333, with bond lengths ranging from 2.00 Å to 2.74 Å. Beyond hydrogen bonds, several non-covalent interactions were also observed. Both the ligands, 4g and 7b, exhibited extensive van der Waals interactions with multiple residues, including ASP268, GLY393, ASP265, THR395, GLY391, ALA271, PHE392, VAL270, THR300, GLY305, THR302, and GLY205 for 4g and ALA271, VAL270, THR395, SN396, GLY305, GLU309, HIS298 andGLY391 for 7b. These interactions contribute to the overall stability and binding affinity of these compounds towards FabB. Additionally, pi- alkyl and pi-sulfur interactions were also observed with both ligands, which further enhance their binding affinity to the target protein.

In the PfFabI (1NHG) protein, compound 4g showed binding affinity superior to the control (thiolactomycin). The hydrophobic alkyl/ pi- alkyl and pi-orbital interactions with surrounding residues contributed to the enhanced binding stability, which was further augmented by a significant number of van der Waals interactions (Fig. 4). The amino acid residues that contributed to van der Waals interaction in the active site of the protein were LYS285, THR266, ALA312, ALA217, LEU265, LEU 315, ILE369, PRO314, GLY104, GLY106, GLY110, GLY112, SER215, SER317, TYR111, TYR277, PHE368, and MET281. Interestingly, no hydrogen bond formation was observed between compound 4g and the active site of the protein (Table 3). On the other hand, hydrogen bond formation anchored compound 7b, thiolactomycin, and the naïve ligand (triclosan), along with other non-covalent interactions in the active site of the protein. Compound 7b formed two hydrogen bonds with residues ALA169 and GLY104, with bond lengths of 1.89 Å and 2.57 Å, respectively. On the other hand, thiolactomycin and triclosan formed one and three hydrogen bonds, respectively, with bond lengths between 2.29 and 3.05 Å.

For PfFabI (4IGE), compound 4g exhibited a similar pattern to 1NHG with superior binding affinity compared to thiolactomycin. The ligand formed stabilizing hydrogen bonds along with pi-sulfur, alkyl, and pi-alkyl contacts, facilitating proper accommodation within the binding cavity. A single hydrogen bond formation with the SER317 residue, with a bond length of 3.08 Å, was observed. On the other hand, compound 7b formed two hydrogen bonds with ALA169 and TRP131 in the binding site, in addition to other non-covalent interactions. However, the native chromenone ligand demonstrated stronger binding due to more extensive interactions in the active site. In contrast, PfFabZ (3AZA) showed fewer stabilizing interactions for the test compounds. The active site of FabZ, being a dehydratase, is structurally distinct and less accommodating to the thiolactomycin-based derivatives. The reduced number of hydrogen bonds and weaker hydrophobic interactions resulted in comparatively poor binding affinities.

Compound 4g displayed the highest binding affinity against PfFabI (1ING), despite the absence of hydrogen bonding interactions. The docking pose suggests that the stability results from the thiomorpholine sulfur atom, which is 'cushioned' within the glycine-rich loop, forming van der Waals contacts with Gly104, Gly106, Gly110, and Gly112. Although individually weak, these interactions could contribute significantly to the overall stable protein-ligand stability. Consequently, the docking studies unambiguously revealed that conjugate 4g has a superior binding affinity towards the PfFAbI enzyme, particularly with 1ING, compared to the intermediate 7b and the control, thiolactomycin.

In silico ADMET and lead-likeness profiling serve as critical decision-making filters to prioritize candidates for advanced mechanistic characterization (38). This assessment was performed for 7b and 4g, using thiolactomycin as a control, to select a lead for high-resolution studies (Table 4). While initial biological evaluation identified 7b as the most potent phenotypic hit (IC₅₀ = 0.2 μM), toxicological screening revealed an Ames-positive result. This mutagenic liability likely stems from the propensity of its primary alkyl-bromide moiety for non-specific macromolecular alkylation, a known genotoxic risk. In contrast, the thiomorpholine-alkyl conjugate 4g was confirmed as Ames-negative, exhibiting high GI absorption and a favorable bioavailability score (0.55). Although the increased molecular weight and lipophilicity of 4g resulted in moderate solubility and a predictive CYP-inhibition profile common to larger heterocyclic leads, it remained well within the acceptable boundaries for lead-likeness. Consequently, based on this toxicity profile and superior target affinity toward PfFabI in docking, 4g was prioritized over the mutagenic intermediate 7b for detailed 200 ns MD simulations to elucidate the structural basis of its antimalarial activity

A 200 ns MD simulation was performed to transition the initial rigid “lock and key” docking model to a more realistic, dynamic illustration of the PfFabI-4g complex. While initial docking suggested that 4g stabilized primarily through van der Waals contacts of the thiomorpholine sulfur against the glycine-rich loop (Gly104- Gly112) without hydrogen bonding, the MD trajectory revealed a more nuanced induced-fit mechanism.

In the MD simulation of the PfFabI-4g complex, the thermal energy (300 K) and explicit solvent environment cause subtle rearrangements within the binding pocket, thereby relaxing the initial docking pose. Because of this structural relaxation, two persistent hydrogen-bonding interactions emerged that remained stable throughout the simulation trajectory. Moreover, the transition of the PfFabI-4g complex from a static pose to a dynamic one suggests that the thiomorpholine sulfur acts as a critical hydrophobic anchor that stabilizes the ligand in the glycine-rich region to orient the structure in a way that maximizes these late-emerging polar interactions.

A comparison between 4g and thiolactomycin (control) revealed a significant correlation between structural dynamics and thermodynamic affinity. The thiolactomycin- protein complex was more rigid structurally, as evidenced by the lower RMSD and considerable reduction in RMSF values, particularly of residues 32-370 in the catalytic loop. However, the MM-PBSA analysis suggested that structural rigidity does not necessarily translate to potency. The structurally less rigid conjugate 4g displayed a superior total free binding energy value of -22.14 kcal/mol compared to -20.27 kcal/mol exhibited by thiolactomycin. Notably, the contribution of van der Waals enthalpy to the total binding energy was exceptional (ΔV_dwaals= -41.81 kcal/mol) in the PfFabI-4g complex, which quantitatively validates the importance of the thiomorpholine-sulfur interactions. These results underscore the superior binding profile of 4g and support its development as a potent lead inhibitor of the P. falciparum FAS-II pathway, particularly Fabl.

CONCLUSION

The emerging trend of drug-resistant Plasmodium spp. strains highlights the urgent need for new and safer antimalarial agents. In this study, we synthesized and evaluated a series of thiolactomycin-inspired thiomorpholine and piperidine conjugates. In vitro antimalarial evaluation identified 7b and 4g as the most potent compounds, with IC₅₀ values of 0.2 and 1.81 μM, respectively. However, the higher phenotypic potency of 7b was offset by toxicological profiling, which revealed it to be an Ames-positive mutagenic agent. Multi-target docking studies against FAS-II enzymes indicated the superior binding affinity of 4g compared to 7b and thiolactomycin, particularly toward PfFabI. Interestingly, the thiomorpholine sulfur atom was instrumental in anchoring and stabilizing the ligand at a glycine ring loop in the binding pocket through predominant van der Waals interactions. The 200 ns MD simulation demonstrated that 4g achieves superior binding through an 'induced-fit' mechanism that accounts for its antimalarial activity. Intriguingly, the transition from a static docking pose to dynamic MD trajectories revealed the emergence of persistent hydrogen bonds (absent in the initial docking) that stabilized the 4g- PfFabI complex, alongside a robust network of van der Waals interactions. The thiomorpholine sulfur atom was instrumental in anchoring and stabilizing the ligand at a glycine ring loop in the binding pocket. Furthermore, the complex achieved a superior binding free energy (ΔG_Total = -22.14 kcal/mol) compared to thiolactomycin, providing a thermodynamic rationale for its potency. Ultimately, this work establishes 4g as a promising candidate for hit-to-lead optimization and further development.

Funding statement

This research work was funded by Umm Al- Qura University, Saudi Arabia, under grant number: 26UQU4280406GSSR10

Authors’ contributions

S. Azeeza, M.S. Malik, S.A. Ahmed, and A. Kamal were responsible for the conceptualization of the study. S. Azeeza, M.S. Malik, S.T. Basha, S.A. Ahmed, and A. Kamal developed the research methodology. Q.M.S. Jamal and M.A. Mohammad performed the software development and implementation. S. Azeeza and Q.M.S. Jamal managed data curation. S. Azeeza, M.S. Malik, Q.M.S. Jamal, M.A. Mohammad, M.M. Al-Rooqi, S.S. Samman, Z. Moussa, and S.A. Ahmed conducted the formal analysis of the results. S. Azeeza, M.S. Malik, M.A. Mohammed, M.M. Al-Rooqi, S.S. Samman, S.T. Basha, Z. Moussa, and S.A. Ahmed performed the laboratory investigations. S. Azeeza, M.S. Malik, Q.M.S. Jamal, M.A. Mohammad, M.M. Al-Rooqi, S.S. Samman, S.T. Basha, Z. Moussa, and S.A. Ahmed were responsible for the validation of the experimental data. Q.M.S. Jamal, M.A. Mohammad, and S.T. Basha created the visualizations and figures. S. Azeeza, M.S. Malik, Q.M.S. Jamal, M.M. Al-Rooqi, S.T. Basha, Z. Moussa, and S.A. Ahmed prepared the original draft of the manuscript. M.S. Malik, S.A. Ahmed, and A. Kamal were responsible for the review and editing of the final manuscript. S.A. Ahmed and A. Kamal provided overall supervision for the research project. All authors have read and approved the finalized article. Each author has fulfilled the authorship criteria and affirmed that this article represents honest and original work. The authors, S. Azeeza and M. S. Malik, have contributed equally to the paper.

Conflict of interest statements

The authors declared no conflict of interest in this study.

AI declaration

During the preparation of this work, the authors used Gemini 3 Flash to improve readability and language. After using this tool, the authors reviewed and edited the content and take full responsibility for the content of the publication

Data availability

The data supporting the findings of this study are documented and available from the corresponding author upon reasonable request. Researchers interested in accessing the data should contact the corresponding author to discuss the terms and conditions of access.