Piperazine-Thiourea Hybrids as Novel Antiplatelet Agents Targeting COX-1: Synthesis, in Vitro , and in Silico Evaluation

Figures

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  1. Thiourea and piperazine-based derivatives reported in the literature showing in vitro COX-1 inhibition and antiplatelet activity on arachidonic-acid (AA)–induced platelet aggregation.

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  1. Synthesis of Piperazine Thioureas 3a–p

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  2. Antiplatelet effects of piperazine thiourea derivatives at a concentration of 100 μM on platelet aggregation induced by arachidonic acid (AA) (500 μM). Acetylsalicylic acid (ASA) was used as positive control, and DMSO (1%) as a negative control. *p ≤ 0.05 versus negative control (one-way ANOVA, Tukey’s test).

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  3. Concentration required to inhibit 50% of arachidonic acid (AA)-induced platelet aggregation (IC50) for piperazine thiourea derivatives compared to acetylsalicylic acid (ASA). Data are expressed as the mean ± standard deviation (SD) (n = 3). IC50 values (μM) were determined for compounds 3a (80.7 ± 11.0), 3g (83.1 ± 14.7), 3j (88.0 ± 9.9), 3p (74.5 ± 4.8), and ASA (36.3 ± 4.2). *p ≤ 0.05 versus positive control (one-way ANOVA, Tukey’s test)..

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  4. Evaluation of piperazine thiourea derivatives on the in vitro coagulation of human plasma, assessed by (A) prothrombin time (PT) and (B) activated partial thromboplastin time (aPTT). All compounds were tested at a concentration of 100 μM, with 1% DMSO used as a negative control. T1 /T0 represents the ratio of test time to control time (in seconds). The results are expressed as the mean ± standard deviation (SD) (n = 3). *p ≤ 0.05 versus negative control (one-way ANOVA, Tukey’s test).

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  5. Hemocompatibility assessment of piperazine thioureas derivatives (100 μM) using the hemolysis assay after 3 h of incubation. Values below 10% are considered nonhemolytic. DMSO 1% is the negative control and Triton X-100 1% is the positive control. The data were presented as mean ± standard deviation (SD) (n = 3). *p ≤ 0.05 versus negative control (one-way ANOVA, Tukey’s test).

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  6. Structural organization and active-site conservation of cyclooxygenase-1 (COX-1) in ovine and human enzymes. (A) Three-dimensional representation of COX-1 highlighting the spatial organization of its two functionally coupled catalytic regions: the cyclooxygenase catalytic site (orange) and the peroxidase site (purple). Key residues involved in substrate binding, catalytic initiation, and site-specific inhibition are highlighted in pink. (B) Superposition of the cyclooxygenase catalytic site showing identical amino-acid composition and near-perfect spatial overlap between human COX-1 (green) and ovine COX-1 (cyan). Amino acid residues are represented using their standard three-letter codes, as follows: LEU: Leucine, HIS: Histidine, ARG: Arginine, VAL: Valine, TYR: Tyrosine, GLN: Glutamine, ILE: Isoleucine, SER: Serine, MET: Methionine, PHE: Phenylalanine, ALA: Alanine, TRP: Tryptophan, and GLY: Glycine.

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  7. Redocking simulations conducted to validate the Gold algorithm in the reference site of oCOX-1 receptor. (A) Experimentally determined binding modes of the COX-1–mofezolac complex, as recorded in the PDB structure 5WBE. Mofezolac is represented by green sticks, color-coded by atom type. The residues interacting with the ligand are shown as purple sticks, color-coded by atom type, and labeled according to the corresponding amino acid. Hydrogen bonds are depicted as green dashed lines, while salt bridges are represented by pink dashed lines. Residues involved in hydrophobic contacts with the inhibitor are also displayed. A complementary 2D interaction diagram is also provided to facilitate visualization of protein–ligand interactions, with all interaction distances displayed. (B) Redocking of the COX-1-mofezolac complex (5WBE). The experimental pose is represented by green sticks, while the docked pose is shown as cyan sticks, both color-coded by atom type. Amino acid residues are represented using their standard three-letter codes, as follows: ALA: Alanine, ARG: Arginine, GLY: Glycine, HIS: Histidine, ILE: Isoleucine, LEU: Leucine, PHE: Phenylalanine, SER: Serine, TRP: Tryptophan,…

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  8. Docking of active thioureas 3a, 3g, 3j, and 3p on the COX-1 receptor. The ligands are represented by green sticks and colored by atom type. The residues involved in interactions with the ligand are shown as purple sticks, colored by atom type, and labeled according to the corresponding amino acid. Hydrogen bonds are represented by dashed green lines, salt bridges by dashed pink lines, π–π interactions by dashed purple lines, and cation−π interactions by dashed orange lines. Residues involved in hydrophobic contacts with the inhibitor are also shown. Complementary 2D interaction diagrams are also provided to facilitate visualization of protein–ligand interactions, with all interaction distances displayed. Amino acid residues are represented using their standard three-letter codes, as follows: ALA: Alanine, ARG: Arginine, ILE: Isoleucine, LEU: Leucine, MET: Methionine, PHE: Phenylalanine, SER: Serine, TRP: Tryptophan, TYR: Tyrosine, and VAL: Valine.

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  9. Analysis of contact number, minimum distance, and binding energy of hCOX1 complexes with antiplatelet thiourea derivatives. (A) Minimum distances established between the COX1 protein and the thiourea derivatives 3a, 3g, 3j, and 3p. (B) Number of contacts established between the COX1 protein and the thiourea derivatives 3a, 3g, 3j, and 3p. (C) Binding energy computed for the complexes formed between the hCOX1 protein and the thiourea derivatives 3a, 3g, 3j, and 3p.

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  10. Protein–ligand interaction occupancy profiles for COX-1 complexes with thiourea derivatives 3a, 3g, 3j, and 3p obtained from molecular dynamics simulations. Panels show the percentage occupancy of key protein–ligand interactions along the simulation time for (A) 3a, (B) 3g, (C) 3j, and (D) 3p. Only residues exhibiting significant interaction occupancies (≥5%) are shown. Amino acid residues are represented using their standard three-letter codes, as follows: ALA: Alanine, ARG: Arginine, GLU: Glutamic Acid, GLY: Glycine, ILE: Isoleucine, LEU: Leucine, PHE: Phenylalanine, PRO: Proline, SER: Serine, THR: Threonine, TRP: Tryptophan, TYR: Tyrosine, and VAL: Valine.

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  11. Per-residue Root-Mean-Square Fluctuation (RMSF) of hCOX-1 in apo and ligand-bound states over the simulations. For each panel, the RMSF of apo hCOX-1 (purple) is superimposed on those of the ligand-bound complexes 3a–hCOX1, 3g–hCOX1, and 3j-hCOX1. Vertical red dashed lines indicate key cyclooxygenase residues: Arg120, Tyr355, Tyr385, and Ser530. Amino acid residues are represented using their standard three-letter codes, as follows: ARG: Arginine, TYR: Tyrosine, and SER: Serine.