Molecular Requirements for the Expression of Antiplatelet Effects by Synthetic Structural Optimized Analogues of the Anticancer Drugs Imatinib and Nilotinib

Figures & data

Figure 1 Modifications of prepared imatinib and nilotinib derivatives. Upper panel left: Following removal of the piperazinyl ring of imatinib, modifications were made to the final phenyl moiety as shown (I–IV). Upper panel right: Removal of the imidazolyl ring, modifications were made to the final phenyl ring (V, VI). Lower panel: Structure of the imatinib or nilotinib analogues by replacement of the amide bond with the urea moiety (VII, VIII).

Scheme 1 Reactions and conditions for the synthesis of the imatinib I–IV and of the imatinib/nilotinib analogues VII and VIII.

Scheme 2 Reactions and conditions for the synthesis of the nilotinib analogues V and VI.

Table 1 The Inhibitory Effect of Imatinib, Nilotinib and Their Synthetic Analogues on AA-Induced Platelet Aggregation in PRP

Tyrosine Kinase Inhibitors	IC50-Values (μΜ) (5 min Incubation)	IC50-Values (μΜ) (1 min Incubation)
Imatinib	13.30	21.71
I	4.30	n.d.
II	6.88	n.d.
III	30.39	n.d.
IV	23.77	n.d.
Nilotinib	3.91	11.75
V	0.41	2.94
VI	4.67	n.d.
VII	31.90	n.d.
VIII	3.47	n.d.

Notes: Platelet aggregation in PRP was performed in the presence of 500 μΜ AA. IC₅₀ values (concentrations that induce 50% inhibition of platelet aggregation) are the mean from at least 3 different experiments.

Abbreviations: AA, Arachidonic Acid; n.d., not determined; PRP, Platelet-Rich Plasma.

Figure 2 (A). Bar graph illustrating the % inhibition induced by imatinib (Im), nilotinib (N) and related synthetic analogues (at a concentration of 100 μΜ) on platelet aggregation induced by ADP or TRAP-6. Values represent the Mean±SD from three different platelet preparations. (B). Representative aggregation curves illustrating the inhibitory effect of compound V, at different concentrations, on platelet aggregation induced by i. AA ii. ADP, iii. TRAP-6.

Abbreviations: ADP, Adenosine Diphosphate; SD, Standard Deviation; TRAP-6, Thrombin Receptor Activating Peptide-6.

Figure 3 Bar graphs illustrating the effect of imatinib, nilotinib and synthetic compound V on P-selectin membrane expression induced by the platelet agonists (A) AA, (B) ADP and (C) TRAP-6. Platelets in PRP were labeled with anti-CD62P-PE monoclonal antibody and analyzed by flow cytometry to determine the membrane expression of P-selectin on activated platelets. The compounds imatinib, nilotinib and V were used at the concentration of 100 μΜ.

Notes: Results are the mean±SD from at least three independent experiments. *p<0.01 compared with activated platelets, ^$p<0.05 compared with imatinib, ^#p<0.01 compared with nilotinib. **p<0.05 compared with respective activated platelets, ^&p<0.05 compared with nilotinib.
Abbreviations: AA, Arachidonic Acid; ADP, Adenosine Diphosphate; MFI, Mean Fluorescence Intensity; PRP, Platelet-Rich Plasma; SD, Standard Deviation; TRAP-6, Thrombin Receptor Activating Peptide-6.

Figure 4 Models of imatinib, nilotinib and of the most active analogue V bound to c-Src enzyme based on PDB (ID: 1Υ57): (A) Superposition of the model of the V-c-Src (in green) and the structures of nilotinib (in brown), imatinib (in pink) at the active site of the kinase c-Src. The differentiations are focused mainly on the final phenyl moiety. (B, C, D) Modeled binding modes presented as 2D ligand-interaction ligand (LID) of imatinib, nilotinib and compound V bound, respectively, positioned at the active site of the kinase c-Src, as derived from flexible docking calculations.

Note: Protein residues forming hydrogen bonds (dotted green lines) are labelled.