Identification of a New Morpholine Scaffold as a P2Y12 Receptor Antagonist

Abstract

The P2Y12 receptor is critical for platelet activation and is an attractive drug target for the prevention of atherothrombotic events. Despite the proven antithrombotic efficacy of P2Y12 inhibitors, these thienopyridine scaffolds are prodrugs that lack important features of the ideal antithrombotic agent. For this reason, ticagrelor-a new chemical class of P2Y12 receptor antagonist-was developed, but it can cause shortness of breath and various types of bleeding. Moreover, ticagrelor is a cytochrome P450 3A4 substrate/inhibitor and, therefore, caution should be exercised when it is used concomitantly with strong CYP3A4 inducers/inhibitors. There is a need for novel P2Y12 receptor antagonist scaffolds that are reversible and have high efficacy without associated side effects. Here, we describe a novel antagonist containing a morpholine moiety that was identified by screening libraries of commercially available compounds. The molecule, Compound E, acted on P2Y12, but not P2Y1 and P2Y13, and exhibited pharmacological characteristics that were distinct from those of ticagrelor, acting instead on P2Y12 via an allosteric mechanism. These results provide a basis for the development/optimization of a new class of P2Y12 antagonists.

Keywords: P2Y12 receptor antagonist; antiplatelet; morpholine moiety; ticagrelor.

Figure 1

High-throughput screen of anti-platelet compounds. Commercially available compound libraries were screened to identify antagonists of the ADP receptor P2Y12. Six compounds were found to inhibit 20 μM ADP-induced platelet aggregation in a 96-well format. Compounds A and C contain a pyridazinone moiety. Compounds B, D, E, and F contain sulfonamide, triazoline, morpholine, and pyrazine moieties, respectively.

Figure 2

Inhibition of ADP-induced platelet aggregation by six candidate antagonists of P2Y12. The reference compounds ATP, MRS2179, 2-MeSAMP, and AZD6140 were used as ADP, P2Y1, and P2Y12 receptor antagonists. (A) references and (B) the six candidate compounds inhibited platelet aggregation induced by 20 μM ADP. Data are representative of two experiments performed in triplicate and are expressed as mean ± SD (see also Table 1).

Figure 3

Binding characteristics of rhP2Y12. Saturation curves and Scatchard plots of (A) [³H]-2-MeSADP and (B) [³H]ADP. Non-specific binding was determined using 25 µM 2-MeSADP. Membranes prepared from HEK293T cells stably expressing human P2Y12 receptor were incubated with (C) 1 nM [³H]-2-MeSADP and (D) 3 nM [³H]ADP with increasing concentrations of test compound to determine IC₅₀. Compound E (morpholine moiety) selectively inhibited the binding of both radio-ligands. Data are representative of three experiments performed in duplicate or triplicate and are expressed as mean ± SD (see also Table 2).

Figure 4

Comparison of pharmacological characteristics of Compound E and ticagrelor. Competitive binding was determined using (A) [³H]-2-MeSADP and (B) [³H]ADP as radio-ligands. Membranes prepared from HEK293T-rhP2Y12 cells were incubated with ticagrelor, 2-MeSADP, ADP, and Compound E with increasing concentrations of test compound to determine IC₅₀. Data are representative of three experiments performed in duplicate or triplicate and are expressed as mean ± SD (see also Table 3).

Figure 5

Selectivity for P2Y12 over P2Y1 and P2Y13 receptors (A,B) Changes in cytosolic Ca²⁺ concentration in NIH3T3-rhP2Y1 + Gq cells. (A) Representative Ca²⁺ responses triggered by serially diluted MRS2365 in NIH3T3 cells co-transfected with human P2Y1 receptor and Gq/o; (B) FLIPR Ca²⁺ dye-loaded NIH3T3-P2Y1 + Gq cells were preincubated with indicated concentrations of MRS2179, 2-MeSAMP, ticagrelor, and Compound E (0.03–100 μM). The inhibition of Ca²⁺ increases induced by 100 nM MRS2365 was measured as an increase in relative fluorescence units (RFUs) and is plotted against the log concentration of test compounds; (C) Selectivity for rhP2Y13 receptor. Membranes were prepared from COS-7 cells stably expressing the human P2Y13 receptor. Competitive binding was determined using 1 nM [3H]2-MeSADP and Non-specific binding was determined using 25 µM 2-MeSADP. Data are expressed as mean ± SD of one experiment performed in triplicate.

Figure 6

Binding modes of Compound E, AZD1283, and 2-MeSADP to hP2Y12. (A) Binding mode of AZD1283 (green, stick style) co-crystallized with hP2Y12 (blue ribbon style; PDB code 4NTJ); (B) Docking model of Compound E (grey, ball-and-stick style) with hP2Y12 (blue ribbon style; PDB code 4NTJ); (C) Docking model of Compound E overlapping AZD1283 co-crystallized with hP2Y12; (D) Binding mode of 2-MeSADP (pink, stick style) co-crystallized with hP2Y12 (orange ribbon style; PDB code 4PXZ); (E) Overlay of proposed binding model of Compound E with 2-MeSADP co-crystallized with hP2Y12. Hydrogen bonds are displayed as yellow-green dashed lines. For clarity, only key residues are indicated in line-and-stick models and are labeled using the one-letter amino acid code.