Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)-1H-indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ-Thrombin

Abstract

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based 3. Of these, 9j (PAR4 IC50 = 445 nM, PAR1 response IC50 > 30 μM) and 10h (PAR4 IC50 = 179 nM, PAR1 response IC50 > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin.

Figure 1

Structures and properties of reported PAR-4 antagonists.

Figure 2

Library optimization strategy for 3 to improve PAR4 antagonist activity, selectivity, and the DMPK profile.

Figure 3

Structures of 10h and 12, PAR4 anatgonists containing the–methoxy-6-methylimidazo[2,1-b][1,3,4]thiadiazole bicyclic ring system.

Figure 4

Molecular pharmacology profile of 9j and 10h. (A) PAR4-AP concentration–response curves (n = 3) with equivalent inhibition of both PAC-1 (IC₅₀ = 445 nM) and P-Selectin (IC₅₀ = 435 nM). (B) Progressive fold-shift inhibition assay with 9j, showing complex mode of PAR4 inhibition. (C) PAR4-AP concentration–response curves (n = 3) with comparable inhibition of both PAC-1 (IC₅₀ = 179 nM) and P-Selectin (IC₅₀ = 132 nM). (D) Progressive fold-shift inhibition assay with 10h, also showing a complex mode of PAR4 inhibition. Note: compound solubility was not an issue at the concentrations evaluated, i.e., no precipitate was observed.

Figure 5

Molecular pharmacology profile of (A) 9j and (B) 10h against activation of PAR4 with the tethered ligand, 100 nM γ-thrombin.

Scheme 1

Synthesis of PAR4 Antagonist Analogues 8^{a a}Reagents and conditions: (a) THF, NBS (1.0 equiv), 0 °C, 1 h, 90–99%; (b) aryl/heteroarylboronic acid (1.5 equiv), Pd(PPh₃)₄ (0.1 equiv), Na₂CO₃ (2.0 equiv), DMF–H₂O (4:1), µw, 120 °C, 15 min, 76–95%; (c) K₂CO₃ (2 equiv), benzyl or alkyl bromide (2 equiv, DMF, 45 °C, 16 h, 60–90%; (d) LAH, THF, 0 °C, 1 h, 50–85%.