Discovery of brain-penetrant, orally bioavailable aminothienopyridazine inhibitors of tau aggregation

Abstract

Agents capable of preventing the misfolding and sequestration of the microtubule-stabilizing protein tau into insoluble fibrillar aggregates hold considerable promise for the prevention and/or treatment of neurodegenerative tauopathies such as Alzheimer's disease. Because tauopathies are characterized by amyloidosis that is restricted to the central nervous system (CNS), plausible candidate compounds for in vivo evaluation must both prevent tau fibrillization and achieve significant brain levels. Recently, we reported the discovery of the aminothienopyridazine (ATPZ) class of tau aggregation inhibitors and now describe a series of new analogues that are both effective inhibitors of tau fibrillization and display significant brain-to-plasma exposure ratios after administration to mice. Further, two of the most promising examples, 15 and 16, were found to reach significant brain exposure levels following oral administration. Taken together, these results suggest that examples from the ATPZ class hold promise as candidates for in vivo efficacy studies in animal models of neurodegenerative tauopathies.

Figure 1

Figure 2

Summary of structure tau anti-fibrillization activity of ATPZs.

Figure 3

(A) Representative dose-response curves in the heparin-induced K18PL fibrillization assay; (B) SDS-PAGE analysis of supernatant (S) or pellet (P) samples obtained after centrifugation of fibrillizing mixtures incubated in the presence of DMSO vehicle, or 100 μM of test compound (11 and 16).

Figure 4

(A) Comparison of maximal percent inhibition of K18PL fibrillization caused by test compounds in which the fibrillizing mixtures did (sup) or did not (tot) undergo centrifugation at time zero to remove insoluble material. Compounds were added at 50 μM to the tau fibrillization mixture and incubated for 30 min. Samples were then divided in two halves, one of which was incubated without further treatment (“tot”), and one that was centrifuged at 186,000g for 30 min. After centrifugation of the latter sample, the supernatants (“sup”) was collected and incubated as per the fibrillization protocol. (B) Representative examples of SDS-PAGE analysis of supernatant (S) or pellet (P) samples obtained after centrifugation of fibrillizing mixtures prior to incubation; (C) Representative dose-response curves with (“sup”) and without (“tot”) initial centrifugation.

Figure 5

MT-assembly assay. Tubulin (30 μM) and tau40 (20 μM) were incubated with the test compound (50 μM; e.g., 16 [green triangles]) or DMSO (open and black squares) to evaluate possible interference in the tau-promoted MT-polymerization that may be caused by the ATPZ inhibitors of tau aggregation. As control experiment, MT-polymerization assay was also conducted in the absence of tau40 (open blue circles). Additional results are presented in the Supporting Information.

Figure 6

(A) Brain and plasma level of 16 after i.v. administration of 2 mg/Kg; (B) Brain and plasma level of 16 after oral (p.o.) administration of 5 mg/Kg; (C) Brain and plasma level of 15 after oral administration of 5 mg/Kg.

Scheme 1. Reagents and Reaction conditions

a) (i) NaNO₂, 37% HCl, ethanol, water, 0 °C, 20 min; (ii) ethyl acetoacetate, sodium acetate, ethanol, water, 0 °C, 2 h; b) X = Cl: ethyl cyanoacetate, ammonium acetate, acetic acid, 170 °C (microwave irradiation), 4 min; c) X = Br: ethyl cyanoacetate, 4-aminobutyric acid, 160 °C, 2.5 h; d) S₈, morpholine, ethanol, 150 °C (microwave irradiation), 15 min; e) titanium (IV) isopropoxide, isopropanol, 170 °C (microwave irradiation), 40 min. f) LiOH·H₂O, tetrahydrofuran, water, rt, 16 h; g) appropriate amine, BOP reagent, N,N-diisopropylethylamine, dimethylsulfoxide, rt, 4 h.