Discovery of Potent and Brain-Penetrant Tau Tubulin Kinase 1 (TTBK1) Inhibitors that Lower Tau Phosphorylation In Vivo

Abstract

Structural analysis of the known NIK inhibitor 3 bound to the kinase domain of TTBK1 led to the design and synthesis of a novel class of azaindazole TTBK1 inhibitors exemplified by 8 (cell IC50: 571 nM). Systematic optimization of this series of analogs led to the discovery of 31, a potent (cell IC50: 315 nM) and selective TTBK inhibitor with suitable CNS penetration (rat Kp,uu: 0.32) for in vivo proof of pharmacology studies. The ability of 31 to inhibit tau phosphorylation at the disease-relevant Ser 422 epitope was demonstrated in both a mouse hypothermia and a rat developmental model and provided evidence that modulation of this target may be relevant in the treatment of Alzheimer’s disease and other tauopathies.

Identification and Utilization of a Chemical Probe to Interrogate the Roles of PIKfyve in the Lifecycle of β-Coronaviruses

“…We began our campaign with a subset of analogs that probed the importance of the sevenmembered ring system in AMG28. Based upon a recently solved co-crystal structure of AMG28 bound to TTBK1 (PDB: 7JXX) 16 and our own kinase inhibitor design experience, we hypothesized that the aminopyrimidine ring makes essential hydrogen bonds with the hinge of PIKfyve. It was suggested by the authors of the TTBK1 paper that the three methylene groups of the sevenmembered ring help maintain the planarity of the molecule rather than making key contacts with the protein.…”

“…Based on the analogs in Tables 1 and 2, we next made a small set of six-membered derivatives bearing an alkyne capped with diverse groups. We designed these to include a fivemembered 1-methylimidazole ( 14), a cyclopropyl ring (15), or a dimethylamino group (16). These substituents represent diversity in size and electronic nature, exploring the size of a ring system tolerated within this portion of the PIKfyve binding pocket as well as nitrogen atoms projected in various directions.…”

“…This places them in the same selectivity range as other six-membered analogs from Tables 1 and 2, which bind between 1 and 20 WT human kinases in the same panel. The PIKfyve potency of these compounds was found to be poor for 1-methylimidazole-bearing analog 14 but improve when this ring system was replaced with the smaller cyclopropyl (15) or dimethylamino (16) groups. The DiscoverX PIKfyve PoC data was found to correlate well with the PIKfyve NanoBRET assay IC50 values for these analogs.…”

“…Analogs bearing larger groups on the terminus of the alkyne (4 and 5, for example) suggest that the pocket accommodates bulk and thus the homologated (22)(23)(24) or sterically encumbered (25 and 26) propargylic alcohol analogs were predicted to be well tolerated by PIKfyve. Finally, the dimethylamino analog (16) indicated that the hydroxyl group is dispensable and thus analog 20 was pursued as a potential alternative.…”

“…The analytical data for 3 matches that previously reported. 16 1 H NMR (400 MHz, CD3OD-d4 δ 8.59 (s, 1H), 8.17 (s, 2H), 8.07 (d, J = 5.8 Hz, 1H), 7.41 (d, J = 8.3 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 6.0 Hz, 1H), 1.60 (s, 6H). HPLC purity: 100%.…”