Discovery of 7-Oxo-2,4,5,7-tetrahydro-6H-pyrazolo[3,4-c]pyridine Derivatives as Potent, Orally Available, and Brain-Penetrating Receptor Interacting Protein 1 (RIP1) Kinase Inhibitors: Analysis of Structure–Kinetic Relationships

Abstract

We report the discovery of 7-oxo-2,4,5,7-tetrahydro-6 H-pyrazolo[3,4- c]pyridine derivatives as a novel class of receptor interacting protein 1 (RIP1) kinase inhibitors. On the basis of the overlay study between HTS hit 10 and GSK2982772 (6) in RIP1 kinase, we designed and synthesized a novel class of RIP1 kinase inhibitor 11 possessing moderate RIP1 kinase inhibitory activity and P-gp mediated efflux. The optimization of the core structure and the exploration of appropriate substituents utilizing SBDD approach led to the discovery of 22, a highly potent, orally available, and brain-penetrating RIP1 kinase inhibitor with excellent PK profiles. Compound 22 significantly suppressed necroptotic cell death both in mouse and human cells. Oral administration of 22 (10 mg/kg, bid) attenuated disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). Moreover, analysis of structure-kinetic relationship (SKR) for our novel chemical series was also discussed.

Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis

“…Further molecular dynamic simulation study revealed that NTB451 indeed interacted with K45 of RIPK1, which mediates kinase activity. NTB451 has a structural similarity at their benzyl-triazol moiety with some known RIPK1 inhibitors, Nec-1, GSK 2982772 [ 23 , 36 ]. However, the functional relationship of such moiety needs to be elaborately studied.…”

“…We used this program to generate the initial docking poses of NTB451 and U0126, which were needed for the following molecular dynamics (MD) simulation study. The crystal structure of RIP1 kinase, bound to inhibitor (PDB ID: 6C3E) [ 36 ] with a resolution of 2.6 Å, was used for the validation of the docking simulation. The active site of RIPK1 was defined from the bound inhibitor, 2-benzyl-5-nitro-1H-benzimidazole, within a radius of 10 Å.…”

Receptor-interacting protein kinase 1 (RIPK1) as a therapeutic target

“…Decreased cFLIP and reduced levels of activated Casp8 in patients with MS, and thus decreased Casp8 inactivating cleavage of RIPK1, likely explain the increased RIPK1 kinase activity and necroptosis seen in MS. Genetic ( Ripk1 D138N/D138N ) and pharmacological (Nec-1s) inhibition of RIPK1 kinase ameliorated disease pathology, improved animal behaviour, attenuated the production of pro-inflammatory cytokines and decreased recruitment of immune cells in two animal models of MS 3 . Another inhibitor of RIPK1 kinase developed by Takeda also showed efficacy in an animal model of MS 98 .…”

“…A subsequent family of benzoxazepinone RIPK1 inhibitors was published by GlaxoSmithKline (GSK), which included GSK′481 and the clinical candidate GSK′772 (refs 173 , 174 ), which was followed by the identification of a dihydropyrazole chemotype of RIPK1 inhibitors including GSK′963 and GSK′547 (refs 175 , 176 ). Published and patented compounds generated by Takeda, Genentech and Rigel are all from the same benzoxazepinone family as the GSK′481 series 177 – 179 .

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Molecular design opportunities presented by solvent‐exposed regions of target proteins

“…The 7‐ and 8‐positions on the benzoxazepinone core of receptor interacting protein 1 (RIP1) kinase inhibitor 94 are exposed to the solvent‐accessible region. Consequently, optimization of the center platform and careful modification of neighboring substituents by utilizing structure‐based design resulted in the identification of the cyanosubstituted derivative 95 as a highly active and brain‐penetrating RIP1 kinase inhibitor with favorable oral bioavailability and excellent ADMET properties ( t 1/2 = 210 minutes, in human and mouse liver microsomes; high plasma exposure (AUC = 658 ng/h/mL) and a moderate plasma duration (mean residence time, MRT = 3.1 hour in mice) (Figure E) …”