Substituted pyrazolones require N² hydrogen bond donating ability to protect against cytotoxicity from protein aggregation of mutant superoxide dismutase 1

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal neurodegenerative disease. Although the cause remains unknown, misfolded protein aggregates are seen in neurons of sporadic ALS patients, and familial ALS mutations, including mutations in superoxide dismutase 1 (SOD1), produce proteins with an increased propensity to misfold and aggregate. A structure activity relationship of a lead scaffold exhibiting neuroprotective activity in a G93A-SOD1 mouse model for ALS has been further investigated in a model PC12 cellular assay. Synthesis of biotinylated probes at the N¹ nitrogen of the pyrazolone ring gave compounds (5d-e) that retained activity within 10-fold of the proton-bearing lead compound (5a) and were equipotent with a sterically less cumbersome N¹-methyl substituted analogue (5b). However, when methyl substitution was introduced at N¹ and N ² of the pyrazolone ring, the compound was inactive (5c). These data led us to investigate further the pharmacophoric nature of the pyrazolone unit. A range of N¹ substitutions were tolerated, leading to the identification of an N¹-benzyl substituted pyrazolone (5m), equipotent with 5a. Substitution at N² or excision of N², however, removed all activity. Therefore, the hydrogen bond donating ability of the N²-H of the pyrazolone ring appears to be a critical part of the structure, which will influence further analogue synthesis.