A novel series of 6-benzhydryl-4-amino-quinolin-2-ones was discovered as cannabinoid type 1 receptor (CB 1 R) inverse agonists based on the high-throughput screening hit, compound 1a. Structure−activity relationships were studied to improve in vitro/in vivo pharmacology and restrict distribution to the peripheral circulation. We adopted several strategies such as increasing topological polar surface area, incorporating discrete polyethylene glycol side chains, and targeting P-glycoprotein (P-gp) to minimize access to the brain. Compound 6a is a P-gp substrate and a potent and highly selective CB 1 R inverse agonist, demonstrating excellent in vivo metabolic stability and a low brain to plasma ratio. However, brain receptor occupancy studies showed that compound 6a may accumulate in brain with repeat dosing. This was evidenced by compound 6a inhibiting food intake and inducing weight loss in diet-induced obese mice. Thus, a strategy based on P-gp efflux may not be adequate for peripheral restriction of the disclosed quinolinone series.

Aim: To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. Materials & methods: An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism in vivo by middle-down approach. Results & Conclusion: For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.

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