Characterization and Chemical Optimization of the D2 Dopamine Receptor‐Selective Antagonist, ML321, Identifies Lead Compounds for the Clinical Treatment of Neuropsychiatric Disorders

Abstract

Schizophrenia is a devastating illness characterized by both positive (hallucinations, delusions) and negative (flat affect, decreased motivation) symptoms coupled with cognitive impairment. Current antipsychotic medications are effective in treating the positive symptoms through antagonism of the D2 dopamine receptor (D2R). However, antipsychotic treatment is also hindered by side‐effects due to off‐target activities at other GPCRs and unfavorable binding kinetics at the D2R. We have identified and characterized a novel D2R antagonist with high selectivity against other GPCRs – ML321. In functional profiling screens of up to 168 different GPCRs, ML321 showed little activity beyond potent inhibition of the D2R, and to a lesser extent the D3R, demonstrating exceptional GPCR selectivity. Schild‐type functional assays revealed that ML321 acts as a competitive antagonist of the D2R while kinetic studies showed that ML321 exhibits slow‐on and fast‐off receptor binding rates; properties that are believed to limit extrapyramidal side‐effects that are commonly observed with antipsychotics. In fact, using doses that were maximally effective in antipsychotic‐predictive behavioral assays in rodents, ML321 promoted little to no catalepsy, suggesting that ML321 may produce less extrapyramidal side‐effects in patients. Importantly, no other D2R antagonist exhibits this pharmacological and behavioral profile supporting its development into an advanced drug lead. While a promising therapeutic, ML321 has a short metabolic half‐life, impeding its clinical development. A metabolite study revealed that the primary site of metabolism involves oxidation of the alkyl‐thiophene portion of ML321. To create more metabolically stable derivatives, we iteratively designed and synthesized over 100 analogs with modifications focused on the alkyl‐thiophene moiety. These analogs were pharmacologically characterized for both D2R binding affinity and function, and were also tested for metabolic stability, permeability, and solubility. These efforts have led to the optimization of ML321 into a collection of lead candidates that show similar pharmacological characteristics of ML321, but with marked increases in metabolic stability and ADME properties. Molecular docking and mutagenesis studies have led to a better understanding of how ML321 binds to the D2R, which will further assist in analog design and development. Together, these findings have advanced our understanding of ML321 structure‐activity relationships, particularly around the alkyl‐thiophene moiety, and have identified lead candidates for in vivo pharmacokinetic studies, thus representing substantial progress in the development of a new antipsychotic treatment.