Alzheimer's disease (AD) is a devastating dementia without a disease-modifying therapy. According to FDA, AD clinical trials are over 200 but so far have made zero success in slowing AD progression. This continuous failure speaks for the difficulty of this task and calls for re-evaluating the existing therapeutic strategies. As a neurodegenerative disorder, neuronal death is fundamental to AD pathogenesis. Current therapeutic strategies concentrate on combating neurotoxic insults implicated in AD (i.e., Aβ, phospho-tau, free radicals, inflammatory toxins, etc.). Less attention has been paid to strengthening neuronal defense given that neuronal death is an imbalance between its defense and insult. Studies in our lab revealed that defense mechanism of central cholinergic system in AD is compromised in subjects with G protein-coupled receptor (GPCR) kinase-5 (GRK5) deficiency; the latter appears during aging and worsens in AD. The compromised neuronal defense leads to susceptibility to degeneration triggered by excessive Aβ or hypoxia whereas an active anthranilamide derivative CN168 was able to prevent the neurodegeneration and the cognitive impairments in GRK5-deficient Swedish APP transgenic (GAP) mice. Therefore, emerging evidence supports the idea that strengthening neuronal defense raises neuronal death threshold thus increasing the difficulty for various neurotoxic insults to trigger neuronal death. This new therapeutic strategy focuses on the defense rather than individual insult but works against multiple insults simultaneously. Such a therapeutic effort will not replace, but rather complement, the existing therapeutic effort, therefore bringing new hope for discovering disease-modifying therapy for AD. While CN168 was proven to be effective at the level of proof-of-concept (PoC) in preventing cognitive decline in GAP mice, we also noticed that CN168 has two violations of the five drug-likeness rules (MW>500; LogP>5), with a short duration of central action. This requires further optimization of the pharmacokinetic (PK) properties of CN168 prior to future clinical studies. Because CN168 has proven in vivo efficacy and previously demonstrated superb selectivity and sensitivity towards muscarinic receptor-2, we propose to use CN168 as the active pharmaceutical ingredient (API) and to improve its brain to plasma (B2P) ratio using prodrug approach. In this project, we will characterize three carefully selected prodrugs of CN168 by comparing their PK properties, and study their pharmacodynamics (PD) and toxicology before preparing the investigational new drug (IND) application for the finally selected prodrug(s). We will achieve our goals with the following three Specific Aims: Aim 1. Pharmacokinetics of the API (CN168) and its prodrugs: in this aim we will (1) synthesize CN168 and its prodrugs in quantities sufficient for their PK profiling in vivo; and then (2) carry out their PK characterization and select the prodrug(s) outperforming CN16...