This talk will discuss the discovery and development of M1 positive allosteric modulators (PAMs) for the treatment of cognitive dysfunction in schizophrenia and AD. From a weak and non-selective HTS hit, we developed highly M1 selective in vivo tool compounds that culminate in the discovery of VU0467319, an M1 PAM clinical candidate that had successfully completed a Phase I Single Ascending Dose clinical trial. Pharmacokinetic assessment revealed that, in humans upon increasing dose, a circulating, inactive metabolite constituted a major portion of the total drug-related AUC. One approach the team employed to reduce inactive metabolite formation in the back-up program was the kinetic isotope effect, replacing the metabolically labile C-H bonds with shorter, more stable C-D bonds. The C-D dipole afforded VU6045422, a more potent M1 PAM (human EC50 = 192 nM, 80% ACh Max) than its proteo-congener VU0467319 (human EC50 = 492 nM, 71% ACh Max), and retained the desired profile of minimal M1 agonism. Overall, the profile of VU6045422 supported advancement, as did greater in vitro metabolic stability in both microsomes and hepatocytes than VU0467319. In both rat and dog in vivo, low doses proved to mirror the in vitro profile; however, at higher doses in 14-day exploratory toxicology studies, the amount of the same undesired metabolite derived from VU6045422 was equivalent to that produced from VU0467319. This unexpected IVIVC result, coupled with less than dose-proportional increases in exposure and no improvement in solubility, led to discontinuation of VU0467319/ VU6045422 development. Attempts to block metabolism with the incorporation of deuterium atoms proved successful in vitro, and in vivo at low exposures; however, in high-dose toxicology studies, the degree of oxidative metabolism was comparable to the proteo-congener. Here, we describe a second-generation back-up effort based on the VU0467319 scaffold wherein strategic placement of a tertiary hydroxyl moiety afforded VU6052254, a potent M1 PAM (EC50 = 59 nM, 79% ACh max), with high CNS exposure (rat Kp = 1.07; Kp,uu = 1.27; P-gp ER = 1.97, Papp = 23 x 10-6 cm/s), reduced metabolism across species, excellent pharmacodynamic responses (MED in rat NOR = 1 mg/kg PO; MED in rat CFC = 0.3 mg/kg PO), excellent multi-species PK (Clps < 10 mL/min/kg, %F >65) and favorable human PK and dose projections. However, an in vitro CYP450 induction liability, confirmed in chronic dosing studies, precluded further development.