Optimization of benzo[d]oxazol-2(3H)-one activators of Slack potassium channels




Mishra, Nigam
Du, Yu
Spitznagel, Brittany
Weaver, C.
Emmitte, Kyle


0000-0003-2695-6695 (Mishra, Nigam)

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Introduction: Fragile X syndrome (FXS) is an inherited genetic disorder that causes intellectual disability (ID) and other comorbid conditions, including cognitive disabilities, behavioral conditions, and autism spectrum disorder (ASD). FXS is caused by silencing of the FMR1 gene, which leads to an absence of the fragile X mental retardation protein 1 (FMRP). FMRP has multiple important functions and among these is activation of Slack potassium (K+) channels. Slack is a member of the Slo family of channels, which are critical regulators of electrical activity in the nervous system. Thus, dysregulation of Slack activity leads to abnormal neuronal activity. Preclinical studies have shown that Slack activity is critical for higher brain functions including cognitive flexibility, learning, and memory. Hence, selective activation of Slack by a small molecule may represent a viable approach to treating certain cognitive deficits in FXS and potentially other forms of ID. Objective: Currently there are no available selective small-molecule activators of Slack, so the objective of our work is to develop such tools to investigate them as a potential new therapeutic approach to FXS. Methods: Hit compound VU0609159 was identified via high-throughput screening (HTS) using thallium (Tl+) flux assay in HEK-293 cells stably expressing Slack and confirmed in an automated electrophysiology assay. Library synthesis based, iterative hit optimization strategy around hit VU0609159 was employed. Classical and state-of-the-art synthetic chemistry techniques including microwave assisted organic synthesis and flow chemistry were employed for synthesis of target compounds. Purification was done by automated liquid chromatography. Bruker Fourier 300HD and Agilent 6230 time-of-flight LC/MS were utilized to obtain NMR and HRMS, respectively. Results: VU0609159 is a member of a series of benzo[d]oxazol-2(3H)-ones and has multiple regions amenable to rapid Structure-activity relationship (SAR) development through the preparation of small libraries of compounds. During SAR study, we found cyclic and acyclic branched linkers optimal for the activity. Substitution of benzo[d]oxazol-2(3H)-one ring with benzo[d]thiazol-2(3H)-one gave encouraging results. Conclusion: SAR for multiple regions around Slack activity in the VU0609159 series has been identified utilizing this approach and our Tl+ flux assay. Studies combining optimal functional groups within each region of the scaffold are ongoing.