Synthesis and Characterization of Fluorinated Cytosporone-Like Activators of NR4A1

Cocaine Use Disorder (CUD) is a chronic, relapsing condition marked by disrupted dopamine signaling and long-term changes in neural plasticity. Current treatments are limited, and relapse rates remain high. Recent studies identify NR4A1/NUR77, an orphan nuclear receptor and immediate-early gene, as a key regulator of the brain’s response to cocaine. CART (cocaine- and amphetamine-regulated protein transcript, encoded by CARTPT) is a neuropeptide that plays a crucial role in mediating satiety and reward in mesolimbic dopaminergic circuitry. Activation of Nr4a1 using small molecules such as Cytosporone B (Csn-B) has been shown to selectively reduce drug-seeking behavior in drug-primed animals through increased transactivation at the Cartpt gene promoter, highlighting Nr4a1 as a potential therapeutic target for CUD. This project focused on synthesizing and characterizing analogs of Csn-B with improved pharmacokinetic properties, especially blood-brain barrier (BBB) permeability, and metabolic stability. Analogs were prioritized based on their predicted binding and drug-like properties using in silico docking (AutoDock Vina) and pharmacokinetic prediction tools (SwissADME). Key synthetic strategies included late-stage fluorination and macrocyclization to enhance lipophilicity and molecular rigidity. A new compound, KMH-0046, was synthesized via Friedel–Crafts acylation of a fluoro-aryl ester followed by selective demethylation. Biological assays in SH-SY5Y neuroblastoma cells showed that KMH-0046 significantly increased NR4A1 expression at non-toxic doses, matching the efficacy of Csn-B but with more robust activation of genes associated with dopamine homeostasis, such as CARTPT and TH (tyrosine hydroxylase). These findings support the use of fluorination to enhance both biological activity and pharmacological potential. Overall, the project advances the understanding of NR4A1 as a drug target in addiction and identifies KMH-0046 as a lead compound for further development. Future directions include expanding compound libraries, performing direct receptor-binding studies, and evaluating in vivo efficacy in mouse models of CUD.