5-HT1A targeting PARCEST agent DO3AM-MPP with potential for receptor imaging: Synthesis, physico-chemical and MR studies

Abstract

Contrast enhancement in MRI using magnetization or saturation transfer techniques promises better sensitivity, and faster acquisition compared to T₁ or T₂ contrast. This work reports the synthesis and evaluation of 5-HT_1A targeted PARACEST MRI contrast agent using 1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide (DO3AM) as the bifunctional chelator, and 5-HT_1A-antagonist methoxyphenyl piperazine (MPP) as a targeting unit. The multi-step synthesis led to the MPP conjugated DO3AM with 60% yield. CEST-related physicochemical parameters were evaluated after loading DO3AM-MPP with paramagnetic MRI active lanthanides: Gadolinium (Gd-DO3AM-MPP) and Europium (Eu-DO3AM-MPP). Luminescence lifetime measurements with Eu-DO3AM-MPP and computational DFT studies using Gd-DO3AM-MPP revealed the coordination of one water molecule (q = 1.43) with metal-water distance (r_M-H₂O) of 2.7 Å and water residence time (τ_m) of 0.23 ms. The dissociation constant of K_d 62 ± 0.02 pM as evaluated from fluorescence quenching of 5-HT_1A (protein) and docking score of -4.81 in theoretical evaluation reflect the binding potential of the complex Gd-DO3AM-MPP with the receptor 5-HT_1A. Insights of the docked pose reflect the importance of NH₂ (amide) and aromatic ring in Gd-DO3AM-MPP while interacting with Ser 374 and Phe 370 in the antagonist binding pocket of 5-HT_1A. Gd-DO3AM-MPP shows longitudinal relaxivity 5.85 mM^-1s^-1 with a water residence lifetime of 0.93 ms in hippocampal homogenate containing 5-HT_1A. The potentiometric titration of DO3AM-MPP showed strong selectivity for Gd³⁺ over physiological metal ions such as Zn²⁺ and Cu²⁺. The in vitro and in vivo studies confirmed the minimal cytotoxicity and presential binding of Gd-DO3AM-MPP with 5-HT_1A receptor in the hippocampus region of the mice. Summarizing, the complex Gd-DO3AM-MPP can have a potential for CEST imaging of 5-HT_1A receptors.

Keywords: Chemical Exchange Saturation Transfer; Computational Studies; Density Functional Theory; Fluorescence quenching; Longitudinal and Transverse relaxivity; Water Residence Lifetime.