Fluorine-18 Labeling of the MDM2 Inhibitor RG7388 for PET Imaging: Chemistry and Preliminary Evaluation.

Abstract

RG7388 (Idasanutlin) is a potent inhibitor of oncoprotein murine double minute 2 (MDM2). Herein we investigated the feasibility of developing ¹⁸F-labeled RG7388 as a radiotracer for imaging MDM2 expression in tumors with positron emission tomography (PET). Two fluorinated analogues of RG7388, <b>6</b> and <b>7</b>, were synthesized by attaching a fluoronicotinyl moiety to RG7388 via a polyethylene glycol (PEG₃) or a propyl linker. The inhibitory potency (IC₅₀) of <b>6</b> and <b>7</b> against MDM2 was determined by a fluorescence polarization (FP)-based assay. Next, compound <b>6</b> was labeled with ¹⁸F using a trimethylammonium triflate precursor to obtain [¹⁸F]FN-PEG₃-RG7388 ([¹⁸F]<b>6</b>), and its properties were evaluated in MDM2 expressing wild-type p53 tumor cell lines (SJSA-1 and HepG2) in vitro and in tumor xenografts in vivo. The FP assays revealed an IC₅₀ against MDM2 of 119 nM and 160 nM for <b>6</b> and <b>7</b>, respectively. ¹⁸F-labeling of <b>6</b> was achieved in 50.3 ± 7.5% radiochemical yield. [¹⁸F]<b>6</b> exhibited a high uptake (∼70% of input dose) and specificity in SJSA-1 and HepG2 cell lines. Saturation binding assays revealed a binding affinity (<i>K</i>_d) of 128 nM for [¹⁸F]<b>6</b> on SJSA-1 cells. In mice, [¹⁸F]<b>6</b> showed fast clearance from blood with a maximum tumor uptake of 3.80 ± 0.85% injected dose per gram (ID/g) in HepG2 xenografts at 30 min postinjection (p.i.) and 1.32 ± 0.32% ID/g in SJSA-1 xenografts at 1 h p.i. Specificity of [¹⁸F]<b>6</b> uptake in tumors was demonstrated by pretreatment of mice with SJSA-xenografts with a blocking dose of RG7388 (35 mg/kg body weight, i.p.). In vivo stability studies in mice using HPLC showed ∼60% and ∼30% intact [¹⁸F]<b>6</b> remaining in plasma at 30 min and 1 h p.i., respectively, with the remaining activity attributed to polar peaks. Our results suggest that RG7388 is a promising molecular scaffold for ¹⁸F-labeled probe development for MDM2. Additional labeling strategies and functionalizing locations on RG7388 are under development to improve binding affinity and in vivo stability of the ¹⁸F-labeled compound to make it more amenable for PET imaging of MDM2 in vivo.