Synthesis and evaluation of [¹⁸F]mIpoPET-1, a fluorine-18 labelled mTOR inhibitor derivative based on an imidazo[4,5-b]pyrazin-2-one backbone.

Objectives: The mammalian target of rapamycin (mTOR) is a prominent target for drug development in the oncological space. Although first generation inhibitors are being used clinically second and third generation mTOR inhibitors designed to have improved efficacy are being developed. A key factor with mTOR inhibitors is that not all patients respond to treatment, and hence careful patient selection is necessary for optimal results. This work aims to develop a molecular imaging probe for the non-invasive selection of patients receptive to treatment with everolimus (RAD001) or second generation dual mTORC1/2 inhibitors. Based on a dual mTORC1/2 inhibitor with imidazo[4,5-b]pyrazin-2-one scaffold, we designed, synthesised, and characterised a novel molecular imaging probe for positron emission tomography (PET).^[1-4]

Methods: Triazole 1 was prepared in a two-step one-pot reaction by first converting the commercially available carbamoyl to an amidine which was subsequently reacted with hydrazine (Figure 1). Nucleophilic aromatic substitution of 3,5-dibromopyrazin-2-amine with the TBDMS-protected building block 2 gave amine 3 which was converted to imidazole 4a using CDI. Deprotection of 4a and Suzuki coupling with 1 completed the core scaffold 5 which, following activation in the form of its tosylate, served as the precursor for radioactive and non-radioactive syntheses of 6-(4-(1H-1,2,4-triazol-3-yl)phenyl)-1-(2-fluoroethyl)-1H-imidazo[4,5-b]pyrazin-2(3H)-one (mIpoPET-1) using different reaction conditions. Radiochemical purity of resulting [¹⁸F]mIpoPET-1 was determined via radio-HPLC and its identity was confirmed via co-injection with fully characterised mIpoPET-1. The biological profile of [¹⁸F]mIpoPET-1 was assessed in vitro in a RAD001 sensitive and insensitive model system using previously characterised breast cancer cell lines.^[5,6]

Results: Synthesis of the tosylate precursor 6 proceeded in 6.6% yield over 6 linear steps. Fluorination of 6 using TBAF gave the non-radioactive standard mIpoPET-1 in 44% yield. Fully automated radiosynthesis of [¹⁸F]mIpoPET-1 was achieved in 5.3 ± 0.4% radiochemical yield (RCY) via direct nucleophilic radiofluorination of 6 with [¹⁸F]KF/K₂₂₂ using an iPHASE FlexLab module. Semi-preparative RP-HPLC purification and reformulation gave [¹⁸F]mIpoPET-1 in 1.6% ± 0.2% overall RCY with a process time of 70 minutes. Radiochemical purity was >99% at end-of-synthesis (EOS) and the retention times of co-injected [¹⁸F]mIpoPET-1 and mIpoPET-1 were identical. Typical molar activity was 22.1 GBq/µmol (EOS). Cell uptake assays showed 5.0-fold increased uptake of [¹⁸F]mIpoPET-1 in RAD001 sensitive BT-474 cells compared to RAD001 insensitive MDA-MB-231 cells. Blocking studies with excess mIpoPET-1 confirmed target specific cell uptake of [¹⁸F]mIpoPET-1. Lack of reduced cell uptake of [¹⁸F]mIpoPET-1 at 4°C compared to 37°C indicates dependence on passive cell membrane transport. Overall uptake on cell surfaces was low at 0.5% - 0.8%. Conclusion: Automated radiolabelling of [¹⁸F]mIpoPET-1 was achieved in sufficient quantities for biological evaluation and with high radiochemical purity. In vitro uptake assays showed increased uptake of [¹⁸F]mIpoPET-1 in RAD001 sensitive compared to insensitive cells.

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