Targeted therapy of HER2 positive breast cancer has led to clinical success in some cases with primary and secondary resistance being major obstacles. Due to the substantial involvement of mTOR kinase in cell growth and proliferation pathways it is now targeted in combination treatments to counteract HER2 targeted therapy resistance. However, the selection of receptive patient populations for a specific drug combination is crucial. This work aims to develop a molecular probe capable of identifying patients with tumour populations which are receptive to RAD001 combination therapy. Based on the structure of a mTOR inhibitor specific for mTORC1, we designed, synthesised and characterised a novel benzofuran based molecular probe which suits late stage fluorination via Click chemistry. Synthesis of the alkyne precursor 5 proceeded in 27.5% yield over 7 linear steps. Click derivatisation gave the non-radioactive standard in 25% yield. Radiosynthesis of [18F]1-((1-(2-Fluoroethyl)-1H-1,2,3-triazol-4-yl) methyl)-4-((5-methoxy-2-phenylbenzofuran-4-yl) methyl) piperazine ([18F]mBPET-1) proceeded over two steps which were automated on an iPhase FlexLab synthesis module. In the first step, 2-[18F]fluoroethylazide ([18F]6) was produced, purified by automated distillation in 60% non-decay-corrected yield and subjected to Click conditions with 5. Semi-preparative RP-HPLC purification and reformulation gave [18F]mBPET-1 in 40% ± 5% (n = 6) overall RCY with a process time of 90 min. Radiochemical purity was ≥99% at end of synthesis (EOS) and ≥ 98% after 4 h at room temperature. Molar activities ranged from typically 24.8 GBq/μmol (EOS) to a maximum of 78.6 GBq/μmol (EOS). Lipophilicity of [18F]mBPET-1 was determined at pH 7.4 (logD7.4 = 0.89). [18F]mBPET-1 showed high metabolic stability when incubated with mouse S9 liver fractions which resulted in a 0.8% drop in radiochemical purity after 3 h. Cell uptake assays showed 1.3–1.9-fold increased uptake of the [18F]mBPET-1 in RAD001 sensitive compared to insensitive cells across a panel of 4 breast cancer cell lines. Molecular targeting of mTOR with [18F]mBPET-1 distinguishes mTOR inhibitor sensitive and insensitive cell lines. Future studies will explore the ability of [18F]mBPET-1 to predict response to mTOR inhibitor treatment in in vivo models.

中文翻译：

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[18F] mBPET-1的合成和验证，一种基于苯并呋喃骨架的氟18标记的mTOR抑制剂衍生物。

HER2阳性乳腺癌的靶向治疗在某些情况下已取得临床成功，主要和次要耐药性是主要障碍。由于mTOR激酶在细胞生长和增殖途径中的大量参与，现在将其作为联合治疗的靶标以抵消HER2靶向的治疗抗性。但是，为特定药物组合选择可接受的患者人群至关重要。这项工作的目的是开发一种分子探针，该探针能够鉴定患有RAD001联合疗法的肿瘤人群。基于对mTORC1特异的mTOR抑制剂的结构，我们设计，合成和表征了一种新型的基于苯并呋喃的分子探针，该探针适合通过Click化学作用进行后期氟化。炔烃前体5的合成在27中进行。7个线性步骤的产率为5％。单击衍生化得到非放射性标准品，产率为25％。[18F] 1-（（1-（2-氟乙基）-1H-1,2,3-三唑-4-基）甲基）-4-（（5-甲氧基-2-苯基苯并呋喃-4-基）的放射性合成甲基）哌嗪（[18F] mBPET-1）分两步进行，这些步骤在iPhase FlexLab合成模块上实现了自动化。第一步，制备2- [18F]氟乙基叠氮化物（[18F] 6），通过自动蒸馏以60％的非衰减校正产率纯化，并在5的Click条件下进行。半制备RP-HPLC纯化和重新配制产生[18F] mBPET-1的总RCY为40％±5％（n = 6），处理时间为90分钟。合成结束时（EOS）的放射化学纯度为≥99％，室温4 h后的放射化学纯度为≥98％。摩尔活动范围从典型的24.8 GBq /μmol（EOS）到最大78.6 GBq /μmol（EOS）。在pH 7.4（logD7.4 = 0.89）下测定[18F] mBPET-1的亲脂性。[18F] mBPET-1与小鼠S9肝馏分温育时显示出高代谢稳定性，导致3小时后放射化学纯度下降0.8％。细胞摄取分析显示，与不敏感的细胞相比，跨越4组乳腺癌细胞系，RAD001敏感的[18F] mBPET-1的摄取增加了1.3-1.9倍。[18F] mBPET-1对mTOR的分子靶向作用可区分mTOR抑制剂敏感和不敏感细胞系。未来的研究将探索[18F] mBPET-1在体内模型中预测对mTOR抑制剂治疗的反应的能力。细胞摄取分析显示，与不敏感的细胞相比，跨越4组乳腺癌细胞系，RAD001敏感的[18F] mBPET-1的摄取增加了1.3-1.9倍。[18F] mBPET-1对mTOR的分子靶向作用可区分mTOR抑制剂敏感和不敏感细胞系。未来的研究将探索[18F] mBPET-1在体内模型中预测对mTOR抑制剂治疗的反应的能力。细胞摄取分析表明，与不敏感的细胞相比，跨越4种乳腺癌细胞系，RAD001敏感的[18F] mBPET-1的摄取增加了1.3-1.9倍。[18F] mBPET-1对mTOR的分子靶向作用可区分mTOR抑制剂敏感和不敏感细胞系。未来的研究将探索[18F] mBPET-1在体内模型中预测对mTOR抑制剂治疗的反应的能力。