Generalization of Rapid C-[¹¹C]Methylation Reactions for the Synthesis of Short-Lived ¹¹C-Labeled Chemical Probes

In this study, we achieved one of the most challenging transformations in our long-term collaborator Suzuki’s unique “Rapid C-[¹¹C]methylation reaction,” namely, the cross-coupling of saturated alkyl carbons. We successfully established conditions for introducing the [¹¹C]methyl group at benzylic and cinnamyl positions, which are widely encountered in drug molecules. Because the initially selected benzylic tributylstannane substrates suffered from undesired side reactions, we employed boronic ester substrates as alternatives. Furthermore, the introduction of sterically demanding, strongly basic phosphine ligands into the palladium(0) catalyst system dramatically accelerated the reaction. We revealed that the yield exhibited a bell-shaped dependence on temperature, underscoring the importance of precise temperature control.

The developed conditions were proven to be highly effective for practical ¹¹C radiolabeling. Importantly, we also succeeded in ¹¹C-labeling of heteroaromatic rings, which had previously been considered difficult to access, thereby enabling rapid synthesis of a wide range of short-lived ¹¹C-labeled PET probes. These advances have been comprehensively summarized in recent review articles.¹–⁴

¹¹C-Labeling of O⁶-Benzylguanine, an MGMT Inhibitor Involved in Sensitivity to Alkylating Anticancer Agents

O⁶-Methylguanine-DNA methyltransferase (MGMT) repairs TMZ-induced methylated DNA lesions in malignant gliomas, thereby conferring resistance to temozolomide (TMZ) and serving as a major prognostic factor for poor therapeutic outcomes. This study aims to develop diagnostic molecular probes targeting MGMT by exploiting positron emission tomography (PET) imaging, which enables real-time three-dimensional visualization of target molecules. In this work, we report the creation of a ¹¹C-labeled PET probe based on the MGMT inactivator O⁶-benzylguanine (O⁶-BG).⁵

By applying our original rapid coupling methodology, we established an efficient one-pot synthesis of ¹¹C-labeled O⁶-BG. Initial attempts using an unprotected stannane precursor as the coupling substrate failed to afford the desired product in sufficient yield. To overcome this limitation, we designed a novel precursor in which the guanine moiety was protected. We then applied the palladium-catalyzed rapid C-[¹¹C]methylation of heteroaromatics, previously generalized in our earlier studies, which enabled smooth incorporation of the ¹¹C-methyl group. Furthermore, by integrating a rapid deprotection step under strongly basic conditions, we succeeded in synthesizing the ¹¹C-labeled O⁶-BG scaffold efficiently and with high radiochemical purity.

This work represents an interdisciplinary and translational research effort, supported by public funding, with the overarching goal of bridging basic methodology to clinical application.

Clinical-Grade PET Formulation and Imaging of (R,S)-Isoproterenol

This study was conducted in preparation for first-in-human PET imaging of (R,S)-isoproterenol (ISO), a catecholamine derivative reported to suppress aggregation of hyperphosphorylated tau protein implicated in Alzheimer’s disease progression. We established a manufacturing process for producing a high-quality PET probe suitable for human administration and successfully performed preclinical brain PET imaging in rats.⁶

The (R,S)-ISO PET probe was synthesized via reductive alkylation using [2-¹¹C]acetone as a precursor. Although no prior examples of this methodology applied to catecholamine scaffolds had been reported and the initial radiochemical yields were very low, systematic optimization was carried out:

1. [2-¹¹C]Acetone synthesis - fine-tuning of reaction parameters to suppress side reactions.
2. Reductive alkylation - optimization of acid-catalyzed conditions and elimination of ether contamination.
3. Purification and stabilization - selection of HPLC fractionation columns tailored for highly polar compounds and development of formulation strategies to improve stability.

Through these improvements, we achieved PET formulations meeting clinical standards and demonstrated effective brain imaging at the preclinical level. Moreover, analysis of brain kinetics and in vivo metabolism under these optimized conditions provided fundamental safety insights to support future clinical application.⁷

¹¹C-Labeling of the Acyclic Retinoid (ACR) and PET Imaging for Brain Kinetics Analysis

This study focused on the acyclic retinoid (ACR), a synthetic derivative originally developed in Japan as a chemopreventive agent against hepatocellular carcinoma recurrence. ACR exhibits high chemical stability and shares functional similarities with all-trans retinoic acid (ATRA). To date, we have achieved the following results:⁷˒⁸

1. Development of a ¹¹C-labeled ACR PET probe using our original rapid carbon-carbon coupling methodology (Figure A).⁷
2. In vivo brain PET imaging in rats and monkeys, demonstrating efficient blood-brain barrier penetration and widespread distribution throughout the brain (Figure B).⁷
3. Metabolite profiling in plasma and brain, which unexpectedly revealed increased brain accumulation of highly polar metabolites. These findings suggest that metabolically generated “polar retinoid carriers” may contribute to brain uptake (Figures C and D).⁷
4. Discovery of novel neuroprotective functions of ACR, including induction of neural differentiation from stem cells and attenuation of neurological deficits in a rat intracerebral hemorrhage model—effects that had not previously been reported for this compound.⁸

¹¹C-Labeling of Protopanaxadiol (PPD), a Bioactive Constituent of Ginseng, and PET Imaging

Ginsenosides, the major bioactive constituents of ginseng, exhibit diverse pharmacological effects, yet their in vivo pharmacokinetics remain largely unexplored. In this study, we focused on 20(S)-protopanaxadiol (PPD), a principal metabolite of ginsenosides. By achieving a controlled cross-metathesis reaction at an internal olefin site within the complex dammarane skeleton, we successfully synthesized a boronic ester precursor for radiolabeling.¹⁰

Subsequently, rapid Pd(0)-catalyzed ¹¹C-methylation was applied to this precursor. By suppressing peroxide formation at the lipidic allylic carbon, we accomplished efficient ¹¹C-labeling of PPD with high radiochemical purity and high specific activity. PET imaging revealed low brain uptake in rats, while mice exhibited pronounced hepatobiliary excretion. These results establish a versatile methodological platform for the ¹¹C-labeling of ginsenosides.