Synthesis of cold standards
The synthesis of cold standards was carried out for spectroscopic characterization and identification, as well as a comparison with radiolabel products for confirmation of product by radio HPLC. Synthetic procedures were undertaken as outlined in Scheme 2.
Iodonium salt precursors
The initial synthetic route to an iodonium salt precursor was envisioned as shown in Scheme 1, with a sodium hydride coupling between commercially available indole-3-carbaldehyde and the appropriately substituted iodo benzyl bromide. This intermediate would then undergo a one-pot reaction to form an iodonium salt suitable for radiolabelling. In order to determine optimal conditions, initial reactions were carried out with the para substituted material.
Synthesis of Compound 1 was undertaken as described in Scheme 2 and the product was obtained in quantitative yield. With Compound 1 in hand, a one-pot synthesis of the iodonium salt was trialled using reaction conditions described by Zhu, Jalalian and Olofsson(9). The resultant reaction mixture showed no indication of product formation. The characteristic proton signal correlating to the aldehyde proton at approximately 10 ppm was absent and the expected mass for the desired product was not detected with high resolution mass spectrometry, suggesting an incompatibility between these reaction conditions and the aldehyde functional group. Literature indicated that sodium periodate(10) and peroxide(11) could also be employed as oxidants and were trialled with similar results. Other counter ions and aryl systems were also investigated, with a summary of reactants attempted with Compound 1 shown in Table 1 with none of the combinations resulting in any identifiable products other than starting material.
Table 1
Reaction conditions trialled with for iodonium salt synthesis.
Oxidant | Counter ion | Aryl System |
MCPBA | OTf/OTs | p-Methoxybenzene |
Peroxide | OTs | 1,3, Dimethoxy Benzene |
Oxone | OTs | 1,3,5 Trimethoxy Benzene |
Sodium Periodate | Acetic acid/H2SO4 | 1,3,5 Trimethoxy Benzene |
Attempts to carry out iodonium salt forming reactions in a two-step process shown in Scheme 4 also yielded no identifiable compounds. After determining iodonium salt conditions were not compatible with this molecule, a major product from the oxidation step of the two-step reaction was isolated via column chromatography and crystallized using vapour diffusion methods for x-ray crystallography, with the structure shown in Fig. 2.
Boronic acid pinacol ester precursors
With an iodine containing intermediate already in hand, a Miyarua borylation reaction was undertaken to provide the para substituted precursor in a 72% yield. As previous coupling reactions had been undertaken successfully, commercially available sources appropriately substituted boronic acid pinacol ester containing benzyl bromides were used to investigate a one-step coupling synthetic route. This method produced the desired product in yields 78%, 76% and 58% yields of the para, meta, and ortho precursors respectively. Product identity was confirmed through spectroscopy as well as x-ray crystal structures.
With three precursors in hand, radiolabelling experiments were undertaken.
Radiochemistry
Reaction conditions from Tredwell et al.(12) were utilized for initial radiolabelling experiments, as shown in Scheme 6. Under these conditions, no radiolabelled products were isolated.
As initial radiolabelling attempts yielded no discernible radiolabel products and previous literature suggested that these reactions may not be suitable for automated synthesis(12), possibly due to the inert gas systems they often operate under; the mechanism of catalysis for these reactions is unclear but may operate through a Cham-Lam coupling-like oxidation cycle, which would require atmospheric oxygen that is not present in standard, inert gas flushed automated systems. The reaction was attempted again with air being purged into the reaction vessel throughout the labelling, with no improvement in radiolabel incorporation.
A 4-Methoxycarbonylphenylboronic acid, pinacol ester was utilized as a model for trouble shooting as it is chemically similar to reagents used in both the Tredwell paper and another paper authored by Mossine and coworkers(13), which had resulted in excellent yields, however under the previously stated conditions, no radiolabelling was observed.
As the catalyst system described by Treadwell was not able to produce radiolabelled products in our hands, another system, described by Mossine and shown in Scheme 7 was investigated.
This system also produced no discernible radiolabelled products at the expected retention time. Mossine and co-workers had noted poor radiochemical yields prior to their own optimization for boronic acid labelling with regards to eluents. Development of a new eluent was required for successful synthesis, which utilized a minimized quantity of potassium carbonate and potassium triflate in combination with [2.2.2] Cryptand.
Attempts to carry out the radiosynthesis with the model system utilizing other standard eluents such as bicarbonate and tertbutyl amine were unsuccessful.
To determine if the eluent was the limiting factor in the radiosynthesis, a synthesis was carried out without fluoride isolation, with evaporation of the 18O water being performed prior to the labelling reaction, which yielded minor new radiolabelling products.
Adoption of a potassium triflate eluent system afforded radiolabelling of the model system as the major radiolabel product. Further optimization of the eluent showed that the preconditioned QMA cartridge used for an 18F-FDG synthesis contained enough bicarbonate for labelling and so this was removed from the eluent. When using QMA cartridges which had been reconditioned after initial use, significant variability was observed, so this was avoided for future synthesis.
Having successfully produced a radiolabelled molecule in the model system, the BpinKAM001 system was revisited, utilizing the revised catalyst system and new eluent, with successful product formation being achieved. HPLC purification of the radio peak from the reaction mixture was undertaken in the Flexlab module and confirmed to be the desired radiotracer by registration with the cold standard peak retention time, as shown in Fig. 4. Using these conditions radiolabelling of the remaining BpinKAM002 and BpinKAM003 compounds was undertaken successfully, with HPLC traces shown in Fig. 5 and Fig. 6
Decay corrected yields for the purified tracers were 10–12% (n = 5), 10–25% (n = 3) and 12–18% (n = 3) for 18F KAM001, 18F KAM002 and 18F KAM003 respectively.