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 1 to yield cold standards in a good yield.
Synthesis of precursors
Coupling of indole-3-carbaldehyde and benzyl halides
A general sodium hydride deprotonation of the indole followed by addition of a benzyl halide of the appropriate substituent and substitution pattern was used for generation of cold standards as well as precursors and iodinated intermediates, as shown in Scheme 1. Yields for these compounds were typically above 70%, with the ortho substituted boronic acid pinacol ester derivative yielding lower at 58%.
A standard Miyaura borylation was utilized for generation of the parasubstituted boronic acid pinacol ester precursor from an iodine substituted intermediate to afford the product in a 72% yield.
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. To determine optimal conditions, initial reactions were carried out with the para substituted material, as shown in Scheme 2.
Synthesis of 1-(4-iodo-benzyl)-1H-indole-3-carbaldehyde (Compound 1) was undertaken as described in Scheme 1 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(16), shown in Scheme 3. The resultant reaction mixture showed no indication of product formation. The characteristic proton signal correlating to the aldehyde proton at approximately 10ppm 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(17) and peroxide(18) could also be employed as oxidants and were trialled with similar results. 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.
Attempts to carry out iodonium salt forming reactions in a two-step process shown in Scheme 4 also yielded no identifiable compounds. Being unable to isolate a satisfactory salt from these conditions, a two-step reaction(20) produced a major product from the initial oxidation step and was isolated via column chromatography and crystallized using vapour diffusion methods for x-ray crystallography, with the structure shown in Figure 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, shown in Scheme 5. As previous coupling reactions had been undertaken successfully, commercially available sources of para, meta or ortho substituted boronic acid pinacol ester containing benzyl bromides were used to investigate a one-step coupling synthetic route. This method utilizing the standard benzyl halide coupling reaction conditions produced the desired product in yields 78%, 76% and 58% yields of the para, meta, and ortho boronic acid precursors respectively. Product identity was confirmed through spectroscopy as well as x-ray crystal structures.
With three precursors in hand, radiolabelling experiments were undertaken.
Reaction conditions adapted from Tredwell et al.(15) were utilized for initial radiolabelling experiments with the para precursor, as shown in Scheme 6. Under these conditions, no radiolabelled products were isolated, with the majority of the starting material being unreacted.
As initial radiolabelling attempts yielded no discernible radiolabel products and previous literature suggested that these reactions may not be suitable for automated synthesis(15), 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(21), 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 et al. (10), which had resulted in excellent yields, however under the previously stated conditions, no radiolabelling was observed.
As the system described by Tredwell was not able to produce radiolabelled products in our hands, another similar system, described by Mossine et al. 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] kryptofix.
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 QMA cartridge facilitated fluoride isolation, with evaporation of the 18O water being performed in the absence of additives such as kryptofix prior to the labelling reaction. This system yielded small amounts of previously unobserved 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. A summary of conditions trialled with the model compound is shown in Table 3
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, with conditions shown in Scheme 8. HPLC purification of the radio peak from the reaction mixture was undertaken in the Flexlab module, with a representative trace shown in the supplementary information, and confirmed to be the desired radiotracer by registration with the cold standard peak retention time, as shown in Figure 4. Using these conditions radiolabelling of the remaining BpinKAM002 and BpinKAM003 compounds was undertaken successfully, with HPLC traces shown in Figure 5 and Figure 6.
Decay corrected yields for the purified tracers were 10.76% ± 0.96% (n=5), 14.7% ±8.58% (n=3) and 14.92% ±3.9% (n=3) for 18F KAM001, 18F KAM002 and 18F KAM003 respectively. All tracers were shown to have a radiochemical purity greater than 99%, with a representative run of the 18F KAM002 producing a molar activity of 1.09GBq/μmol.