7 April 2024
Neuropeptide Y (NPY) is one of the most abundant peptides in the central nervous system of mammals and is involved in several physiological processes through NPY Y1, Y2, Y4 and Y5 receptors. Of those, the Y2 receptor has particular relevance for its autoreceptor role in inhibiting the release of NPY and other neurotransmitters and for its involvement in relevant mechanisms such as feeding behaviour, cognitive processes, emotion regulation, circadian rhythms and disorders such as epilepsy and cancer. PET imaging of the Y2 receptor can provide a valuable platform to understand this receptor’s functional role and evaluate its potential as a therapeutic target. In this work, we set out to refine the chemical and radiochemical synthesis of the Y2 receptor antagonist N-[11 C]Me-JNJ31020028 for in vivo PET imaging studies. The non-radioactive reference compound, N-Me-JNJ-31020028, was synthesised through batch synthesis and continuous flow methodology, with 43% and 92% yields, respectively. N-[11 C]Me-JNJ-31020028 was obtained with a radiochemical purity > 99%, RCY of 31% and molar activity of 156 GBq/μmol. PET imaging clearly showed the tracer’s biodistribution in several areas of the mouse brain and gut where Y2 receptors are known to be expressed.
Neuropeptide Y (NPY) is a conserved 36-amino acid peptide belonging to a family of regulatory peptides, including peptide YY (PYY) and pancreatic peptide (PP). It stands as one of the most prevalent peptides within the central nervous system of mammals. NPY is involved in processes of energy homeostasis, water consumption, circadian rhythms, sleep, learning, memory, emotional regulation and angiogenesis. NPY’s physiological processes are mediated through the activation of different subtypes of NPY receptors (Y1, Y2, Y4 and Y5) that are expressed in both central and peripheral nervous systems and belong to the G-protein coupled receptors (GPCR) superfamily. Notably, the Y2 receptor has gained some interest over the years. It has been linked to the physiological processes of feeding behaviour, anxiety, neuronal excitability, angiogenesis, circadian rhythm, alcohol dependence, cognitive processes and locomotor activity. Additionally, the Y2 receptor is mainly pre-synaptic and has an autoreceptor role, as it inhibits the release of NPY and other neurotransmitters. Despite the extensive study and characterisation of the Y2 receptor, its neuromolecular implication in several diseases is not yet fully understood. Developing a suitable PET imaging biomarker could provide a platform to study the underlying mechanisms of these and other diseases and identify possible drug targets. Winterdahl et al. reported the development of an N-[11 C]Me-JNJ-31020028 PET tracer for the Y2 receptor and brain imaging in living pigs. The favourable attributes of the Y2 receptor antagonist JNJ-31020028 were leveraged, such as its high affinity and selectivity towards the Y2 receptors, and the radiolabelling was performed using [11 C]CH3 I. The radiotracer permeated the blood–brain barrier, and its binding was in agreement with the anatomical distribution of the Y2 receptors described in reference. It also showed rapid distribution throughout the brain and slow metabolization in the bloodstream. However, these studies were mainly focused on the binding affinity of N-[11 C]Me-JNJ-31020028 and its interaction with the Y2 receptor and were not continued. Despite the importance of these studies, we aimed to deepen the knowledge of the Y2 receptor’s biodistribution as it can provide insights into its relevance in health and disease.
The development of PET tracers involves the optimisation of several key steps, such as the radiolabelling reaction, purification and reformulation conditions and the quality control of the final product. A non-radioactive reference compound is needed for the labelled product’s radiochemical identification and HPLC control analysis. Moreover, this standard compound is required to calculate the labelled product’s molar activity, which is relevant for potential receptor occupancy and saturation and is defined as the measured radioactivity per mole of compound. When this standard compound is not available commercially, its synthesis is required. Thus, one can obtain the desired non-radioactive reference compound by conventional batch synthesis or use a continuous flow methodology to produce the compound on a larger scale. Continuous flow or microfluidic technology is based on conducting chemical reactions in a continuous stream within narrow channels that allow unique control over the reaction conditions. This technology allows the use of lower reaction volumes, higher pressures and better temperature control, leading to reduced reaction times and better product selectivity/yield with the benefit of being easy to automate. Not only is this technology highly used in organic synthesis but also in PET radiochemical preparations. The characteristics of this methodology present an improvement in PET chemistry in several key steps that often are problematic when conducting radiosynthesis, such as the need for quick reactions due to short-lived radionuclides, namely 18 F (110 min half-life) and 11 C (20 min half-life), and reduced consumption of necessary and expensive reagents. Consequently, continuous flow technology has been implemented to synthesise different radiopharmaceuticals. For instance, in carbon-11 radiochemistry, microfluidics has been used in 11 C-methylations and 11 C-carbonylations reactions with some promising results and radiopharmaceuticals like 11 C-DASB, 11 C-raclopride and 11 C-flumazenil have been prepared through this technology, highlighting the potential of the use of microfluidics in PET radiotracer synthesis.
Hereby, we present the chemical and radiochemical developments of the Y2 receptor antagonist N-Me-JNJ31020028. First, N-Me-JNJ31020028 was synthesised, isolated, characterised and used as the analytic standard for the following studies. Then, the development of a continuous flow synthetic methodology for the preparation of the non-radioactive compound was performed, which enabled synthesising the reference compound on a larger scale and represented a valuable preliminary study to implement this type of technology in radiotracer production. Subsequently, we described the development and implementation of a new process of radiosynthesis of the PET tracer N-[11 C]Me-JNJ31020028 and the first PET images and biodistribution studies, to our knowledge, in healthy C57BL/6N mice representing a solid baseline of expertise for future research within mouse models.
Aiming to prepare pure N-Me-JNJ31020028 2 as an analytic standard, the studies began with the batch N-methylation of precursor JNJ-31020028 1. The synthesis was performed via JNJ-31020028 1 reaction with 1.5 equiv. of iodomethane and 1.5 equiv. of sodium hydride (60% mineral oil dispersion) in DMF at room temperature for 1 h and 45 min. The needed product was isolated by flash chromatography using a gradient of dichloromethane and MeOH/NEt3 mixture as eluent. N-Me-JNJ-31020028 2 was obtained with a 42% isolated yield. The purity of the N-methylated compound 2 was analysed through HPLC, where it was observed to have chemical purity > 99%. It was also fully characterised by 1D/2D NMR spectroscopies and HRMS; the data are available in the Supplementary Materials.
Continuous flow methylation of precursor 1 was performed using the same synthetic strategy used for the batch preparation but taking two approaches: one using a tubular reactor, ideal for scaling-up of reactions, and the other using a chip reactor, more suitable for transposition of the process to the radiolabelling facility. The general apparatus configuration consisted of an E-Series Vapourtec’s flow chemistry apparatus equipped with an inert gas kit, V3 pumps, a continuous flow reactor (either a 10 mL tubular or a 1.5 mL chip reactor) and a collection valve. Two peristaltic pumps were used to pump a 0.025 M solution of (1) with 1.5 equiv. of sodium hydride (flask A) and an iodomethane 0.038 or 0.075 M solution (flask B), as depicted in Figure 2.
Each experiment was performed by pumping the solution in flasks A and B at a constant flow rate through the flow chemistry reactor (thermostated at 25 or 40 °C). When the steady state was reached, the reaction crude flow stream was collected, and the conversion and yield of 2 were determined by HPLC analysis. The reaction parameters regarding the equivalents of CH3 I, temperature and residence time were optimised and the results are presented in Table 1 and Table 2.
The studies started by using a tubular flow reactor (10 mL). Firstly, we tested different residence times of CH3 I (1.5 equiv., 0.038 M), such as 25, 10, 5 and 3 min, at 25 °C. The best result was achieved when the residence time was set to 5 min with complete conversion of precursor 1 and 90% yield of compound 2. Increasing the residence time to 10 min did not improve the yield, and at 25 min, the conversion and yield decreased to 82% and 64%, respectively. This result may be attributed to the degradation of 2 at higher residence times. Complete conversion was obtained with 3 min of residence time, but a decrease in yield to 88% was observed. Then, with 5 min of residence time, the temperature was increased to 40 °C, and a slight yield improvement (92%) was obtained. Next, we evaluated the increase in CH3 I from 1.5 (0.038 M) to 3 equiv. (0.075 M) at either 25 or 40 °C. In this case, an increase in side product formation was observed through the decree of product yield of 81 and 77%, respectively. To summarise, 5 min of residence time, 25 °C and 1.5 equiv. of CH3 I were shown to be the best reaction conditions to perform the methylation reaction in which it was possible to obtain N-Me-JNJ-31020028 2 with a productivity of 1.4 mmol/h.
Aiming to transpose the continuous flow synthesis conditions of 2 to the radiosynthesis protocols that use small-scale reactors and loops, we also evaluated the use of a chip flow reactor using the standard apparatus configuration equipped with a 1.5 mL chip reactor. Using the previous best reaction conditions (1.5 equiv. of CH3 I, at 25 °C and 5 min of residence time), compound 2 was obtained with 66% conversion and 60% yield. A significant improvement was achieved by increasing the temperature to 40 °C, and 2 was obtained with 94% conversion and 88% yield. The best results were achieved when using 3.0 equiv. of CH3 I at 40 °C, as the N-methylated product 2 was obtained with 95% conversion and 89% yield. Furthermore, it should be noted that in PET chemistry, the methylating agent is in the gas phase instead of iodomethane
