One-pot synthesis of high molar activity 6-[18F]fluoro-l-DOPA by Cu-mediated fluorination of a BPin precursor.

A one-pot two-step synthesis of 6-[18F]fluoro-l-DOPA ([18F]FDOPA) has been developed involving Cu-mediated radiofluorination of a pinacol boronate ester precursor. The method is fully automated, provides [18F]FDOPA in good activity yield (104 ± 16 mCi, 6 ± 1%), excellent radiochemical purity (>99%) and high molar activity (3799 ± 2087 Ci mmol-1), n = 3, and has been validated to produce the radiotracer for human use.

[18F]BTK-1: A Novel Positron Emission Tomography Tracer for Imaging Bruton's Tyrosine Kinase.

PURPOSE: Bruton's tyrosine kinase (BTK) is a key component of B cell receptor (BCR) signaling, and as such a critical regulator of cell proliferation and survival. Aberrant BCR signaling is important in the pathogenesis of various B cell malignancies and autoimmune disorders. Here, we describe the development of a novel positron emission tomography (PET) tracer for imaging BTK expression and/or occupancy by small molecule therapeutics. METHODS: Radiochemistry was carried out by reacting the precursor with [18F]fluoride on a GE FX-FN TracerLab synthesis module to produce [18F]BTK-1 with a 6% decay-corrected radiochemical yield, 100 ± 6 GBq/µmol molar activity, and a radiochemical purity of 99%. Following intravenous administration of [18F]BTK-1 (3.63 ± 0.59 MBq, 0.084 ± 0.05 µg), 60-min dynamic images were acquired in two xenograft models: REC-1, an efficacious mantle cell lymphoma model, and U87MG, a non-efficacious glioblastoma model. Subsequent studies included vehicle, pretreatment (10 min prior to tracer injection), and displacement (30 min post-tracer injection) studies with different reversible BTK inhibitors to examine BTK binding. Human radiation dosimetry was estimated based on PET imaging in healthy rats. RESULTS: Uptake of [18F]BTK-1 was significantly higher in BTK expressing REC-1 tumors than non-BTK expressing U87MG tumors. Administration of BTK inhibitors prior to tracer administration blocked [18F]BTK-1 binding in the REC-1 tumor model consistent with [18F]BTK-1 binding to BTK. The predicted effective dose in humans was 0.0199 ± 0.0007 mSv/MBq. CONCLUSION: [18F]BTK-1 is a promising PET tracer for imaging of BTK, which could provide valuable information for patient selection, drug dose determination, and improving our understanding of BTK biology in humans.

Strategies for PET imaging of the receptor for advanced glycation endproducts (RAGE).

The implication of the receptor for advanced glycation end-products (RAGE) in numerous diseases and neurodegenerative disorders makes it interesting both as a therapeutic target and as an inflammatory biomarker. In the context of investigating RAGE as a biomarker, there is interest in developing radiotracers that will enable quantification of RAGE using positron emission tomography (PET) imaging. We have synthesized potential small molecule radiotracers for both the intracellular ([18F]InRAGER) and extracellular ([18F]RAGER) domains of RAGE. Herein we report preclinical evaluation of both using in vitro (lead panel screens) and in vivo (rodent and nonhuman primate PET imaging) methods. Both radiotracers have high affinity for RAGE and show good brain uptake, but suffer from off-target binding. The source of the off-target PET signal is not attributable to binding to melatonin receptors, but remains unexplained. We have also investigated use of lipopolysaccharide (LPS)-treated mice as a possible animal model with upregulated RAGE for evaluation of new imaging agents. Immunoreactivity of the mouse brain sections revealed increases in RAGE in the male cohorts, but no difference in the female groups. However, it proves challenging to quantify the changes in RAGE due to off-target binding of the radiotracers. Nevertheless, they are appropriate lead scaffolds for future development of 2nd generation RAGE PET radiotracers because of their high affinity for the receptor and good CNS penetration.

Synthesis of high-molar-activity [18F]6-fluoro-L-DOPA suitable for human use via Cu-mediated fluorination of a BPin precursor.

[18F]6-fluoro-L-DOPA ([18F]FDOPA) is a diagnostic radiopharmaceutical for positron emission tomography (PET) imaging that is used to image Parkinson's disease, brain tumors, and focal hyperinsulinism of infancy. Despite these important applications, [18F]FDOPA PET remains underutilized because of synthetic challenges associated with accessing the radiotracer for clinical use; these stem from the need to radiofluorinate a highly electron-rich catechol ring in the presence of an amino acid. To address this longstanding challenge in the PET radiochemistry community, we have developed a one-pot, two-step synthesis of high-molar-activity [18F]FDOPA by Cu-mediated fluorination of a pinacol boronate (BPin) precursor. The method is fully automated, has been validated to work well at two separate sites (an academic facility with a cyclotron on site and an industry lab purchasing [18F]fluoride from an outside vendor), and provides [18F]FDOPA in reasonable radiochemical yield (2.44 ± 0.70 GBq, 66 ± 19 mCi, 5 ± 1%), excellent radiochemical purity (>98%) and high molar activity (76 ± 30 TBq/mmol, 2,050 ± 804 Ci/mmol), n = 26. Herein we report a detailed protocol for the synthesis of [18F]FDOPA that has been successfully implemented at two sites and validated for production of the radiotracer for human use.

The Discovery of a Novel Phosphodiesterase (PDE) 4B-Preferring Radioligand for Positron Emission Tomography (PET) Imaging.

As part of our effort in identifying phosphodiesterase (PDE) 4B-preferring inhibitors for the treatment of central nervous system (CNS) disorders, we sought to identify a positron emission tomography (PET) ligand to enable target occupancy measurement in vivo. Through a systematic and cost-effective PET discovery process, involving expression level (Bmax) and biodistribution determination, a PET-specific structure-activity relationship (SAR) effort, and specific binding assessment using a LC-MS/MS "cold tracer" method, we have identified 8 (PF-06445974) as a promising PET lead. Compound 8 has exquisite potency at PDE4B, good selectivity over PDE4D, excellent brain permeability, and a high level of specific binding in the "cold tracer" study. In subsequent non-human primate (NHP) PET imaging studies, [18F]8 showed rapid brain uptake and high target specificity, indicating that [18F]8 is a promising PDE4B-preferring radioligand for clinical PET imaging.

Discovery and Characterization of (R)-6-Neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894), an Alkyne-Lacking Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator Profiled in both Rat and Nonhuman Primates.

We previously observed a cutaneous type IV immune response in nonhuman primates (NHP) with the mGlu5 negative allosteric modulator (NAM) 7. To determine if this adverse event was chemotype- or mechanism-based, we evaluated a distinct series of mGlu5 NAMs. Increasing the sp3 character of high-throughput screening hit 40 afforded a novel morpholinopyrimidone mGlu5 NAM series. Its prototype, (R)-6-neopentyl-2-(pyridin-2-ylmethoxy)-6,7-dihydropyrimido[2,1-c][1,4]oxazin-4(9H)-one (PF-06462894, 8), possessed favorable properties and a predicted low clinical dose (2 mg twice daily). Compound 8 did not show any evidence of immune activation in a mouse drug allergy model. Additionally, plasma samples from toxicology studies confirmed that 8 did not form any reactive metabolites. However, 8 caused the identical microscopic skin lesions in NHPs found with 7, albeit with lower severity. Holistically, this work supports the hypothesis that this unique toxicity may be mechanism-based although additional work is required to confirm this and determine clinical relevance.

Preclinical Evaluation of 18F-PF-05270430, a Novel PET Radioligand for the Phosphodiesterase 2A Enzyme.

UNLABELLED: The enzyme phosphodiesterase 2A (PF-05270430) is a potential target for development of novel therapeutic agents for the treatment of cognitive impairments. The goal of the present study was to evaluate the PDE2A ligand (18)F-PF-05270430, 4-(3-fluoroazetidin-1-yl)-7-methyl-5-(1-methyl-5-(4-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)imidazo[1,5-f][1,2,4]triazine, in nonhuman primates. METHODS: (18)F-PF-05270430 was radiolabeled by 2 methods via nucleophilic substitution of its tosylate precursor. Tissue metabolite analysis in rodents and PET imaging in nonhuman primates under baseline and blocking conditions were performed to determine the pharmacokinetic and binding characteristics of the new radioligand. Various kinetic modeling approaches were assessed to select the optimal method for analysis of imaging data. RESULTS: (18)F-PF-05270430 was synthesized in greater than 98% radiochemical purity and high specific activity. In the nonhuman primate brain, uptake of (18)F-PF-05270430 was fast, with peak concentration (SUVs of 1.5-1.8 in rhesus monkeys) achieved within 7 min after injection. The rank order of uptake was striatum > neocortical regions > cerebellum. Regional time-activity curves were well fitted by the 2-tissue-compartment model and the multilinear analysis-1 (MA1) method to arrive at reliable estimates of regional distribution volume (VT) and binding potential (BPND) with 120 min of scan data. Regional VT values (MA1) ranged from 1.28 mL/cm(3) in the cerebellum to 3.71 mL/cm(3) in the putamen, with a BPND of 0.25 in the temporal cortex and 1.92 in the putamen. Regional BPND values estimated by the simplified reference tissue model (SRTM) were similar to those from MA1. Test-retest variability in high-binding regions (striatum) was 4% ± 6% for MA1 VT, 13% ± 6% for MA1 BPND, and 13% ± 7% SRTM BPND, respectively. Pretreatment of animals with the PDE2A inhibitor PF-05180999 resulted in a dose-dependent reduction of (18)F-PF-05270430 specific binding, with a half maximal effective concentration of 69.4 ng/mL in plasma PF-05180999 concentration. CONCLUSION: (18)F-PF-05270430 displayed fast and reversible kinetics in nonhuman primates, as well as specific binding blockable by a PDE2A inhibitor. This is the first PET tracer with desirable imaging properties and demonstrated ability to image and quantify PDE2A in vivo.

Stoichiometric and catalytic deuterium and tritium labeling of "unactivated" organic substrates with cationic Ir(III) complexes.

[reaction: see text] Ir(III) complex [Cp(PMe(3))IrMe(CH(2)Cl(2))][BAr(f)] (1) was used to introduce deuterium stoichiometrically into substituted naphthalene/benzene templates and several "drug-like" entities. The exchange process is tolerant of a wide array of functional groups. Labeling of warfarin using subatmospheric pressures of T(2) led to specific activities and total activities rivaling current functional group directed tritium labeling methods. When paired with the appropriate deuterium donor, Cp(PMe(3))Ir(H(3))OTf (4) was found to deuterate a number of organic compounds catalytically.

Synthesis and Binding Affinities of Novel Re-Containing 7alpha-Substituted Estradiol Complexes: Models for Breast Cancer Imaging Agents.

The diagnosis and staging of breast cancer could be improved by the development of imaging radiopharmaceuticals that provide a noninvasive determination of the estrogen receptor status in the tumor cells. Toward this goal, we have synthesized a number of novel Re-containing 7alpha-substituted estradiol complexes. The introduction of the 7alpha side chain involves the alkylation of tetrahydropyranyloxy-protected 6-keto estradiol. The methods used to introduce the rhenium metal involve "3 + 1" and "4 + 1" mixed ligand complexes (2a-c and 5, respectively), tricarbonyl dithioether complexes (3), and the cyclopentadienyltricarbonylmetal organometallic system (4ab, 6, 7). These complexes showed binding affinities for the estrogen receptor (as high as 45% for the "3 + 1" complex 2c) when compared to the native ligand estradiol. The polarity of some complexes (4ab) was modified to improve biodistribution properties by introducing (poly)ether linkages into the 7alpha side chain (6, 7). These complexes provide a further refinement of our understanding of ligand structure-binding affinity correlations for the estrogen receptor, and they furnish the synthetic groundwork for the synthesis of the analogous Tc-99m complexes for evaluation as breast tumor imaging agents.

Discovery and preclinical characterization of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo-[3,4-b]pyrazine (PF470): a highly potent, selective, and efficacious metabotropic glutamate receptor 5 (mGluR5) negative allosteric modulator.

A novel series of pyrazolopyrazines is herein disclosed as mGluR5 negative allosteric modulators (NAMs). Starting from a high-throughput screen (HTS) hit (1), a systematic structure-activity relationship (SAR) study was conducted with a specific focus on balancing pharmacological potency with physicochemical and pharmacokinetic (PK) properties. This effort led to the discovery of 1-methyl-3-(4-methylpyridin-3-yl)-6-(pyridin-2-ylmethoxy)-1H-pyrazolo[3,4-b]pyrazine (PF470, 14) as a highly potent, selective, and orally bioavailable mGluR5 NAM. Compound 14 demonstrated robust efficacy in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-rendered Parkinsonian nonhuman primate model of l-DOPA-induced dyskinesia (PD-LID). However, the progression of 14 to the clinic was terminated because of a potentially mechanism-mediated finding consistent with a delayed-type immune-mediated type IV hypersensitivity in a 90-day NHP regulatory toxicology study.

Design and selection parameters to accelerate the discovery of novel central nervous system positron emission tomography (PET) ligands and their application in the development of a novel phosphodiesterase 2A PET ligand.

To accelerate the discovery of novel small molecule central nervous system (CNS) positron emission tomography (PET) ligands, we aimed to define a property space that would facilitate ligand design and prioritization, thereby providing a higher probability of success for novel PET ligand development. Toward this end, we built a database consisting of 62 PET ligands that have successfully reached the clinic and 15 radioligands that failed in late-stage development as negative controls. A systematic analysis of these ligands identified a set of preferred parameters for physicochemical properties, brain permeability, and nonspecific binding (NSB). These preferred parameters have subsequently been applied to several programs and have led to the successful development of novel PET ligands with reduced resources and timelines. This strategy is illustrated here by the discovery of the novel phosphodiesterase 2A (PDE2A) PET ligand 4-(3-[(18)F]fluoroazetidin-1-yl)-7-methyl-5-{1-methyl-5-[4-(trifluoromethyl)phenyl]-1H-pyrazol-4-yl}imidazo[5,1-f][1,2,4]triazine, [(18)F]PF-05270430 (5).

The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH).

INTRODUCTION: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo. We report here the synthesis and in vivo evaluation of [(18)F]PF-9811, a novel PET ligand for non-invasive imaging of FAAH in the brain. METHODS: The potency and selectivity of unlabeled PF-9811 were determined by activity-based protein profiling (ABPP) both in vitro and in vivo. [(18)F]PF-9811 was synthesized in a 3-step, one-pot reaction sequence, followed by HPLC purification. Biological evaluation was performed by biodistribution and dynamic PET imaging studies in male rats. The specificity of [(18)F]PF-9811 uptake was evaluated by pre-administration of PF-04457845, a potent and selective FAAH inhibitor, 1h prior to radiotracer injection. RESULTS: Biodistribution studies show good uptake (SUV~0.8 at 90 min) of [(18)F]PF-9811 in rat brain, with significant reduction of the radiotracer in all brain regions (37%-73% at 90 min) in blocking experiments. Dynamic PET imaging experiments in rat confirmed the heterogeneous uptake of [(18)F]PF-9811 in brain regions with high FAAH enzymatic activity, as well as statistically significant reductions in signal following pre-administration of the blocking compound PF-04457845. CONCLUSIONS: [(18)F]PF-9811 is a promising PET imaging agent for FAAH. Biodistribution and PET imaging experiments show that the tracer has good uptake in brain, regional heterogeneity, and specific binding as determined by blocking experiments with the highly potent and selective FAAH inhibitor, PF-04457845.

Pancreatic beta cell mass PET imaging and quantification with [11C]DTBZ and [18F]FP-(+)-DTBZ in rodent models of diabetes.

PURPOSE: The aim of this study is to compare the utility of two positron emission tomography (PET) imaging ligands ((+)-[(11)C]dihydrotetrabenazine ([(11)C]DTBZ) and the fluoropropyl analog ([(18)F]FP-(+)-DTBZ)) that target islet ß-cell vesicular monoamine transporter type II to measure pancreatic ß-cell mass (BCM). PROCEDURES: [(11)C]DTBZ or [(18)F]FP-(+)-DTBZ was injected, and serial PET images were acquired in rat models of diabetes (streptozotocin-treated and Zucker diabetic fatty) and ß-cell compensation (Zucker fatty). Radiotracer standardized uptake values (SUV) were correlated to pancreas insulin content measured biochemically and histomorphometrically. RESULTS: On a group level, a positive correlation of [(11)C]DTBZ pancreatic SUV with pancreas insulin content and BCM was observed. In the STZ diabetic model, both [(18)F]FP-(+)-DTBZ and [(11)C]DTBZ correlated positively with BCM, although only ∼25% of uptake could be attributed to ß-cell uptake. [(18)F]FP-(+)-DTBZ displacement studies indicate that there is a substantial fraction of specific binding that is not to pancreatic islet ß cells. CONCLUSIONS: PET imaging with [(18)F]FP-(+)-DTBZ provides a noninvasive means to quantify insulin-positive BCM and may prove valuable as a diagnostic tool in assessing treatments to maintain or restore BCM.

Stoichiometric and catalytic H/D incorporation by cationic iridium complexes: a common monohydrido-iridium intermediate.

A mechanistic study of the stoichiometric and catalytic H/D exchange reactions involving cationic iridium complexes is presented. Strong evidence suggests that both stoichiometric and catalytic reactions proceed via a monohydrido-iridium species. Stoichiometric deuterium incorporation reactions introduce multiple deuterium atoms into the organic products when aryliridium compounds CpPMe(3)Ir(C(6)H(4)X)(OTf) (X = H, o-CH(3), m-CH(3), p-CH(3)) react with D(2). Multiple deuteration occurs at the unhindered positions (para and meta) of toluene, when X = CH(3). The multiple-deuteration pathway is suppressed in the presence of an excess of the coordinating ligand, CH(3)CN. The compound CpPMe(3)IrH(OTf) (1-OTf) is observed in low-temperature, stoichiometric experiments to support a monohydrido-iridium intermediate that is responsible for catalyzing multiple deuteration in the stoichiometric system. When paired with acetone-d(6)(), [CpPMe(3)IrH(3)][OTf] (4) catalytically deuterates a wide range of substrates with a variety of functional groups. Catalyst 4 decomposes to [CpPMe(3)Ir(eta(3)-CH(2)C(OH)CH(2))][OTf] (19) in acetone and to [CpPMe(3)IrH(CO)][OTf] (1-CO) in CH(3)OH. The catalytic H/D exchange reaction is not catalyzed by simple H(+) transfer, but instead proceeds by a reversible C-H bond activation mechanism.

(R)-N-[11C]methyl-3-pyrrolidyl benzilate, a high-affinity reversible radioligand for PET studies of the muscarinic acetylcholine receptor.

We recently reported the synthesis and binding affinity of ligands for the muscarinic acetylcholine receptor (mAChR) based on both the pyrrolidyl and piperidyl benzilate scaffold. One of these, (R)-3-pyrrolidyl benzilate, was successfully radiolabeled with [(11)C]methyl triflate and the resulting compound, (R)-N-[(11)C]methyl-3-pyrrolidyl benzilate (3-[(11)C]NMPYB), was evaluated as a reversible, acetylcholine-sensitive tracer for the mAChR (K(i) of unlabeled 3-NMPYB is 0.72 nM). This compound displayed high, receptor-mediated retention in regions of the mouse and rat brain known to have high concentrations of mAChRs. Moreover, bolus studies in a pigtail monkey showed that this compound had superior clearance from the brain when compared to muscarinic radiotracers previously employed in human PET studies. Infusion studies in the same monkey revealed that it was possible to achieve equilibrium of radiotracer distribution for 3-[(11)C]NMPYB in both the striatum and cortex. Sensitivity to endogenous acetylcholine levels was evaluated by injecting phenserine (5 mg/kg) into rats prior to administration of 3-[(11)C]NMPYB in an equilibrium infusion protocol. This pretreatment produced a modest, statistically significant decrease (9-11%) in the distribution volume ratios for muscarinic receptor rich regions of the rat brain as compared to controls.