An in-loop 11 C-carbonylation process for the radiosynthesis of 11 C-carboxylic acids and esters from halide precursors has been developed. The reaction proceeds at room temperature under mild conditions and enables 11 C-carbonylation of both electron deficient and electron rich (hetero)aromatic halides to provide 11 C-carboxylic acids and esters in good to excellent radiochemical yields, high radiochemical purity, and excellent molar activity. The process has been fully automated using commercial radiochemistry synthesis modules, and application to clinical production is demonstrated via validated cGMP radiosyntheses of [11 C]bexarotene and [11 C]acetoacetic acid.
This report describes a comparison of four different routes for the clinical-scale radiosynthesis of the κ-opioid receptor antagonist [11C]LY2795050. Palladium-mediated radiocyanation and radiocarbonylation of an aryl iodide precursor as well as copper-mediated radiocyanation of an aryl iodide and an aryl boronate ester have been investigated. Full automation of all four methods is reported, each of which provides [11C]LY2795050 in sufficient radiochemical yield, molar activity, and radiochemical purity for clinical use. The advantages and disadvantages of each radiosynthesis method are compared and contrasted.
Mitochondrial complex I (MC-I) is an essential component of brain bioenergetics and can be quantified and studied using positron emission tomography (PET). A specific high affinity 18F radiotracer for MC-I enables monitoring of neurodegenerative disease progression and pathology via PET imaging. To facilitate clinical research studies tracking MC-I activity in Parkinson's disease and other neurodegenerative diseases, a fully automated synthesis of the recently described 2-tert-butyl-4-chloro-5-{6-[2-(2[18F]fluoroethoxy)-ethoxy]-pyridin-3-ylmethoxy}-2H-pyridazin-3-one ([18F] BCPP-EF, [ 18 F]1) was developed. We report the first automated synthesis [18F]BCPP-EF using a green radiochemistry approach. The radiotracer was synthesized with good radiochemical yield, excellent radiochemical purity, and high molar activity.
This report describes a copper-mediated radiocyanation of aryl halides that is applicable to complex molecules. This transformation tolerates an exceptionally wide range of functional groups, including unprotected amino acids. As such, it enables the site-specific introduction of [11C]CN into peptides at an iodophenylalanine residue. The use of a diamine-ligated copper(I) mediator is crucial for achieving high radiochemical yield under relatively mild conditions, thus limiting racemization and competing side reactions of other amino acid side chains. The reaction has been scaled and automated to deliver radiolabeled peptides, including analogues of adrenocorticotropic hormone 1-27 (ACTH) and nociceptin (NOP). For instance, this Cu-mediated radiocyanation was leveraged to prepare >40 mCi of [11C]cyano-NOP to evaluate biodistribution in a primate using positron emission tomography. This investigation provides preliminary evidence that nociceptin crosses the blood-brain barrier and shows uptake across all brain regions (SUV > 1 at 60 min post injection), consistent with the known distribution of NOP receptors in the rhesus brain.
Amino Acids , Copper , Amines , Animals , Positron-Emission Tomography/methods , Radiopharmaceuticals , Tissue Distribution
Melatonin is a neurohormone that modulates several physiological functions in mammals through the activation of melatonin receptor type 1 and 2 (MT1 and MT2). The melatonergic system is an emerging therapeutic target for new pharmacological interventions in the treatment of sleep and mood disorders; thus, imaging tools to further investigate its role in the brain are highly sought-after. We aimed to develop selective radiotracers for in vivo imaging of both MT1 and MT2 by positron emission tomography (PET). We identified four previously reported MT ligands with picomolar affinities to the target based on different scaffolds which were also amenable for radiolabeling with either carbon-11 or fluorine-18. [11C]UCM765, [11C]UCM1014, [18F]3-fluoroagomelatine ([18F]3FAGM), and [18F]fluoroacetamidoagomelatine ([18F]FAAGM) have been synthesized in high radiochemical purity and evaluated in wild-type rats. All four tracers showed moderate to high brain permeability in rats with maximum standardized uptake values (SUVmax of 2.53, 1.75, 3.25, and 4.47, respectively) achieved 1-2 min after tracer administration, followed by a rapid washout from the brain. Several melatonin ligands failed to block the binding of any of the PET tracer candidates, while in some cases, homologous blocking surprisingly resulted in increased brain retention. Two 18F-labeled agomelatine derivatives were brought forward to PET scans in non-human primates and autoradiography on human brain tissues. No specific binding has been detected in blocking studies. To further investigate pharmacokinetic properties of the putative tracers, microsomal stability, plasma protein binding, log D, and membrane bidirectional permeability assays have been conducted. Based on the results, we conclude that the fast first pass metabolism by the enzymes in liver microsomes is the likely reason of the failure of our PET tracer candidates. Nevertheless, we showed that PET imaging can serve as a valuable tool to investigate the brain permeability of new therapeutic compounds targeting the melatonergic system.
Melatonin , Animals , Brain/diagnostic imaging , Brain/metabolism , Fluorine Radioisotopes/metabolism , Ligands , Mammals/metabolism , Melatonin/metabolism , Positron-Emission Tomography/methods , Radiopharmaceuticals , Rats , Receptors, Melatonin/metabolism
Mutations in the huntingtin gene (HTT) triggers aggregation of huntingtin protein (mHTT), which is the hallmark pathology of neurodegenerative Huntington's disease (HD). Development of a high affinity 18F radiotracer would enable the study of Huntington's disease pathology using a non-invasive imaging modality, positron emission tomography (PET) imaging. Herein, we report the first synthesis of fluorine-18 imaging agent, 6-(5-((5-(2,2-difluoro-2-(fluoro-18F)ethoxy)pyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one ([18F]1), a radioligand for HD and its preclinical evaluation in vitro (autoradiography of post-mortem HD brains) and in vivo (rodent and non-human primate brain PET). [18F]1 was synthesized in a 4.1% RCY (decay corrected) and in an average molar activity of 16.5 ± 12.5 GBq/µmol (445 ± 339 Ci/mmol). [18F]1 penetrated the blood-brain barrier of both rodents and primates, and specific saturable binding in post-mortem brain slices was observed that correlated to mHTT aggregates identified by immunohistochemistry.
A new method for the synthesis of the highly selective delta opioid receptor (DOR) antagonist radiotracer N1 '-([11 C]methyl)naltrindole ([11 C]MeNTI) is described. The original synthesis required hydrogenation of a benzyl protecting group after 11 C-labeling, which is challenging in modern radiochemistry laboratories that tend to be heavily automated and operate according to current good manufacturing practice. To address this challenge, we describe development of a novel MeNTI precursor bearing a methoxymethyl acetal (MOM) protecting group, which is easily removed with HCl, and employ it in an updated synthesis of [11 C]MeNTI. The new synthesis is fully automated and validated for clinical use. The total synthesis time is 45 min and provides [11 C]MeNTI in good activity yield (49 ± 8 mCi), molar activity (3,926 ± 326 Ci/mmol) and radiochemical purity (97% ± 2%).
Indoles/chemistry , Morphinans/chemistry , Positron-Emission Tomography/methods , Radiopharmaceuticals/chemical synthesis , Receptors, Opioid, delta/metabolism , Carbon Radioisotopes/chemistry
The radiotracers [11C]COU and [11C]PHXY are potential PET imaging agents for in vivo studies of monoamine oxidases (MAOs), as previously shown in rodent and primate brain. One-pot, automated methods for the radiosynthesis of [11C]PHXY and [11C]COU were developed to provide reliable and improved radiochemical yields. Although derived from the structure of the neurotoxin MPTP, COU did not exhibit in vivo neurotoxicity to dopaminergic nerve terminals in the mouse brain as assayed by losses of VMAT2 radioligand binding. PET imaging studies in rats demonstrated that both [11C]COU and [11C]PHXY exhibit retention in cardiac tissues that can be blocked by pretreatment with the MAO inhibitors deprenyl (MAO-B) and pargyline (MAO-A and -B). In addition to prior neuroimaging applications, [11C]COU and [11C]PHXY are thus also of interest for studies of MAO enzymatic activity and imaging of sympathetic nerve density in heart.
Imaging of the opioid system was one of the earliest applications of Positron Emission Tomography (PET) imaging in neuroscience that remains in widespread use today and in the age of the opioid crisis the technique is as important as ever. In this viewpoint the rich history of opioid imaging using PET is highlighted, including discussion of the preferred radiotracers for imaging of µ, δ, κ and ORL-1 receptors in clinical applications. We also draw attention to key innovations that were essential to development of radiotracers for imaging opioid receptors including production of high molar activity PET radionuclides and new approaches to radiochemistry.
Positron-Emission Tomography , Receptors, Opioid , Radiochemistry , Radioisotopes , Receptors, Opioid, mu
PURPOSE: Copper-mediated radiofluorination (CMRF) is emerging as the method of choice for the formation of aromatic C-18F bonds. This minireview examines proof-of-concept, pre-clinical, and in-human imaging studies of new and established imaging agents containing aromatic C-18F bonds synthesized with CMRF. An exhaustive discussion of CMRF methods is not provided, although key developments that have enabled or improved upon the syntheses of fluorine-18 imaging agents are discussed. METHODS: A comprehensive literature search from April 2014 onwards of the Web of Science and PubMed library databases was performed to find reports that utilize CMRF for the synthesis of fluorine-18 radiopharmaceuticals, and these represent the primary body of research discussed in this minireview. Select conference proceedings, previous reports describing alternative methods for the synthesis of imaging agents, and preceding fluorine-19 methodologies have also been included for discussion. CONCLUSIONS: CMRF has significantly expanded the chemical space that is accessible to fluorine-18 radiolabeling with production methods that can meet the regulatory requirements for use in Nuclear Medicine. Furthermore, it has enabled novel and improved syntheses of radiopharmaceuticals and facilitated subsequent PET imaging studies. The rapid adoption of CMRF will undoubtedly continue to simplify the production of imaging agents and inspire the development of new radiofluorination methodologies.
In a high-throughput screening campaign, we recently discovered the rRNA-binding tetracyclines, methacycline and meclocycline, as inhibitors of Dicer-mediated processing of microRNAs. Herein, we describe our biophysical and biochemical characterization of these compounds. Interestingly, although direct, albeit weak, binding to the pre-microRNA hairpins was observed, the inhibitory activity of these compounds was not due to RNA binding. Through additional biochemical and chemical studies, we revealed that metal chelation likely plays a principle role in their mechanism of inhibition. By exploring the activity of other known RNA-binding scaffolds, we identified additional disconnections between direct RNA interaction and inhibition of Dicer processing. Thus, the results presented within provide a valuable case study in the complexities of targeting RNA with small molecules, particularly with weak binding and potentially promiscuous scaffolds.
Eukaryotic translation initiation factor 4E (eIF4E) is a key player in the initiation of cap-dependent translation through recognition of the m7GpppX cap at the 5' terminus of coding mRNAs. As eIF4E overexpression has been observed in a number of human diseases, most notably cancer, targeting this oncogenic translation initiation factor has emerged as a promising strategy for the development of novel anti-cancer therapeutics. Toward this end, in the present study, we have rationally designed a series of Bn7GxP-based PROTACs for the targeted degradation of eIF4E. Herein we describe our synthetic efforts, in addition to biochemical and cellular characterization of these compounds.
Drug Design , Eukaryotic Initiation Factor-4E/metabolism , Guanosine/analogs & derivatives , Proteolysis/drug effects , Cell Line, Tumor , Chemistry Techniques, Synthetic , Eukaryotic Initiation Factor-4E/chemistry , Guanosine/chemical synthesis , Guanosine/chemistry , Guanosine/pharmacology , Humans , Models, Molecular , Protein Conformation
Proteins containing intrinsic disorder often form secondary structure upon interaction with a binding partner. Modulating such structures presents an approach for manipulating the resultant functional outcomes. Translational repressor protein 4E-BP1 is an example of an intrinsically disordered protein that forms an α-helix upon binding to its protein ligand, eIF4E. Current biophysical methods for analyzing binding-induced structural changes are low-throughput, require large amounts of sample, or are extremely sensitive to signal interference by the ligand itself. Herein, we describe the discovery and development of a conditionally fluorescent 4E-BP1 peptide that reports structural changes of its helix in high-throughput format. This reporter peptide is based on conditional quenching of fluorescein by thioamides. In this case, fluorescence signal increases as the peptide becomes more ordered. Conversely, destabilization of the α-helix results in decreased fluorescence signal. The low concentration and low volume of peptide required make this approach amenable for high-throughput screening to discover ligands that alter peptide secondary structure.
Carrier Proteins/metabolism , Fluorescent Dyes/chemistry , Peptides/metabolism , Thioamides/chemistry , Amino Acid Sequence , Carrier Proteins/chemical synthesis , Carrier Proteins/chemistry , Eukaryotic Initiation Factor-4E/metabolism , Fluorescein-5-isothiocyanate/chemistry , Humans , Peptides/chemical synthesis , Peptides/chemistry , Protein Conformation, alpha-Helical , Protein Folding
Dysregulation of microRNA (miRNA) expression has been linked to many human diseases; however, because of the challenges associated with RNA-targeted drug discovery, additional approaches are needed for probing miRNA biology. The emerging regulatory role of miRNA-binding proteins in miRNA maturation presents such an alternative strategy. Exploiting our laboratory's click chemistry-based high-throughput screening (HTS) technology, catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a modular method by which to discover new chemical tools for manipulating pre-miRNA-miRNA-binding protein interactions. Using the pre-let-7d-Lin28 interaction as proof-of-concept, the results presented demonstrate how HTS using cat-ELCCA can enable the discovery of small molecules targeting RNA-protein interactions.
In the present report, the role of computationally estimated efficiency indices and pose clustering has been demonstrated in effective decision making, resource management and chemical prioritization. As an example, 720 annulated furanones from six different scaffold classes were computationally docked against Pf-DHFR active site using AutoDock 4.2. Many trends were established by navigating efficiency indices (BEI and SEI) in 2D planes. These trends were then explained by comparing interaction profiles of docked poses with that of known actives/inhibitors. Cases where trends emerged from efficiency plots resonated well with the pattern of a particular cluster diagram were considered as guidelines for optimization purpose. These kind of guidelines can help medicinal chemists in prioritization their work and in effective management of time, energy and chemical resources.
A facile route for the assembly of new bis-heterocyclic imidazo[2,1-b][1,3]thiazinyl/benzothiazoyl-phenyl)benzamide scaffolds through a two-step Groebke-Blackburn-Bienaymé (GBB)/Ugi reaction sequence is reported, which establishes multiple points of diversity in the final products. The highlights of this procedure are the survival of the aldehyde group following the GBB reaction without the need for additional protection/deprotection steps. Moreover, the reaction is operationally simple, with the absence of any catalyst, and exhibits excellent functional-group tolerance under minutes of microwave irradiation.
The advent of cycloaddition reactions in the synthesis of heterocycles and their ever burgeoning applications in the fields of material chemistry, catalysis and drugs have been a profound scientific development. In particular, isocyanide based cycloaddition reactions have been harbingers of an exciting new chapter in the realms of organic synthesis. The emergence of numerous synthetic protocols utilizing formal cycloaddition of isocyanides with conjugated heterodienes has unleashed countless opportunities to design and synthesize diverse heterocyclic scaffolds. To date, there has not been any exclusive review on a formal [4+1] cycloaddition involving isocyanides. The present review highlights the journey of formal [4+1] cycloaddition reactions of isocyanides with diverse electrophilic substrates viz. oxadienes, azadienes, thioacyl imines, alkylidene amides, alkylidene hydrazines, α,ß-unsaturated nitro compounds, α-thioxothioamides, nitroso alkenes, acyl imines, vinyl ketenes, vinyl isocyanates, etc. to afford functionalized pyrroles, imidazoles, furans, oxazoles, pyrazoles, etc.
The needles of Taxus wallichiana gave a taxoid 1-hydroxy- 2-deacetoxy-5-decinnamoyl-taxinine j, whose structure has been established by spectroscopic data and confirmed by X-ray crystallography. The taxoid possesses significant cytotoxic and immunomodulatory activity.
Antineoplastic Agents/pharmacology , Immunologic Factors/pharmacology , Taxoids/pharmacology , Taxus/chemistry , Antineoplastic Agents/chemistry , Antineoplastic Agents/isolation & purification , Cell Line, Tumor , Cell Proliferation/drug effects , Concanavalin A/pharmacology , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Immunologic Factors/chemistry , Immunologic Factors/isolation & purification , Lymphocytes/drug effects , Models, Molecular , Molecular Conformation , Taxoids/chemistry , Taxoids/isolation & purification , Vinblastine/pharmacology , Vincristine/pharmacology
