Intramolecular Friedel-Crafts Acylation of [11C]Isocyanates Enabling the Radiolabeling of [carbonyl-11C]DPQ.

α,ß-aromatic lactams are highly abundant in biologically active molecules, yet so far they cannot be radiolabeled with short-lived (t1/2=20.3 min), ß+-decaying carbon-11, which has prevented their application as positron emission tomography tracers. Herein, we developed, optimized, and applied a widely applicable, one-pot, quick, robust and automatable radiolabeling method for α,ß-aromatic lactams starting from [11C]CO2 using the reagent POCl3⋅AlCl3. This method proceeds via intramolecular Friedel-Crafts acylation of in situ formed [11C]isocyanates and shows a broad substrate scope for the formation of five- and six-membered rings. We implemented our developed labeling method for the radiosynthesis of the potential PARP1 PET tracer [carbonyl-11C]DPQ in a clinical radiotracer production facility following the standards of the European Pharmacopoeia.

Synthesis and Evaluation of [11C]MCC950 for Imaging NLRP3-Mediated Inflammation in Atherosclerosis.

Overexpression of the NLRP3 inflammasome has been attributed to the progressive worsening of a multitude of cardiovascular inflammatory diseases such as myocardial infarction, pulmonary arterial hypertension, and atherosclerosis. The recently discovered potent and selective NLRP3 inhibitor MCC950 has shown promise in hindering disease progression, but NLRP3-selective cardiovascular positron emission tomography (PET) imaging has not yet been demonstrated. We synthesized [11C]MCC950 with no-carrier-added [11C]CO2 fixation chemistry using an iminophosphorane precursor (RCY 45 ± 4%, >99% RCP, 27 ± 2 GBq/µmol, 23 ± 3 min, n = 6) and determined its distribution both in vivo and ex vivo in C57BL/6 and atherogenic ApoE-/- mice. Small animal PET imaging was performed in both strains following intravenous administration via the lateral tail vein and revealed considerable uptake in the liver that stabilized by 20 min (7-8.5 SUV), coincident with secondary renal excretion. Plasma metabolite analysis uncovered excellent in vivo stability of [11C]MCC950 (94% intact). Ex vivo autoradiography performed on excised aortas revealed heterogeneous uptake in atherosclerotic plaques of ApoE-/- mice in comparison to C57BL/6 controls (48 ± 17 %ID/m2 vs 18 ± 8 %ID/m2, p = 0.002, n = 4-5). Treatment of ApoE-/- mice with nonradioactive MCC950 (5 mg/kg, iv) 10 min prior to radiotracer administration increased uptake in the intestine (5.3 ± 1.8 %ID/g vs 11.0 ± 3.7 %ID/g, p = 0.04, n = 4-6) and in aortic lesions (48 ± 17 %ID/m2 vs 104 ± 15 %ID/m2, p = 0.0002, n = 5) by 108% and 117%, respectively, without significantly increasing plasma free fraction (fp, 1.3 ± 0.4% vs 1.7 ± 0.8%, n = 2). These results suggest that [11C]MCC950 uptake demonstrates specific binding and may prove useful for in vivo NLRP3 imaging in atherosclerosis.

Pharmacological and metabolic parameters of [18F]flubrobenguane in clinical imaging populations.

BACKGROUND: Cardiac sympathetic nervous system molecular imaging has demonstrated prognostic value. Compared with meta-[11C]hydroxyephedrine, [18F]flubrobenguane (FBBG) facilitates reliable estimation of SNS innervation using similar analytical methods and possesses a more convenient physical half-life. The aim of this study was to evaluate pharmacokinetic and metabolic properties of FBBG in target clinical cohorts. METHODS: Blood sampling was performed on 20 participants concurrent to FBBG PET imaging (healthy = NORM, non-ischemic cardiomyopathy = NICM, ischemic cardiomyopathy = ICM, post-traumatic stress disorder = PTSD). Image-derived blood time-activity curves were transformed to plasma input functions using cohort-specific corrections for plasma protein binding, plasma-to-whole blood distribution, and metabolism. RESULTS: The plasma-to-whole blood ratio was 0.78 ± 0.06 for NORM, 0.64 ± 0.06 for PTSD and 0.60 ± 0.14 for (N)ICM after 20 minutes. 22 ± 4% of FBBG was bound to plasma proteins. Metabolism of FBBG in (N)ICM was delayed, with a parent fraction of 0.71 ± 0.05 at 10 minutes post-injection compared to 0.53 ± 0.03 for PTSD/NORM. While there were variations in metabolic rate, metabolite-corrected plasma input functions were similar across all cohorts. CONCLUSIONS: Rapid plasma clearance of FBBG limits the impact of disease-specific corrections of the blood input function for tracer kinetic modeling.

Photocatalyzed radiosynthesis of 11 C-phenylacetic acids.

Fast and straightforward incorporation of radionuclides into pharmaceutically relevant molecules is one of the main barriers to preclinical and clinical tracer research. Late-stage direct incorporation of cyclotron-produced [11 C]CO2 to afford carbon-11-labeled radiopharmaceuticals has the potential to provide ready-to-inject positron emission tomography agents in less than an hour. The present work describes photocatalyzed carboxylation of alkylbenzene derivatives to afford 11 C-phenylacetic acids. Reaction conditions and scope are investigated followed by application of this methodology to the preparative radiosynthesis of [11 C]fenoprofen, a nonsteroidal anti-inflammatory drug.

Fast Carbon Isotope Exchange of Carboxylic Acids Enabled by Organic Photoredox Catalysis.

Carbazole/cyanobenzene photocatalysts promote the direct isotopic carboxylate exchange of C(sp3) acids with labeled CO2. Substrates that are not compatible with transition-metal-catalyzed degradation-reconstruction approaches or prone to thermally induced reversible decarboxylation undergo isotopic incorporation at room temperature in short reaction times. The radiolabeling of drug molecules and precursors with [11C]CO2 is demonstrated.

Fluorine-18-Labeled Fluorescent Dyes for Dual-Mode Molecular Imaging.

Recent progress realized in the development of optical imaging (OPI) probes and devices has made this technique more and more affordable for imaging studies and fluorescence-guided surgery procedures. However, this imaging modality still suffers from a low depth of penetration, thus limiting its use to shallow tissues or endoscopy-based procedures. In contrast, positron emission tomography (PET) presents a high depth of penetration and the resulting signal is less attenuated, allowing for imaging in-depth tissues. Thus, association of these imaging techniques has the potential to push back the limits of each single modality. Recently, several research groups have been involved in the development of radiolabeled fluorophores with the aim of affording dual-mode PET/OPI probes used in preclinical imaging studies of diverse pathological conditions such as cancer, Alzheimer's disease, or cardiovascular diseases. Among all the available PET-active radionuclides, 18F stands out as the most widely used for clinical imaging thanks to its advantageous characteristics (t1/2 = 109.77 min; 97% ß+ emitter). This review focuses on the recent efforts in the synthesis and radiofluorination of fluorescent scaffolds such as 4,4-difluoro-4-bora-diazaindacenes (BODIPYs), cyanines, and xanthene derivatives and their use in preclinical imaging studies using both PET and OPI technologies.

(11)C[double bond, length as m-dash]O bonds made easily for positron emission tomography radiopharmaceuticals.

The positron-emitting radionuclide carbon-11 ((11)C, t1/2 = 20.3 min) possesses the unique potential for radiolabeling of any biological, naturally occurring, or synthetic organic molecule for in vivo positron emission tomography (PET) imaging. Carbon-11 is most often incorporated into small molecules by methylation of alcohol, thiol, amine or carboxylic acid precursors using [(11)C]methyl iodide or [(11)C]methyl triflate (generated from [(11)C]carbon dioxide or [(11)C]methane). Consequently, small molecules that lack an easily substituted (11)C-methyl group are often considered to have non-obvious strategies for radiolabeling and require a more customized approach. [(11)C]Carbon dioxide itself, [(11)C]carbon monoxide, [(11)C]cyanide, and [(11)C]phosgene represent alternative reactants to enable (11)C-carbonylation. Methodologies developed for preparation of (11)C-carbonyl groups have had a tremendous impact on the development of novel PET tracers and provided key tools for clinical research. (11)C-Carbonyl radiopharmaceuticals based on labeled carboxylic acids, amides, carbamates and ureas now account for a substantial number of important imaging agents that have seen translation to higher species and clinical research of previously inaccessible targets, which is a testament to the creativity, utility and practicality of the underlying radiochemistry.

Preclinical PET Neuroimaging of [11C]Bexarotene.

Activation of retinoid X receptors (RXRs) has been proposed as a therapeutic mechanism for the treatment of neurodegeneration, including Alzheimer's and Parkinson's diseases. We previously reported radiolabeling of a Food and Drug Administration-approved RXR agonist, bexarotene, by copper-mediated [(11)C]CO2 fixation and preliminary positron emission tomography (PET) neuroimaging that demonstrated brain permeability in nonhuman primate with regional binding distribution consistent with RXRs. In this study, the brain uptake and saturability of [(11)C]bexarotene were studied in rats and nonhuman primates by PET imaging under baseline and greater target occupancy conditions. [(11)C]Bexarotene displays a high proportion of nonsaturable uptake in the brain and is unsuitable for RXR occupancy measurements in the central nervous system.

Practical Radiosynthesis and Preclinical Neuroimaging of [11C]isradipine, a Calcium Channel Antagonist.

In the interest of developing in vivo positron emission tomography (PET) probes for neuroimaging of calcium channels, we have prepared a carbon-11 isotopologue of a dihydropyridine Ca2+-channel antagonist, isradipine. Desmethyl isradipine (4-(benzo[c][1,2,5]oxadiazol-4-yl)-5-(isopropoxycarbonyl)-2,6-dimethyl-1,4-dihydropyridine -3-carboxylic acid) was reacted with [11C]CH3I in the presence of tetrabutylammonium hydroxide in DMF in an HPLC injector loop to produce the radiotracer in a good yield (6 ± 3% uncorrected radiochemical yield) and high specific activity (143 ± 90 GBq·µmol-1 at end-of-synthesis). PET imaging of normal rats revealed rapid brain uptake at baseline (0.37 ± 0.08% ID/cc (percent of injected dose per cubic centimeter) at peak, 15-60 s), which was followed by fast washout. After pretreatment with isradipine (2 mg·kg-1, i.p.), whole brain radioactivity uptake was diminished by 25%-40%. This preliminary study confirms that [11C]isradipine can be synthesized routinely for research studies and is brain penetrating. Further work on Ca2+-channel radiotracer development is planned.

Ortho-Stabilized (18) F-Azido Click Agents and their Application in PET Imaging with Single-Stranded DNA Aptamers.

Azido (18) F-arenes are important and versatile building blocks for the radiolabeling of biomolecules via Huisgen cycloaddition ("click chemistry") for positron emission tomography (PET). However, routine access to such clickable agents is challenged by inefficient and/or poorly defined multistep radiochemical approaches. A high-yielding direct radiofluorination for azido (18) F-arenes was achieved through the development of an ortho-oxygen-stabilized iodonium derivative (OID). This OID strategy addresses an unmet need for a reliable azido (18) F-arene clickable agent for bioconjugation reactions. A ssDNA aptamer was radiolabeled with this agent and visualized in a xenograft mouse model of human colon cancer by PET, which demonstrates that this OID approach is a convenient and highly efficient way of labeling and tracking biomolecules.

PET imaging of fatty acid amide hydrolase with [(18)F]DOPP in nonhuman primates.

Fatty acid amide hydrolase (FAAH) regulates endocannabinoid signaling. [(11)C]CURB, an irreversibly binding FAAH inhibitor, has been developed for clinical research imaging with PET. However, no fluorine-18 labeled radiotracer for FAAH has yet advanced to human studies. [(18)F]DOPP ([(18)F]3-(4,5-dihydrooxazol-2-yl)phenyl (5-fluoropentyl)carbamate) has been identified as a promising (18)F-labeled analogue based on rodent studies. The goal of this work is to evaluate [(18)F]DOPP in nonhuman primates to support its clinical translation. High specific activity [(18)F]DOPP (5-6 Ci·µmol(-1)) was administered intravenously (iv) to three baboons (2M/1F, 3-4 years old). The distribution and pharmacokinetics were quantified following a 2 h dynamic imaging session using a simultaneous PET/MR scanner. Pretreatment with the FAAH-selective inhibitor, URB597, was carried out at 200 or 300 µg/kg iv, 10 min prior to [(18)F]DOPP administration. Rapid arterial blood sampling for the first 3 min was followed by interval sampling with metabolite analysis to provide a parent radiotracer plasma input function that indicated ∼95% baseline metabolism at 60 min and a reduced rate of metabolism after pretreatment with URB597. Regional distribution data were analyzed with 1-, 2-, and 3-tissue compartment models (TCMs), with and without irreversible trapping since [(18)F]DOPP covalently links to the active site of FAAH. Consistent with previous findings for [(11)C]CURB, the 2TCM with irreversible binding was found to provide the best fit for modeling the data in all regions. The composite parameter λk3 was therefore used to evaluate whole brain (WB) and regional binding of [(18)F]DOPP. Pretreatment studies showed inhibition of λk3 across all brain regions (WB baseline: 0.112 mL/cm(3)/min; 300 µg/kg URB597: 0.058 mL/cm(3)/min), suggesting that [(18)F]DOPP binding is specific for FAAH, consistent with previous rodent data.

Shifting the energy landscape of multicomponent reactions using aziridine aldehyde dimers: a mechanistic study.

A multicomponent reaction between an aziridine aldehyde dimer, isocyanide, and l-proline to afford a chiral piperazinone was studied to gain insight into the stereodetermining and rate-limiting steps of the reaction. The stereochemistry of the reaction was found to be determined by isocyanide addition, while the rate-limiting step was found to deviate from traditional isocyanide-based multicomponent reactions. A first-order rate dependence on aziridine aldehyde dimer and a zero-order rate dependence on all other reagents have been obtained. Computations at the MPWPW91/6-31G(d) level supported the experimental kinetic results and provide insight into the overall mechanism and the factors contributing to stereochemical induction. These factors are similar to traditional isocyanide-based multicomponent reactions, such as the Ugi reaction. The computations revealed that selective formation of a Z-iminium ion plays a key role in controlling the stereoselectivity of isocyanide addition, and the carboxylate group of l-proline mediates stereofacial addition. These conclusions are expected to be applicable to a wide range of reported stereoselective Ugi reactions and provide a basis for understanding the related macrocyclization of peptides with aziridine aldehydes.

Stereocontrolled disruption of the Ugi reaction toward the production of chiral piperazinones: substrate scope and process development.

The factors determining diastereoselectivity observed in the multicomponent conversion of amino acids, aziridine aldehyde dimers, and isocyanides into chiral piperazinones have been investigated. Amino acid-dependent selectivity for either trans- or cis-substituted piperazinone products has been achieved. An experimentally determined diastereoselectivity model for the three-component reaction driven by aziridine aldehyde dimers has predictive value for different substrate classes. Moreover, this model is useful in reconciling the previously reported observations in multicomponent reactions between isocyanides, α-amino acids, and monofunctional aldehydes.

Alternative approaches for PET radiotracer development in Alzheimer's disease: imaging beyond plaque.

Alzheimer's disease (AD) and related dementias show increasing clinical prevalence, yet our understanding of the etiology and pathobiology of disease-related neurodegeneration remains limited. In this regard, noninvasive imaging with radiotracers for positron emission tomography (PET) presents a unique tool for quantifying spatial and temporal changes in characteristic biological markers of brain disease and for assessing potential drug efficacy. PET radiotracers targeting different protein markers are being developed to address questions pertaining to the molecular and/or genetic heterogeneity of AD and related dementias. For example, radiotracers including [(11) C]-PiB and [(18) F]-AV-45 (Florbetapir) are being used to measure the density of Aß-plaques in AD patients and to interrogate the biological mechanisms of disease initiation and progression. Our focus is on the development of novel PET imaging agents, targeting proteins beyond Aß-plaques, which can be used to investigate the broader mechanism of AD pathogenesis. Here, we present the chemical basis of various radiotracers which show promise in preclinical or clinical studies for use in evaluating the phenotypic or biochemical characteristics of AD. Radiotracers for PET imaging neuroinflammation, metal ion association with Aß-plaques, tau protein, cholinergic and cannabinoid receptors, and enzymes including glycogen-synthase kinase-3ß and monoamine oxidase B amongst others, and their connection to AD are highlighted.

A practical guide for the preparation of C1-labeled α-amino acids using aldehyde catalysis with isotopically labeled CO2.

Isotopically carbon-labeled α-amino acids are valuable synthetic targets that are increasingly needed in pharmacology and medical imaging. Existing preparations rely on early stage introduction of the isotopic label, which leads to prohibitive synthetic costs and time-intensive preparations. Here we describe a protocol for the preparation of C1-labeled α-amino acids using simple aldehyde catalysts in conjunction with [*C]CO2 (* = 14, 13, 11). This late-stage labeling strategy is enabled by the one-pot carboxylate exchange of unprotected α-amino acids with [*C]CO2. The protocol consists of three separate procedures, describing the syntheses of (±)-[1-13C]phenylalanine, (±)-[1-11C]phenylalanine and (±)-[1-14C]phenylalanine from unlabeled phenylalanine. Although the delivery of [*C]CO2 is operationally distinct for each experiment, each procedure relies on the same fundamental chemistry and can be executed by heating the reaction components at 50-90 °C under basic conditions in dimethylsulfoxide. Performed on scales of up to 0.5 mmol, this methodology is amenable to C1-labeling of many proteinogenic α-amino acids and nonnatural derivatives, which is a breakthrough from existing methods. The synthesis of (±)-[1-13C]phenylalanine requires ~2 d, with product typically obtained in a 60-80% isolated yield (n = 3, µ = 71, σ = 8.3) with an isotopic incorporation of 70-88% (n = 18, µ = 72, σ = 9.0). Starting from the preformed imino acid (~3 h preparation time), rapid synthesis of (±)-[1-11C]phenylalanine can be completed in ~1 h with an isolated radiochemical yield of 13%. Finally, (±)-[1-14C]phenylalanine can be accessed in ~2 d with a 51% isolated yield and 11% radiochemical yield.

PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Quinazoline-2-Carboxamides as Selective PET Radiotracers for Matrix Metalloproteinase-13 Imaging in Atherosclerosis.

Matrix metalloproteinase-13 (MMP-13) plays a critical role in the progression of unstable atherosclerosis. A series of highly potent and selective MMP-13 inhibitors were synthesized around a quinazoline-2-carboxamide scaffold to facilitate radiolabeling with fluorine-18 or carbon-11 positron-emitting nuclides and visualization of atherosclerotic plaques. In vitro enzyme inhibition assays identified three compounds as promising radiotracer candidates. Efficient automated radiosyntheses provided [11C]5b, [11C]5f, and [18F]5j and enabled pharmacokinetic characterization in atherosclerotic mice. The radiotracers displayed substantial differences in their distribution and excretion. Most favorably for vascular imaging, [18F]5j exhibited low uptake in metabolic organs with minimal retention of myocardial radioactivity, substantial renal clearance, and high metabolic stability in plasma. Ex vivo aortic autoradiography and competition studies revealed that [18F]5j specifically binds to MMP-13 within atherosclerotic plaques and localizes to lipid-rich regions. This study demonstrates the utility of the quinazoline-2-carboxamide scaffold for MMP-13 selective positron emission tomography (PET) radiotracer development and identifies [18F]5j for imaging atherosclerosis.

Conformational modulation of in vitro activity of cyclic RGD peptides via aziridine aldehyde-driven macrocyclization chemistry.

Here, we demonstrate a conjugation strategy whereby cyclic RGD-containing macrocycles are prepared using aziridine aldehydes, isocyanides, and linear peptides, followed by conjugation to a cysteamine linker. Our method involves site-selective aziridine ring-opening with the nucleophilic sulfhydryl group of cysteamine. Fluorescein was then efficiently conjugated to the primary amine of cysteamine by NHS-chemistry. This strategy may be expanded to provide easy access to a wide variety of fluorescent dyes or radiometal chelators. Modeling studies showed that aziridine aldehyde cyclization chemistry stabilized the RGD motif into the required bioactive conformation and that this cyclization chemistry modulated the geometry of macrocycles of different residue lengths. In vitro studies showed that cPRGDA and cPRGDAA both selectively bound to α(V)ß(3)-overexpressing U87 glioblastoma cells, and that cPRGDA had a better binding affinity compared to cPRGDAA. The improved binding affinity of cPRGDA was attributed to the fixed Pro-C(α)-Asp-C(α) distance surrounding the stabilized RGD motif in cPRGDA.

Selective Imaging of Matrix Metalloproteinase-13 to Detect Extracellular Matrix Remodeling in Atherosclerotic Lesions.

PURPOSE: Overexpression and activation of matrix metalloproteinase-13 (MMP-13) within atheroma increases susceptibility to plaque rupture, a major cause of severe cardiovascular complications. In comparison to pan-MMP targeting [18F]BR-351, we evaluated the potential for [18F]FMBP, a selective PET radiotracer for MMP-13, to detect extracellular matrix (ECM) remodeling in vascular plaques possessing markers of inflammation. PROCEDURES: [18F]FMBP and [18F]BR-351 were initially assessed in vitro by incubation with en face aortae from 8 month-old atherogenic ApoE-/- mice. Ex vivo biodistributions, plasma metabolite analyses, and ex vivo autoradiography were analogously performed 30 min after intravenous radiotracer administration in age-matched C57Bl/6 and ApoE-/- mice under baseline or homologous blocking conditions. En face aortae were subsequently stained with Oil Red O (ORO), sectioned, and subject to immunofluorescence staining for Mac-2 and MMP-13. RESULTS: High-resolution autoradiographic image analysis demonstrated target specificity and regional concordance to lipid-rich lesions. Biodistribution studies revealed hepatobiliary excretion, low accumulation of radioactivity in non-excretory organs, and few differences between strains and conditions in non-target organs. Plasma metabolite analyses uncovered that [18F]FMBP exhibited excellent in vivo stability (≥74% intact) while [18F]BR-351 was extensively metabolized (≤37% intact). Ex vivo autoradiography and histology of en face aortae revealed that [18F]FMBP, relative to [18F]BR-351, exhibited 2.9-fold greater lesion uptake, substantial specific binding (68%), and improved sensitivity to atherosclerotic tissue (2.9-fold vs 2.1-fold). Immunofluorescent staining of aortic en face cross sections demonstrated elevated Mac-2 and MMP-13-positive areas within atherosclerotic lesions identified by [18F]FMBP ex vivo autoradiography. CONCLUSIONS: While both radiotracers successfully identified atherosclerotic plaques, [18F]FMBP showed superior specificity and sensitivity for lesions possessing features of destructive plaque remodeling. The detection of ECM remodeling by selective targeting of MMP-13 may enable characterization of high-risk atherosclerosis featuring elevated collagenase activity.

Aldehyde-catalysed carboxylate exchange in α-amino acids with isotopically labelled CO2.

The isotopic labelling of small molecules is integral to drug development and for understanding biochemical processes. The preparation of carbon-labelled α-amino acids remains difficult and time consuming, with established methods involving label incorporation at an early stage of synthesis. This explains the high cost and scarcity of C-labelled products and presents a major challenge in 11C applications (11C t1/2 = 20 min). Here we report that aldehydes catalyse the isotopic carboxylate exchange of native α-amino acids with *CO2 (* = 14, 13, 11). Proteinogenic α-amino acids and many non-natural variants containing diverse functional groups undergo labelling. The reaction probably proceeds via the trapping of *CO2 by imine-carboxylate intermediates to generate iminomalonates that are prone to monodecarboxylation. Tempering catalyst electrophilicity was key to preventing irreversible aldehyde consumption. The pre-generation of the imine carboxylate intermediate allows for the rapid and late-stage 11C-radiolabelling of α-amino acids in the presence of [11C]CO2.