L-[methyl-(11C)]-methionine of high enantiomeric purity production via on-line 11C-methylation of L-homocysteine thiolactone hydrochloride

L-[methyl-(11C)]-methionine ([11C]MET), labelled with carbon-11 (T1/2 = 20 min), is the most commonly used amino acid radiotracer for PET diagnostics of brain tumors. The production of [11C]MET via on-line 11C-methylation of L-homocysteine thiolactone hydrochloride (lactone) on C18 solid-phase extraction cartridge creates a problem of insufficient enantiomeric purity (content of L-isomer) of the product. The results of a systematic study of the influence of reaction parameters (lactone/base and the EtOH/H20 ratios, time of 11C-methylation) on the content of L-isomer in the preparation are presented. The developed method of on-line [11C]MET synthesis allows to obtain a product with a sufficiently high radiochemical yield (75 +/- 3%, n = 100, based on [11C]CH3I) and reliably high content of L-isomer (93.7 +/- 0.5%) satisfying the requirements of clinical applications. [11C] MET synthesis was performed on a fully automated module designed by the Institute of Human Brain (IHB RAS).     

Direct one-step labeling of cysteine residues on peptides with [11C]methyl triflate for the synthesis of PET radiopharmaceuticals

Radiolabeled peptides have emerged as an attractive platform for the diagnostic and therapeutic oncology. However, the ¹¹C-radiolabeling of peptides for positron emission tomography (PET) has been poorly explored, owing to the relatively short half-life of carbon-11 (t _1/2 = 20.3 min) and time-consuming multi-step radiochemical reactions. Existing methods have found limited use and are not routinely encountered in the production of radiotracers. Herein, we propose a facile one-step direct ¹¹C-methylation of cysteine residues in peptides using [¹¹C]methyl triflate under ambient temperatures (20 °C) and short reaction times, on the order of seconds. Good regioselectivity of this method was demonstrated by HPLC in a simple peptide (glutathione, GSH) and a more complex test decapeptide (Trp-Tyr-Trp-Ser-Arg-Cys-Lys-Trp-Thr-Gly) bearing multiple nucleophilic sites. In addition, we extend this method towards the synthesis of [¹¹C]Cys(Me)-[Tyr³-octreotate] as a demonstration of applicability for peptides of biological interest. This octreotate derivative was obtained in non-decay-corrected radiochemical yields of 11 ± 2 % (n = 3) with a synthesis time of approx. 30 min.     

Carbon‐11 radiolabeling of an oligopeptide containing tryptophan hydrochloride via a Pictet‐Spengler reaction using carbon‐11 formaldehyde

A procedure for the synthesis of a¹¹C‐labeled oligopeptide containing [1‐¹¹C]1,2,3,4‐tetrahydro‐β‐carboline‐3‐carboxylic acid ([1‐¹¹C]Tpi) from the corresponding Trp?HCl‐containing peptides has been developed involving a Pictet‐Spengler reaction with [¹¹C]formaldehyde. The synthesis of [1‐¹¹C]Tpi from Trp and [¹¹C]formaldehyde was examined as a model reaction with the aim of developing a facile and effective method for the labeling of peptides with carbon‐11. The Pictet‐Spengler reaction of Trp and [¹¹C]formaldehyde in acidic media (TsOH or HCl) afforded the desired [1‐¹¹C]Tpi in a moderate radiochemical yield. Herein, the application of a Pictet‐Spengler reaction to an aqueous solution of Trp?HCl gave the desired product with a radiochemical yield of 45.2%. The RGD peptide cyclo[Arg‐Gly‐Asp‐D‐Tyr‐Lys] was then selected as a substrate for the labeling reaction with [¹¹C]formaldehyde. The radiolabeling of a Trp?HCl‐containing RGD peptide using the Pictet‐Spengler reaction was successful. Furthermore, the remote‐controlled synthesis of a [1‐¹¹C]Tpi‐containing RGD peptide was attempted by using an automatic production system to generate [¹¹C]CH₃I. The radiochemical yield of the [1‐¹¹C]Tpi‐containing RGD at the end of synthesis (EOS) was 5.9 ± 1.9% (n = 4), for a total synthesis time of about 35 min. The specific activity was 85.7 ± 9.4 GBq/µmol at the EOS. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of (R,S)-[4-11C]baclofen via Michael addition of nitromethane labeled with short-lived 11C

The synthesis of (R,S)-[4-¹¹C]baclofen, the first ¹¹C-labeled GABA_B agonist, was demonstrated via Michael addition of nitro[¹¹C]methane as a key step. A tetrabutylammonium fluoride promoted Michael addition of nitro[¹¹C]methane to methyl p-chlorocinnamate, followed by the nitro-group reduction in the presence of NiCl₂ and NaBH₄ in aqueous MeOH and alkaline hydrolysis yielded (R,S)-[4-¹¹C]baclofen in 36.4 ± 1.8% radiochemical conversion in three steps within 20 min.     

Radiosynthesis and evaluation of [11C]CMP,a high affinity GSK3 ligand

Dysfunction of GSK3 is implicated in the etiology of many brain, inflammatory, cardiac diseases, and cancer. PET imaging would enable in vivo detection and quantification of GSK3 and can impact the choice of therapy, allow non-invasive monitoring of disease progression and treatment effects. In this report, the synthesis and evaluation of a high affinity GSK3 ligand, [¹¹C]2-(cyclopropanecarboxamido)-N-(4-methoxypyridin-3-yl)isonicotinamide, ([¹¹C]CMP, (3), (IC₅₀?=?3.4?nM, LogP?=?1.1) is described. [¹¹C]CMP was synthesized in 25?±?5% yield by radiomethylating the corresponding phenolate using [¹¹C]CH₃I. The radioligand exhibited modest uptake in U251 human glioblastoma cell lines with ～50% specific binding. MicroPET studies in rats indicated negligible blood–brain barrier (BBB) penetration of [¹¹C]CMP, despite its high affinity and suitable logP value for BBB penetration. However, administration of cyclosporine prior to [¹¹C]CMP injection showed significant improvement in brain radioactivity uptake and the tracer binding. This finding indicates that [¹¹C]CMP might be a P-gp efflux substrate and therefore has some limitations for routine in vivo PET evaluations in brain.     

Synthesis of carbon-11-labeled aminoalkylindole derivatives as new candidates of cannabinoid receptor radioligands for PET imaging of alcohol abuse

Carbon-11-labeled aminoalkylindole derivatives (1-butyl-7-[¹¹C]methoxy-1H-indol-3-yl)(naphthalene-1-yl)methanone ([¹¹C]3), 1-butyl-7-[¹¹C]methoxy-3-(naphthalene-1-ylmethyl)-1H-indole ([¹¹C]5), and 1-butyl-7-[¹¹C]methoxy-3-(naphthalene-2-yl)-1H-indole ([¹¹C]8) were prepared by O-[¹¹C]methylation of their corresponding precursors with [¹¹C]CH₃OTf under basic condition (2 N NaOH) and isolated by a simplified solid-phase extraction (SPE) method in 50–60% radiochemical yields based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 23 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 185–555 GBq/μmol.     

[11C]PR04.MZ,a promising DAT ligand for low concentration imaging: Synthesis,efficient 11C-O-methylation and initial small animal PET studies

PR04.MZ was designed as a highly selective dopamine transporter inhibitor, derived from natural cocaine. Its binding profile indicates that [¹¹C]PR04.MZ may be suited as a PET radioligand for the non-invasive exploration of striatal and extrastriatal DAT populations. As a key feature, its structural design facilitates both, labelling with fluorine-18 at its terminally fluorinated butynyl moiety and carbon-11 at its methyl ester function. The present report concerns the efficient [¹¹C]MeI mediated synthesis of [¹¹C]PR04.MZ from an O-desmethyl precursor trifluoroacetic acid salt with Rb₂CO₃ in DMF in up to 95 ± 5% labelling yield. A preliminary μPET-experiment demonstrates the reversible, highly specific binding of [¹¹C]PR04.MZ in the brain of a male Sprague–Dawley rat.     

Synthesis of (R,S)-isoproterenol,an inhibitor of tau aggregation,as an 11C-labeled PET tracer via reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone

(R,S)-Isoproterenol inhibits the formation of toxic granular tau oligomers associated with neuronal loss and development of cognitive disorders, and is an attractive drug candidate for Alzheimer’s disease. To elucidate its behavior in the brain by positron emission tomography, we synthesize (R,S)-[¹¹C]isoproterenol by reductive alkylation of (R,S)-norepinephrine with [2-¹¹C]acetone, which was in turn synthesized in situ under improved conditions afforded a decay-corrected radiochemical yield of 54%. The reductive alkylation using NaBH(OAc)₃ as reducing agent in the presence of benzoic acid in DMSO/DMF (60:40 v/v) at 100 °C for 10 min gave (R,S)-[¹¹C]isoproterenol in an 87% radio-high performance liquid chromatography (HPLC) analytical yield. HPLC separation using a strong cation exchange column, followed by pharmaceutical formulation in the presence of d/l-tartaric acid, afforded (R,S)-[¹¹C]isoproterenol with a total radioactivity of 2.0 ± 0.2 GBq, a decay-corrected radiochemical yield of 19 ± 2%, chemical and radiochemical purities of 71% and >99%, respectively, and a molar activity of 100 ± 13 GBq/μmol (n = 3). The overall synthesis time from the end of the bombardment to pharmaceutical formulation was 48 min. A preliminary preclinical PET study in a rat demonstrated the potential of the radioligand for the evaluation of the penetration of (R,S)-isoproterenol in human brain.     

Radiosynthesis of N-(4-chloro-3-[11C]methoxyphenyl)-2-picolinamide ([11C]ML128) as a PET radiotracer for metabotropic glutamate receptor subtype 4 (mGlu4)

N-(Chloro-3-methoxyphenyl)-2-picolinamide (3, ML128, VU0361737) is an mGlu₄ positive allosteric modulator (PAM), which is potent and centrally penetrating. 3 is also the first mGlu₄ PAM to show efficacy in a preclinical Parkinson disease model upon systemic dosing. As a noninvasive medical imaging technique and a powerful tool in neurological research, positron emission tomography (PET) offers a possibility to investigate mGlu₄ expression in vivo under physiologic and pathological conditions. We synthesized a carbon-11 labeled ML128 ([¹¹C]3) as a PET radiotracer for mGlu₄, and characterized its biological properties in Sprague Dawley rats. [¹¹C]3 was synthesized from N-(4-chloro-3-hydroxyphenyl)-2-picolinamide (2) using [¹¹C]CH₃I. Total synthesis time was 38 ± 2.2 min (n = 7) from the end of bombardment to the formulation. The radioligand [¹¹C]3 was obtained in 27.7 ± 5.3% (n = 5) decay corrected radiochemical yield based on the radioactivity of [¹¹C]CO₂. The radiochemical purity of [¹¹C]3 was >99%. Specific activity was 188.7 ± 88.8 GBq/mol (n = 4) at the end of synthesis (EOS).PET images were conducted in 20 normal male Sprague Dawley rats including 11 control studies, 6 studies blocking with an mGlu₄ modulator (4) to investigate specificity and 3 studies blocking with an mGlu₅ modulator (MTEP) to investigate selectivity. These studies showed fast accumulation of [¹¹C]3 (peak activity between 1–3 min) in several brain areas including striatum, thalamus, hippocampus, cerebellum, and olfactory bulb following with fast washout. Blocking studies with the mGlu₄ modulator 4 showed 22–28% decrease of [¹¹C]3 accumulation while studies of selectivity showed only minor decrease supporting good selectivity over mGlu₅. Biodistribution studies and blood analyses support fast metabolism. Altogether this is the first PET imaging ligand for mGlu₄, in which the labeled ML128 was used for imaging its in vivo distribution and pharmacokinetics in brain.     

Facile synthesis of carbon-11-labeled cholesterol-based cationic lipids as new potential PET probes for imaging of gene delivery in cancer

Gene therapy based on gene delivery is a promising strategy for the treatment of various human diseases such as cancer. Cationic lipids represent one of the important synthetic gene delivery systems. There is a great interest in imaging of gene therapy using the biomedical imaging technique positron emission tomography (PET). Carbon-11-labeled cholesterol-based cationic lipids were first designed and synthesized as new potential PET probes for imaging of gene delivery in cancer. The [¹¹C-methyl]quaternary amine target tracers, N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]pyrrolidinium iodide ([¹¹C]4a), N-[¹¹C]methyl-N′-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]imidazolium iodide ([¹¹C]4b), N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]piperidinium iodide ([¹¹C]4c), N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]-4-methylpiperidinium iodide ([¹¹C]4d), and N-[¹¹C]methyl-N-[4-(cholest-5-en-3β-yloxycarbonyl)butyl]morpholinium iodide ([¹¹C]4e), were prepared from their corresponding tertiary amine precursors with [¹¹C]methyl iodide ([¹¹C]CH₃I) through N-[¹¹C]methylation and isolated by a simplified solid-phase extraction (SPE) method using a Silica Sep-Pak cartridge in 50-60% radiochemical yields decay corrected to end-of-bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).     

Synthesis and evaluation of 4-(2-fluoro-4-[11C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide for PET imaging of the metabotropic glutamate receptor 2 in the rat brain

Metabotropic glutamate receptor 2 (mGluR2) has been suggested as a therapeutic target for treating schizophrenia-like symptoms arising from increased glutamate transmission in the human forebrain. However, no reliable positron emission tomography (PET) radiotracer allowing for in vivo visualization of mGluR2 in the human brain is currently available. In this study, we synthesized 4-(2-fluoro-4-[¹¹C]methoxyphenyl)-5-((2-methylpyridin-4-yl)methoxy)picolinamide ([¹¹C]1) and evaluated its potential as a PET tracer for imaging mGluR2 in the rodent brain. Compound 1, a negative allosteric modulator (NAM) of mGluR2, showed high in vitro binding affinity (IC₅₀: 26?nM) for mGluR2 overexpressed in human cells. [¹¹C]1 was synthesized by O-[¹¹C]methylation of the phenol precursor 2 with [¹¹C]methyl iodide. After the reaction, HPLC purification and formulation, [¹¹C]1 of 7.4?±?2.8?GBq (n?=?8) was obtained from [¹¹C]carbon dioxide of 22.5?±?4.8?GBq (n?=?8) with >99% radiochemical purity and 70?±?32?GBq/μmol (n?=?8) molar activity at the end of synthesis. In vitro autoradiography for rat brains showed that [¹¹C]1 binding was heterogeneously distributed in the cerebral cortex, striatum, hippocampus, and cerebellum. This pattern is consistent with the regional distribution pattern of mGluR2 in the rodent brain. The radioactivity was significantly reduced by self- or MNI-137 (a mGluR2 NAM) blocking. Small-animal PET studies indicated a low in vivo specific binding of [¹¹C]1 in the rat brain. The brain uptake was increased in a P-glycoprotein and breast cancer resistant protein double knockout mouse, when compared to a wild-type mouse. While [¹¹C]1 presented limited potential as an in vivo PET tracer for mGluR2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.     

Radiosynthesis and evaluation of [11C]EMPA as a potential PET tracer for orexin 2 receptors

EMPA is a selective antagonist of orexin 2 (OX₂) receptors. Previous literature with [³H]-EMPA suggest that it may be used as an imaging agent for OX₂ receptors; however, brain penetration is known to be modest. To evaluate the potential of EMPA as a PET radiotracer in non-human primate (as a step to imaging in man), we radiolabeled EMPA with carbon-11. Radiosynthesis of [¹¹C]N-ethyl-2-(N-(6-methoxypyridin-3-yl)-2-methylphenylsulfonamido)-N-(pyridin-3-ylmethyl)acetamide ([¹¹C]EMPA), and evaluation as a potential PET tracer for OX₂ receptors is described. Synthesis of an appropriate non-radioactive O-desmethyl precursor was achieved from EMPA with sodium iodide and chlorotrimethylsilane. Selective O-methylation using [¹¹C]CH₃I in the presence of cesium carbonate in DMSO at room temp afforded [¹¹C]EMPA in 1.5–2.5% yield (non-decay corrected relative to trapped [¹¹C]CH₃I at EOS) with ?95% chemical and radiochemical purities. The total synthesis time was 34–36 min from EOB. Studies in rodent suggested that uptake in tissue was dominated by nonspecific binding. However, [¹¹C]EMPA also showed poor uptake in both rats and baboon as measured with PET imaging.     

Synthesis of carbon-11-labeled casimiroin analogues as new potential PET agents for imaging of quinone reductase 2 and aromatase expression in breast cancer

Carbon-11-labeled casimiroin analogues were first designed and synthesized as new potential PET agents for imaging of quinone reductase (QR) 2 and aromatase expression in breast cancer. [¹¹C]casimiroin (6-[¹¹C]methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one, [¹¹C]11) and its carbon-11-labeled analogues 5,6,8-trimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]17), 8-methoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21a), 6,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21b), and 5,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21c), were prepared from their corresponding precursors with [¹¹C]methyl triflate ([¹¹C]CH₃OTf) under basic conditions (NaH) through either O- or N-[¹¹C]methylation and isolated by semi-preparative HPLC method in 40-50% radiochemical yields decay corrected to end of bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).     

Radiosynthesis of [thiocarbonyl-11C]disulfiram and its first PET study in mice

[Thiocarbonyl-¹¹C]disulfiram ([¹¹C]DSF) was synthesized via iodine oxidation of [¹¹C]diethylcarbamodithioic acid ([¹¹C]DETC), which was prepared from [¹¹C]carbon disulfide and diethylamine. The decay-corrected isolated radiochemical yield (RCY) of [¹¹C]DSF was greatly affected by the addition of unlabeled carbon disulfide. In the presence of carbon disulfide, the RCY was increased up to 22% with low molar activity (A_m, 0.27 GBq/μmol). On the other hand, [¹¹C]DSF was obtained in 0.4% RCY with a high A_m value (95 GBq/μmol) in the absence of carbon disulfide. The radiochemical purity of [¹¹C]DSF was always >98%. The first PET study on [¹¹C]DSF was performed in mice. A high uptake of radioactivity was observed in the liver, kidneys, and gallbladder. The uptake level and distribution pattern in mice were not significantly affected by the A_m value of the [¹¹C]DSF sample used. In vivo metabolite analysis showed the rapid decomposition of [¹¹C]DSF in mouse plasma.     

Radiosynthesis and evaluation of a novel monoacylglycerol lipase radiotracer: 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[11C]carboxylate

Monoacylglycerol lipase (MAGL) is a major serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid (AA) and glycerol in the brain. Because 2-AG and AA are endogenous biologically active ligands in the brain, the inhibition of MAGL is an attractive therapeutic target for neurodegenerative diseases. In this study, to visualize MAGL via positron emission tomography (PET), we report a new carbon-11-labeled radiotracer, namely 1,1,1,3,3,3-hexafluoropropan-2-yl-3-(1-benzyl-1H-pyrazol-3-yl)azetidine-1-[¹¹C]carboxylate ([¹¹C]6). Compound 6 exhibited high in vitro binding affinity (IC₅₀ = 0.41 nM) to MAGL in the brain with a suitable lipophilicity (cLogD = 3.29). [¹¹C]6 was synthesized by reacting 1,1,1,3,3,3-hexafluoropropanol (7) with [¹¹C]phosgene ([¹¹C]COCl₂), followed by a reaction with 3-(1-benzyl-1H-pyrazol-3-yl)azetidine hydrochloride (8), which resulted in a 15.0 ± 6.8% radiochemical yield (decay-corrected, n = 7) based on [¹¹C]CO₂ and a 45 min synthesis time from the end of bombardment. A biodistribution study in mice showed high uptake of radioactivity in MAGL-rich organs, including the lungs, heart, and kidneys. More than 90% of the total radioactivity was irreversibly bound in the brain homogenate of rats 5 min and 30 min after the radiotracer injection. PET summation images of rat brains showed high radioactivity in all brain regions. Pretreatment with 6 or MAGL-selective inhibitor JW642 significantly reduced the uptake of radioactivity in the brain. [¹¹C]6 is a promising PET tracer which offers in vivo specific binding and selectivity for MAGL in rodent brains.     

Fully automated synthesis and initial PET evaluation of [11C]PBR28

Fully automated synthesis and initial PET evaluation of a TSPO radioligand, [¹¹C]PBR28 (N-(2-[¹¹C]methoxybenzyl)-N-(4-phenoxypyridin-3-yl)acetamide), are reported. These results facilitate the potential preclinical and clinical PET studies of [¹¹C]PBR28 in animals and humans.     

[11C]BCTC: Radiosynthesis and in vivo binding to transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in the mouse trigeminal nerve

The purpose of this study was to synthesize a new positron emission tomography radiotracer, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-[¹¹C]carboxamide ([¹¹C]BCTC, [¹¹C]1), and assess its in vivo binding to the transient receptor potential vanilloid subfamily member 1 (TRPV1) receptor in mice. [¹¹C]BCTC was synthesized by reacting the hydrochloride of 4-tertiarybutylaniline (2·HCl) with [¹¹C]phosgene ([¹¹C]COCl₂) to give isocyanate [¹¹C]4, followed by reaction with 4-(3-chloropyridin-2-yl)tetrahydropyrazine (3). [¹¹C]BCTC was obtained at a 16–20% radiochemical yield, based on the cyclotron-produced [¹¹C]CO₂ (decay-corrected to the end of bombardment), with >98% radiochemical purity and 65–110 GBq/μmol specific activity at the end of synthesis. An ex vivo biodistribution study in mice confirmed the specific binding of [¹¹C]BCTC to TRPV1 in the trigeminal nerve, which is a tissue with high TRPV1 expression.     

In vivo comparison of N-11CH3 vs O-11CH3 radiolabeled microtubule targeted PET ligands

Altered dynamics of microtubules (MT) are implicated in the pathophysiology of a number of brain diseases. Therefore, radiolabeled MT targeted ligands that can penetrate the blood brain barrier (BBB) may offer a direct and sensitive approach for diagnosis, and assessing the clinical potential of MT targeted therapeutics using PET imaging. We recently reported two BBB penetrating radioligands, [¹¹C]MPC-6827 and [¹¹C]HD-800 as specific PET ligands for imaging MTs in brain. The major metabolic pathway of the above molecules is anticipated to be via the initial labeling site, O-methyl, compared to the N-methyl group. Herein, we report the radiosynthesis of N-¹¹CH₃-MPC-6827 and N-¹¹CH₃-HD-800 and a comparison of their in vivo binding with the corresponding O-¹¹CH₃ analogues using microPET imaging and biodistribution methods. Both O-¹¹CH₃ and N-¹¹CH₃ labeled MT tracers exhibit high specific binding and brain. The N-¹¹CH₃ labeled PET ligands demonstrated similar in vivo binding characteristics compared with the corresponding O-¹¹CH₃ labeled tracers, [¹¹C]MPC-6827 and [¹¹C]HD-800 respectively.     

Metabolism of 2'-carboxyarabinitol in leaves

Results presented here indicate that 2′-carboxyarabinitol (CA) is the in vivo precursor and product of 2′-carboxyarabinitol 1-phosphate (CA1P) metabolism in leaves. When [2-¹⁴C]CA was fed in the light to leaves of five species known to be highly active in CA1P metabolism (Phaseolus vulgaris, Lycopersicon esculentum, Helianthus annuus, Petunia hybrida, and Beta vulgaris), [¹⁴C]CA1P was formed in the dark. Reillumination of a Phaseolus leaf caused this [¹⁴C]CA1P to be rapidly metabolized to [¹⁴C]CA (t_½ = 1 min). The epimer 2′-carboxyribitol could not substitute for CA in the dark synthesis of CA1P, and CA in the anionic form was a better substrate than CA in the lactone form. In leaves of Phaseolus vulgaris, the active CA pool size used in the dark synthesis of CA1P is between about 70 and 110 nanomoles per milligram of chlorophyll. The photosynthetic electron transport inhibitor diuron did not affect the dark synthesis of [¹⁴C]CA1P, but did greatly reduce the rate of its subsequent light degradation (t_½ = approximately 10 min). Dark synthesis of [¹⁴C]CA1P was inhibited by dithiothreitol and NaF. From the present data, we suggest that CA1P and CA participate in a metabolic substrate cycle in vivo.     

A high-yield route to synthesize the P-glycoprotein radioligand [11C]N-desmethyl-loperamide and its parent radioligand [11C]loperamide

N-Desmethyl-loperamide and loperamide were synthesized from α,α-diphenyl-γ-butyrolactone and 4-(4-chlorophenyl)-4-hydroxypiperidine in five and four steps with 8% and 16% overall yield, respectively. The amide precursor was synthesized from 4-bromo-2,2-diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypiperidine in 2 steps with 21–57% overall yield. [¹¹C]N-Desmethyl-loperamide and [¹¹C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 20–30% and 10–15% radiochemical yields, respectively, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB.