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.     

Radiosynthesis of [11C]Vandetanib and [11C]chloro-Vandetanib as new potential PET agents for imaging of VEGFR in cancer

Vandetanib (ZD6474) and its chlorine analogue chloro-Vandetanib are potent and selective vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors with low nanomolar IC₅₀ values. [¹¹C]Vandetanib and [¹¹C]chloro-Vandetanib, new potential PET agents for imaging of VEGFR in cancer, were first designed, synthesized and labeled at nitrogen and oxygen positions from their corresponding N- and O-des-methylated precursors, in 40-50% decay corrected radiochemical yield and 370-555 GBq/μmol specific activity at end of bombardment (EOB).     

Radiosynthesis of three [11C]ureido-substituted benzenesulfonamides as PET probes for carbonic anhydrase IX in tumors

Three ureido-substituted benzenesulfonamides 1a-c have been developed as potent inhibitors for carbonic anhydrase IX, which is overexpressed in hypoxic tumors. In this study, we labeled these unsymmetrical ureas 1a-c using [(11)C]phosgene ([(11)C]COCl(2)) as a labeling agent with the expectation that [(11)C]1a-c could become promising positron tomography probes for imaging carbonic anhydrase IX in tumors. The strategy for radiosynthesis of [(11)C]1a-c was to react hydrochloride of anilines 2a-c with [(11)C]COCl(2) to give isocyanate [(11)C]4a-c, followed by a reaction with 4-aminobenzenesulfonamide (3).     

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 IGF1R PET ligand [11C]GSK1838705A

Radiosynthesis and evaluation of [¹¹C]GSK1838705A in mice using microPET and determination of specificity in human GBM UG87MR cells are described herein. The radioligand was synthesized by reacting desmethyl-GSK1838705A with [¹¹C]CH₃I using GE FX2MeI module in ～5% yield (EOS), >95% radiochemical purity and a specific activity of 2.5 ± 0.5 Ci/μmol. MicroPET imaging in mice indicated that [¹¹C]GSK1838705A penetrated blood brain barrier (BBB) and showed retention of radiotracer in brain. The radioligand exhibited high uptake in U87MG cells with >70% specific binding to IGF1R. Our experiments suggest that [¹¹C]GSK-1838705A can be a potential PET radiotracer for the in vivo quantification of IGF1R expression in GBM and other brain tumors.     

Evaluation of [11C]hemicholinium-15 and [18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart

[(11)C]Hemicholinium-15 ([(11)C]HC-15) and [(18)F]hemicholinium-15 ([(18)F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [(11)C]HC-15 was prepared by N-[(11)C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [(11)C]CH(3)OTf in 55-70% radiochemical yield decay corrected to end of bombardment (EOB) and 2-3Ci/mumol specific activity at end of synthesis (EOS). [(18)F]HC-15 was prepared by N-[(18)F]fluoromethylation of the precursor using [(18)F]FCH(2)OTf in 20-30% radiochemical yield decay corrected to EOB and >1.0Ci/mumol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20min post-intravenous injection, and the results show the heart region uptakes 1.32+/-0.75%ID/g in R-ventricle for [(11)C]HC-15 and 1.28+/-0.81%ID/g in L-ventricle for [(18)F]HC-15, respectively. The dynamic PET imaging studies of [(11)C]HC-15 in rats were acquired 60min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0mg/kg of unlabeled HC-15 prior to [(11)C]HC-15 injection. [(11)C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [(13)N]NH(3), [(11)C]HC-15, and [(18)F]HC-15. PET studies in dogs of both [(11)C]HC-15 and [(18)F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [(11)C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.     

Synthesis and in vivo evaluation of [11C]MPTQ: A potential PET tracer for alpha2A-adrenergic receptors

Radiosynthesis and in vivo evaluation of [N-methyl-¹¹C] 5-methyl-3-[4-(3-phenylallyl)-piperazin-1-ylmethyl]-3,3a,4,5-tetrahydroisoxazolo[4,3-c]quinoline (1), a potential PET tracer for alpha2-adrenergic receptors is described. Syntheses of nonradioactive standard 1 and corresponding desmethyl precursor 2 were achieved from 2-aminobenzaldehyde in 40% and 65% yields, respectively. Methylation using [¹¹C]CH₃I in presence of aqueous potassium hydroxide in DMSO afforded [¹¹C]1 in 25% yield (EOS) with >99% chemical and radiochemical purities with a specific activity ranged from 3–4 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. PET studies in anesthetized baboon show that [¹¹C]1 penetrates BBB and accumulates in alpha2A-AR enriched brain areas.     

Radiosynthesis and pharmacological evaluation of [11C]EMD-95885: a high affinity ligand for NR2B-containing NMDA receptors

EMD-95885, 6-[3-[4-(4-fluorobenzyl)piperidino]propionyl]-3H-benzoxazol-2-one (1) has been described as a selective antagonist for the NMDA receptors containing NR2B subunits, displaying an IC50 of 3.9 nM for this subtype. EMD-95885 (1) has been synthesized in good overall yield and labelled with carbon-11 ( T1/2 : 20.4 min) at its benzoxazolinone moiety using [11C]phosgene. The pharmacological profile of [11C]EMD-95885 ([11C]-1) was evaluated in vivo in rats with biodistribution studies and brain radioactivity monitored with intracerebral radiosensitive beta-microprobes. The brain uptake of [11C]-1 was homogeneous (0.4-0.6%ID/mL) across the different brain structures studied. This in vivo brain regional distribution of [11C]-1 was not consistent with the known distribution of NR2B subunits. Also as a measure of specificity the hippocampus/cerebellum ratio reached 0.8 throughout the time course of the experiment supporting the lack of specificity. Competition studies with the NR2B prototypic ligand ifenprodil and EMD-95885 (1), 30 min before the radioligand injection, displayed homogeneous reduction of [11C]-1 uptake of 40-60%. Pre-treatment of rats with DTG (sigma ligand), MDL105519 (glycine site antagonist) and MK801 (ion channel blocker) had no inhibitory effect on [11C]-1 uptake. Use of haloperidol as a blocking drug also resulted in a homogeneous inhibition of [11C]-1 uptake by 66-60%, which does not reflect binding to dopamine or sigma receptors. Due to the homogeneous radioligand uptake and inhibition and no measure of cerebral blood flow effects during these blocking studies it is uncertain whether any specific binding is observed. In view of these results, [11C]EMD-95885 ([11C]-1) does not have the required properties for imaging NR2B containing NMDA receptors using positron emission tomography.     

Radiosynthesis and preclinical evaluation of [18F]FEM as a potential novel PET probe for tumor imaging

In the 21st century, the incidence and mortality of cancer, one of the most challenging diseases in the world, have rapidly increased. The purpose of this study was to develop 2-(2-[¹⁸F]fluoroethoxy)ethyl 4-methylbenzenesulfonate ([¹⁸F]FEM) as a positron emission tomography (PET) agent for tumor imaging. In this study, [¹⁸F]FEM was synthesized with a good radiochemical yield (45.4 ± 5.8%), high specific radioactivity (over 25 GBq/μmol), and commendable radiochemical purity (over 99%). The octanol/water partition coefficient of [¹⁸F]FEM was 1.44 ± 0.04. The probe demonstrated good stability in vitro (phosphate-buffered saline (PBS) and mouse serum (MS)), and binding specificity to five different tumor cell lines (A549, PC-3, HCC827, U87, and MDA-MB-231). PET imaging of tumor-bearing mice showed that [¹⁸F]FEM specifically accumulated at the tumor site of the five different tumor cell lines. The average tumor-to-muscle (T/M) ratio was over 2, and the maximum T/M values reached about 3.5. The biodistribution and dynamic PET imaging showed that most probes were metabolized by the liver, whereas a small part was metabolized by the kidney. Moreover, dynamic brain images and quantitative data showed [¹⁸F]FEM can quickly cross the blood brain barrier (BBB) and quickly fade out, thereby suggesting it may be a promising candidate probe for the imaging of brain tumors. The presented results demonstrated that [¹⁸F]FEM is a promising probe for tumor PET imaging.     

Synthesis and in vivo evaluation of [11C]SN003 as a PET ligand for CRF1 receptors

Synthesis and evaluation of [O-methyl-11C](4-methoxy-2-methylphenyl)[1-(1-methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-yl]amine or [11C]SN003 ([11C]6), as a PET imaging agent for CRF1 receptors, in baboons is described. 4-[1-(1-Methoxymethylpropyl)-6-methyl-1H-[1,2,3]triazolo[4,5-c]pyridin-4-ylamino]-3-methylphenol (5), the precursor molecule for the radiolabeling, was synthesized from 2,4-dichloro-6-methyl-3-nitropyridine in seven steps with 20% overall yield. The total time required for the synthesis of [11C]SN003 is 30 min from EOB using [11C]methyl triflate in the presence of NaOH in acetone. The yield of the synthesis is 22% (EOS) with >99% chemical and radiochemical purities and a specific activity of >2000 Ci/mmol. PET studies in baboon show that [11C]6 penetrates the BBB and accumulates in brain. No detectable specific binding was observed, likely due to the rapid metabolism or low density of CRF1 receptors in primate brain.     

Radiosynthesis and in vivo evaluation of [11C]MC80 for P-glycoprotein imaging

P-glycoprotein (P-gp) is an ATP-dependent efflux pump protecting the body against xenobiotics. The in vitro characterized modulator 6,7-dimethoxy-2-(6-methoxy-naphthalen-2-ylmethyl)-1,2,3,4-tetrahydroisoquinoline (MC80) of the P-gp pump was labelled with ¹¹C and evaluated in vivo for its potential to image P-gp function and expression. Radiochemical pure (>98%) [¹¹C]MC80 was obtained within 25 min starting from [¹¹C]methyl iodide with radiochemical yield of 26%. Biodistribution studies in FVB mice demonstrated a high baseline brain uptake (7.66 ± 1.38%ID/g at 1 min pi). Cerebral uptake was increased in mdr1a knock-out mice as well as after CsA pretreatment. Pre-administration of an excess of non-radioactive MC80 caused a reduced uptake in several target organs including brain, pancreas and intestines. The results indicate that [¹¹C]MC80 kinetics are modulated by P-gp. Reversed phase-HPLC analysis of brain revealed an excellent metabolic profile (>90% intact [¹¹C]MC80).     

Radiosynthesis and in vivo evaluation of [11C]MOV as a PET imaging agent for COX-2

Radiosynthesis and in vivo evaluation of [¹¹C]4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (methoxy analogue of valdecoxib, [¹¹C]MOV), a COX-2 inhibitor, was conducted in rat and baboon. Synthesis of the reference standard MOV (3), and its desmethyl precursor 2 for radiolabeling were performed using 1,2-diphenylethan-1-one as the starting material in five steps with 15% overall yield. Radiosynthesis of [¹¹C]MOV was accomplished in 40?±?10% yield and?>99% radiochemical purity by reacting the precursor 2 in dimethyl formamide (DMF) with [¹¹C]CH₃I followed by removal of the dimethoxytrityl (DMT) protective group using trifluroacetic acid. PET studies in anesthetized baboon showed very low uptake and homogeneous distribution of [¹¹C]MOV in brain. The radioligand underwent rapid metabolism in baboon plasma. MicroPET studies in male Sprague Dawley rats revealed [¹¹C]MOV binding in lower thorax. The tracer binding in rats was partially blocked in heart and duodenum by the administration of 1?mg/kg oral dose of COX-2 inhibitor valdecoxib.     

Facile synthesis of [11C]edrophonium and its analogues as new potential PET imaging agents for heart acetylcholinesterase

[11C]Edrophonium and its analogues have been synthesized for evaluation as new potential positron emission tomography (PET) imaging agents for heart acetylcholinesterase. The tracers were prepared by N-[11C]methylation of precursors using [11C]methyl triflate and isolated by solid-phase extraction (SPE) purification procedure in 50-65% radiochemical yields.     

Synthesis and biodistribution of [11C]A-836339, a new potential radioligand for PET imaging of cannabinoid type 2 receptors (CB2)

Recently, A-836339 [2,2,3,3-tetramethylcyclopropanecarboxylic acid [3-(2-methoxyethyl)-4,5-dimethyl-3H-thiazol-(2Z)-ylidene]amide] (1) was reported to be a selective CB₂ agonist with high binding affinity. Here we describe the radiosynthesis of [¹¹C]A-836339 ([¹¹C]1) via its desmethyl precursor as a candidate radioligand for imaging CB₂ receptors with positron-emission tomography (PET). Whole body and the regional brain distribution of [¹¹C]1 in control CD1 mice demonstrated that this radioligand exhibits specific uptake in the CB₂-rich spleen and little specific in vivo binding in the control mouse brain. However, [¹¹C]1 shows specific cerebral uptake in the lipopolysaccharide (LPS)-induced mouse model of neuroinflammation and in the brain areas with Aβ amyloid plaque deposition in a mouse model of Alzheimer’s disease (APPswe/PS1dE9 mice). These data establish a proof of principle that CB₂ receptors binding in the neuroinflammation and related disorders can be measured in vivo.     

Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain

2-((4-(1-[(11)C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC(50)=0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [(11)C]CH(3)I, ～45% yield, >92% radiochemical purity, >370 GBq/μmol specific activity at end of bombardment (EOB). Evaluation in Sprague-Dawley rats revealed that [(11)C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [(11)C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood-brain-barrier may limit the clinical utility of [(11)C]MP-10 as a PDE10A PET tracer.     

Synthesis,in vitro and in vivo evaluation of [11C]MMTP: A potential PET ligand for mGluR1 receptors

Synthesis, in vitro and in vivo evaluation of [O-methyl-¹¹C]dimethylamino-3(4-methoxyphenyl)-3H-pyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4-one (1), a potential imaging agent for mGluR1 receptors using PET are described. Synthesis of the corresponding desmethyl precursor 2 was achieved by demethylation of the methoxyphenyl compound 1 in 90% yield. Methylation using [¹¹C]MeOTf in presence of NaOH afforded [¹¹C]1 in 30% yield (EOS) with >99% chemical and radiochemical purities and with a specific activity of 3–5 Ci/μmol (n = 6). The total synthesis time was 30 min from EOB. The radiotracer selectively labeled mGluR1 receptors in slide-mounted sections of postmortem human brain containing cerebellum, hippocampus, prefrontal cortex and striatum as demonstrated by in vitro autoradiography using phosphor-imaging. PET studies in anesthetized baboon show that [¹¹C]1 penetrates the BBB and accumulates in cerebellum, a region reported to have higher expression of mGluR1. These findings suggest [¹¹C]1 is a promising PET radiotracer candidate for mGluR1.     

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.     

Radiosynthesis of carbon-11-labeled camptothecin derivatives as potential positron emission tomography tracers for imaging of topoisomerase I in cancers

Four carbon-11-labeled camptothecin derivatives, 9-[11C]methoxy-20(S)-camptothecin ([11C]5), 10-[11C]methoxy-20(S)-camptothecin ([11C]7), 9-nitro-10-[11C]methoxy-20(S)-camptothecin ([11C]9), and 9-[([11C]trimethylamino)methyl]-10-hydroxy-20(S)-camptothecin ([11C]11), have been synthesized as potential positron emission tomography tracers for imaging of topoisomerase I in cancers.     

[11C]Olanzapine,radiosynthesis and lipophilicity of a new potential PET 5-HT2 and D2 receptor radioligand

Olanzapine and its precursor desmethyl-Olanzapine were synthesized from malononitrile, propionaldehyde, 1-fluoro-2-nitrobenzene, and substituted piperazine in 4, 4, 5, and 5 steps with 35%, 32%, 26%, and 32% overall chemical yield, respectively. [¹¹C]Olanzapine was prepared from desmethyl-Olanzapine with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 40–50% radiochemical yield based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB. The calculated Log P (C Log P) value of [¹¹C]Olanzapine is 3.39.     

Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation

Synthesis of [(11)C]celecoxib, a selective COX-2 inhibitor, and [(11)C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug transporter in biliary excretion was evaluated. The synthesis of [(11)C]celecoxib was achieved in one-pot by reacting [(11)C]methyl iodide with an excess of the corresponding pinacol borate precursor using Pd(2)(dba)(3), P(o-tolyl)(3), and K(2)CO(3) (1:4:9) in DMF. The radiochemical yield of [(11)C]celecoxib was 63±23% (decay-corrected, based on [(11)C]CH(3)I) (n=7) with a specific radioactivity of 83±23GBq/μmol (n=7). The average time of synthesis from end of bombardment including formulation was 30min with >99% radiochemical purity. [(11)C]SC-62807 was synthesized from [(11)C]celecoxib by further rapid oxidation in the presence of excess KMnO(4) with microwave irradiation. The radiochemical yield of [(11)C]SC-62807 was 55±9% (n=3) (decay-corrected, based on [(11)C]celecoxib) with a specific radioactivity of 39±4GBq/μmol (n=3). The average time of synthesis from [(11)C]celecoxib including formulation was 20min and the radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [(11)C]celecoxib and [(11)C]SC-62807 showed largely different excretion processes, and consequently, [(11)C]SC-62807 was rapidly excreted via hepatobiliary excretion without further metabolism. [(11)C]SC-62807 was shown to have a high potential as a PET probe for evaluating drug transporter function in biliary excretion.