David L. Gildersleeve

High yield synthesis of high specific activity R-(−)-[11C]epinephrine for routine PET studies in humans

R-(-)-[11C]Epinephrine ([11C]EPI) has been synthesized from R-(-)-norepinephrine by direct methylation with [11C]methyl iodide or [11C]methyl triflate. The total synthesis time including HPLC purification was 35-40 min. The radiochemical yields (EOB) were 5-10% for [11C]methyl iodide and 15-25% for [11C]methyl triflate. Radiochemical purity was > 98%; optical purity determined by radio-chiral HPLC was > 97%. The [11C]methyl triflate technique produces R-(-)-[11C]epinephrine in quantities (80-170 mCi) sufficient for multiple positron emission tomography studies in humans. The two synthetic methods are generally applicable to the production of other N-[11C]methyl phenolamines and N-[11C]methyl catecholamines.

Mouse brain distribution of a carbon-11 labeled vesamicol derivative: Presynaptic marker of cholinergic neurons

The regional mouse brain distribution of a new carbon-11 labeled derivative of vesamicol, [11C]-5-(N-methylamino)benzovesamicol [( 11C]MABV) is reported. Radiotracer concentrations in vivo are in the rank order of striatum greater than cortex greater than hippocampus greater than hypothalamus greater than cerebellum, consistent with reported distributions of other presynaptic cholinergic neuronal markers. In time course studies, striatum/cerebellum and cortex/cerebellum ratios for (-)-[11C]MABV continue to increase to values of 13 and 5, respectively, 75 min after i.v. injection of [11C]MABV. The specific binding in striatum and cortex is lowered by pretreatment with (+/-)-vesamicol, and shows stereoselectivity with lower uptake and lower ratios for the (+)-enantiomer. (-)-enantiomer. (-)-[11C]MABV is proposed as a positron-emitting radioligand for the in vivo study of presynaptic cholinergic neurons.

Synthesis and evaluation of [123I]-Iodo-PK11195 for mapping peripheral-type benzodiazepine receptors (ω3) in heart

Abstract An iodinated analog of PK11195, 1-(2-chlorophenyl)- N -methyl- N -(1-methylpropyl)isoquinoline-3-carboxamide, a specific antagonist of the peripheral-type benzodiazepine receptor (ω 3 ), has been synthesized in three steps with an overall chemical yield of 40%. Both [ 123 I]- and [ 125 I]-Iodo-PK11195 have been synthesized by solid-state isotopic exchange in >60% isolated radiochemical yield and specific activity of 233–348 mCi/mmol. Tissue distribution studies in rats indicate a high uptake of radioactivity in adrenal glands, heart, lung and kidneys, which was blocked 63–87% by preadministration of cold PK11195. Single photon emission computer tomography (SPECT) imaging of the canine heart has been accomplished with [ 123 I]PK11195. These results suggest that [ 123 I]PK11195 has potential as a SPECT radiotracer for studying the ω 3 receptor in humans.

Synthesis of a high specific activity 125I-labeled analog of PK 11195, potential agent for SPECT imaging of the peripheral benzodiazepine binding site

The peripheral benzodiazepine binding site ligand PK 11195 has been 125I-labeled by direct displacement of aromatic chlorine under solid-state conditions in 50-76% radiochemical yield and greater than 94% radiochemical purity. Purification by high pressure liquid chromatography increased the specific activity of the product from an initial 15-17 Ci/mmol to a final activity of 260-910 Ci/mmol. To determine the affinity of this [125I]PK 11195 analog for human glioma cells, saturation experiments were performed on monolayers of U251 human glioblastoma cells. Scatchard analysis of saturation data demonstrated that the [125I]PK 11195 analog binds to a single class of sites with a KD of 8.0 +/- 1.7 nM and maximal binding of 3.8 +/- 0.1 pmol/mg protein. These values are similar to those obtained when [3H]PK 11195 was assayed in U251 cells (KD = 14 +/- 3.4, Bmax = 4.1 +/- 1.3) suggesting that iodination does not appreciably alter the binding of PK 11195 to human glioma cells. In vivo autoradiographic studies of brain in C6 glioma bearing rats demonstrate selective binding of the radioligand to the tumor. These results suggest that this [125I]PK 11195 analog may be a useful radiotracer for the study of peripheral benzodiazepine binding sites.

Synthesis of the 123I- and 125I-labeled cholinergic nerve marker (-)-5-iodobenzovesamicol

The highly toxic curraremimetic and cholinergic neuron marker (-)-5-iodobenzovesamicol (IBVM) has been labeled with iodine-125 and iodine-123. [125I]IBVM, suitable for animal distribution and ex vivo autoradiographic studies, was synthesized by solid-state exchange; isolated yields were 65-89% with specific activities in the range of 130-200 Ci/mmol. The synthesis of no-carrier-added (-)-5-[125I]IBVM from the corresponding chiral (-)-5-(tri-n-butyltin) derivative using Na125I was evaluated using the oxidants H2O2, peracetic acid and chloramine-T. Both peracetic acid and chloramine-T gave good yields (70-95%). However, when Na123I was utilized, acceptable yields of [123I]IBVM were obtained only with chloramine-T. Use of the latter oxidant did produce 5-chlorobenzovesamicol which was eliminated during HPLC purification. After optimization of the reaction parameters, [123I]IBVM in batch sizes of 10-27 mCi, is routinely obtained with a specific activity of 30-70,000 Ci/mmol, radiochemical purity (> 97%) and chiral purity (> 98%). Isolated radiochemical yields have averaged 71% (N = 40). Distribution analyses of [125I]IBVM and [123I]IBVM in mice 4 h following intravenous administration show essentially equivalent concentrations of the two tracers in the four brain regions sampled. The exceptionally high specific activity of [123I]IBVM has made possible the evaluation of this radiotracer in humans.

NMDA receptor channels: Labeling of MK-801 with iodine-125 and fluorine-18

Methods for labeling the glutamate channel blocking agent MK-801 with iodine-125 (125I) and fluorine-18 (18F) are described. Radioiodine was incorporated in the 1- or 3-positions of the aromatic ring of (+/-)MK-801 by solid-state halogen exchange techniques. Attachment of the [18F]fluoromethyl group to the bridgehead methyl position was achieved by reaction of [18F]fluoride with the triflamide alcohol 8 or the novel cyclic sulfamate 9 recently reported by Merck chemists. Radiochemical yields of (+/-) 13-[18F]-fluoromethyl-MK-801 were greater than 72%, EOB; radiochemical purity greater than 99%. In competitive binding studies using rat brain homogenates, (+/-)3-bromo-MK-801 showed greater affinity than (+/-)MK-801 for the glutamate-linked channel. The experimental log P (2.1 +/- 0.1) of MK-801 is optimal for transit of the blood-brain barrier. These preliminary findings support further testing of 3-[123 I]iodo-MK-801 and (+/-)13-[18F]fluoromethyl-MK-801 as possible agents for in vivo mapping of the glutamate receptor complex.

Adrenal cortical 11β-hydroxylase and side-chain cleavage enzymes. requirement for the A- or B-pyridyl ring in metyrapone for inhibition

The adrenal cortical enzyme systems, 11 beta-hydroxylase, P-450 11 beta, and the side-chain cleavage complex, P-450 scc, differ only in their cytochrome P-450s. Structural modifications of metyrapone, an inhibitor of cytochrome P-450 enzyme systems, have been made to determine the requirement for the A- or B-pyridyl ring for inhibition of P-45011 beta and P-450 scc activities. Three new analogs of metyrapone (A-phenylmetyrapone, B-phenylmetyrapone and diphenylmetyrapone) were synthesized and evaluated as inhibitors using a crude, defatted bovine adrenal cortical mitochondrial preparation. Characterization of the mitochondrial preparation demonstrated: enhancement of both activities by the addition of 15.0 microM adrenodoxin, the addition of 1% ethanol decreased both activities less than 10%, and the apparent Km of deoxycorticosterone for P-45011 beta was 6.8 microM and the apparent Km of cholesterol for P-450 scc was 21.6 microM. Inhibition of P-45011 beta and P-450 scc activities with these compounds demonstrated: the B-pyridyl ring of metyrapone is required for inhibition of both activities whereas requirement for the A-ring is less stringent, and the four metyrapone analogs were more selective inhibitors of P-45011 beta activity. These studies suggest that the A-phenyl metyrapone analog is a good candidate for further development of a selective adrenocortical radiopharmaceutical.

Synthesis of a nonpeptide carbon-11 labeled substance P antagonist for PET studies

CP 96,345 is a nonpeptide high affinity antagonist of the substance P (NK1) receptor. The radiosynthesis of [11C]CP 96,345 suitable for Positron Emission Tomography (PET) applications is described. [11C]CP 96,345 was prepared by O-methylation of a desmethyl precursor via in situ generation of its phenolate salt. The in vivo tissue distribution of [11C]CP 96,345 in guinea pigs (n = 2) at 5 and 30 min was determined. Uptake was low in brain (approximately 0.04% dose/g) and highest (approximately 1-2% dose/g) in the spleen and lungs. The present findings indicate that the use of [11C]CP 96,345 in PET might be more applicable to the study of substance P receptors in peripheral tissues involved with inflammatory disease and arthritis.

Metabolic Fate of the Heart Agent (18F)6-Fluorometaraminol

Studies were performed to determine whether [18F]6-fluorometaraminol (18F-FMR), a new neuronal heart radiopharmaceutical, is metabolized in vivo and if the metabolites are taken up in heart. Rat, dog, baboon and guinea pig were injected with 18F-FMR and tissue samples were analyzed for metabolites by HPLC. Liver contained the most metabolites of the tissues studied with 25-90% of the radioactivity present as metabolites at 1 h in all the species studied. While metabolites of 18F-FMR are found in blood, no significant accumulation of these metabolites is found in heart (less than or equal to 0.3%) 1 h after i.v. administration in any species except rat. These studies suggest that 18F-FMR is a suitable agent for quantitative imaging of the heart by positron emission tomography.

Direct optical resolution of vesamicol and a series of benzovesamicol analogues by high-performance liquid chromatography

The direct optical resolution of the vesicular acetylcholine uptake inhibitors vesamicol and benzovesamicol and nine benzovesamicol analogues were performed by HPLC on a commercially available cellulose tris(3,5-di-methylphenyl carbamate) chiral stationary phase. Separation of each enantiomeric pair was optimized with respect to solvent strength and flow-rate, using mobile phase mixtures of hexane-2-propanol-diethylamine. The method has been successfully applied to the analysis of the optical purity of benzovesamicol intermediates and products, including (-)-5-[123I]iodobenzovesamicol which is currently undergoing clinical evaluation as a tracer for mapping central cholinergic neurons, and the purification of both antipodes of (+/-)-7-[125I]iodobenzovesamicol.