Synthesis and evaluation of novel radioligands for positron emission tomography imaging of metabotropic glutamate receptor subtype 1 (mGluR1) in rodent brain

We designed three novel positron emission tomography ligands, N-(4-(6-(isopropylamino)pyrimidin-4-yl)-1,3-thiazol-2-yl)-4-[(11)C]methoxy-N-methylbenzamide ([(11)C]6), 4-[(18)F]fluoroethoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([(18)F]7), and 4-[(18)F]fluoropropoxy-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide ([(18)F]8), for imaging metabotropic glutamate receptor type 1 (mGluR1) in rodent brain. Unlabeled compound 6 was synthesized by benzoylation of 4-pyrimidinyl-2-methylaminothiazole 10, followed by reaction with isopropylamine. Removal of the methyl group in 6 gave phenol precursor 12 for radiosynthesis. Two fluoroalkoxy analogues 7 and 8 were prepared by reacting 12 with tosylates 13 and 14. Radioligands [(11)C]6, [(18)F]7, and [(18)F]8 were synthesized by O-[(11)C]methylation or [(18)F]fluoroalkylation of 12. Compound 6 showed high in vitro binding affinity for mGluR1, whereas 7 and 8 had weak affinity. Autoradiography using rat brain sections showed that [(11)C]6 binding is aligned with the reported distribution of mGluR1 with high specific binding in the cerebellum and thalamus. PET study with [(11)C]6 in rats showed high brain uptake and a similar distribution pattern to that in autoradiography, indicating the usefulness of [(11)C]6 for imaging brain mGluR1.     

Synthesis and simple 18F-labeling of 3-fluoro-5-(2-(2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile as a high affinity radioligand for imaging monkey brain metabotropic glutamate subtype-5 receptors with positron emission tomography

2-fluoromethyl analogs of (3-[(2-methyl-1,3-thiazol-4yl)ethynyl]pyridine) were synthesized as potential ligands for metabotropic glutamate subtype-5 receptors (mGluR5s). One of these, namely, 3-fluoro-5-(2-(2-(fluoromethyl)thiazol-4-yl)ethynyl)benzonitrile (3), was found to have exceptionally high affinity (IC50 = 36 pM) and potency in a phosphoinositol hydrolysis assay (IC50 = 0.714 pM) for mGluR5. Compound 3 was labeled with fluorine-18 (t1/2 = 109.7 min) in high radiochemical yield (87%) by treatment of its synthesized bromomethyl analog (17) with [18F]fluoride ion and its radioligand behavior was assessed with positron emission tomography (PET). Following intravenous injection of [18F]3 into rhesus monkey, radioactivity was avidly taken up into brain with high uptake in mGluR5 receptor-rich regions such as striata. [18F]3 was stable in monkey plasma and human whole blood in vitro and in monkey and human brain homogenates. In monkey in vivo, a single polar radiometabolite of [18F]3 appeared rapidly in plasma. [18F]3 merits further evaluation as a PET radioligand for mGluR5 in human subjects.     

Synthesis and nicotinic acetylcholine receptor in vivo binding properties of 2-fluoro-3-[2(S)-2-azetidinylmethoxy]pyridine: a new positron emission tomography ligand for nicotinic receptors.

The lead compound of a new series of 3-pyridyl ethers, the azetidine derivative A-85380 (3-[(S)-2-azetidinylmethoxy]pyridine), is a potent and selective ligand for the human alpha4beta2 nicotinic acetylcholine receptor (nAChR) subtype. In vitro, the fluoro derivative of A-85380 (2-fluoro-3-[(S)-2-azetidinylmethoxy]pyridine or F-A-85380) competitively displaced [3H]cytisine or [3H]epibatidine with Ki values of 48 and 46 pM, respectively. F-A-85380 has been labeled with the positron emitter fluorine-18 (t1/2 (half-life) = 110 min) by no-carrier-added nucleophilic aromatic substitution by K[18F]F-K222 complex with (3-[2(S)-N-(tert-butoxycarbonyl)-2-azetidinylmethoxy]pyridin-2-yl) tri methylammonium trifluoromethanesulfonate as a highly efficient labeling precursor, followed by TFA removal of the Boc protective group. The total synthesis time was 50-53 min from the end of cyclotron fluorine-18 production (EOB). Radiochemical yields, with respect to initial [18F]fluoride ion radioactivity, were 68-72% (decay-corrected) and 49-52% (non-decay-corrected), and the specific radioactivities at EOB were 4-7 Ci/micromol (148-259 GBq/micromol). In vivo characterization of [18F]F-A-85380 showed promising properties for PET imaging of central nAChRs. This compound does not bind in vivo to alpha7 nicotinic or 5HT3 receptors. Moreover, its cerebral uptake can be modulated by the synaptic concentration of the endogenous ligand acetylcholine. The preliminary PET experiments in baboons with [18F]F-A-85380 show an accumulation of the radiotracer in the brain within 60 min. In the thalamus, a nAChR-rich area, uptake of radioactivity reached a maximum at 60 min (4% I.D./100 mL of tissue). [18F]F-A-85380 appears to be a suitable radioligand for brain imaging nAChRs with PET.     

(3)H]ZM241385--an antagonist radioligand for adenosine A(2A) receptors in rat brain

Binding of the novel adenosine A(2A) receptor-selective antagonist radioligand [2-(3)H]-4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5,)triazin-5-yl amino]ethyl)phenol ([(3)H]ZM241385) was examined using particulate preparations and frozen sections of rat brain. In membranes from the rat striatum, binding was saturable, reversible and temperature-dependent. Analysis of saturation isotherms indicated that [(3)H]ZM241385 bound with high affinity (K(d) of 0.84 nM), high density (1680 fmol mg protein(-1)) and with a high proportion of specific binding (93% at 1 nM radioligand). Examination of competition profiles indicated that [(3)H]ZM241385 bound to sites with an A(2A) adenosine receptor-like rank order. The presence of guanosine 5'-(3-thio)-triphosphate failed to alter either [(3)H]ZM241385 binding or agonist competition for [(3)H]ZM241385 binding. Autoradiographic analysis of [(3)H]ZM241385 binding to frozen sections of rat brain indicated specific binding to the rat striatum of similar affinity (K(d) of 0.43 nM) and susceptibility to adenosine receptor ligands. At 2 nM [(3)H]ZM241385, specific binding comprised 95+/-1% total binding. In the hippocampus and frontal cortex, binding of [(3)H]ZM241385 failed to saturate and was of lower density. Taken together, these results indicate that [(3)H]ZM241385 should prove to be a useful radioligand in the characterisation of adenosine A(2A) receptors.     

Development of a new radioligand, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[18F]fluoroethyl-5-methoxybenzyl)acetamide, for pet imaging of peripheral benzodiazepine receptor in primate brain

To develop a positron emission tomography (PET) ligand for imaging the 'peripheral benzodiazepine receptor' (PBR) in brain and elucidating the relationship between PBR and brain diseases, four analogues (4-7) of N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide (2) were synthesized and evaluated as ligands for PBR. Of these compounds, fluoromethyl (4) and fluoroethyl (5) analogues had similar or higher affinities for PBR than the parent compound 2 (K(i) = 0.16 nM for PBR in rat brain sections). Iodomethyl analogue 6 displayed a moderate affinity, whereas tosyloxyethyl analogue 7 had weak affinity. Radiolabeling was performed for the fluoroalkyl analogues 4 and 5 using fluorine-18 ((18)F, beta(+); 96.7%, T(1/2) = 109.8 min). Ligands [(18)F]4 and [(18)F]5 were respectively synthesized by the alkylation of desmethyl precursor 3 with [(18)F]fluoromethyl iodide ([(18)F]8) and 2-[(18)F]fluoroethyl bromide ([(18)F]9). The distribution patterns of [(18)F]4 and [(18)F]5 in mice were consistent with the known distribution of PBR. However, compared with [(18)F]5, [(18)F]4 displayed a high uptake in the bone of mice. The PET image of [(18)F]4 for monkey brain also showed significant radioactivity in the bone, suggesting that this ligand was unstable for in vivo defluorination and was not a useful PET ligand. Ligand [(18)F]5 displayed a high uptake in monkey brain especially in the occipital cortex, a region with richer PBR than the other regions in the brain. The radioactivity level of [(18)F]5 in monkey brain was 1.5 times higher than that of [(11)C]2, and 6 times higher than that of (R)-(1-(2-chlorophenyl)-N-[(11)C]methyl,N-(1-methylpropyl)isoquinoline ([(11)C]1). Moreover, the in vivo binding of [(18)F]5 was significantly inhibited by PBR-selective 2 or 1, indicating that the binding of [(18)F]5 in the monkey brain was mainly due to PBR. Metabolite analysis revealed that [(18)F]4 was rapidly metabolized by defluorination to [(18)F]F(-) in the plasma and brain of mice, whereas [(18)F]5 was metabolized by debenzylation to a polar product [(18)F]13 only in the plasma. No radioactive metabolite of [(18)F]5 was detected in the mouse brain. The biological data indicate that [(18)F]5 is a useful PET ligand for PBR and is currently used for imaging PBR in human brain.     

5-substituted derivatives of 6-halogeno-3-((2-(S)-azetidinyl)methoxy)pyridine and 6-halogeno-3-((2-(S)-pyrrolidinyl)methoxy)pyridine with low picomolar affinity for alpha4beta2 nicotinic acetylcholine receptor and wide range of lipophilicity: potential probes for imaging with positron emission tomography

Potential positron emission tomography (PET) ligands with low picomolar affinity at the nicotinic acetylcholine receptor (nAChR) and with lipophilicity (log D) ranging from -1.6 to +1.5 have been synthesized. Most members of the series, which are derivatives of 5-substituted-6-halogeno-A-85380, exhibited a higher binding affinity at alpha4beta2-nAChRs than epibatidine. An analysis, by molecular modeling, revealed an important role of the orientation of the additional heterocyclic ring on the binding affinity of the ligands with nAChRs. The existing nicotinic pharmacophore models do not accommodate this finding. Two compounds of the series, 6-[(18)F]fluoro-5-(pyridin-3-yl)-A-85380 ([(18)F]31) and 6-chloro-3-((2-(S)-azetidinyl)methoxy)-5-(2-[(18)F]fluoropyridin-5-yl)pyridine) ([(18)F]35), were radiolabeled with (18)F. Comparison of PET data for [(18)F]31 and 2-[(18)F]FA shows the influence of lipophilicity on the binding potential. Our recent PET studies with [(18)F]35 demonstrated that its binding potential values in Rhesus monkey brain were ca. 2.5 times those of 2-[(18)F]FA. Therefore, [(18)F]35 and several other members of the series, when radiolabeled, will be suitable for quantitative imaging of extrathalamic nAChRs.     

Synthesis of three novel fluorine-18 labeled analogues of L-deprenyl for positron emission tomography (PET) studies of monoamine oxidase B (MAO-B)

The aim in this project was to synthesize and to study fluorine-18 labeled analogues of l-deprenyl which bind selectively to the enzyme monoamine oxidase B (MAO-B). Three fluorinated l-deprenyl analogues have been generated in multistep organic syntheses. The most promising fluorine-18 compound N-[(2S)-1-[(18)F]fluoro-3-phenylpropan-2-yl]-N-methylprop-2-yn-1-amine (4c) was synthesized by a one-step fluorine-18 nucleophilic substitution reaction. Autoradiography on human brain tissue sections demonstrated specific binding for compound 4c to brain regions known to have a high content of MAO-B. In addition, the corresponding nonradioactive fluorine-19 compound (13) inhibited recombinant human MAO-B with an IC(50) of 170.5 ± 29 nM but did not inhibit recombinant human MAO-A (IC(50) > 2000 nM), demonstrating its specificity. Biodistribution of 4c in mice showed high initial brain uptake leveling at 5.2 ± 0.04%ID/g after 2 min post injection. In conclusion, compound 4c is a specific inhibitor of MAO-B with high initial brain uptake in mice and is, therefore, a candidate for further investigation in PET.     

Evaluation of 18F-labeled benzodioxine piperazine-based dopamine D4 receptor ligands: lipophilicity as a determinate of nonspecific binding

Derivatization of the putative neuroleptic 1-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-4-(4-fluorobenzyl)piperazine (3a) led to a series of new dopamine receptor D4 ligands displaying high affinity (Ki=1.1-15 nM) and D2/D4 subtype selectivities of about 800-6700. These ligands were labeled with the short-lived positron emitter fluorine-18 and analyzed for their potential application for imaging studies by positron emission tomography (PET). In vitro autoradiography was used to determine their nonspecific binding behavior as a result of their structural and thus physicochemical properties. The biodistribution, in vivo stability, and brain uptake of the most promising D4 radioligand candidate were determined. This proved to be 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4-((6-fluoropyridin-3-yl)methyl)piperazine ([18F]3d), which revealed an excellent binding pattern with a high selectivity and limited nonspecific binding in vitro. This analogue also exhibited a high stability and an extremely high brain uptake in vivo with specific binding in hippocampus, cortex, colliculus, and cerebellum as determined by ex vivo autoradiography. Thus, [18F]3d appears as a suitable D4 radioligand for in vivo imaging, encouraging continued evaluation by PET studies.     

Synthesis and evaluation of no-carrier-added 8-cyclopentyl-3-(3-[(18)F]fluoropropyl)-1-propylxanthine ([(18)F]CPFPX): a potent and selective A(1)-adenosine receptor antagonist for in vivo imaging

This report describes the precursor synthesis and the no-carrier-added (nca) radiosynthesis of the new A(1) adenosine receptor (A(1)AR) antagonist [(18)F]8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine (CPFPX), 3, with fluorine-18 (half-life = 109.6 min). Nucleophilic radiofluorination of the precursor tosylate 8-cyclopentyl-3-(3-tosyloxypropyl)-7-pivaloyloxymethyl-1-propylxanthine, 2, with nca [(18)F]KF under aminopolyether-mediated conditions (Kryptofix 2.2.2/K(2)CO(3)) followed by deprotection was straightforward and, after formulation, gave the radioligand ready for injection with a radiochemical yield of 45 +/- 7%, a radiochemical purity of >98% and a specific radioactivity of >270 GBq/micromol (>7.2 Ci/micromol). Preparation time averaged 55 min. The synthesis proved reliable for high batch yields ( approximately 7.5 GBq) in routine production (n = 120 runs). The radiotracer was pharmacologically evaluated in vitro and in vivo and its pharmacokinetics in rodents determined in detail. After iv injection a high accumulation of radioactivity occurred in several regions of mouse brain including thalamus, striatum, cortex, and cerebellum. Antagonism by the specific A(1)AR antagonists 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N(6)-cyclopentyl-9-methyladenine (N-0840), but not with the A(2)AR antagonist 3,7-dimethyl-1-propargylxanthine (DMPX), indicated specific and reversible binding of the radioligand to A(1)AR in cortical and subcortical regions of interest. In mouse blood at least two polar metabolites formed rapidly (50% at 5 min after tracer application). However, chromatographic analyses of brain homogenate extracts taken 60 min pi showed that >98% of radioactivity was unchanged radioligand. Chromatographic isolation and reinjection of peripherally formed radioactive metabolites revealed no accumulation of radioactivity in mouse brain, probably due to the polarity of the metabolites. These preliminary results suggest that nca [(18)F]CPFPX is a useful radioligand for the noninvasive imaging of the brain A(1)AR.     

Discovery of (-)-7-methyl-2-exo-[3'-(6-[18F]fluoropyridin-2-yl)-5'-pyridinyl]-7-azabicyclo[2.2.1]heptane, a radiolabeled antagonist for cerebral nicotinic acetylcholine receptor (alpha4beta2-nAChR) with optimal positron emission tomography imaging properties

Several isomers of 7-methyl-2-exo-([(18)F]fluoropyridinyl-5'-pyridinyl)-7-azabicyclo[2.2.1]heptane have been developed as radioligands with optimized brain kinetics for PET imaging of nAChR. The binding assay demonstrated that all isomers are beta-nAChR selective ligands with Ki = 0.02-0.3 nM. The experimental lipophilicity values of all isomers were in the optimal range for the cerebral radioligands (log D7.4= 0.67-0.99). The isomers with higher binding affinity manifested slow baboon brain kinetics, whereas the isomer with the lowest binding affinity (Ki = 0.3 nM) ((-)-7-methyl-2- exo-[3'-(6-[(18)F]fluoropyridin-2-yl)-5'-pyridinyl]-7-azabicyclo[2.2.1]heptane, [(18)F](-)-6c) and greatest lipophilicity (log D 7.4 = 0.99) exhibited optimal brain kinetics. [(18)F](-)-6c manifests a unique combination of the optimally rapid brain kinetics, high BP and brain uptake, and favorable metabolic profile. Pharmacological studies showed that (-)-6c is an alpha4beta2-nAChR antagonist with low side effects in mice. This combination of imaging properties suggests that [(18)F]-(-)- 6c is a potentially superior replacement for 2-[(18)F]fluoro-A-85380 and 6-[(18)F]fluoro-A-85380, the only available nAChR PET radioligands for humans.     

Regional brain uptake of the muscarinic ligand, [18F]FP-TZTP,is greatly decreased in M2 receptor knockout mice but not in M1, M3 and M4 receptor knockout mice

A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.     

N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[(131)I]iodo-5-me thoxybenzyl)acetamide: a potent iodinated radioligand for the peripheral-type benzodiazepine receptor in brain

To image the peripheral-type benzodiazepine receptor (PBR) in vivo, we previously developed two positron emission tomography (PET) ligands, N-(2-[11C],5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl)acetamide ([11C]1a) and its [18F]fluoroethyl analogue ([18F]1b), for the investigation of PBR in the living human brain. This time, using 1a as a leading compound, we designed two novel iodinated analogues, N-(5-fluoro-2-phenoxyphenyl)-N-(2-iodo-5-methoxybenzyl)acetamide (3a) and N-(2,5-dimethoxybenzyl)-N-(5-iodo-2-phenoxyphenyl)acetamide (3b) for the PBR imaging. Ligands 3 were synthesized by the iodination of tributystannyl precursors 10. Radiolabeling for 3 with 131I was carried out by the reaction of 10 with [131I]NaI using H2O2 as an oxidizing agent. In vitro competition experiments determined that 3a exhibited both high affinity and selectivity for PBR (IC50: 7.8 nM) vs CBR (>1 microM). Biodistribution study in mice determined that [131I]3a had a high radioactivity level (1.69% dose/g) in the brain, and its distribution pattern in the brain was consistent with the known distribution of PBR in rodents. Ex vivo autoradiography of the rat brain gave visual evidence that [131I]3a was a potent and specific radioligand for PBR.     

The discovery of epidepride and its analogs as high-affinity radioligands for imaging extrastriatal dopamine D(2) receptors in human brain

[(123)I]Epidepride, [(18)F]fallypride, and [(76)Br]isoremoxipride (FLB-457) and their corresponding [(11)C]labeled derivatives belong to a class of high-affinity radioligands for SPECT or PET imaging of dopamine D(2) receptors in the human brain. In contrast to previously used imaging agents, these ligands are capable of identifying extrastriatal dopamine D(2) receptors. The design of these substituted benzamides derive its origin from the atypical antipsychotic agent, remoxipride. Starting in the late 1970's, halogenated analogs of (S)-sulpiride were evaluated in binding assays and behavioral studies, leading to the discovery of remoxipride. Remoxipride was 10 times weaker than sulpiride in vitro but 50 times more potent in vivo. Search for a putative active metabolite of remoxipride led to the discovery of raclopride and eticlopride, the former becoming a useful radioligand as tritium or carbon-11 labeled form for receptor binding and PET studies, respectively. In the US, the mono-iodine analog of raclopride, [(123)I]iodobenzamide (IBZM), was found to have moderate putamen-to-cerebellum ratio in rat and human brain. Continued search for metabolites of remoxipride led to the discovery of its 3,6-dihydroxy derivative, NCQ-344, with an extremely potent in vivo activity in the rat. SAR studies of the metabolites of remoxipride led to the discovery of the 3-methoxy isomer, isoremoxipride (FLB-457) and its corresponding 6-hydroxy analog, FLB-463, both having affinities for the dopamine D(2) receptor in the 20-30 pM range. Later, the 5-[(123)I]iodo analog of FLB-463, [(123)I]ioxipride ([(123)I]NCQ-298), became a potential SPECT imaging agent. In the mean time, the deshydroxy analog of IBZM, [(125)I]iodopride, showed binding potential in the rat similar to [(125)I]IBZM. Epidepride was designed by combining the structure of isoremoxipride with that of iodopride. In 1988, epidepride was independently prepared and radiolabeled in three separate laboratories in Stockholm, Berkeley, and Nashville. Evaluation of seven [(125)I]iodine substituted analogs of raclopride, including IBZM, revealed the unusual high striatum-to-cerebellum ratio of 234 of [(125)I]epidepride in the rat. Subsequent SPECT images with [(123)I]epidepride demonstrated its ability to identify extrastriatal dopamine D(2) receptors in the human brain. Exploration of the structure of epidepride confirmed its exceptional properties, to be exceeded only by its N-allyl homolog, [(125)I]nalepride. The design by others of a series of potent 5-(3-[(18)F]fluoropropyl) substituted analogs of epidepride for PET imaging, lead to the discovery of [(18)F]fallypride. By elucidating the role of lipophilicity in the substituted benzamides, the excellent imaging characteristics of [(11)C]/[(123)I]epidepride, [(11)C]/[(76)Br]isoremoxipride and [(18)F]fallypride, could not only be explained but predicted with remarkable accuracy. By using the inverse product of the receptor affinity (K(D)), and the apparent partition constant of the radioligand (P((7.4))), estimates of maximal binding potential of any radioligand for imaging of any neurotransmitter receptor or transporter site seem possible.     

Development of LC-MS/MS-based receptor occupancy tracers and positron emission tomography radioligands for the nociceptin/orphanin FQ (NOP) receptor

Currently, a lack of sufficient tools has limited the understanding of the relationship between neuropsychiatric disorders and the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor. Herein, we describe the discovery and development of an antagonist NOP receptor occupancy (RO) tracer and a novel positron emission tomography (PET) radioligand suitable to probe the NOP receptor in human clinical studies. A thorough structure-activity relationship (SAR) around the high-affinity 3-(2'-fluoro-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)-2-(2-halobenzyl)-N-alkylpropanamide scaffold identified a series of subnanomolar, highly selective NOP antagonists. Subsequently, these unlabeled NOP ligands were evaluated in vivo by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in rat to determine brain uptake, kinetics and specific binding. (S)-27 was identified as a suitable unlabeled preclinical RO tracer to accurately quantify NOP receptor engagement in rat brain. Three compounds were selected for evaluation in nonhuman primates as PET tracers: (-)-26, (-)-30, and (-)-33. Carbon-11 labeling of (+)-31 yielded [(11)C]-(S)-30, which exhibited minimal generation of central nervous system (CNS) penetrant radiometabolites, improved brain uptake, and was an excellent PET radioligand in both rat and monkey. Currently [(11)C]-(S)-30 is being evaluated as a PET radiotracer for the NOP receptor in human subjects.     

2-(4,5-dihydro-1H-imidazol-2-yl)indazole (indazim) derivatives as selective I(2) imidazoline receptor ligands.

A series of variously substituted 2-(4,5-dihydro-1H-imidazol-2-yl)indazoles 3a-j and 2-(4,5-dihydro-1H-imidazol-2-yl)-4,5,6,7-tetrahydroindazole 6 were prepared by the regiospecific heteroalkylation of corresponding indazoles 1a-k with 2-chloro-4,5-dihydroimidazole (2). Their affinity to imidazoline I(2) receptors and alpha(2)-adrenergic receptors was determined by radioligand binding assay carried out on P(2) membrane preparations obtained from rat whole brains. 4-Chloro-2-(4,5-dihydro-1H-imidazol-2-yl)indazole (3f, 4-Cl-indazim) showed a 3076-fold difference in affinity for the [(3)H]2BFI-labeled imidazoline I(2) receptors relative to the [(3)H]RX821001-labeled alpha(2)-adrenergic receptors. This highly selective compound should prove to be useful tool in further understanding the functions of the imidazoline I(2) receptors.     

Development and evaluation of endothelin-A receptor (radio)ligands for positron emission tomography

The expression and function of endothelin (ET) receptors are abnormal in cardiovascular diseases, tumor progression, and tumor metastasis. A previously reported promising radioligand for positron emission tomography (PET) based on the non-peptide ET(A) receptor antagonist PD 156707 showed specific binding to target receptors in the myocardium but high accumulation in bile and intestine, probably because of its high lipophilicity. In this study we describe the synthesis of a series of fluorinated derivatives with hydrophilic building blocks. All compounds were evaluated as high affinity ET(A) receptor ligands (16, 17, 23-26, K(i) = 1.4-7.9 nM) with high subtype selectivity over the ET(B) receptor. [(18)F]3-Benzo[1,3]dioxol-5-yl-4-{3-[1-(2-{2-[2-(2-fluoroethoxy)ethoxy]ethoxy}ethyl)-1H-[1,2,3]triazol-4-ylmethoxy]-4,5-dimethoxybenzyl}-5-hydroxy-5-(4-methoxyphenyl)-5H-furan-2-one ([(18)F]17) was synthesized as one of the radioligands of this series that possesses a higher hydrophilicity and an excellent stability in human serum. Improved clearance properties and specific uptake in target organs have been confirmed by biodistribution studies and small animal PET imaging.     

Synthesis of a potent and selective (18)F-labeled delta-opioid receptor antagonist derived from the Dmt-Tic pharmacophore for positron emission tomography imaging

Identification and pharmacological characterization of two new selective delta-opioid receptor antagonists, derived from the Dmt-Tic pharmacophore, of potential utility in positron emission tomography (PET) imaging are described. On the basis of its high delta selectivity, H-Dmt-Tic--Lys(Z)-OH (reference compound 1) is a useful starting point for the synthesis of (18)F-labeled compounds prepared by the coupling of N-succinimidyl 4-[ (18)F]fluorobenzoate ([(18)F]SFB) with Boc-Dmt-Tic--Lys(Z)-OH under slightly basic conditions at 37 degrees C for 15 min, deprotection with TFA, and HPLC purification. The total synthesis time was 120 min, and the decay-corrected radiochemical yield of [(18)F]- 1 was about 25-30% ( n = 5) starting from [(18)F]SFB ( n = 5) with an effective specific activity about 46 GBq/micromol. In vitro autoradiography studies showed prominent uptake of [ (18)F]- 1 in the striatum and cortex with significant blocking by 1 and UFP-501 (selective delta-opioid receptor antagonist), suggesting high specific binding of [(18)F]- 1 to delta-opioid receptors. Noninvasive microPET imaging studies revealed the absence of [(18)F]- 1 in rat brain, since it fails to cross the blood-brain barrier. This study demonstrates the suitability of [ (18)F]- 1 for imaging peripheral delta-opioid receptors.     

In vivo measurement of the serotonin transporter with (S)-([18F]fluoromethyl)-(+)-McN5652.

The radiolabeled serotonin transporter (SERT) ligand [(11)C](+)-McN5652 has recently been used in clinical positron emission tomography (PET) studies for SERT imaging. However, this radioligand offers disadvantages in routine clinical settings because of its short radioisotope half-life (eg PET facilities within hospitals without a cyclotron need to acquire such radioligands from distant cyclotron units for clinical use). S-([(18)F]fluoromethyl)-(+)-McN5652 ([(18)F](+)-FMe-McN5652) is an analogue which has been synthesized newly, and has a significantly longer radioisotope half-life. In the porcine brain, it demonstrates the same characteristic distribution pattern of serotonin-uptake sites like the (11)C-labeled congener with the highest binding in the midbrain and thalamus and the lowest in the cerebellum and occipital cortex. It shows a 30% higher blood-brain transfer and a slower peripheral metabolism than [(11)C](+)-McN5652. Rather uniform brain binding was observed after injection of the pharmacologically inactive radiolabeled enantiomer, or after pretreatment with the highly selective SERT inhibitor citalopram. The norepinephrine uptake inhibitor maprotiline did not show any inhibitory effect. Using a one-tissue compartment model (K(1), k"(2)) or a two-tissue compartment model (K(1) to k(4)) with or without constraints for calculation, the regional binding parameters of [(11)C](+)-McN5652 and [(18)F](+)-FMe-McN5652 are highly correlated among each other and with the SERT density, as determined by in vitro binding of [(3)H]citalopram. Using constraints to correct for the free fraction and nonspecific binding of the radiotracers, a considerable increase of the midbrain-occipital cortex ratios with higher values for [(18)F](+)-FMe-McN5652 compared to [(11)C](+)McN5652 was revealed. It is concluded that [(18)F](+)-FMe-McN5652 has better features than [(11)C](+)McN5652 for SERT imaging with PET.     

Novel fluorinated derivatives of the broad-spectrum MMP inhibitors N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](benzyl)- and (3-picolyl)-amino]-3-methyl-butanamide as potential tools for the molecular imaging of activated MMPs with PET

An approach to the in vivo imaging of locally upregulated and activated matrix metalloproteinases (MMPs) found in many pathological processes is offered by positron emission tomography (PET). Hence, appropriate PET radioligands for MMP imaging are required. Here, we describe the syntheses of novel fluorinated MMP inhibitors (MMPIs) based on lead structures of the broad-spectrum inhibitors N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](benzyl)-amino]-3-methyl-butanamide (CGS 25966) and N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)-amino]-3-methyl-butanamide (CGS 27023A). Additionally, tailor-made precursor compounds for radiolabeling with the positron-emitter 18F were synthesized. All prepared hydroxamate target compounds showed high in vitro MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13. As a consequence, the promising fluorinated hydroxamic acid derivative 1f was resynthesized in its 18F-labeled version via two different procedures yielding the potential PET radioligand [18F]1f. As expected, the biodistribution behavior of this novel compound and that of the more hydrophilic variant [18F]1j, also developed by our group, indicates that there was no tissue specific accumulation in wild-type (WT) mice.     

Synthesis, radiofluorination, and in vitro evaluation of pyrazolo[1,5-a]pyridine-based dopamine D4 receptor ligands: discovery of an inverse agonist radioligand for PET

A series of fluoro-substituted analogs structurally derived from the aminomethyl-substituted pyrazolo[1,5- a]pyridine lead compounds 9 (FAUC 113) and 10 (FAUC 213) were synthesized and evaluated as high-affinity D 4 receptor (D 4R) ligands ( 3a- 3h, K i = 1.3-28 nM). The para-fluoroethoxy-substituted derivatives 3f and 3h revealed an outstanding D 4 subtype selectivity of more than 3 orders of magnitude over both congeners D 2 and D 3 combined with inverse agonism at D 4R. The corresponding (18)F-labeled radioligands revealed high serum stability in vitro and log P values of 2-3. In vitro rat brain autoradiography showed specific binding of [ (18)F]3h in distinct brain regions, including the gyrus dentate of the hippocampus, that were inhibited by both eticlopride (65-80%) and the selective D 4R antagonist 10 (78-93%). The observed binding pattern was mainly consistent with the known D 4R distribution in the rat brain. Thus, [(18)F]3h (FAUC F41) represents a potential radioligand for studying the D 4R in vivo by positron emission tomography (PET).