Synthesis and in vitro evaluation of small-molecule [18F] labeled gonadotropin-releasing hormone (GnRH) receptor antagonists as potential PET imaging agents for GnRH receptor expression

Two novel small molecule gonadotropin-releasing hormone (GnRH) receptor antagonists (12 and 13) of the furamide-class were synthesized and evaluated in vitro for their receptor binding affinities for the rat GnRH receptor. Radiolabeling with no carrier added fluorine-18 of the appropriate precursors was investigated in a one-step reaction. Log P (Octanol/PBS pH 7.4) and serum stability of the compounds were investigated. The antagonists showed low nM affinity for the rat GnRH receptor. ¹⁸F-radiolabled compounds were obtained in high radiochemical purity (>95%) and specific activity (>75 GBq/μmol). These findings suggest this class of compounds holds promise as potential probes for PET targeting of GnRH-receptor expression.     

Synthesis and evaluation of radiolabeled antagonists for imaging of beta-adrenoceptors in the brain with PET.

Five potent, lipophilic beta-adrenoceptor antagonists (carvedilol, pindolol, toliprolol and fluorinated analogs of bupranolol and penbutolol) were labeled with either carbon-11 or fluorine-18 and evaluated for cerebral beta-adrenoceptor imaging in experimental animals. The standard radioligand for autoradiography of beta-adrenoceptors, [125I]-iodocyanopindolol, was also included in this survey. All compounds showed either very low uptake in rat brain or a regional distribution that was not related to beta-adrenoceptors, whereas some ligands did display specific binding in heart and lungs. Apparently, the criteria of a high affinity and a moderately high lipophilicity were insufficient to predict the suitability of beta-adrenergic antagonists for visualization of beta-adrenoceptors in the central nervous system.     

Fluorine-18 radiolabelling, biodistribution studies and preliminary PET evaluation of a new memantine derivative for imaging the NMDA receptor

A synthetic method has been established for preparing [18F]1-amino-3-fluoromethyl-5-methyl-adamantane ([18F]AFA). Biodistribution of the radiotracer in mice showed high brain uptake. The peak uptake (3.7% I.D/g organ) for the brain occurred at 30 min after injection. Accumulation of radioactivity in mouse brain was consistent with the known distribution of the NMDA receptors. The binding of [18F]AFA to the phencyclidine (PCP) binding sites of the NMDA receptor complex and the sigma recognition sites in a Rhesus monkey was also examined using positron emission tomography (PET). The regional brain distribution of [18F]AFA was changed by memantine and by (+)-MK-801, indicating competition for the same binding sites. Treatment with haloperidol caused a marked reduction of radioactivity uptake in all the brain regions examined. (-)-Butaclamol, which has pharmacological specificity for sigma sites, did not have any significant effects.     

Synthesis, in vitro and in vivo evaluation of fluorine-18 labelled FE-GW405833 as a PET tracer for type 2 cannabinoid receptor imaging

The type 2 cannabinoid receptor (CB?R) is part of the endocannabinoid system and is expressed in tissues related to the immune system. As the CB?R has a very low brain expression in non-pathological conditions, but is upregulated in activated microglia, it is an interesting target for visualization of neuroinflammation using positron emission tomography with a suitable radiolabeled CB?R ligand. In this study, we radiolabelled a fluoroethyl derivative of GW405833, a well known CB?R partial agonist, with fluorine-18 (half-life 109.8 min) by alkylation of the phenol precursor with 1-bromo-2-[1?F]fluoroethane. In vitro studies showed that FE-GW405833 behaved as a selective high affinity (27 nM) inverse agonist for hCB?R. [1?F]FE-GW405833 showed moderate initial brain uptake in mice and rats, but a slow washout from brain and plasma due to retention of a radiometabolite. Specific binding of the tracer to human CB?R was demonstrated in vivo in a rat model with local CB?R overexpression in the brain. Optimized derivatives of GW405833 that are less susceptible to metabolism will need to be developed in order to provide a useful tracer for CB?R quantification with PET.     

Synthesis and evaluation of 18F-labeled dopamine D3 receptor ligands as potential PET imaging agents

A series of fluoro substituted aryl carboxamides was synthesized revealing high affinity for the dopamine D3 receptor. In contrast to 2-methoxy substitution, a 2,3-dichloro substitution pattern at the phenylpiperazine moiety induces a 10-fold increase of D3 affinity which is expressed by Ki values of 0.53, 1.1, and 9.0 nM for 8b, 8d, and 8f. Applying aromatic 18F-for-Br(Cl) substitution, high radiochemical yields between 76-82% were obtained for [18F]8c-f. The most promising ligand, [18F]8d, was used as imaging agent of the D3 receptor in vitro. However, due to the lack of specific binding, further studies should aim at the development of radioligands with improved D3 receptor selectivity.     

Novel analogues of ketamine and phencyclidine as NMDA receptor antagonists

The identification of structurally novel analogues of ketamine and phencyclidine (PCP), as NMDA receptor antagonists, with low to moderate potency at GluN2A and GluN2B receptors is discussed. In particular, some examples, such as compounds 6 and 10, shows decreased calculated lipophilicity, when compared to PCP, while retaining moderate activity. Moreover, the germinal aryl amino substituted lactam ring, as exemplified in compounds 7-10 and 11-13, constitutes a novel scaffold with potential application in the design of biologically active compounds.     

Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.     

Synthesis and in vitro affinities of various MDL 100907 derivatives as potential 18F-radioligands for 5-HT2A receptor imaging with PET

Radiolabelled piperidine derivatives such as [¹¹C]MDL 100907 and [¹⁸F]altanserin have played an important role in diagnosing malfunction in the serotonergic neurotransmission. A variety of novel piperidine MDL 100907 derivatives, possible to label with ¹⁸F-fluorine, were synthesized to improve molecular imaging properties of [¹¹C]MDL 100907. Their in vitro affinities to a broad spectrum of neuroreceptors and their lipophilicities were determined and compared to the clinically used reference compounds MDL 100907 and altanserin. The novel compounds MA-1 (53) and (R)-MH.MZ (56) show K_i-values in the nanomolar range towards the 5-HT_2A receptor and insignificant binding to other 5-HT receptor subtypes or receptors. Interestingly, compounds MA-1 (53), MH.MZ (55) and (R)-MH.MZ (56) provide a receptor selectivity profile similar to MDL 100907. These compounds could possibly be preferable antagonistic ¹⁸F-tracers for visualization of the 5-HT_2A receptor status. Medium affine compounds (VK-1 (32), (51), (52), (54)) were synthesized and have K_i values between 30 and 120 nM. All promising compounds show log P values between 2 and 3, that is, within the range of those for the established radiotracers altanserin and MDL 100907. The novel compounds MA-1 (53) and (R)-MH.MZ (56) thus appear to be promising high affine and selective tracers of ¹⁸F-labelled analogues for 5-HT_2A imaging with PET.     

Novel bisbenzamidines and bisbenzimidazolines as noncompetitive NMDA receptor antagonists.

A series of novel bisbenzamidines and bisbenzimidazolines with different linkers connecting the aromatic groups was tested in vitro for NMDA receptor antagonist activity. IC50 values for these compounds ranged from 1.2 to >200 microM. The bisbenzamidine with a homopiperazine ring as the central linker was found to be the most potent NMDA receptor antagonist among all the pentamidine analogues tested so far.     

Synthesis and preliminary evaluation of a new fluorine-18 labelled triazine derivative for PET imaging of cannabinoid CB2 receptor

Cannabinoid CB2 PET tracers are considered as a promising alternative to PBR/TSPO tracers for the in-vivo imaging of neuroinflammation. We describe here the synthesis and characterization of compound 3, a new potent and brain penetrating CB2 ligand based on an original triazine template. The PET tracer [¹⁸F]-dideutero-3 was prepared in a three steps radiosynthesis, and demonstrated significant uptake in rhesus macaque and baboon brain with a maximum SUV of about 0.7–0.9 g/mL, followed by a moderate washout over time.     

Synthesis and in vitro evaluation of ambrisentan analogues as potential endothelin receptor antagonists

A series of novel 2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3,3-diphenyl butyric acid derivatives were synthesized and evaluated for their antagonistic activity for endothelin-1-induced contraction in rabbit aorta. Within this series of compounds, 2-[(4,6-dimethylpyrimidin-2-yl)oxy]-3-cyano-3,3-diphenylpropionic acid (4) displays comparable potency with ambrisentan (1), and warrants further investigation.     

Synthesis and evaluation of novel F-18 labeled fluoroarylvaline derivatives: Potential PET imaging agents for tumor detection

Two F-18 labeled fluoroarylvaline derivatives, methyl 2-(2-[¹⁸F]fluoro-4-nitrobenzamido)-3-methylbutanoate ([¹⁸F]1, [¹⁸F]MFNBMB) and its corresponding acid 2-(2-[¹⁸F]fluoro-4-nitrobenzamido)-3-methylbutanoic acid ([¹⁸F]2, [¹⁸F]FNBMBA), have been designed and synthesized, respectively, by our team. Meanwhile, we research on their biodistributions in mice model bearing S 180 tumor. Furthermore, we also carried out the biological evaluations of 2-[¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) and O-2-[¹⁸F]fluoroethyl-l-tyrosine (l-[¹⁸F]FET) in the same model for comparison with our targeting molecules [¹⁸F]1 and [¹⁸F]2. Excitingly, the tumor/blood (T/Bl) and tumor/brain (T/Br) ratios were 2.91, 7.06 at 30 min, 3.44, 5.61 at 60 min post injection for [¹⁸F]1, 2.32, 13.30 for [¹⁸F]2 at 30 min post injection, which were obviously superior to [¹⁸F]FDG and l-[¹⁸F]FET in the same model and demonstrated that [¹⁸F]1 and [¹⁸F]2, especially [¹⁸F]2, were potential PET imaging agents for tumor detection.     

Subtype selective NMDA receptor antagonists: evaluation of some novel alkynyl analogues

A benzylpiperidine analogue with an acetylenic linker, 5-(3-[4-(4-fluorobenzyl)-piperidin-1-yl]-prop-1-ynyl)-1,3-dihydrobenzimidazol-2-one (3), was identified as a chemical lead with excellent activity at the NR1A/2B receptor (IC50=3 nM). Efforts to optimize this activity led to focused modifications around the structural motif of 3. The synthesis and SAR studies are discussed.     

Synthesis and resolution of racemic eliprodil and evaluation of the enantiomers of eliprodil as NMDA receptor antagonists

A short synthesis of the NMDA receptor antagonist (rac)-Eliprodil (9) and its resolution into the enantiomers by chiral HPLC is described. The enantiomers (R)-9 and (S)-9 were found to exhibit markedly different affinities for NR2B subunit containing NMDA receptors.     

Synthesis of a fluorine-18 labeled derivative of epibatidine for in vivo nicotinic acetylcholine receptor PET imaging

Epibatidine (exo-2-(2'-chloro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a natural compound isolated from the skin of the Ecuadorian poison frog Epipedobates tricolor, is the most potent nicotinic acetylcholine receptor (nAChR) agonist reported to date. In order to visualize and quantify in vivo these receptors in human brain using Positron Emission Tomography (PET), [18F]norchlorofluoroepibatidine (exo-2-(2'-[18F]fluoro-5'-pyridyl)-7-azabicyclo[2.2.1]heptane), a fluorine-18 (t(1/2): 110 min) radiolabeled derivative of epibatidine has been designed. The corresponding 2'-bromo-, 2'-iodo- and 2'-nitro exo-2-(5'-pyridyl)-7-azabicyclo[2.2.1]heptane analogues as labeling precursors, as well as norchlorofluoroepibatidine as a reference compound have been synthesized by reductive, stereoselective, palladium-catalyzed Heck-type coupling between an N-Boc protected azanorbornene and the corresponding halopyridine. [18F]Norchlorofluoroepibatidine has been radiolabeled with fluorine-18 by nucleophilic aromatic substitution from the corresponding Boc-protected halo- and nitro precursors using [18F]FK-K222 complex in DMSO by conventional heating (at 150-180 degrees C for 10 min) or microwave activations (at 100 Watt, for 1 to 2.5 min), followed by TFA-removal of the protective group. Typically, using the microwave activation procedure, 60-80 mCi (2.22-2.96 GBq) of pure [18F]norchlorofluoroepibatidine could be obtained in less than 2 h (110-115 min) from the bromo labeling precursor, with specific radioactivities of 1.5-2.5 Ci/micromol (55.5-92.5 GBq/micromol) calculated for End of Bombardment. The preliminary PET experiments in baboon (Papio papio) with [18F]norchlorofluoroepibatidine show a high uptake and a rapid accumulation of the radiotracer into the brain within 30 min. In the thalamus, a nAChR rich area, uptake of radioactivity reached a maximum at 40 min (10% I.D./100 mL tissue). The ratio of radioactivity thalamus/cerebellum (the latter being a nAChR poor area) was 2 at 40 min and increased with time, up to 4.3 at 160 min. Its specific regiodistribution and its high ratio of specific-to-nonspecific binding confirm the ideal profile of [18F]norchlorofluoroepibatidine as a suitable radioligand for PET imaging of nAChRs in the brain.     

Synthesis of a new fluorine-18-labeled bexarotene analogue for PET imaging of retinoid X receptor

The reference standard 2-fluoro-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzoic acid was synthesized from 2,5-dimethyl-2,5-hexanediol and 2-fluoro-4-methylbenzoic acid in 10 steps with 3% overall chemical yield. The precursor 2-nitro-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzoic acid was synthesized from 2,5-dimethyl-2,5-hexanediol and dimethyl-2-nitroterephthalate in seven steps with 2% overall chemical yield. The target tracer 2-[¹⁸F]fluoro-4-(1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)vinyl)benzoic acid was synthesized from its nitro-precursor by the nucleophilic substitution with K[¹⁸F]F/Kryptofix 2.2.2 and isolated by HPLC combined with solid-phase extraction (SPE) purification in 20–30% radiochemical yield with 37–370 GBq/μmol specific activity at end of bombardment (EOB).     

Synthesis and in vitro evaluation of 18F labeled tyrosine derivatives as potential positron emission tomography (PET) imaging agents

Three new ¹⁸F labeled fluoroalkyl tyrosine derivatives, O-(2-[¹⁸F]fluoroethyl)-α-methyltyrosine (FEMT, [¹⁸F]2), O-(2-[¹⁸F]fluoroethyl)-2-l-azatyrosine (FEAT, [¹⁸F]3), O-(2-[¹⁸F]fluoroethyl)-l-tyrosineamide (FETA, [¹⁸F]4) have been synthesized and radiofluorinated with 5–34% decay-corrected yield. In vitro studies were carried out in U-138 MG human glioblastoma. Cellular uptake of new tracers was compared to clinically utilized imaging agent O-(2-[¹⁸F]fluoroethyl)-l-tyrosine (FET, [¹⁸F]1). The uptake of tracers followed the order of FET ([¹⁸F]1) > FEAT([¹⁸F]3) > FEMT ([¹⁸F]2) ≈ FETA ([¹⁸F]4).     

Synthesis and preliminary evaluation of 18F-icotinib for EGFR-targeted PET imaging of lung cancer

Epidermal growth factor receptor (EGFR) has emerged as an attracting target in the field of imaging and treatment for non-small cell lung cancer (NSCLC). Radiolabeled EGFR-tyrosine kinase inhibitors (EGFR-TKIs) specifically targeting EGFR are deemed as promising probes for the imaging of NSCLC. This study aimed to label icotinib (one kind of EGFR-TKI) with ¹⁸F through click reaction to develop a new EGFR-targeting PET probe-¹⁸F-icotinib. ¹⁸F-icotinib was obtained in 44.81% decay-corrected yield in 100?min synthesis time with 34?GBq/μmol specific activity and >99% radiochemical purity at the end of synthesis. The identity of the product was confirmed by co-injection with ¹⁸F-icotinib and ¹⁹F-icotinib. The Log P was 1.28?±?0.04 (n?=?6). The tracer displayed excellent stability after incubation for 4?h in vitro. ¹⁸F-icotinib showed satisfying binding ability to A549 NSCLC cells, which could be inhibited by icotinib. PET imaging studies demonstrated a specific uptake of the radiotracer (0.90?±?0.24% ID/g) in A549 tumor-bearing mice, while lower uptake was observed in heart, lung and spleen at 1.5?h post injection. Inmunohistochemical staining confirmed that the A549 tumor was EGFR-positive. Therefore, we considered that ¹⁸F-icotinib was a highly promising compound for EGFR-based tumor PET imaging.     

Synthesis and evaluation of novel F-18 labeled 4-aminoquinazoline derivatives: potential PET imaging agents for tumor detection

Three novel (18)F-labeled 4-aminoquinazoline derivatives, N-(3-chloro-4-fluorophenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]1), N-(3-ethynylphenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]2), and N-(3-bromophenyl)-6-(2-[(18)F]fluoroethoxy)-7-methoxyquinazolin-4-amine([(18)F]3) were synthesized and radiolabeled by two-step reaction with overall radiochemical yield of 21-24% (without decay corrected). Then we carried out their biodistribution experiments in S180 tumor-bearing mice. Results showed that they had certain concentration accumulation in tumor and fast clearance from muscle and blood. It was encouraging that [(18)F]3 was competitive among three (18)F-labeled 4-aminoquinazoline derivatives in some aspects such as tumor/muscle uptake ratio reaching 7.70 at 60 min post-injection, tumor/blood uptake ratio reaching 6.61 at 120 min post-injection. So we compared radioactivity characteristics of [(18)F]3 with those of [(18)F]-FDG and L-[(18)F]-FET in the same animal model. The absolute radioactivity uptake of [(18)F]3 in tumor reached 3.31 at 60 min p.i., which was slightly higher than [(18)F]-FDG (2.16) and L-[(18)F]-FET (2.75) at the same time phase. For [(18)F]3, tumor/muscle uptake ratio peaked 7.70 at 60 min, which was obviously superior to those of [(18)F]-FDG and L-[(18)F]-FET at all time points. The tumor/brain uptake ratios of [(18)F]3 were 10.36, 17.42, 41.11 at 30 min, 60 min and 120 min post-injection, respectively, and are much higher than those of L-[(18)F] FET (2.54, 2.92 and 2.95) and [(18)F]-FDG (0.61, 1.02 and 1.33) at the same time points. All these results indicate that [(18)F]3 is promising to become a potential PET tumor imaging agent.