Synthesis and in vivo evaluation of a new PET radioligand for studying sigma-2 receptors

The cyclohexyl piperazine 1 (1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-1-yl)-propyl]-piperazine) has been shown to be a potent and selective sigma-2 receptor ligand. In the present study, we prepared [(11)C]1 by O-alkylation of the phenolic precursor 2 with [(11)C]CH(3)I. [(11)C]1 was obtained in a 29% non-decay corrected yield and specific activity of 9299 mCi/micromol calculated at end-of-synthesis. The biodistribution of [(11)C]1 in mouse brain demonstrated rapid and homogenous concentration in all brain structures, which included the cortex, thalamus, cerebellum and striatum. Co-administration of unlabelled 1 (1 mg/kg) or the sigma-2 selective ligand SM-21 (1 mg/kg) failed to show any significant inhibition of [(11)C]1 uptake in the mouse brain. The evaluation of this radioligand in vivo in the mouse clearly indicates that it does not possess the required properties for studying sigma-2 receptors in the brain using PET.     

A novel nicotinic acetylcholine receptor antagonist radioligand for PET studies

Using positron emission tomography (PET) with a specific and selective radioligand targeting nicotinic acetylcholine receptor (nAChR) would allow us to better understand various nAChR related CNS disorders. The use of radiolabeled nAChR antagonists would provide a much safer pharmacological profile, avoiding most peripheral side effects that might be generated from radiolabeled nAChR agonists even at the tracer level; thus, PET imaging with nAChR antagonists would facilitate clinical application. A potent and selective nAChR antagonist was labeled and characterized with PET in non-human primates. Its high brain uptake, high signal-to-noise ratio, and high specific binding strongly suggest a great potential to carry out imaging studies in humans. In addition, the use of a C-11 radiotracer would allow us to perform multiple PET studies in the same individual within a short time frame. The presence of an iodine atom in the molecule also allows the possibility to label with radioiodine for SPECT studies.     

Synthesis,tissue distribution in rats and PET studies in baboon brain of no-carrier-added [18F]RU 52461: in vivo evaluation as a brain glucocorticoid receptor radioligand

11,17β-Dihydroxy-6-methyl-17α -(3-[¹⁸F]fluoro-prop-1 -ynyl)androsta-1,4,6-trien-3-one ([¹⁸F]RU 52461), an ¹⁸F-analog of RU 28362, was synthesized by bromide displacement with [¹⁸F]fluoride in 12–30% overall radiochemical yield (decay-corrected) within 140 min from end of bombardment (EOB). The specific activity was 900–1500 mCi/μmol (33.3–55.5 GBq/μmol) at the end of synthesis (EOS). Biodistribution studies indicated high adrenal and pituitary retention, and uniformly low uptake of [¹⁸F]RU 52461 in all other brain regions of the rat. Except for the pituitary, no specific receptor-mediated uptake of [¹⁸F]RU 52461 could be demonstrated using saturating doses of unlabeled RU 52461 in rat brain. While no change was observed throughout the brain areas in adrenalectomized rats and in animals coinjected with dexamethasone, when compared to controls. PET studies revealed extremely low levels of radioactivity in baboon brain. Therefore, [¹⁸F]RU 52461 does not appear to cross the blood-brain barrier, suggesting that this radiopharmaceutical is not suitable to visualize the brain glucocorticoid binding sites by PET.     

11C-labeling and preliminary evaluation of vortioxetine as a PET radioligand

Vortioxetine is a new multi-modal drug against major depressive disorder with high affinity for a range of different serotonergic targets in the CNS. We report the ¹¹C-labeling of vortioxetine with [¹¹C]MeI using a Suzuki-protocol that allows for the presence of an unprotected amine. Preliminary evaluation of [¹¹C]vortioxetine in a Danish Landrace pig showed rapid brain uptake and brain distribution in accordance with the pharmacological profile, all though an unexpected high binding in cerebellum was also observed. [¹¹C]vortioxetine displayed slow tracer kinetics with peak uptake after 60 min and with limited wash-out from the brain. Further studies are needed but this radioligand may prove to be a valuable tool in unraveling the clinical effects of vortioxetine.     

Radiosynthesis and in vivo evaluation of a fluorine-18 labeled pyrazine based radioligand for PET imaging of the adenosine A2B receptor

On the basis of a pyrazine core structure, three new adenosine A_2B receptor ligands (7a–c) were synthesized containing a 2-fluoropyridine moiety suitable for ¹⁸F-labeling. Compound 7a was docked into a homology model of the A_2B receptor based on X-ray structures of the related A_2A receptor, and its interactions with the adenosine binding site were rationalized. Binding affinity data were determined at the four human adenosine receptor subtypes. Despite a rather low selectivity regarding the A₁ receptor, 7a was radiolabeled as the most suitable candidate (K_i(A_2B)?=?4.24?nM) in order to perform in vivo studies in mice with the aim to estimate fundamental pharmacokinetic characteristics of the compound class. Organ distribution studies and a single PET study demonstrated brain uptake of [¹⁸F]7a with a standardized uptake value (SUV) of ≈1 at 5?min post injection followed by a fast wash out. Metabolism studies of [¹⁸F]7a in mice revealed the formation of a blood–brain barrier penetrable radiometabolite, which could be structurally identified. The results of this study provide an important basis for the design of new derivatives with improved binding properties and metabolic stability in vivo.     

Dopaminergic and benzodiazepine receptors studied in vivo by PET

Using Positron Emission Tomography (PET) and specific radioligands, dopaminergic D2 (DA-D2 receptors) and benzodiazepine receptors (BZ-receptors) were studied in living animals during normal and pathological conditions. In vivo characterization of both receptors was performed using two highly specific antagonists namely: 11C-Ro 15 1788 for BZ-receptors and 76 Br-Bromospiperone for DA-D2 receptors. Changes in 11c-Ro 15 1788 specific binding to BZ-receptors were observed during convulsive seizures. After MPTP treatment, a decrease in the 76 Br-Bromospiperone striatal specific binding was observed, correlated with the establishment of a Parkinson-like syndrome.     

Synthesis and structure-activity relationship studies in peripheral benzodiazepine receptor ligands related to alpidem

The exploration of the structure-affinity relationships concerning a new class of peripheral benzodiazepine receptor (PBR) ligands related to alpidem has been pursued in order to evaluate the consistency of the structure-affinity relationships among different classes (and subclasses) of PBR ligands. The target amide derivatives were prepared following a previously published procedure based on the condensation of pyrrolo[3,4-b]quinoline derivatives 11a,b with glyoxylic acid mono-hydrate and the subsequent amidation of the acids obtained via mixed anhydride. On the other hand, the preparation of compound 9g lacking the pharmacophoric (delta1) carbonyl group involved: (a) the double sequential attack of the dimethylmethyleneammonium salt obtained from bis(dimethylamino)methane and acetyl chloride to pyrrolo[3,4-b]quinoline derivative 11b, (b) the quaternization of the obtained allylamine derivative 13 with methyl iodide, and (c) the palladium-catalyzed allylation of N-methyl-p-anisidine by quaternary allylammonium cation 14. The structure-affinity relationship trends observed in this subclass of tricyclic alpidem-related PBR ligands find correlations in other classes (or subclasses) of PBR ligands. This result supports the initial pharmacophoric hypothesis and suggests a common mode of interaction at the PBR binding site.     

A benzenesulfonamide derivative as a novel PET radioligand for CXCR4

CXCR4 is involved in various diseases such as inflammation, tumor growth, and cancer metastasis through the interaction with its natural endogenous ligand, chemokine CXCL12. In an effort to develop imaging probes for CXCR4, we developed a novel small molecule CXCR4-targeted PET agent (compound 5) by combining our established benzenesulfonamide scaffold with a labeling component by virtue of click chemistry. 5 shows nanomolar affinity (IC₅₀ = 6.9 nM) against a known CXCR4 antagonist (TN14003) and inhibits more than 65% chemotaxis at 10 nM in vitro assays. Radiofluorinated compound 5 ([¹⁸F]5) demonstrates a competitive cellular uptake against CXCL12 in a dose-dependent manner. Further, microPET images of [¹⁸F]5 exhibits preferential accumulation of radioactivity in the lesions of λ-carrageenan-induced paw edema, human head and neck cancer orthotopic xenograft, and metastatic lung cancer of each mouse model.     

Synthesis and biological evaluation of [18F]fluorovinpocetine,a potential PET radioligand for TSPO imaging

Despite of various PET radioligands targeting the translocator protein TSPO 18-KDa are used for the investigations of neuroinflammatory conditions associated with neurological disorders, development of new TSPO radiotracers is still an active area of the researches with a major focus on the ¹⁸F-labelled radiotracers. Here, we report the radiochemical synthesis of [¹⁸F]vinpocetine, fluorinated analogue of previously reported TSPO radioligand, [¹¹C]vinpocetine. Radiolabeling was achieved by [¹⁸F]fluoroethylation of apovincaminic acid with [¹⁸F]fluoroethyl bromide. [¹⁸F]vinpocetine was obtained in quantities >2.7 GBq in RCY of 13% (non–decay corrected), and molar activity >60 GBq/µmol within 95 min synthesis time. Preliminary PET studies in a cynomolgus monkey and metabolite studies by HPLC demonstrated similar results by [¹⁸F]vinpocetine as for [¹¹C]vinpocetine, including high blood-brain barrier permeability, regional uptake pattern and fast washout from the NHP brain. These results demonstrate that [¹⁸F]fluorovinpocetine warrants further evaluation as an easier accessible alternative to [¹¹C]vinpocetine.     

Synthesis and preclinical evaluation of [18F]FSL25.1188, a reversible PET radioligand for monoamine oxidase-B

Carbon-11 labeled SL25.1188 is a promising reversible monoamine oxidase-B (MAO-B) radioligand that was recently translated for human positron emission tomography (PET) imaging. Herein, we report the development of a novel fluorinated derivative, namely, [¹⁸F](S)-3-(6-(3-fluoropropoxy)benzo[d]isoxazol-3-yl)-5-(methoxymethyl)oxazolidin-2-one ([¹⁸F]FSL25.1188; [¹⁸F]6), as a candidate ¹⁸F-labeled MAO-B radioligand, and, its subsequent preclinical evaluation in non-human primates (NHP). [¹⁸F]6 was produced and isolated (>6 GBq) with high radiochemical purity (>99%), and molar activity (>100 GBq/µmol at time of injection). Autoradiography studies conducted in post-mortem human brain sections revealed [¹⁸F]6 binding in MAO-B rich regions. PET imaging study of [¹⁸F]6 in NHP showed high brain uptake (SUV > 2.5) as well as a regional brain radioactivity distribution in accordance with MAO-B expression. [¹⁸F]6 displayed favorable in vivo kinetics, with an early peak in the time-activity curve followed by progressive wash-out from the NHP brain. Specificity of [¹⁸F]6 was investigated in a pre-treatment study with l-deprenyl (1.0 mg/kg) wherein reduced radioligand uptake was observed in all MAO-B rich regions. Results from the current preclinical investigation suggests [¹⁸F]6 is a promising MAO-B PET radioligand. Further evaluation of [¹⁸F]6 and structurally related ¹⁸F-analogs are underway to identify an optimized candidate for clinical research studies.     

Synthesis, radiosynthesis and in vivo preliminary evaluation of [11C]LBT-999, a selective radioligand for the visualisation of the dopamine transporter with PET

LBT-999 (8-((E)-4-fluoro-but-2-enyl)-3beta-p-tolyl-8-aza-bicyclo[3.2.1]octane-2beta-carboxylic acid methyl ester), a cocaine derivative belonging to a new generation of highly selective dopamine transporter (DAT) ligands, and its corresponding carboxylic acid derivative, the latter used as precursor for labelling both with tritium and the positron-emitter carbon-11 (half-life: 20.38 min), were synthesized from (R)-cocaine. [(3)H]LBT-999 (>99% radiochemically pure, specific radioactivity of 3.1 TBq/mmol) was prepared from [(3)H]methyl iodide, allowing its in vitro pharmacological evaluation (K(D): 9 nM for DAT and IC(50) > 1000 nM for SERT and NET). Routine production batches of 4.5-9.0 GBq of iv injectable solutions of [(11)C]LBT-999 (with specific radioactivities ranging from 30 to 45 GBq/mumol) were prepared in 25-30 min (HPLC purification and formulation included) using the efficient methylation reagent [(11)C]methyl triflate. The preliminary in vivo pharmacological evaluation of [(11)C]LBT-999, using both biodistributions in rats and brain imaging in monkeys with positron emission tomography (PET), clearly illustrates that this ligand is an excellent candidate for quantification with PET of DAT in humans.     

Development of a new radioligand for cholecystokinin receptor subtype 2 scintigraphy: From molecular modeling to in vivo evaluation

To improve the targeting to tumors expressing the cholecystokinin receptor subtype 2 (CCK2R) with limited kidney uptake, we synthesized a novel cholecystokinin C-terminal tetrapeptide (CCK4)-based derivative conjugated to an original bipyridine-chelator (BPCA), ¹¹¹In-BPCA-(Ahx)₂-CCK4. To our knowledge this is the first CCK4-based radioligand that presents a high affinity for the CCK2R, a high and specific tumor uptake, a low renal accumulation and a very good visualization of tumors in vivo compared with an internal control, ¹¹¹Indium-trans-cyclohexyldiethylenetriaminepenta-acetic acid-cholecystokinin octapeptide (¹¹¹In-CHX-A″-DTPA-CCK8). These properties make ¹¹¹In-BPCA-(Ahx)₂-CCK4, a promising candidate for imaging and peptide receptor radionuclide therapy of CCK2R positive tumors.     

Design, synthesis and structure-affinity relationships of aryloxyanilide derivatives as novel peripheral benzodiazepine receptor ligands

Since the peripheral benzodiazepine receptor (PBR) has been primarily found as a high-affinity binding site for diazepam in rat kidney, numerous studies of it have been performed. However, the physiological role and functions of PBR have not been fully elucidated. Currently, we presented the pharmacological profile of two high and selective PBR ligands, N-(2,5-dimethoxybenzyl)-N-(4-fluoro-2-phenoxyphenyl)acetamide (7-096, DAA1106) (PBR: IC(50)=0.28 nM) and N-(4-chloro-2-phenoxyphenyl)-N-(2-isopropoxybenzyl)acetamide (7-099, DAA1097) (PBR: IC(50)=0.92 nM). The compounds are aryloxyanilide derivatives, and identified with known PBR ligands such as benzodiazepine (1, Ro5-4864), isoquinoline (2, PK11195), imidazopyridine (3, Alpidem), and indole (5, FGIN-1-27) derivatives. The aryloxyanilide derivatives, which have been derived by opening the diazepine ring of 1, are a novel class as PBR ligands and have exhibited high and selective affinity for peripheral benzodiazepine receptors (PBRs). These novel derivatives would be useful for exploring the functions of PBR. In this paper, the design, synthesis and structure-affinity relationships of aryloxyanilide derivatives are described.     

Synthesis of a fluorine-18-labelled derivative of 6-nitroquipazine,as a radioligand for the in vivo serotonin transporter imaging with PET

Considerable efforts have been engaged in the design, synthesis and pharmacological characterization of radioligands for imaging the serotonin transporter, based on its implication in several neuropsychiatric diseases, such as depression, anxiety and schizophrenia. In the 5-halo-6-nitroquipazine series, the fluoro derivative has been designed for positron emission tomography (PET). The corresponding 5-iodo-, 5-bromo- and 5-chloro N-Boc-protected quipazines as labelling precursors, as well as 5-fluoro-6-nitroquipazine as a reference compound have been synthesized. 5-[(18)F]Fluoro-6-nitroquipazine has been radiolabelled with fluorine-18 (positron-emitting isotope, 109.8 min half-life) by nucleophilic aromatic substitution from the corresponding N-Boc protected 5-bromo- and 5-chloro-precursors using K[(18)F]F-K(222) complex in DMSO by conventional heating (145 degrees C, 2 min) or microwave activation (50 W, 30-45 s), followed by removal of the protective group with TFA. Typically, 15-25 mCi (5.5-9.2 GBq) of 5-[(18)F]fluoro-6-nitroquipazine (1-2 Ci/micromol or 37-72 GBq/micromol) could be obtained in 70-80 min starting from a 550-650 mCi (20.3-24.0 GBq) aliquot of a cyclotron [(18)F]F(-) production batch (2.7-3.8% non decay-corrected yield based on the starting [(18)F]fluoride). Ex vivo studies (biodistribution in rat), as well as PET imaging (in monkey) demonstrated that 5-[(18)F]fluoro-6-nitroquipazine ([(18)F]-1d) readily crossed the blood brain barrier and accumulated in the regions rich in 5-HT transporter (frontal- and posterial cortex, striata). However, the low accumulation of the tracer in the thalamus (rat and monkey) as well as the comparable displacement of the tracer observed with both citalopram, a -HT re-uptake inhibitor and maprotiline, a norepinephrine re-uptake inhibitor (rat), indicate that 5-[(18)F]fluoro-6-nitroquipazine ([(18)F]-1d) does not have the suggested potential for PET imaging of the serotin transporter (SERT).     

PBRL, a putative peripheral benzodiazepine receptor, in primitive erythropoiesis

Benzodiazepines are a class of psychoactive drugs widely used for their anxiolytic, anticonvulsant, muscle relaxant and hypnotic properties. Although the benzodiazepine receptor in the central nervous system has been well studied, the role of peripheral type benzodiazepine receptor, PBR, remains elusive. Here, we show that there are two PBR homologous genes in amniotes, PBR and PBRL, based on phylogenetic analysis. In chickens, PBRL is exclusively expressed during early development in differentiating primitive erythrocytes and this expression is tightly correlated with that of hemoglobin genes. PBR is not expressed in hematopoietic system during this period and is weakly expressed in developing central nervous system. Because one of PBRs' known functions is to regulate heme transport between the mitochondria and cytoplasm, we investigated expression profiles of heme biosynthesis genes. Seven of the eight enzymes involved in heme biosynthesis, with the exception of protoporphyrinogen oxidase, are present in chicken genome. Five of them, delta-aminolevulinate synthase, delta-aminolevulinic acid dehydrogenase, porphobilinogen deaminase, coproporphyrinogen decarboxylase and ferrochelatase, show stage-specific increase in gene expression correlated with primitive hematopoiesis, but not with primitive erythrocyte differentiation. PBRL protein is localized to the mitochondria in culture cells, and pharmacological inhibition of PBRL activity results in a decrease in globin protein levels during primitive erythropoiesis. Our data suggest a developmental role of PBRs in erythropoiesis in chickens, possibly via the regulation of heme availability for the assembly of functional hemoglobins.     

Synthesis and evaluation of N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide: a deuterium-substituted radioligand for peripheral benzodiazepine receptor

N-(5-Fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]2) is a potent ligand (IC(50): 1.71 nM) for peripheral benzodiazepine receptor (PBR). However, in vivo evaluation on rodents and primates showed that this ligand was unstable and rapidly metabolized to [(18)F]F(-) by defluorination of the [(18)F]fluoromethyl moiety. In this study, we designed a deuterium-substituted analogue, N-(5-fluoro-2-phenoxyphenyl)-N-(2-[(18)F]fluoromethoxy-d(2)-5-methoxybenzyl)acetamide ([(18)F]5) as a radioligand for PBR to reduce the in vivo metabolic rate of the non-deuterated [(18)F]2. The design principle was based on the hypothesis that the deuterium substitution may reduce the rate of defluorination initiated by cleavage of the C-H bond without altering the binding affinity for PBR. The non-radioactive 5 was prepared by reacting diiodomethane-d(2) (CD(2)I(2), 6) with a phenol precursor 7, followed by treatment with tetrabutylammonium fluoride. The ligand [(18)F]5 was synthesized by the alkylation of 7 with [(18)F]fluoromethyl iodide-d(2) ([(18)F]FCD(2)I, [(18)F]9). Compound 5 displayed a similar in vitro affinity to PBR (IC(50): 1.90 nM) with 2. In vivo evaluation demonstrated that [(18)F]5 was metabolized by defluorination to [(18)F]F(-) as a main radioactive component, but its metabolic rate was slower than that of [(18)F]2 in the brain of mice. The deuterium substitution decreased the radioactivity level of [(18)F]5 in the bone of mouse, augmented by the percentage of specific binding to PBR in the rat brain determined by ex vivo autoradiography. However, the PET image of [(18)F]5 for monkey brain showed high radioactivity in the brain and skull, suggesting a possible species difference between rodents and primates.     

In vivo evaluation of [18F]FECIMBI-36, an agonist 5-HT2A/2C receptor PET radioligand in nonhuman primate

We recently reported the radiosynthesis and in vitro evaluation of [¹⁸F]-2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-(2-fluoroethoxy)benzyl)ethanamine, ([¹⁸F]FECIMBI-36) or ([¹⁸F]1), an agonist radioligand for 5HT_2A/2C receptors in postmortem samples of human brain. Herein we describe the in vivo evaluation of [¹⁸F]FECIMBI-36 in vervet/African green monkeys by PET imaging. PET images show that [¹⁸F]FECIMBI-36 penetrates the blood-brain barrier and a low retention of radioactivity is observed in monkey brain. Although the time activity curves indicate a somehow heterogeneous distribution of the radioligand in the brain, the low level of [¹⁸F]FECIMBI-36 in brain may limit the use of this tracer for quantification of 5-HT_2A/2C receptors by PET.     

Radioiodinated 2'-iodospiperone: a new radioligand for in vivo dopamine receptor study

In vivo dopamine receptor binding of the newly synthesized ligand, 125I-2'-iodospiperone (125I-2'-ISP), was studied in mouse brain. The highest accumulation was found in the striatum. Analysis of the striatal homogenate showed the 125I-2'-ISP to be metabolically stable. Furthermore, this striatal binding was saturable and displaced only by dopaminergic drugs. On the other hand, the accumulation in the cortex was as low as that of the cerebellum and uneffected by the administration of serotoninergic drugs and dopaminergic drugs; results assessed by macroautoradiographic studies. Thus, the newly synthesized 125I-2'-ISP presented high affinity for dopamine receptors in vivo and therefore, holds great potential for the in vivo dopamine receptor studies, provided 123I becomes readily available.     

Design, synthesis, and structure-activity relationships of novel tetracyclic compounds as peripheral benzodiazepine receptor ligands

The peripheral benzodiazepine receptor (PBR) is pharmacologically distinct from the central benzodiazepine receptor (CBR) and has been identified in a wide range of peripheral tissues as well as in the central nervous system. Although numerous studies have been performed of it, the physiological roles and functions of the PBR are still unclear. In the present study, in exploring new types of ligands for PBR, we found that a new series of compounds having a tetracyclic ring system, which were designed from FGIN-1-27, exhibited high affinities for PBR. We prepared and evaluated them for PBR affinities. The results of binding tests showed that 12e and 12f were the most potent PBR ligands among them (12e: IC(50)=0.44nM, 12f: IC(50)=0.37nM). In this paper, we present the design, synthesis, and structure-activity relationships (SARs) of novel tetracyclic compounds.