Synthesis and evaluation of a 18F-labeled spirocyclic piperidine derivative as promising σ1 receptor imaging agent.

Several spirocyclic piperidine derivatives were designed and synthesized as σ1 receptor ligands. In vitro competition binding assays showed that the fluoroalkoxy analogues with small substituents possessed high affinity towards σ1 receptors and subtype selectivity. Particularly for ligand 1'-((6-(2-fluoroethoxy)pyridin-3-yl)methyl)-3H-spiro[2-benzofuran-1,4'-piperidine] (2), high σ1 receptor affinity (Ki=2.30 nM) and high σ1/σ2 subtype selectivity (142-fold) as well as high σ1/VAChT selectivity (234-fold) were observed. [18F]2 was synthesized using an efficient one-pot, two-step reaction method in a home-made automated synthesis module, with an overall isolated radiochemical yield of 8-10%, a radiochemical purity of higher than 99%, and specific activity of 56-78GBq/µmol. Biodistribution studies of [18F]2 in ICR mice indicated high initial brain uptake and a relatively fast washout. Administration of haloperidol, compound 1 and different concentrations of SA4503 (3, 5, or 10 µmol/kg) 5 min prior to injection of [18F]2 significantly decreased the accumulation of radiotracer in organs known to contain σ1 receptors. Ex vivo autoradiography in Sprague-Dawley rats demonstrated high accumulation of radiotracer in brain areas with high expression of σ1 receptors. These encouraging results prove that [18F]2 is a suitable candidate for σ1 receptor imaging with PET in humans.

Synthesis and biological evaluation of ¹8F labeled fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives for sigma-1 receptor imaging.

We report the synthesis and evaluation of a series of fluoro-oligo-ethoxylated 4-benzylpiperazine derivatives as potential σ(1) receptor ligands. In vitro competition binding assays showed that 1-(1,3-benzodioxol-5-ylmethyl)-4-(4-(2-fluoroethoxy)benzyl)piperazine (6) exhibits low nanomolar affinity for σ(1) receptors (K(i)=1.85 ± 1.59 nM) and high subtype selectivity (σ(2) receptor: K(i)=291 ± 111 nM; K(i)σ(2)/K(i)σ(1)=157). [(18)F]6 was prepared in 30-50% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via nucleophilic (18)F(-) substitution of the corresponding tosylate precursor. The logD(pH 7.4) value of [(18)F]6 was found to be 2.57 ± 0.10, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiotracers in organs known to contain σ(1) receptors, including the brain, lungs, kidneys, heart, and spleen. Administration of haloperidol 5 min prior to injection of [(18)F]6 significantly reduced the concentration of radiotracers in the above-mentioned organs. The accumulation of radiotracers in the bone was quite low suggesting that [(18)F]6 is relatively stable to in vivo defluorination. The ex vivo autoradiography in rat brain showed high accumulation of radiotracers in the brain areas known to possess high expression of σ(1) receptors. These findings suggest that [(18)F]6 is a suitable radiotracer for imaging σ(1) receptors with PET in vivo.

Synthesis and biological evaluation of a novel 99mTc cyclopentadienyl tricarbonyl complex ([(Cp-R)99mTc(CO)3]) for sigma-2 receptor tumor imaging.

We report the design, synthesis and biological evaluation of a novel (99m)Tc 4-(4-cyclohexylpiperazine-1-yl)-butan-1-one-1-cyclopentadienyltricarbonyl technetium ([(99m)Tc]5) as a potential SPECT tracer for imaging of σ(2) receptors in tumors. [(99m)Tc]5 was prepared in 25±5% isolated radiochemical yield with radiochemical purity of >99% via double-ligand transfer (DLT) reaction from the ferrocene precursor 2b (4-(4-cyclohexylpiperazine-1-yl)-1-ferrocenylbutan-1-one). The corresponding Re-complex 4 and the ferrocenyl complex 2b showed relatively high affinity towards σ(2) receptors in in vitro competition binding assay (K(i) values of 4 and 2b were 64.4±18.5 nM and 43.6±21.3 nM, respectively) and moderate to high selectivity versus σ(1) receptors (K(i)σ(1)/K(i)σ(2) ratios were 12.5 and 95.5, respectively). The logD value of [(99m)Tc]5 was determined to be 2.52±0.33. Biodistribution studies in mice revealed comparably high initial brain uptake of [(99m)Tc]5 and slow washout. Administration of haloperidol 5 min prior to injection of [(99m)Tc]5 significantly reduced the radiotracer uptake in brain, heart, lung, and spleen by 40-50% at 2h p.i.. Moreover, [(99m)Tc]5 showed high uptake in C6 glioma cell lines (8.6%) after incubation for 1h. Blocking with haloperidol to compete with [(99m)Tc]5 significantly reduced the cell uptake. Preliminary blocking study in C6-brain-tumor bearing rats showed that [(99m)Tc]5 binds to σ receptors in the brain-tumor specifically. These results are encouraging for further exploration of (99m)Tc-labeled probes for σ(2) receptor tumor imaging in vivo.

(E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives serve as probes for ß-amyloid plaques.

We report the synthesis and biological evaluation of novel (E)-5-styryl-1H-indole and (E)-6-styrylquinoline derivatives as probes for imaging ß-amyloid (Aß) plaques. These derivatives showed binding affinities for Aß1₋40 aggregates with K(i) values varying from 4.1 to 288.4 nM. (E)-5-(4-iodostyryl)-1H-indole (8) clearly stained Aß plaques in the brain sections of Alzheimer's disease (AD) model mice (APP/PS1). Furthermore, autoradiography for [¹²5I]8 displayed intense and specific labeling of Aß plaques in the brain sections mentioned above with low background. In biodistribution experiments using normal mice [¹²5I]8 showed high initial brain uptake followed by rapid washout (4.27 and 0.64% ID/g at 2 and 30 min post injection, respectively). These findings suggests that [¹²³I]8 may be a potential SPECT imaging agent for detecting Aß plaques in AD brain.

Synthesis and biological evaluation of indole-chalcone derivatives as ß-amyloid imaging probe.

A series of chaclone derivatives containing an indole moiety were evaluated in competitive binding assays with Aß(1-42) aggregates versus [(125)I]IMPY. The affinity of these compounds ranged from 4.46 to >1008 nM, depending on the substitution on the phenyl ring. Fluorescent staining in vitro showed that one compound with a N,N-dimethylamino group intensely stained Aß plaques within brain sections of AD transgenic mice. The radioiodinated probe [(125)I]-(E)-3-(1H-indol-5-yl)-1-(4-iodophenyl)prop-2-en-1-one, [(125)I]4, was prepared and autoradiography in sections of brain tissue from an animal model of AD showed that it labeled Aß plaques specifically. However, experiments with normal mice indicated that [(125)I]4 exhibited a low uptake into the brain in vivo (0.41% ID/g at 2 min). Additional chemical modifications of this indole-chalcone structure may lead to more useful imaging agents for detecting ß-amyloid plaques in the brains of AD patients.

Novel imaging agents for ß-amyloid plaque based on the N-benzoylindole core.

We report the synthesis and evaluation of a series of N-benzoylindole derivatives as novel potential imaging agents for ß-amyloid plaques. In vitro binding studies using Aß(1-40) aggregates versus [(125)I]TZDM showed that all these derivatives demonstrated high binding affinities (K(i) values ranged from 8.4 to 121.6 nM). Moreover, two radioiodinated compounds [(125)I]7 and [(125)I]14 were prepared. Autoradiography for [(125)I]14 displayed intense and specific labeling of Aß plaques in the brain sections of AD model mice (C57, APP/PS1) with low background. In vivo biodistribution in normal mice exhibited sufficient initial brain uptake for imaging (2.19% and 2.00%ID/g at 2 min postinjection for [(125)I]7 and [(125)I]14, respectively). Although additional modifications are necessary to improve brain uptake and clearance from the brain, the N-benzoylindole may be served as a backbone structure to develop novel ß-amyloid imaging probes.

Synthesis and biological evaluation of novel 4-benzylpiperazine ligands for sigma-1 receptor imaging.

We report the synthesis and evaluation of 4-benzylpiperazine ligands (BP-CH(3), BP-F, BP-Br, BP-I, and BP-NO(2)) as potential σ(1) receptor ligands. The X-ray crystal structure of BP-Br, which crystallized with monoclinic space group P2(1)/c, has been determined. In vitro competition binding assays showed that all the five ligands exhibit low nanomolar affinity for σ(1) receptors (K(i)=0.43-0.91nM) and high subtype selectivity (σ(2) receptor: K(i)=40-61nM; K(i)σ(2)/K(i)σ(1)=52-94). [(125)I]BP-I (1-(1,3-benzodioxol-5-ylmethyl)-4-(4-iodobenzyl)piperazine) was prepared in 53±10% isolated radiochemical yield, with radiochemical purity of >99% by HPLC analysis after purification, via iododestannylation of the corresponding tributyltin precursor. The logD value of [(125)I]BP-I was found to be 2.98±0.17, which is within the range expected to give high brain uptake. Biodistribution studies in mice demonstrated relatively high concentration of radiolabeled substances in organs known to contain σ(1) receptors, including the brain, lung, kidney, heart, and spleen. Administration of haloperidol 5min prior to injection of [(125)I]BP-I significantly reduced the concentration of radioactivity in the above-mentioned organs. The accumulation of radiolabeled substance in the thyroid was quite low suggesting that [(125)I]BP-I is relatively stable to in vivo deiodination. These findings suggest that the binding of [(125)I]BP-I to σ(1) receptors in vivo is specific.

Synthesis and evaluation of novel benzothiazole derivatives based on the bithiophene structure as potential radiotracers for beta-amyloid plaques in Alzheimer's disease.

In this study, six novel benzothiazole derivatives based on the bithiophene structure were developed as potential beta-amyloid probes. In vitro binding studies using Abeta aggregates showed that all of them demonstrated high binding affinities with K(i) values ranged from 0.11 to 4.64nM. In vitro fluorescent staining results showed that these compounds can intensely stained Abeta plaques within brain sections of APP/PS1 transgenic mice, animal model for AD. Two radioiodinated compounds [(125)I]-2-(5'-iodo-2,2'-bithiophen-5-yl)-6-methoxybenzo[d]thiazole [(125)I]10 and [(125)I]-2-(2,2'-bithiophen-5-yl)-6-iodobenzo[d]thiazole [(125)I]13 were successfully prepared through an iododestannylation reaction. Furthermore, in vitro autoradiography of the AD model mice brain sections showed that both [(125)I]10 and [(125)I]13 labeled the Abeta plaques specifically with low background. In vivo biodistribution studies in normal mice indicated that [(125)I]13 exhibited high brain uptake (3.42% ID/g at 2min) and rapid clearance from the brain (0.53% ID/g at 60min), while [(125)I]10 showed lower brain uptake (0.87% ID/g at 2min). In conclusion, these preliminary results of this study suggest that the novel radioiodinated benzothiazole derivative [(125)I]13 may be a candidate as an in vivo imaging agent for detecting beta-amyloid plaques in the brain of AD patients.

Novel anilinophthalimide derivatives as potential probes for beta-amyloid plaque in the brain.

A group of novel 4,5-dianilinophthalimide derivatives has been synthesized in this study for potential use as beta-amyloid (Abeta) plaque probes. Staining of hippocampus tissue sections from Alzheimer's disease (AD) brain with the representative compound 9 indicated selective labeling of it to Abeta plaques. The binding affinity of radioiodinated [(125)I]9 for AD brain homogenates was 0.21 nM (K(d)), and of other derivatives ranged from 0.9 to 19.7 nM, except for N-methyl-4,5-dianilinophthalimide (K(i)>1000 nM). [(125)I]9 possessed the optimal lipophilicity with LogP value of 2.16, and its in vivo biodistribution in normal mice exhibited excellent initial brain uptake (5.16% ID/g at 2 min after injection) and a fast washout rate (0.56% ID/g at 60 min). The encouraging results suggest that this novel derivative of [(123)I]9 may have potential as an in vivo SPECT probe for detecting amyloid plaques in the brain.

[Spectroscopic study on binding of folic acid to human serum albumin].

The interaction of human serum albumin and folic acid was studied using fluorescence spectroscopy, UV absorption and synchronous fluorescence spectroscopy in the pH 7.4 Tris-HCl buffer system at different temperatures. The research shows that these interactions result in the endogenous fluorescence quenching of HSA, which belongs to a static quenching mechanism. The quenching rate constants, the binding constants and the binding sites of the static quenching were calculated. The distance between the body (HSA) and receptor (folic acid) and the efficiency of energy transfer were obtained to be 1.77 nm and 0. 052 65 respectively, based on the theory of Forster nonradiative energy transfer. And according to the thermodynamic parameters calculated the binding of HSA and folic acid is mainly attributed to the hydrophobic interaction, partly static force. Further more the synchronous fluorescence spectrum was utilized to investigate the conformational transformation; The decline result of the hydrophobic nature around Trp demonstrates that the folic acid is in the hydrophobic cavity of HSA.

[Electronic spectral study on salicylaldehyde-amino acid Schiff bases in water].

Seven salicylaldehyde-amino acid Schiff bases in water were investigated by means of electronic spectra, which is helpful to investigate the label of radionuclides with the seven ligands in water for radiopharmaceutical study. Except 10(-5) mol x L(-1) for Sal-tyr, the minimum concentration of formation is 10(-4) mol x L(-1) for all other Sal-aas. Only Sal-his is generated at pH 9-10, while the minimum pH conditions for formation are 8 for Sal-gly and Sal-tyr, and 9 for other four. All Sal-aa but Sal-cys, whose concentration at 36 h is higher than that of newly produced one, decompose gradually since generation, with existing time of 24 h for Sal-gly and Sal-glu, and 48 h for other four.

Radioiodinated benzimidazole derivatives as single photon emission computed tomography probes for imaging of ß-amyloid plaques in Alzheimer's disease.

Five iodinated 2-phenyl-1H-benzo[d]imidazole derivatives were synthesized and evaluated as potential probes for ß-amyloid (Aß) plaques. One of the compounds, 4-(6-iodo-1H-benzo[d]imidazol-2-yl)-N,N-dimethylaniline (12), showed excellent affinity for Aß(1-42) aggregates (K(i) = 9.8 nM). Autoradiography with sections of postmortem Alzheimer's disease (AD) brain revealed that a radioiodinated probe [(125)I]12, labeled Aß plaques selectively with low nonspecific binding. Biodistribution experiments with normal mice injected intravenously with [(125)I]12 showed high uptake [4.14 percent injected dose per gram (% ID/g) at 2 min] into and rapid clearance (0.15% ID/g at 60 min) from the brain, which may bring about a good signal-to-noise ratio and therefore achieve highly sensitive detection of Aß plaques. In addition, [(125)I]12 labeled amyloid plaques in vivo in an AD transgenic model. The preliminary results strongly suggest that [(125)I]12 bears characteristics suitable for detecting amyloid plaques in vivo. When labeled with (123)I, it may be a useful SPECT imaging agent for Aß plaques in the brain of living AD patients.