Pyrrolovesamicols--synthesis, structure and VAChT binding of two 4-fluorobenzoyl regioisomers

This Letter describes the synthesis of two regioisomers of a new class of vesamicol analogs as possible ligands for imaging the vesicular acetylcholine transporter in future PET studies. The two pyrrolovesamicols (±)-6a and (±)-6b were synthesized by nucleophilic ring opening reaction of a tetrahydroindole epoxide precursor with 4-phenylpiperidine. The reaction mechanism of the synthesis was studied by HPLC and the molecular structures were determined by X-ray structure analysis. Unexpected low binding affinities to VAChT (K(i)=312±73 nM for (±)-6a and K(i)=7320±1840 nM for (±)-6b) were determined by competitive binding analysis using a cell line stably transfected with ratVAChT and (-)-[(3)H]vesamicol.     

3D QSAR study, synthesis, and in vitro evaluation of (+)-5-FBVM as potential PET radioligand for the vesicular acetylcholine transporter (VAChT)

Located in presynaptic cholinergic nerve terminals, the vesicular acetylcholine transporter (VAChT) represents a potential target for quantitative visualization of early degeneration of cholinergic neurons in Alzheimer's disease using PET. Benzovesamicol derivatives are proposed as radioligands for this purpose. We report QSAR studies of vesamicol and benzovesamicol derivatives taking into account the stereoselectivity of the VAChT binding site. Use of different data sets and different models in this study revealed that both enantiomers of 5-fluoro-3-(4-phenyl-piperidin-1-yl)-1,2,3,4-tetrahydro-naphthalen-2-ol (5-FBVM) are promising candidates, with predicted VAChT affinities between 6.1 and 0.05 nM. The synthesis of enantiopure (R,R)- and (S,S)-5-FBVM and their corresponding triazene precursors for future radiofluorination is reported. Both enantiomers exhibited high in vitro affinity for VAChT [(+)-5-FBVM: K(i)=6.95 nM and (-)-5-FBVM: K(i)=3.68 nM] and were selective for σ(2) receptors (～70-fold), only (+)-5-FBVM is selective for σ(1) receptors (～fivefold). These initial results suggest that (+)-(S,S)-5-FBVM warrants further investigation as a potential radioligand for in vivo PET imaging of cholinergic nerve terminals.     

Syntheses and in vitro evaluation of decalinvesamicol analogues as potential imaging probes for vesicular acetylcholine transporter (VAChT)

A series of vesamicol analogues, o-iodo-trans-decalinvesamicol (OIDV) or o-bromo-trans-decalinvesamicol (OBDV), were synthesized and their affinities to vesicular acetylcholine transporter (VAChT) and σ receptors (σ-1, σ-2) were evaluated by in vitro binding assays using rat cerebral or liver membranes. OIDV and OBDV showed greater binding affinity to VAChT (K(i)=20.5±5.6 and 13.8±1.2nM, respectively) than did vesamicol (K(i)=33.9±18.1nM) with low affinity to σ receptors. A saturation binding assay in rat cerebral membranes revealed that [(125)I]OIDV had a single high affinity binding site with a K(d) value of 1.73nM and a B(max) value of 164.4fmol/mg protein. [(125)I]OIDV revealed little competition with inhibitors, which possessed specific affinity to each σ (σ-1 and σ-2), serotonin (5-HT(1A) and 5-HT(2A)), noradrenaline, and muscarinic acetylcholine receptors. In addition, BBB penetration of [(125)I]OIDV was verified in in vivo. The results of the binding studies indicated that OIDV and OBDV had great potential to be VAChT imaging probes with high affinity and selectivity.     

Synthesis and in vitro evaluation of new benzovesamicol analogues as potential imaging probes for the vesicular acetylcholine transporter

Our goal was to synthesize new stereospecific benzovesamicol analogues, which could potentially be used as SPECT or PET radioligands for the vesicular acetylcholine transporter (VAChT). This paper describes the chemical synthesis, resolution and determination of binding affinity for four enantiomeric pairs of derivatives. Their intrinsic affinities were determined by competition against binding of [3H]vesamicol to human VAChT. Of the eight enantiomers, (E)-(R,R)-5-AOIBV [(R,R)-3], and (R,R)-5-FPOBV [(R,R)-4] displayed the highest binding affinities for VAChT (Kd=0.45 and 0.77 nM, respectively), which indicated that an elongation of the chain from 5-idodo as in the case of 5-iodobenzovesamicol (5-IBVM), to a 5-(E)-3-iodoallyloxy or 5-fluoropropoxy substituent, as in 5-AOIBV and 5-FPOBV, respectively, was very well tolerated at the vesamicol binding site. The enantiomer (R,R)-4-MAIBV [(R,R)-16], which retains the basic structure of (-)-5-IBVM but possess an additional aminomethyl substituent in the 4-position of the piperidine ring, displayed lower binding affinity (Kd=8.8 nM). Nevertheless, the result suggests that substitution at this position may be an interesting alternative to investigate for development of new benzovesamicol analogues. As expected, the corresponding (S,S) enantiomers displayed lower Kd values, they were approximately 10-fold lower in the case of (S,S)-5-FPOBV (Kd=8.4 nM) and (E)-(S,S)-5-AOIBV (Kd=4.3 nM). (R,R)-3, and (R,R)-4 showed the same high affinity for VAChT as (-)-5-IBVM and may be suitable as imaging agents of cholinergic nerve terminals.     

Synthesis and evaluation of in vitro bioactivity for vesicular acetylcholine transporter inhibitors containing two carbonyl groups

To identify selective high-affinity ligands for the vesicular acetylcholine transporter (VAChT), we have incorporated a carbonyl group into the structures of trozamicol and prezamicol scaffolds, and also converted the secondary amines of the piperidines of trozamicols and prezamicols into amides. Of 18 new racemic compounds, 4 compounds displayed high affinity for VAChT (K(i)=10-20 nM) and greater than 300-fold selectivity for VAChT over σ(1) and σ(2) receptors, namely (4-(4-fluorobenzoyl)-4'-hydroxy-[1,3'-bipiperidin]-1'-yl)(3-methylthiophen-2-yl)methanone oxalate (9g) (K(i-VAChT)=11.4 nM, VAChT/σ(1)=1063, VAChT/σ(2)=370), (1'-benzoyl-4'-hydroxy-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10c) (K(i-VAChT)=15.4 nM, VAChT/σ(1)=374, VAChT/σ(2)=315), (4'-hydroxy-1'-(thiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10e) (K(i-VAChT)=19.0 nM, VAChT/σ(1)=1787, VAChT/σ(2)=335), and (4'-hydroxy-1'-(3-methylthiophene-2-carbonyl)-[1,3'-bipiperidin]-4-yl)(4-methoxyphenyl)methanone oxalate (10g) (K(i-VAChT)=10.2 nM, VAChT/σ(1)=1500, VAChT/σ(2)=2030). These four compounds can be radiosynthesized with C-11 or F-18 to validate their possibilities of serving as PET probes for quantifying the levels of VAChT in vivo.     

Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.     

Mutational and pH analysis of ionic residues in transmembrane domains of vesicular acetylcholine transporter

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.     

Docking study, synthesis, and in vitro evaluation of fluoro-MADAM derivatives as SERT ligands for PET imaging

In order to predict affinity of new diphenylsulfides for the serotonin transporter (SERT), a molecular modeling model was used to compare potential binding affinity of new compounds with known potent ligands. The aim of this study is to identify a suitable PET radioligand for imaging the SERT, new derivatives, and their precursors for a C-11 or F-18 radiolabeling, were synthesized. Two fluorinated derivatives displayed good in vitro affinity for the SERT (K(i)=14.3+/-1 and 10.1+/-2.7 nM) and good selectivity toward the other monoamine transporters as predicted by the docking study.     

Ferulic acid and benzothiazole dimer derivatives with high binding affinity to beta-amyloid fibrils

New ferulic acid and benzothiazole dimer derivatives were synthesized and evaluated by in vitro competition assay using [(125)I]TZDM for their specific binding affinities to Abeta fibrils. In particular, 4a showed the most excellent binding affinity (K(i)=0.53 nM), compared to PIB (K(i)=0.77 nM), for benzothiazole binding sites of Abeta(1-42) fibrils. This result suggests a possibility of a potential AD diagnostic probe for detection of Abeta fibrils.     

Characterization of radioiodinated (-)-ortho-iodovesamicol binding in rat brain preparations

We investigated the binding characteristics of optical isomers of three iodovesamicol analogs to vesicular acetylcholine transporters (VAChT) and to sigma receptors (sigma-1, sigma-2) in rat brains. In competitive inhibition studies, (-)-enantiomers [(-)-ortho-iodovesamicol ((-)-oIV), (-)-meta-iodovesamicol ((-)-mIV), (-)-vesamicol] displayed a higher affinity for VAChT than (+)-enantiomer [(+)-oIV, (+)-mIV, (+)-vesamicol]. (-)-oIV and (-)-mIV showed the same high affinity for VAChT as (-)-vesamicol. For sigma receptors(sigma-1, sigma-2), (-)-oIV (Ki = 62.2 nM (to sigma-1) and 554 nM(to sigma-2)) showed a lower affinity than (-)-mIV (Ki = 4.5 nM (to sigma-1) and 42.9 nM (to sigma-2)). Furthermore, in a saturation binding study, (-)-[125I]-oIV exhibited a Kd of 17.4 +/- 5.1 nM with a maximum number of binding sites, Bmax, of 559 +/- 51 fmol/ mg of protein. These results showed that (-)-oIV binds to vesicular acetylcholine transporters (VAChT) more selectively than (-)-mIV, previously reported as a VAChT mapping agent, and may be suitable for VAChT imaging studies.     

Transmembrane reorientation of the substrate-binding site in vesicular acetylcholine transporter

Active transport of acetylcholine (ACh) by vesicular ACh transporter (VAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [(3)H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat VAChT expressed in PC12(A1237) cells. Equilibrium titrations of [(3)H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the VAChT. NaCl did not mimic ACh chloride, and bafilomycin A(1) and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the VAChT transport cycle.     

Synthesis and nicotinic acetylcholine receptor binding affinities of 2- and 3-isoxazolyl-8-azabicyclo[3.2.1]octanes

A series of epiboxidine homologues, 2- and 3-isoxazole substituted 8-azabicyclo[3.2.1]octane derivatives was synthesized and evaluated as potential ligands for neuronal nicotinic acetylcholine receptors in [(3)H]cytisine labeled rat brain. The 2beta-isoxazolyl-8-azabicyclo[3.2.1]octane 9b (K(i)=3 nM) was the most potent compound of the series with a binding affinity twice that of nicotine. The 3beta-isoxazolyl-8-azabicyclo[3.2.1]octane 15b (K(i)=148 nM) exhibited moderate affinity while the corresponding 2alpha- and 3alpha-isomers exhibited micromolar binding affinity.     

Influence of retinoic acid and of cyclic AMP on the expression of choline acetyltransferase and of vesicular acetylcholine transporter in NG108-15 cells

Treatment of the cholinergic cell line NG108-15 with retinoic acid or cAMP results in an increase of choline acetyltransferase activity (ChAT) whereas none of these agents influences the amount of the vesicular acetylcholine transporter (VAChT) as judged from vesamicol binding and immunoblot studies. We suggest that immaturity of posttranslational events controlling the expression of VAChT protein is responsible for the apparent absence of coregulation of ChAT and VAChT protein expression.     

Acetylcholine binding site in the vesicular acetylcholine transporter

This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently determined three-dimensional structures for other transporters in the major facilitator superfamily.     

Synthesis and evaluation of novel tropane derivatives as potential PET imaging agents for the dopamine transporter

A novel series of tropane derivatives containing a fluorinated tertiary amino or amide at the 2β position was synthesized, labeled with the positron-emitter fluorine-18 (t(1/2)=109.8 min), and tested as potential in vivo dopamine transporter (DAT) imaging agents. The corresponding chlorinated analogs were prepared and employed as precursors for radiolabeling leading to the fluorine-18-labeled derivatives via a one-step nucleophilic aliphatic substitution reaction. In vitro binding results showed that the 2β-amino compounds 6b, 6d and 7b displayed moderately high affinities to DAT (K(i)<10nM). Biodistribution studies of [(18)F]6b and [(18)F]6d showed that the brain uptakes in rats were low. This is likely due to their low lipophilicities. Further structural modifications of these tropane derivatives will be needed to improve their in vivo properties as DAT imaging agents.     

Regulation of vesicular acetylcholine transporter by the activation of excitatory amino acid receptors in the avian retina

1. Previous studies have shown that phorbol esters induce protein kinase C (PKC) mediated phosphorylation of the vesicular acetylcholine transporter (VAChT) and change its interaction with vesamicol. However, it is not clear whether physiological activation of receptors coupled to PKC activation can alter VAChT behavior.2. Here we tested whether activation of kaianate (KA) receptors alters VAChT. Several studies suggest that the cholinergic amacrine cells display KA/AMPA receptors that mediate excitatory input to these neurons. In addition, KA in the chicken retina can generate intracellular messengers with the potential to regulate cellular functions.3. In cultured chicken retina (E8C11) KA reduced vesamicol binding to VAChT by 53%. This effect was potentiated by okadaic acid, a protein phosphatase inhibitor, and was totally prevented by BIM, a PKC inhibitor.4. Phorbol myristate acetate (PMA), but not -PMA, reduced in more than 85% the number of L-[³H]-vesamicol-specific binding sites in chicken retina, confirming that activation of PKC can influence vesamicol binding to chicken VAChT.5. The data show that activation of glutamatergic receptors reduces [³H]-vesamicol binding sites (VAChT) likely by activating PKC and increasing the phosphorylation of the ACh carrier.     

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 5: 2'-Substituted 6-nitroquipazines

Five C2'-substituted 6-nitroquipazine (6-NQ) derivatives were prepared and evaluated in terms of their biological abilities (K(i)) to displace [(3)H]citalopram binding to serotonin transporter. The relationship between their structure and biological activities revealed that shorter alkyl groups tend to possess higher binding affinity. Both compounds 12a and 12c were found to have the equally highest binding affinity (K(i)=0.43+/-0.02 nM).     

Synthesis and evaluation of 1-(4-[¹⁸F]fluoroethyl)-7-(4'-methyl)curcumin with improved brain permeability for β-amyloid plaque imaging

Alzheimer's disease is characterized by the accumulation of β-amyloid (Aβ) plaques and neurofibrillary tangles (NFTs) in the brain. We previously developed [(18)F]fluoropropylcurcumin ([(18)F]FP-curcumin), which demonstrated excellent binding affinity (K(i)=0.07 nM) for Aβ(1-40) aggregates and good pharmacokinetics in normal mouse brains. However, its initial brain uptake was poor (0.52% ID/g at 2 min post-injection). Therefore, in the present study, fluorine-substituted 4,4'-bissubstituted or pegylated curcumin derivatives were synthesized and evaluated. Their binding affinities for Aβ(1-42) aggregates were measured and 1-(4-fluoroethyl)-7-(4'-methyl)curcumin (1) had the highest binding affinity (K(i)=2.12 nM). Fluorescence staining of Tg APP/PS-1 mouse brain sections demonstrated high and specific labeling of Aβ plaques by 1 in the cortex region, which was confirmed with thioflavin-S staining of the same spots in the adjacent brain sections. Radioligand [(18)F]1 was found to have an appropriate partition coefficient (logP(o/w)=2.40), and its tissue distribution in normal mice demonstrated improved brain permeability (1.44% ID/g at 2 min post-injection) compared to that of [(18)F]FP-curcumin by a factor of 2.8 and fast wash-out from mouse brains (0.45% ID/g at 30 min post-injection). These results suggest that [(18)F]1 may hold promise as a PET radioligand for Aβ plaque imaging.     

Syntheses and binding affinities of 6-nitroquipazine analogues for serotonin transporter. Part 2: 4-substituted 6-nitroquipazines

Eleven 4-substituted derivatives of 6-nitroquipazine were synthesized and evaluated for their abilities to displace [3H]citalopram binding to the rat cortical synaptic membranes. Among them, 4-chloro-6-nitroquipazine was shown to possess the highest binding affinity (K(i=)0.03 nM) which was approximately 6 times higher than that of 6-nitroquipazine (K(i)=0.17 nM) itself. In this paper, we describe the syntheses of 4-substituted 6-nitroquipazine derivatives, the results of corresponding biological evaluation and the SAR study.