Synthesis of novel 4- and 5-substituted benzyl ether derivatives of vesamicol and in vitro evaluation of their binding properties to the vesicular acetylcholine transporter site

Detection of the central cholinergic deficits, a consistent feature of Alzheimer's disease, is essential to allow preventive measures and/or symptomatic treatment already at a very early stage of the disease. The vesicular acetylcholine transporter (VAChT) represents an appropriate target to establish PET radiotracer that are adequate for brain imaging the loss of cholinergic terminals. Here we describe the synthesis and binding characteristics of novel derivatives of vesamicol, known to represent a specific antagonist of VAChT sites. Novel benzyl ether derivatives of vesamicol either 4- or 5-substituted at the cyclohexylring have been synthesized by different regioselective ring opening reactions of a same epoxide precursor. The affinity and selectivity of the novel compounds to VAChT sites were analyzed by competitive radioligand binding studies in rat brain and liver membrane preparations using tritium labeled radioligands. The 4-substituted fluorobenzylether of vesamicol 10b was shown to exhibit a high affinity to VAChT sites (K(i)-value(10b)=10.7+/-1.7 nM), but demonstrated also binding capacities to sigma receptors (K(i-)value(10b)=18.5+/-6.9 nM, [(3)H]DTG; K(i)-value(10b)=30.6+/-9.6 nM, [(3)H]haloperidol). The data suggest the potential of vesamicol derivatives to design appropriate radiotracer for PET imaging of central cholinergic deficits.     

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.     

Conformationally constrained ethylenediamines: synthesis and receptor binding of 6,8-diazabicyclo[3.2.2]nonanes

The synthesis and receptor affinity of 6,8-diazabicyclo[3.2.2]nonanes representing conformationally constrained ethylenediamines are described. The Dieckmann analogous cyclization of the (piperazin-2-yl)propionate 9 provided the bicyclononane 10 only, when the first cyclization product was trapped with chlorotrimethylsilane. 10 was stereoselectively transformed into the bicyclic amines 19a,b and amides 22a,b, which were investigated in competition experiments with radioligands for their sigma(1)-, sigma(2)-, kappa-, and mu-receptor affinities. The (2R)-configured dimethylamine 19a showed promising sigma(1)-receptor affinity (K(i)=23.8 nM) and selectivity, whereas the (2S)-configured (dichlorophenyl)acetamide 22b displayed a sigma-receptor binding profile (sigma(1): K(i)=184 nM; sigma(2): K(i)=263 nM) very similar to the binding profile of the atypical antipsychotic BMY-14802 (26).     

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.     

Synthesis of dopamine transporter selective 3-[2-(diarylmethoxyethylidene)]-8-alkylaryl-8-azabicyclo[3.2.1]octanes

A series of 3-[2-(diarylmethoxyethylidene)]-8-alkylaryl-8-azabicyclo[3.2.1]octanes was synthesized and the binding affinities of the compounds were determined at the dopamine and serotonin transporters. The 8-phenylpropyl analogues 8a (K(i)=4.1 nM) and 8b (K(i)=3.7 nM) were the most potent compounds of the series with binding affinities 3 times greater than GBR-12909. In addition, 8a (SERT/DAT=327) was over 300-fold more selective for the dopamine transporter than the serotonin transporter.     

N1-benzenesulfonylgramine and N1-benzenesulfonylskatole: novel 5-HT6 receptor ligand templates

1-Benzenesulfonyl-5-methoxy-N,N-dimethyltryptamine (3; K(i)=2.3 nM) is a 5-HT(6) receptor antagonist; removal of the 5-methoxy group (i.e., 6; K(i)=4.1 nM) has little impact on receptor affinity. In the present study, it is shown that the aminomethyl portion of 6 can be shortened to gramine analogue 10a (K(i)=3.1 nM); a related skatole derivative 11b (K(i)=12 nM) also binds with high affinity indicating that the aminoethyl portion of the tryptamines is not required for binding. Compounds 10a and 11b represent members of novel classes of 5-HT(6) antagonists.     

Synthesis and structure-affinity relationships of novel spirocyclic sigma receptor ligands with furopyrazole structure

The synthesis of novel spirocyclic sigma receptor ligands with high affinity is described. The cyclization of the hydroxy acetal 8, which represents a key step in the synthesis of the spirocyclic compounds 3, was supported by theoretical considerations. The affinity of the spirocyclic furopyrazoles 3a-c to the sigma receptors was determined in receptor binding studies with radioligands. The N-benzyl (3b) and N-butyl (3c) derivatives display very high sigma(1) receptor affinity (3b, K(i)=0.50 nM; 3c, K(i)=1.28 nM) and high selectivity toward the sigma(2) receptor and some other receptor systems. Calculation of crucial distances of the spirocyclic furopyrazole derivatives 3b and 3c shows good correlation with the pharmacophore model of Glennon.     

Epiboxidine and novel-related analogues: a convenient synthetic approach and estimation of their affinity at neuronal nicotinic acetylcholine receptor subtypes

Racemic exo-epiboxidine 3, endo-epiboxidine 6, and the two unsaturated epiboxidine-related derivatives 7 and 8 were efficiently prepared taking advantage of a palladium-catalyzed Stille coupling as the key step in the reaction sequence. The target compounds were assayed for their binding affinity at neuronal alpha4beta2 and alpha7 nicotinic acetylcholine receptors. Epiboxidine 3 behaved as a high affinity alpha4beta2 ligand (K(i)=0.4 nM) and, interestingly, evidenced a relevant affinity also for the alpha7 subtype (K(i)=6 nM). Derivative 7, the closest analogue of 3 in this group, bound with lower affinity at both receptor subtypes (K(i)=50 nM for alpha4beta2 and K(i)=1.6 microM for alpha7) evidenced a gain in the alpha4beta2 versus alpha7 selectivity when compared with the model compound.     

Synthesis and pharmacological evaluation of pentacyclic 6a,7-dihydrodiindole and 2,3-dihydrodiindole derivatives as novel melatoninergic ligands

The synthesis of novel melatonin analogues 3a and 4a-c designed as melatonin receptor ligands is described. Among the newly synthesized ligands, 2-((S)-2-hydroxymethylindolin-1-ylmethyl)-melatonin 4b displayed the highest affinity for MT(1) receptors (K(i)=9.8 nM) and for MT(2) subtype (K(i)=7.8 nM), whereas the rigid pentacyclic ligand 3 showed the highest selectivity towards the MT(2) receptor subtype (K(i)=319.3 nM for MT(1) and K(i)=65.2 nM for MT(2)).     

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 binding affinity at α4β2 and α7 nicotinic acetylcholine receptors of new analogs of epibatidine and epiboxidine containing the 7-azabicyclo[2.2.1]hept-2-ene ring system

A group of novel racemic nicotinic ligands structurally related to epibatidine or epiboxidine [(±)-10-(±)-17] was synthesized through a palladium-catalyzed cross-coupling between the appropriate vinyl triflate and a range of organometallic heterocycles. The target compounds were evaluated for binding affinity at the α4β2 and α7 neuronal nicotinic receptors (nAChRs). The set of 3-pyridinyl derivatives (±)-10, (±)-11 and (±)-12 exhibited an affinity for the α4β2 nAChR subtype in the subnanomolar range (K(i) values of 0.20, 0.40 and 0.50nM, respectively) and behaved as α4β2 versus α7 subtype selective ligands. Interestingly, the epiboxidine-related dimethylammonium iodide (±)-17, which retained a good affinity for the α4β2 nAChR (K(i)=13.30nM), tightly bound also to the α7 subtype (K(i)=1.60nM), thus displaying a reversal of the affinity trend among the reference and new nicotinic ligands under investigation.     

Enantioselective synthesis of S-(+)-2beta-carboalkoxy-3alpha-[bis(4-fluorophenyl)methoxy]tropanes as novel probes for the dopamine transporter

Synthesis of a series of pure S-(+)-2beta-carboalkoxy-3alpha-[bis(4-fluorophenyl)methoxy]tropanes (>99% ee) was achieved by employing a chiral amine-induced asymmetric reaction of tropinone with methyl cyanoformate as the key step. In this series, all of the S-(+)-enantiomers were 2-fold more potent than their racemic mixtures and all displayed high-affinity binding for DAT (K(i)=13-40 nM). These data support previous findings of significant divergence in structural requirements for high-affinity DAT binding among tropane-based inhibitors. Furthermore, the 2-substituent in the 3alpha-[bis(4-fluorophenyl)methoxy]tropane series is well tolerated at the DAT but not at SERT (K(i)=690-2040 nM), or muscarinic M(1) receptors (K(i)=133-4380 nM) resulting in highly selective DAT ligands that may provide new leads toward a cocaine-abuse therapeutic.     

Synthesis and in vitro evaluation of α-synuclein ligands

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.     

Synthesis and SAR studies of chiral non-racemic dexoxadrol analogues as uncompetitive NMDA receptor antagonists

A series of chiral non-racemic dexoxadrol analogues with various substituents in position 4 of the piperidine ring was synthesized and pharmacologically evaluated. Only the enantiomers having (S)-configuration at the 2-position of the piperidine ring and 4-position of the dioxolane ring were considered. Key steps in the synthesis were an imino-Diels-Alder reaction of enantiomerically pure imine (S)-13, which had been obtained from d-mannitol, with Danishefsky's Diene 14 and the replacement of the p-methoxybenzyl protective group with a Cbz-group. It was shown that (S,S)-configuration of the ring junction (position 2 of the piperidine ring and position 4 of the dioxolane ring) and axial orientation of the C-4-substituent ((4S)-configuration) are crucial for high NMDA receptor affinity. 2-(2,2-Diphenyl-1,3-dioxolan-4-yl)piperidines with a hydroxy moiety ((S,S,S)-5, K(i)=28nM), a fluorine atom ((S,S,S)-6, WMS-2539, K(i)=7nM) and two fluorine atoms ((S,S)-7, K(i)=48nM) in position 4 represent the most potent NMDA antagonists with high selectivity against σ(1) and σ(2) receptors and the polyamine binding site of the NMDA receptor. The NMDA receptor affinities of the new ligands were correlated with their electrostatic potentials, calculated gas phase proton affinities (negative enthalpies of deprotonation) and dipole moments. According to these calculations decreasing proton affinity and increasing dipole moment are correlated with decreasing NMDA receptor affinity.     

Isoindol-1,3-dione and isoindol-1-one derivatives with high binding affinity to beta-amyloid fibrils

Based on the structural features of Indoprofen and PIB, a series of isoindol-1,3-diones 1a-k and isoindol-1-ones 2a-l were designed and synthesized. These 23 compounds were evaluated by competitive binding assay against aggregated Abeta42 fibrils using [(125)I]TZDM. All the isoindolone derivatives showed very good binding affinities with K(i) values in the subnanomolar range (0.42-0.94 nM). Among them, isoindol-1,3-diones 1i and 1k and isoindol-1-ones 2c and 2i exhibited excellent binding affinities (K(i)=0.42-0.44 and 0.46-0.49 nM) than those of Indoprofen (K(i)=0.52 nM) and PIB (K(i)=0.70 nM). These results suggest that isoindolones could be served as a scaffold for potential AD diagnostic probes to monitor Abeta fibrils.     

Synthesis and pharmacological evaluation of bicyclic SNC80 analogues with separated benzhydryl moiety

Directed by molecular modeling studies the pharmacophoric benzhydryl moiety of the delta opioid receptor agonist SNC80 was separated and the two phenyl residues were attached to different positions of the conformationally constrained 6,8-diazabicyclo[3.2.2]nonane framework in order to find novel delta agonists. The crucial reaction step in the chiral pool synthesis was the establishment of the three carbon bridge by a Dieckmann analogous cyclization of the allyl and propyl derivatives 6 and 7 to yield the mixed methyl silyl acetals 8 and 9, respectively. Stereoselective Grignard reaction, dehydration, and introduction of the pharmacophoric (N,N-diethylcarbamoylbenzyl) residue led to the designed delta receptor agonists 3, ent-3, and 20 with a double bond in the bicyclic framework. Hydrogenation of the allyl derivative 14 was performed with ammonium formate and Pd/C to yield the saturated ligands 24a and 24b. Removal of the allyl substituent with RhCl(3), hydrogenation of the ring system, and re-attachment of the allyl moiety provided the allyl derivatives 4a and 4b. In receptor binding studies with the radioligand [(3)H]-deltorphine II only ent-3 showed considerable delta receptor affinity (K(i)=740 nM). Since ent-3 also interacts with mu receptors (K(i)=250 nM) it belongs to the very interesting compound class of mixed delta/mu ligands.     

Binding of isotryptamines and indenes at h5-HT6 serotonin receptors

To determine if the indolic nitrogen atom is required for the binding of N(1)-benzyltryptamines at h5-HT(6) serotonin receptors, several isotryptamines and indene analogs were examined. The affinity of 3-benzyl-N(1)-(N,N-dimethylaminoethyl)indole (5, K(i)=32nM) and 1-benzyl-3-(N,N-dimethylaminoethyl)indene (11, K(i)=3nM) indicates that the indolic nitrogen atom is not essential for binding.     

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.     

Synthesis and dopamine transporter binding affinities of 3alpha-benzyl-8-(diarylmethoxyethyl)-8-azabicyclo[3.2.1]octanes

A series of 3alpha-benzyl-8-(diarylmethoxyethyl)-8-azabicyclo[3.2.1]octanes was synthesized and the binding affinities of the compounds were determined at the dopamine transporter. The unsubstituted analogue 7b (K(i)=98nM) was the most potent compound of the series with binding affinity three-times greater than cocaine and only 5-fold less than GBR-12909. The structure-activity data for 7a-f suggests that these compounds may be binding at the dopamine transporter in a similar fashion to GBR 12909.     

Synthesis, biological evaluation and molecular docking studies of novel 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives

In present study, a series of new 2-(1,3,4-oxadiazol-2-ylthio)-1-phenylethanone derivatives (6a-6x) as potential focal adhesion kinase (FAK) inhibitors were synthesized. The bioassay assays demonstrated that compound 6i showed the most potent activity, which inhibited the growth of MCF-7 and A431 cell lines with IC(50) values of 140 ± 10 nM and 10 ± 1 nM, respectively. Compound 6i also exhibited significant FAK inhibitory activity (IC(50)=20 ± 1 nM). Docking simulation was performed to position compound 6i into the active site of FAK to determine the probable binding model.