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 vivo evaluation of [18F]N-(2-benzofuranylmethyl)-N'-[4-(2-fluoroethoxy)benzyl]piperazine, a novel σ1 receptor PET imaging agent

N-(2-Benzofuranylmethyl)-N'-[4-(2-fluoroethoxy)benzyl]piperazine (6, σ(1)K(i)=2.6 nM) was radiolabeled with fluorine-18 to provide a potential σ(1) receptor radioligand for use in positron emission tomography (PET). Radiofluorination of the appropriate tosylate precursor furnished [(18)F]6 with a specific activity of 45 GBq/μmol, in an average radiochemical yield of 18% and greater than 98% radiochemical purity. MicroPET imaging in Papio hamadryas baboon brain revealed [(18)F]6 uptake consistent with σ receptor distribution, and specificity for σ receptors was demonstrated in a haloperidol pre-treated animal. [(18)F]6 possesses suitable properties for PET imaging of σ(1) receptors, and further investigation of this σ(1) receptor tracer is warranted.     

Effects of linker elongation in a series of N-(2-benzofuranylmethyl)-N'-(methoxyphenylalkyl)piperazine σ₁ receptor ligands

In our continued exploration of disubstituted piperazine derivatives as sigma (σ) receptor ligands with central nervous system (CNS) activity, a series of N-(2-benzofuranylmethyl)-N'-(methoxyphenylalkyl)piperazines (16-21 and 26-31) were synthesized, anticipating that these ligands would better suit the structural requirements of the current σ(1) pharmacophore. Affinities of these ligands for σ(1) and σ(2) receptors were investigated by means of radioligand binding assays, with the identification of N-(2-benzofuranylmethyl)-N'-[3-(4-methoxyphenyl)propyl]piperazine (29, K(i)=3.1 nM, σ(2)/σ(1)=45) as a selective σ(1) ligand. The σ(1) affinities and subtype selectivities of piperazines 16-21 and 26-31 were generally comparable to the corresponding benzylic analogs. Additionally, the affinities of 16-21 and 26-31 for the 5-HT(2B) receptor were much lower than the relatively nonselective methoxybenzylic analogs 2-4, indicating that elongation of the alkyl tether generally improved selectivity for σ(1) receptors.     

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.     

Identification of a potent and selective σ₁ receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells

Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ(1) receptor affinity (K(i) values <25 nM) and good σ(1)/σ(2) selectivity (K(i)σ(2) >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ(1) receptor ligands (K(i)=0.70 and 0.86 nM, respectively) and to display significantly high selectivity over σ(2), μ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ(1) receptor antagonist BD-1063 totally counteracted this effect, confirming that σ(1) receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ(1) agonist profile. As a part of our work, a threedimensional σ(1) pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ(1) receptor subtype.     

Exploration of ring size in a series of cyclic vicinal diamines with σ(1) receptor affinity

Imidazolidine and 1,4-diazepane analogs of N-(2-benzofuranyl)methyl-N'-(4-alkoxybenzyl)piperazines were prepared to explore the effect of ring contraction and expansion on σ receptor affinity and subtype selectivity within a series of cyclic diamines. In vitro receptor binding assays revealed that all cyclic vicinal diamines possessed affinity and selectivity for σ(1) receptors. The imidazolidines possessed nanomolar σ(1) affinities (K(i)=6.45-53.5nM), and relatively low levels of subtype selectivity (σ(2)/σ(1)=58-237). However, the piperazines and diazepanes achieved picomolar σ(1) interactions, with K(i) ranges of 0.05-10.28 and 0.10-0.194nM, respectively. Moreover, the piperazines and diazepanes showed excellent discrimination over the σ(2) receptor, with σ(1) selectivities of 143-16140 and 220-11542, respectively.     

Synthesis and characterization of [¹²⁵I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [(3)H]glycine uptake (IC(50)=2.4 nM). Saturation assay in rat cortical membranes revealed that [(125)I]5 had a single high affinity binding site with a K(d) of 1.54 nM and a B(max) of 3.40 pmol/mg protein. In vitro autoradiography demonstrated that [(125)I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [(125)I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [(125)I]5 showed high blood-brain barrier permeability (1.68-2.17% dose/g at 15-60 min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [(125)I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.     

Binding properties of a highly potent and selective iodinated aminopeptidase N inhibitor appropriate for radioautography

Aminopeptidase N (APN) is a zinc metallopeptidase involved in the inactivation of biologically active peptides. The knowledge of its precise distribution is crucial to investigate its physiological role. This requires the use of appropriate probes such as the recently developed highly potent and selective radiolabeled APN inhibitor 2(S)-benzyl-3-[hydroxy(1'(R)-aminoethyl)phosphinyl]propanoyl-L-3-[ (12 5)I]iodotyrosine ([(125)I]RB 129). Its binding properties were investigated using rat brain homogenates (K(d)=3.4 nM) or APN expressed in COS-7 cells (K(d)=0.9 nM). The specific binding was 95% at [K(d)], and preliminary autoradiography in intestine is promising. The decreased affinity of [(125)I]RB 129 (=10(-6) M) for the E(350)D APN mutant, supports the critical role of E(350) in the amino-exopeptidase action of APN.     

Radioiodinated aza-diphenylacetylenes as potential SPECT imaging agents for beta-amyloid plaque detection

Two new iodinated fluoro- and hydroxy-pegylated aza-diphenylacetylene derivatives, 1 and 2, targeting beta-amyloid (Abeta) plaques have been successfully prepared. In vitro binding carried out in tissue homogenates prepared from postmortem AD brains with [(125)I]IMPY (6-iodo-2-(4'-dimethylamino)phenyl-imidazo[1,2-a]pyridine) as the radioligand indicated good binding affinities (K(i)=9.2 and 16.8 nM for 1 and 2, respectively). Brain penetrations of the corresponding radioiodinated ligands, evaluated in the normal mice, showed good initial brain penetrations (3.55% and 5.67% ID/g for [(125)I]1 and [(125)I]2 at 2 min post-injection). The washout from normal mice brain was relatively fast (0.33% and 0.91% ID/g at 2h post-injection). The specific binding of these radioiodinated ligands to beta-amyloid plaques was clearly demonstrated using film autoradiography of AD brain sections. Taken together, these preliminary results strongly suggest that these novel iodinated aza-diphenylacetylenes may be potentially useful for imaging Abeta plaques in the living human brain.     

Apparent cooperativity in multivalent verotoxin-globotriaosyl ceramide binding: kinetic and saturation binding studies with [(125)I]verotoxin

Verotoxin (VT) binding to the trisaccharide portion of globotriaosyl ceramide (Gb(3)) is believed to be a crucial step in the development of hemolytic uremic syndrome (HUS) commonly known as 'Hamburger disease'. This interaction is the initial step in the binding process and defines the specificity of verotoxin binding to cellular membranes. Although molecular modeling, co-crystallization and co-NMR studies with VT and the trisaccharide moiety of Gb(3) have indicated potential multiple sites for Gb(3) binding, little is known about their direct effects on kinetic and equilibrium binding. Here we describe how the binding of radiolabeled VT ([(125)I]VT1) to Gb(3) in a microtiter well format, is driven by two different association rate constants (k(+1a)=0.0075 and k(+1b)=0.275 min(-1) nM(-1)) with the high affinity site representing 15% of the total specific binding sites. Binding was reversible at room temperature, reached equilibrium after 2-3 h, and non-specific binding was less than 5%. Equilibrium binding studies defined by [(125)I]VT1 saturation binding to 15, 30, 60 and 120 ng Gb(3)/well, showed the presence of a single site with dissociation constants (K(d)s) ranging between 0.5 and 3 nM. However, the maximum density of specific [(125)I]VT1 binding sites (B(max)) did not directly correlate with the Gb(3) concentration per well: the most[(125)I]VT1 binding was observed for 60 ng Gb(3) (B(max)=1.28 nM; compared to 0. 23 nM for 30 ng Gb(3) and 0.65 nM for 120 ng Gb(3)). Furthermore, while Hill coefficients (n(H)) for 15, 30 and 120 ng Gb(3) were close to unity indicating single interactions, for the saturation isotherm for 60 ng Gb(3)/well n(H) was 1.4. Subsequent Scatchard analysis yielded a concave downward curve for [(125)I]VT1 binding to 60 ng Gb(3)/well, suggesting positive co-operativity. We present, for the first time, conclusive binding data confirming the presence of at least two discrete Gb(3) binding sites: these multivalent interactions between verotoxin VT-1 and Gb(3) were described by association reactions driven by two distinct rate constants, as well as by the positive co-operativity governing binding at a restricted receptor concentration. These results imply that the concentration of Gb(3) on the surface of target cells can have a complex, non-linear effect on verotoxin binding and thereby, on sensitivity to cytotoxicity.     

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.     

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.     

Photoaffinity labeling of the alpha 1-adrenergic receptor using an 125I-labeled aryl azide analogue of prazosin

alpha 1-Adrenergic receptor probes, which can be radioiodinated to yield high specific activity radioligands, have been synthesized and characterized. 2-[4-(4-Amino-benzoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquin azoline (CP63,155), an arylamine analogue of the selective alpha 1-adrenergic antagonist prazosin, and its iodinated derivative, 2-[4-(4-amino-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP63,789), bind reversibly and with high affinity (KD = 1 nM and 0.6 nM, respectively) to rat hepatic membrane alpha 1-adrenergic receptors. Conversion of [125I]CP63,789 to the aryl azide yields a photolabile derivative, 2-[4-(4-azido-3-[125I]iodobenzoyl)piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline [( 125I]CP65,526), which prior to photolysis binds competitively and with high affinity (KD = 0.3 nM). Binding of [125I]CP63,789 and [125I]CP65,526 (prior to photolysis) is rapid and saturable. Both ligands identify similar alpha 1-adrenergic receptor binding site concentrations as the parent probe, [3H]prazosin. Specific binding by these iodinated ligands is stereoselective and inhibited by a variety of adrenergic agents with a specificity typical of the alpha 1-adrenergic receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [125I]CP65,526-labeled rat hepatic membranes reveal major protein species with molecular weights of 77K, 68K and 59K. Each protein binds adrenergic ligands with stereoselectivity and with a specificity typical of the alpha 1-adrenergic receptor. Inclusion of multiple protease inhibitors during membrane preparation prior to SDS-PAGE does not alter the labeling of these peptides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of synthetic peptide corresponding to the ACTH-like sequence of human immunoglobulin G1 on proliferation of lymphoblastoid cells

Influence of the ACTH-like peptide H-Val-Lys-Lys-Pro-Gly-Ser-Ser-Val-Lys-Val-OH corresponding to the sequence 11-20 of the variable part of human immunoglobulin G1 (IgG1) heavy chain on growth of MT-4 human T-lymphoblastoid cell line was investigated. It was found that the ACTH-like peptide at concentration range 10(-11) -10(-7) M inhibits the proliferation of MT-4 cells. Labeled ACTH 'address segment' [(125)I]ACTH (13-24) was used to establish that MT-4 cells express specific receptors for ACTH (K(d) = 97 pM). The ACTH-like peptide and human ACTH (but not IgG1 heavy chain) were shown to compete with [(125)I]ACTH (13-24) for binding to these receptors (K(i1) = 0.38 nM and K(i2) = 0.34 nM).     

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, nicotinic acetylcholine receptor binding, antinociceptive and seizure properties of methyllycaconitine analogs

A series of methyllycaconitine (1a, MLA) analogs was synthesized where the (S)-2-methylsuccinimidobenzoyl group in MLA was replaced with a (R)-2-methyl, 2,2-dimethyl-, 2,3-dimethyl, 2-phenyl-, and 2-cyclohexylsuccinimidobenzoyl (1b-f) group. The analogs 1b-f were evaluated for their inhibition of [(125)I]iodo-MLA binding at rat brain alpha7 nicotinic acetylcholine receptors (nAChR). In order to determine selectivity, MLA and the analogs 1b-f were evaluated for inhibition of binding to rat brain alpha,beta nAChR using [(3)H]epibatidine. At the alpha7 nAChR, MLA showed a K(i) value of 0.87 nM, analogs 1b-e possessed K(i) values of 1.67-2.16 nM, and 1f showed a K(i) value of 26.8 nM. Surprisingly, the analog 1e containing the large phenyl substituent (K(i)=1.67 nM) possessed the highest affinity. None of the compounds possessed appreciable affinity for alpha,beta nAChRs. MLA antagonized nicotine-induced seizures with an AD(50)=2 mg/kg. None of the MLA analogs were as potent as MLA in this assay. MLA and all of the MLA analogs, with the exception of 1b, antagonized nicotine's antinociceptive effects in the tail-flick assay. Compound 1c (K(i)=1.78 nM at alpha7 nAChR) with an AD(50) value of 1.8 mg/kg was 6.7 times more potent than MLA (AD(50)=12 mg/kg) in antagonizing nicotine's antinociceptive effects but was 5-fold less potent than MLA in blocking nicotine-induced seizures. Since MLA has been reported to show neuroprotection against beta-amyloid(1-42), these new analogs which have high alpha7 nAChR affinity and good selectivity relative to alpha,beta nAChRs will be useful biological tools for studying the effects of alpha7 nAChR antagonist and neuroprotection.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

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.     

gamma-Carbolines: binding at 5-HT5A serotonin receptors

Screening of various agents resulted in the identification of 5-methyl-1,2,3,4-tetrahydro-gamma-carboline (1; K(i)=5,300 nM) as a compound with modest affinity for mouse 5-HT(5A) receptors. Structure-affinity studies were conducted resulting in 5-methyl-2-[3-(4-fluorophenoxy)propyl]-1,2,3,4-tetrahydro-gamma-carboline (17; K(i)=13 nM). Although 17 also binds at 5-HT(2) receptors, it serves as a novel lead for the further development of 5-HT(5A) ligands.     

Radiosynthesis and in vivo evaluation of [11C]MP-10 as a PET probe for imaging PDE10A in rodent and non-human primate brain

2-((4-(1-[(11)C]Methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl)phenoxy)methyl)-quinoline (MP-10), a specific PDE10A inhibitor (IC(50)=0.18 nM with 100-fold selectivity over other PDEs), was radiosynthesized by alkylation of the desmethyl precursor with [(11)C]CH(3)I, ～45% yield, >92% radiochemical purity, >370 GBq/μmol specific activity at end of bombardment (EOB). Evaluation in Sprague-Dawley rats revealed that [(11)C]MP-10 had highest brain accumulation in the PDE10A enriched-striatum, the 30 min striatum: cerebellum ratio reached 6.55. MicroPET studies of [(11)C]MP-10 in monkeys displayed selective uptake in striatum. However, a radiolabeled metabolite capable of penetrating the blood-brain-barrier may limit the clinical utility of [(11)C]MP-10 as a PDE10A PET tracer.