Synthesis and biological evaluation of 3-alkyl-dihydrotetrabenazine derivatives as vesicular monoamine transporter-2 (VMAT2) ligands

In the search of new probes for in vivo brain imaging of vesicular monoamine transporter type 2 (VMAT2), we have developed an efficient synthesis of a novel series of 3-alkyl-dihydrotetrabenazine (DTBZ) derivatives. The affinity of VMAT2 was evaluated by an in vitro inhibitory binding assay using [¹²⁵I]-iodovinyl-TBZ or [¹⁸F](+)-FP-DTBZ as radioligands in rat striatal tissue homogenates. New DTBZ derivatives exhibited moderate to good binding affinity to VMAT2. Among these new ligands, compound 4b showed the best affinity for VMAT2 (K_i = 5.98 nM) and may be a useful lead compound for future structure-activity studies.     

Synthesis and evaluation of a series of tropane analogues as novel vesicular monoamine transporter-2 ligands

A series of tropane derivatives has been synthesized as lobelane analogues and evaluated for their binding affinity at the vesicular monoamine transporter-2 (VMAT2), and at alpha4beta2* and alpha7* nicotinic acetylcholine receptors. The trop-2-ene analogues 4a and 4b exhibited good affinity and high selectivity for VMAT2.     

Lobelane analogues as novel ligands for the vesicular monoamine transporter-2

A series of lobelane analogues has been synthesized and their structure-activity relationships at the vesicular monoamine transporter-2 (VMAT2) have been evaluated. The most potent analogues in this series were the cis-2,6-piperidino analogues, 25b, 27b, 28b, and 30b, with K(i) values ranging from 430 to 580 nM.     

Synthesis and evaluation of a series of homologues of lobelane at the vesicular monoamine transporter-2

A series of lobelane homologues has been synthesized and evaluated for their [(3)H]DTBZ binding affinity at the vesicular monoamine transporter-2 (VMAT2). The structure-activity relationships (SAR) indicate that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at VMAT2.     

Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.     

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.     

The molecular chaperone Hsc70 interacts with the vesicular monoamine transporter-2

Synaptic transmission depends on the efficient loading of transmitters into synaptic vesicles by vesicular neurotransmitter transporters. The vesicular monoamine transporter-2 (VMAT2) is essential for loading monoamines into vesicles and maintaining normal neurotransmission. In an effort to understand the regulatory mechanisms associated with VMAT2, we have embarked upon a systematic search for interacting proteins. Glutathione-S-transferase pull-down assays combined with mass spectrometry led to the identification of the 70-kDa heat shock cognate protein (Hsc70) as a VMAT2 interacting protein. Co-immunoprecipitation experiments in brain tissue and heterologous cells confirmed this interaction. A direct binding was observed between the amino terminus and the third cytoplasmic loop of VMAT2, as well as, a region containing the substrate binding and the carboxy-terminal domains of Hsc70. Furthermore, VMAT2 and Hsc70 co-fractionated with purified synaptic vesicles obtained from a sucrose gradient, suggesting that this interaction occurs at the synaptic vesicle membrane. The functional significance of this novel VMAT2/Hsc70 interaction was examined by performing vesicular uptake assays in heterologous cells and purified synaptic vesicles from brain tissue. Recombinant Hsc70 produced a dose-dependent inhibition of VMAT2 activity. This effect was mimicked by the closely related Hsp70 protein. In contrast, VMAT2 activity was not altered in the presence of previously denatured Hsc70 or Hsp70, as well as the unrelated Hsp60 protein; confirming the specificity of the Hsc70 effect. Finally, a purified Hsc70 fragment that binds VMAT2 was sufficient to inhibit VMAT2 activity in synaptic vesicles. Our results suggest an important role for Hsc70 in VMAT2 function and regulation.     

Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [³H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [³H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [³H]DA uptake at VMAT2, Ki changes in the nanomolar range (K_i?=?0.014–0.073?µM). Compound 15d exhibited the highest affinity (K_i?=?0.014?µM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K_i?=?0.073?µM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.     

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 biological evaluation of aryl azide derivatives of combretastatin A-4 as molecular probes for tubulin

Two new aryl azides, (Z)-1-(3'-azido-4'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 9 and (Z)-1-(4'-azido-3'-methoxyphenyl)-2-(3",4",5"-trimethoxyphenyl)ethene 5, modeled after the potent antitumor, antimitotic agent combretastatin A-4 (CA-4), have been prepared by chemical synthesis as potentially useful photoaffinity labeling reagents for the colchicine site on beta-tubulin. Aryl azide 9, in which the 3'-hydroxyl group of CA-4 is replaced by an azido moiety, demonstrates excellent in vitro cytotoxicity against human cancer cell lines (NCI 60 cell line panel, average GI50 = 4.07 x 10(-8) M) and potent inhibition of tubulin polymerization (IC50 = 1.4+/-0.1 microM). The 4'-azido analogue 5 has lower activity (NCI 60 cell line panel, average GI50 = 2.28 x 10(-6) M, and IC50 = 5.2+/-0.2 microM for inhibition of tubulin polymerization), suggesting the importance of the 4'-methoxy moiety for interaction with the colchicine binding site on tubulin. These CA-4 aryl azide analogues also inhibit binding of colchicine to tubulin, as does the parent CA-4, and therefore these compounds are excellent candidates for photoaffinity labeling studies.     

In vivo imaging of the vesicular acetylcholine transporter and the vesicular monoamine transporter.

Validation of the vesicular acetylcholine transporter (VAChT) and the neuronal vesicular monoamine transporter (VMAT2) as important molecular targets in the cholinergic and dopamine neurons, respectively, has sparked interest in the development of radiotracers for studying these markers in vitro and in vivo. Currently, a number of selective high-affinity radiotracers are available for studying these targets in vivo with positron emission tomography (PET) or single photon emission computed tomography (SPECT). PET studies of VMAT2 in neuropathology reveal changes in the density of this marker that can be verified independently. Similarly, in vivo studies with VAChT ligands suggest that the latter are potentially useful in detecting cholinergic lesions in vivo; however, additional development is required to fully realize the potential of these radioligands.     

Synthesis ofN-Acetylglucosamine derivatives as probes for specificity of chicken hepatic lectin

Syntheses of the following compounds are described: 6-(Trifluoroacetylamino)hexyl 2-acetamido-2,6-dideoxy--d-glucopyranoside and 2-acetamido-2-deoxy--d-xylopyranoside, two allyl 2-acetamido-2-deoxy--d-glucopyranosiduronic acid derivatives, and several allyl 2-acylamido-2-deoxy--d-glucopyranosides having different acyl groups. These and other compounds were used as inhibitors in the binding assay for the chicken hepatic lectin specific forN-acetylglucosamine. We found that: 1) The inhibitory potency ofN-acylglucosamine derivatives decreased progressively with increase in the size of acyl group, 2) absence of either 3-or 4-OH group ofN-acetylglucosamine lowered the binding affinity more than 100-fold, and 3) the presence of a negatively charged group (carboxylic acid) at the C-6 position did not lower the affinity. The first two items are similar to the mammalian hepatic galactose/N-acetylgalactosamine lectins, but the last item is in a strong contrast to the mammalian lectins.Abbreviations XyLNAc N-acetyl-d-xylosamine - BSA bovine serum albumin - NeuAc N-acetylneuraminic acid - GlcNAc34-BSA amidino-type neoglycoprotein [6] containing on the average 34N-acetylglucosaminyl residues per BSA molecule     

Synthesis and evaluation of 2-substituted 4(3H)-quinazolinone thioether derivatives as monoamine oxidase inhibitors

In the present study, a series of fourteen 2-mercapto-4(3H)-quinazolinone derivatives was synthesised and evaluated as potential inhibitors of the human monoamine oxidase (MAO) enzymes. Quinazolinone is the oxidised form of quinazoline, and although this class has not yet been extensively explored as MAO inhibitors, it has been shown to possess a wide variety of biological activities. Among the quinazolinone derivatives investigated, seven compounds (IC₅₀?<?1?µM) proved to be potent and specific MAO-B inhibitors, with the most potent inhibitor, 2-[(3-iodobenzyl)thio]quinazolin-4(3H)-one, exhibiting an IC₅₀ value of 0.142?μM. Further investigation showed that this inhibitor is a reversible and competitive inhibitor of MAO-B with a K_i value of 0.068?µM. None of the test compounds were MAO-A inhibitors. Analysis of the structure-activity relationships (SARs) for MAO-B inhibition shows that substitution on the C2 position of quinazolinone with a benzylthio moiety bearing a Cl, Br or I on the meta position yields the most potent inhibitors of the series. In contrast, substitution with the unsubstituted benzylthio moiety (IC₅₀?=?3.03?µM) resulted in significantly weaker inhibition activity towards MAO-B. This study suggests that quinazolinones are promising leads for the development of selective MAO-B inhibitors which may be used for the treatment of neurodegenerative disorders such as Parkinson’s disease.     

Genetic deletion of vesicular monoamine transporter-2 (VMAT2) reduces dopamine transporter activity in mesencephalic neurons in primary culture

Our aim was to investigate whether a defect in vesicular monoamine transporter-2 (VMAT2) activities would affect dopaminergic cell functions or not. We examined mesencephalon dopaminergic cultures prepared from VMAT2 wild-type, heterozygous or homozygous knockout (KO) 14-day-old mouse fetuses to determine the number of tyrosine hydroxylase (TH)-positive cells and dopamine transporter activity. The number of TH-positive cells remained unchanged in the VMAT2-KO cultures. Of interest, the dopamine transporter activity in the homozygous cells was significantly decreased, but not in the heterozygous cells, suggesting that complete deletion of VMAT2 inhibited dopamine transporter function. Furthermore, dopamine transporter activity was prominently decreased in the synaptosomal fraction of neonatal homozygous VMAT2-KO mice compared with that of wild-type/heterozygous VMAT2-KO ones, indicating that VMAT2 activity might be one of the factors regulating dopamine transporter activities. To test this possibility, we used reserpine, a VMAT2 inhibitor. Reserpine (1muM) decreased dopamine transporter activity (approx. 50%) in wild-type and heterozygous VMAT2-KO cultures but not in homozygous ones, indicating that blockade of VMAT2 activity reduced dopamine transporter activity. To investigate possible mechanisms underlying the decreased dopamine transporter activity in VMAT2-KO mice, we measured dopamine transporter activities after 24-48h exposure of primary cultures of mesencephalic neurons to dopamine receptor antagonists, PKC inhibitor, PI(3)K inhibitor, and l-DOPA. Among these drugs, l-DOPA slightly reduced the dopamine transporter activities of all genotypes, but the other drugs could not. Since the ratios of reduction in dopamine transporter activity of each genotype treated with l-DOPA were similar, substrate inhibition of dopamine transporters was not the main mechanism underlying the reduced dopamine transporter activity due to genetic deletion of VMAT2. Our results demonstrate that genetic deletion of VMAT2 did not induce immediate cell death but did markedly inhibit dopamine transporter activity.     

Synthesis and biological evaluation of technetium-99m-labeled deoxyglucose derivatives as imaging agents for tumor

Three deoxyglucose (DG) derivatives, S-DG, MAG(3)-DG and MAMA-BA-DG, were synthesized and labeled successfully with high labeling yields and high radio-chemical purities. Biodistribution in tumor-bearing mice demonstrated that these three new (99m)Tc-deoxyglucose derivatives showed accumulation in tumor and high tumor-to-muscle ratios. Among them, the (99m)Tc-MAG(3)-DG showed the best characteristics as a potential tumor marker for single photon emission computed tomography (SPECT).     

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.     

Synthesis and evaluation of stilbene derivatives as a potential imaging agent of amyloid plaques

Fluorescence probes that can detect Aβ (β-amyloid peptide) plaque are important tools for diagnosis of Alzheimer’s disease (AD), and 4-N-methylamino-4′-hydroxystilbene (SB-13) is one of the promising candidate molecules. We report here the synthesis of SB-13 derivatives that consist of various electron donating/withdrawing moieties and distinct size of N-substituents. The synthesized compounds were screened for detection of Aβ40 fibrils in vitro. Four compounds exhibited more than sixfold intensity increase, and they were further analyzed for detail bindings and Aβ plaque imaging. Among these molecules, compound 42 meets two critical requirements for imaging agent; high fluorescence responsiveness and strong binding affinity. This compound showed more than 25-fold increase with the dissociation constant of 1.13 ± 0.37 μM. In AD mouse brain tissue, 42 selectively stained Aβ plaque, more specifically peripheral regions of Aβ plaque. This finding demonstrated its potential use as brain-imaging agents for AD studies.     

Synthesis and evaluation of radioactive/fluorescent peptide probes for imaging of legumain activity

Legumain or asparaginyl endopeptidase is an enzyme overexpressed in some cancers and involved in cancer migration, invasion, and metastasis. We have developed radioiodine- ([¹²⁵I]I-LCP) or fluorescein-labeled peptides (FL-LCP) with a cell-permeable d-Arg nonamer fused to an anionic d-Glu nonamer via a legumain-cleavable linker, to function as peptide probes that measure and monitor legumain activity. Non-cleavable probes of FL-NCP and [¹²⁵I]I-NCP were similarly prepared and evaluated as negative control probes by altering their non-cleavable sequence. Model peptides with the legumain-cleavable or non-cleavable sequence (LCP and NCP, respectively) reacted with recombinant human legumain, and only LCP was digested by this enzyme. [¹²⁵I]I-LCP uptake in legumain-positive HCT116 cells was significantly higher than that of [¹²⁵I]I-NCP (11.2 ± 0.44% vs 1.75 ± 0.06% dose/mg). The accumulation of FL-LCP in the HCT116 cells was rather low (4.75 ± 0.29% dose/mg protein), but not significantly different from the levels of FL-NCP. It is possible that low concentrations of [¹²⁵I]I-LCP (40 pM) can be effectively internalized after legumain cleavage. On the other hand, the cellular uptake of much higher concentrations of the FL-LCP derivative (1 mM) may be restricted by high concentrations of polyanions. The in vivo biodistribution studies in tumor-bearing mice demonstrated that the tumor uptake of [¹²⁵I]I-LCP was 1.34% injected dose per gram (% ID/g) at 30 min. The tumor/blood and tumor/muscle ratios at 30 min were 0.63 and 1.77, respectively, indicating that the [¹²⁵I]I-LCP accumulation in tumors was inadequate for in vivo imaging. Although further structural modifications are necessary to improve pharmacokinetic properties, [¹²⁵I]I-LCP has been demonstrated to be an effective scaffold for the development of nuclear medicine imaging probes to monitor legumain activity in living subjects.     

Synthesis of fluorescent derivatives of wortmannin and demethoxyviridin as probes for phosphatidylinositol 3-kinase

Fluorescent analogs were synthesized of the potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin. The esterification of 11-deacetylwortmannin, 17-hydroxywortmannin, and demethoxyviridin with the fluorescent carboxylic acids NBD-sarcosine and 7-dimethylaminocoumarin-4-acetic acid generated six novel fluorescent esters. Potent inhibition of PI 3-kinase-alpha was observed for the derivatives of 11-desacetylwortmannin and demethoxyviridin.