Synthesis and NMDA receptor affinity of fluorinated dioxadrol analogues

A series of dioxadrol analogues with fluorine substituents in position 4 of the piperidine ring has been synthesized and pharmacologically evaluated. The key step in the synthesis was the fluorination of diastereomeric piperidones 6a and 6c as well as diastereomeric alcohols 9a and 9c with DAST. The reaction of the alcohols 9a and 9c took place with inversion of configuration. After removal of the Cbz-protective group, the NMDA receptor affinities of the resulting secondary amines 8a, 8c, 12b, and 12d were investigated in receptor binding studies. It was shown that the like-configuration of the ring junction was crucial for high NMDA receptor affinity. An axially oriented fluorine atom in position 4 led to 2-(2,2-diphenyl-1,3-dioxolan-4-yl)-4-fluoropiperidine (12d, WMS-2517) with a K_i-value of 27 nM. The NMDA receptor affinity of 8c (WMS-2513) with an additional fluorine atom in equatorial 4-position was slightly reduced (K_i = 81 nM). Both fluorinated dioxadrol derivatives 8c and 12d showed high selectivity against σ₁ and σ₂ receptors as well as the polyamine binding site of NR2B receptors.     

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.     

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.     

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)).     

Conformationally constrained NR2B selective NMDA receptor antagonists derived from ifenprodil: Synthesis and biological evaluation of tetrahydro-3-benzazepine-1,7-diols

NR2B selective NMDA receptor antagonists with tetrahydro-3-benzazepine-1,7-diol scaffold have been designed by formal cleavage and reconstitution of the piperidine ring of the lead compound ifenprodil (1). The secondary amine 10 represents the central building block for the synthesis of more than 25 tetrahydro-3-benzazepin-1-ols. Generally 7-hydroxy derivatives display higher NR2B receptor affinities than the corresponding 7-benzyloxy compounds. A distance of four atoms (five bond lengths) between the basic amino group and the terminal aryl moiety led to highest NR2B affinity. 3-(4-Phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol (WMS-1410, 25) represents the most promising NR2B antagonist of this series showing a K(i)-value of 14nM. Compound 25 reveals excellent selectivity over more than 100 further relevant target proteins, antagonizes glutamate induced excitotoxicity (IC(50)=18.4nM) and is metabolically more stable than ifenprodil. Up to a dose of 100mg/kg 25 is well tolerated by mice and it shows dose dependent analgesic activity in the late neuropathic pain phase of the formalin assay.     

N-[4-(methylsulfonylamino)benzyl]thiourea analogues as vanilloid receptor antagonists: analysis of structure-activity relationships for the "C-Region"

We recently reported that N-(4-t-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl] thiourea (2) was a high affinity antagonist of the vanilloid receptor with a binding affinity of K(i)=63 nM and an antagonism of K(i)=53.9 nM in rat VR1 heterologously expressed in Chinese hamster ovary (CHO) cells (Mol. Pharmacol. 2002, 62, 947-956). In an effort to further improve binding affinity and antagonistic potency, we have modified the C-region of the lead 4-t-butylbenzyl group with diverse surrogates, such as araalkyl, alkyl, 4-alkynylbenzyl, indanyl, 3,3-diarylpropyl, 4-alkoxybenzyl, 4-substituted piperazine and piperidine. The lipophilic surrogates, arylalkyl and alkyl, conferred modest decreases in binding affinities and antagonistic potencies; the groups having heteroatoms resulted in dramatic decreases. Our findings indicate that 4-t-butylbenzyl is one of the most favorable groups for high receptor binding and potent antagonism to VR1 in this structural series.     

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.     

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.     

The Ketamine Analogue Methoxetamine and 3- and 4-Methoxy Analogues of Phencyclidine Are High Affinity and Selective Ligands for the Glutamate NMDA Receptor

In this paper we determined the pharmacological profiles of novel ketamine and phencyclidine analogues currently used as ‘designer drugs’ and compared them to the parent substances via the resources of the National Institute of Mental Health Psychoactive Drug Screening Program. The ketamine analogues methoxetamine ((RS)-2-(ethylamino)-2-(3-methoxyphenyl)cyclohexanone) and 3-MeO-PCE (N-ethyl-1-(3-methoxyphenyl)cyclohexanamine) and the 3- and 4-methoxy analogues of phencyclidine, (1-[1-(3-methoxyphenyl)cyclohexyl]piperidine and 1-[1-(4-methoxyphenyl)cyclohexyl]piperidine), were all high affinity ligands for the PCP-site on the glutamate NMDA receptor. In addition methoxetamine and PCP and its analogues displayed appreciable affinities for the serotonin transporter, whilst the PCP analogues exhibited high affinities for sigma receptors. Antagonism of the NMDA receptor is thought to be the key pharmacological feature underlying the actions of dissociative anaesthetics. The novel ketamine and PCP analogues had significant affinities for the NMDA receptor in radioligand binding assays, which may explain their psychotomimetic effects in human users. Additional actions on other targets could be important for delineating side-effects.     

ssDNA aptamers that selectively bind oxytetracycline

Single stranded DNA aptamers that bind with high affinity and specificity to the oxytetracycline (OTC) were identified by selection from an oligonucleotide library of 10(15) molecules. The binding affinities of four aptamers were in nanomolar range. The aptamers were highly selective in that, lack of -OH group at 5-position in tetracycline and -H group in place of -OH at 6-position in doxycycline determined the specificity of these aptamers to bind OTC. Three aptamers designated as No. 4, 5, and 20 shared strong affinities with K(d)=9.61, 12.08, and 56.84 nM, respectively, as well as selectivity to bind OTC (72-76%). Aptamer No. 4 had strong affinity among all with high selectivity, whereas No. 2 had relatively weak affinity (K(d)=121.1 nM) and moderate selectivity (52%). Our results indicated that the aptamers No. 4, 5, and 20 with variable 40-base oligonucleotides can be good candidates for selectively binding to OTC with high molecular discrimination over its analogs such as tetracycline and doxycycline.     

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.     

N6-Cycloalkyl-2-substituted adenosine derivatives as selective, high affinity adenosine A1 receptor agonists

A series of new selective, high affinity A(1)-AdoR agonists is reported. Compound 23 that incorporated a carboxylic acid functionality in the 4-position of the pyrazole ring displayed K(iL) value of 1 nM for the A(1)-AdoR and >5000-fold selectivity over the A(3) and A(2A)-AdoRs. In addition, compound 19 that incorporated a carboxamide functionality in the 4-position of the pyrazole ring displayed subnanomolar affinity for the A(1)-AdoR (K(iL)=0.6 nM) and >600-fold selectivity over the A(3) and A(2A)-AdoRs.     

Synthesis and structure/NMDA receptor affinity relationships of 1-substituted tetrahydro-3-benzazepines

A novel synthesis of 1-substituted tetrahydro-1H-3-benzazepines 4 is described. Starting with (2-bromophenyl)acetaldehyde acetal 5, the nitrostyrene 9 was prepared in three steps allowing the addition of various nucleophiles to yield the nitroacetals 10. The one-pot Zn/HCl reductive cyclization of the nitroacetals 10 provided the 3-benzazepines 4, which were investigated for their affinity to the phencyclidine binding site of the NMDA receptor. A one-atomic spacer between the 3-benzazepine system and the phenyl residue in position 1 seems to be favorable for high NMDA receptor binding. In this series the benzazepine 4l substituted with the conformationally restricted and H-bond accepting acetanilide substituent in position 1 displays the highest NMDA receptor affinity (K(i)=89 nM).     

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 pharmacology of new enantiopure delta(3)-4-arylkainoids

Seven delta(3)-4-arylkainoids possessing various 4-position aromatic and heteroaromatic groups were synthesized and their apparent affinities were measured in order to explore the influences of 4-position electron density and stereochemistry on receptor affinity and specificity. Kainoids 1a-f were shown to be selective agonists at the NMDA receptor and the electron rich furanyl and thienyl analogues exhibited the highest affinities. Naphthylkainoid 1g proved to be a nonselective antagonist at the iGluRs.     

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).     

Stereochemical selectivity of methanandamides for the CB1 and CB2 cannabinoid receptors and their metabolic stability.

Several chiral, analogues of the endogenous cannabinoid receptor ligand, arachidonylethanolamide (anandamide), methylated at the 2,1' and 2' positions using asymmetric synthesis were evaluated in order to study (a) stereoselectivity of binding to CB1 and CB2 cannabinoid receptors; and (b) metabolic stability with regard to anandamide amidase. Enantiomerically pure 2-methyl arachidonic acids were synthesized through diastereoselective methylation of the respective chiral 2-oxazolidinone enolate derivatives and CB1 and CB2 receptor affinities of the resulting chiral anandamides were evaluated using a standard receptor binding assay. Introduction of a single 2-methyl group increased affinity for CB1, led to limited enantioselectivity and only modestly improved metabolic stability. However, a high degree of enantio- and diastereoselectivity was observed for the 2,1'-dimethyl analogues. (R)-N-(1-methyl-2-hydroxyethyl)-2-(R)-methyl-arachidonamide (4) exhibited the highest CB1 receptor affinity in this series with a K(i) of 7.42 nM, an at least 10-fold improvement on anandamide (K(i)=78.2 nM). The introduction of two methyl groups at the 2-position of anandamide led to no change in affinity for CB1 but somewhat enhanced metabolic stability. Conversely, chiral headgroup methylation in the 2-gem-dimethyl series led to chiral analogues possessing a wide range of CB1 affinities. Of these the (S)-2,2,2'-trimethyl analogue (12) had the highest affinity for CB1 almost equal to that of anandamide. In agreement with our previous anandamide structure-activity relationship work, the analogues in this study showed high selectivity for the CB1 receptor over CB2. The results are evaluated in terms of stereochemical factors affecting the ligand's affinity for CB1 using receptor-essential volume mapping as an aid. Based on the results, a partial CB1 receptor site model is proposed, that bears two hydrophobic pockets capable of accommodating 1'- and 2-methyl groups     

3-Indolyl-1-naphthylmethanes: new cannabimimetic indoles provide evidence for aromatic stacking interactions with the CB(1) cannabinoid receptor

A series of 1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (9-11) and 2-methyl-1-pentyl-1H-indol-3-yl-(1-naphthyl)methanes (12-14) have been synthesized to investigate the hypothesis that cannabimimetic 3-(1-naphthoyl)indoles interact with the CB(1) receptor by hydrogen bonding to the carbonyl group. Indoles 9-11 have significant (K(i)=17-23nM) receptor affinity, somewhat less than that of the corresponding naphthoylindoles (5, 15, 16). 2-Methyl-1-indoles 12-14 have little affinity for the CB(1) receptor, in contrast to 2-methyl-3-(1-naphthoyl)indoles 17-19, which have affinities comparable to those of 5, 15, 16. A cannabimimetic indene hydrocarbon (26) was synthesized and found to have K(i)=26+/-4nM. Molecular modeling and receptor docking studies of naphthoylindole 16, its 2-methyl congener (19) and indolyl-1-naphthylmethanes 11 and 14, combined with the receptor affinities of these cannabimimetic indoles, strongly suggest that these cannabinoid receptor ligands bind primarily by aromatic stacking interactions in the transmembrane helix 3-4-5-6 region of the CB(1) receptor.     

Identification of substituted 4-aminopiperidines and 3-aminopyrrolidines as potent MCH-R1 antagonists for the treatment of obesity

A substituted 4-aminopiperidine was identified as showing activity in an MCH assay from an HTS effort. Subsequent structural modification of the scaffold led to the identification of a number of active MCH antagonists. 3,5-Dimethoxy-N-(1-(naphthalen-2-ylmethyl)piperidin-4-yl)benzamide (5c) was among those with the highest binding affinity to the MCH receptor (K(i)=27nM), when variations were made at benzoyl and naphthylmethyl substitution sites from the initial HTS hit. Further optimization via piperidine ring contraction resulted in enhanced MCH activity in a 3-aminopyrrolidine series, where (R)-3,5-dimethoxy-N-(1-(naphthalen-2-ylmethyl)-pyrrolidin-3-yl)benzamide (10i) was found to be an excellent MCH antagonist (K(i)=7nM).     

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.