Synthesis and pharmacological evaluation of enantiomerically pure endo-configured KOR agonists with 2-azabicyclo[3.2.1]octane scaffold.

Conformationally restricted bicyclic KOR agonists 10 with an endo-configured amino moiety were synthesized to analyze the bioactive conformation of conformationally flexible KOR agonists such as 2-5. A seven-step synthesis starting with (S)-configured 4-oxopiperidine-2-carboxylate 13 was developed. cis- and trans-configured diesters 12 were obtained in a 3 : 1 ratio via hydrogenation of the α,ß-unsaturated ester 14. After establishment of the bicyclic scaffold, a diastereoselective reductive amination of ketone 11 provided exclusively the endo-configured bicyclic amines 10a,b. The 3 : 1 mixtures of enantiomers were separated by chiral HPLC, respectively, leading to enantiomerically pure KOR agonists (1S,5S,7R)-10a,b and (1R,5R,7S)-10a,b (ent-10a,b). The KOR affinity was determined in receptor binding studies with the radioligand [3H]U-69 593. The high KOR affinity of endo-configured amines 10a (Ki = 7 nM) and 10b (Ki = 13 nM) indicates that the dihedral angle of the KOR pharmacophoric element N(pyrrolidine)-C-C-N(phenylacetyl) of 42° is close to the bioactive conformation of more flexible KOR agonists. It should be noted that changing the configuration of potent and selective KOR agonists 10a and 10b led to potent and selective σ1 ligands (e.g. ent-10aKi(σ1) = 10 nM).

Synthesis and Biological Evaluation of Natural-Product-Inspired, Aminoalkyl-Substituted 1-Benzopyrans as Novel Antiplasmodial Agents.

Herein, relationships between the structures of 1-aminoethyl-substituted chromenes and their antimalarial activities were thoroughly investigated. At first, the methyl moiety in the side chain was removed to eliminate chirality. The hydrogenation state of the benzopyran system, the position of the phenolic OH moiety, and the distance of the basic amino moiety toward both aromatic rings were varied systematically. 1-Benzopyran-5-ol 8b (IC50 = 10 nM), 1-benzopyran-7-ol 9c (IC50 = 38 nM), and the aminoalcohol 19c (IC50 = 17 nM) displayed antiplasmodial activity with IC50 values below 50 nM. To identify the mechanism of action, inhibition of three key enzymes by 9c was investigated. 9c was not able to reduce the number of Plasmodia in erythrocytes of mice. This low in vivo activity was explained by fast clearance from blood plasma combined with rapid biotransformation of 9c. Three main metabolites of 9c were identified by liquid chromatography-mass spectrometry (LC-MS) methods.

Ifenprodil Stereoisomers: Synthesis, Absolute Configuration, and Correlation with Biological Activity.

Ifenprodil (1) is a potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonist that is used as a cerebral vasodilator and has been examined in clinical trials for the treatment of drug addiction, idiopathic pulmonary fibrosis, and COVID-19. To correlate biological data with configuration, all four ifenprodil stereoisomers were prepared by diastereoselective reduction and subsequent separation of enantiomers by chiral HPLC. The absolute configuration of ifenprodil stereoisomers was determined by X-ray crystal structure analysis of (1R,2S)-1a and (1S,2S)-1d. GluN2B affinity, ion channel inhibitory activity, and selectivity over α, σ, and 5-HT receptors were evaluated. (1R,2R)-Ifenprodil ((1R,2R)-1c) showed the highest affinity toward GluN2B-NMDA receptors (Ki = 5.8 nM) and high inhibition of ion flux in two-electrode voltage clamp experiments (IC50 = 223 nM). Whereas the configuration did not influence considerably the GluN2B-NMDA receptor binding, (1R)-configuration is crucial for elevated inhibitory activity. (1R,2R)-Configured ifenprodil (1R,2R)-1c exhibited high selectivity for GluN2B-NMDA receptors over adrenergic, serotonergic, and σ1 receptors.

Novel σ1 antagonists designed for tumor therapy: Structure - activity relationships of aminoethyl substituted cyclohexanes.

Depending on the substitution pattern and stereochemistry, 1,3-dioxanes 1 with an aminoethyl moiety in 4-position represent potent σ1 receptor antagonists. In order to increase the stability, a cyclohexane ring first replaced the acetalic 1, 3-dioxane ring of 1. A large set of aminoethyl substituted cyclohexane derivatives was prepared in a six-step synthesis. All enantiomers and diastereomers were separated by chiral HPLC at the stage of the primary alcohol 7, and their absolute configuration was determined by CD spectroscopy. Neither the relative nor the absolute configuration had a large impact on the σ1 affinity. The highest σ1 affinity was found for cis-configured benzylamines (1R,3S)-11 (Ki = 0.61 nM) and (1S,3R)-11 (Ki = 1.3 nM). Molecular dynamics simulations showed that binding of (1R,3S)-11 at the σ1 receptor is stabilized by the typical polar interaction of the protonated amino moiety with the carboxy group of E172 which is optimally oriented by an H-bond interaction with Y103. The lipophilic interaction of I124 with the N-substituent also contributes to the high σ1 affinity of the benzylamines. The antagonistic activity was determined in a Ca2+ influx assay in retinal ganglion cells. The enantiomeric cis-configured benzylamines (1R,3S)-11 and (1S,3R)-11 were able to inhibit the growth of DU145 cells, a highly aggressive human prostate tumor cell line. Moreover, cis-11 could also inhibit the growth of further human tumor cells expressing σ1 receptors. The experimentally determined logD7.4 value of 3.13 for (1R,3S)-11 is in a promising range regarding membrane penetration. After incubation with mouse liver microsomes and NADPH for 90 min, 43% of the parent (1R,3S)-11 remained unchanged, indicating intermediate metabolic stability. Altogether, nine metabolites including one glutathione adduct were detected by means of LC-MS analysis.

Pharmacokinetic properties of enantiomerically pure GluN2B selective NMDA receptor antagonists with 3-benzazepine scaffold.

Recently, the eutomers of highly potent GluN2B selective NMDA receptor antagonists with 3-benzazepine scaffold were identified. Herein, pharmacokinetic properties regarding lipophilicity, plasma protein binding (PPB) and metabolism are analyzed. The logD7.4 values of 1.68 for phenol 1 and 2.46 for methyl ether 2 are in a very good range for CNS agents. A very similar logD7.4 value was recorded for the prototypical GluN2B antagonist ifenprodil (logD7.4 = 1.49). The herein developed high performance affinity chromatography (HPAC) method using human serum albumin as stationary phase led to PPB of 3-benzazepines (R)-1-3 and (S)-1-3 of 76-98%. Upon incubation with mouse liver microsomes, (R)-1-3 and (S)-1-3 showed moderate to high metabolic stability. The (R)-configured eutomers turned out to be metabolically more stable than their (S)-configured distomers. During phase I metabolism of 3-benzazepines 1-3 hydroxylations at both aromatic rings, the aliphatic side chain and the seven-membered ring were observed. O-demethylation of methyl ether (S)-2 was faster than O-demethylation of its enantiomer (R)-2. In phase I biotransformation the phenol eutomer (R)-1 showed comparable stability as ifenprodil. In phase II biotransformation, glucuronidation of the phenolic (only 1) and benzylic hydroxy groups was observed. Both enantiomers formed the same type of metabolites, respectively, but in different amounts. Whereas, the benzylic hydroxy group of (R)-2 was glucuronidated preferably, predominant benzylic glucuronidation of (S)-3 was detected. Mouse liver microsomes produced the glucuronide of phenol 1 (main metabolite) in larger amounts than rat liver microsomes.

Synthesis and Pharmacological Evaluation of Enantiomerically Pure GluN2B Selective NMDA Receptor Antagonists.

To determine the eutomers of potent GluN2B-selective N-methyl-d-aspartate (NMDA) receptor antagonists with a 3-benzazepine scaffold, 7-benzyloxy-3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepin-1-ols (S)-2 and (R)-2 were separated by chiral HPLC. Hydrogenolysis and subsequent methylation of the enantiomerically pure benzyl ethers of (S)-2 and (R)-2 provided the enantiomeric phenols (S)-3 and (R)-3 [3-(4-phenylbutyl)-2,3,4,5-tetrahydro-1H-3-benzazepine-1,7-diol] and methyl ethers (S)-4 and (R)-4. All enantiomers were obtained with high enantiomeric purity (≥99.7 % ee). The absolute configurations were determined by CD spectroscopy. R-configured enantiomers turned out to be the eutomers in receptor binding studies and two-electrode voltage clamp experiments. The most promising ligand of this compound series is the R-configured phenol (R)-3, displaying high GluN2B affinity (Ki =30 nm), high inhibition of ion flux (IC50 =61 nm), and high cytoprotective activity (IC50 =93 nm). Whereas the eudismic ratio in the receptor binding assay is 25, the eudismic ratio in the electrophysiological experiment is 3.

Deconstruction - reconstruction approach to analyze the essential structural elements of tetrahydro-3-benzazepine-based antagonists of GluN2B subunit containing NMDA receptors.

The role of the phenolic and benzylic OH moieties for the interaction of tetrahydro-3-benzazepine-1,7-diol 3d with GluN2B subunit containing NMDA receptors was analyzed by their stepwise removal. Elimination of trifluormethanesulfinate from 10 and 13 represent the key steps in the synthesis. Removal of phenolic OH moiety led to 5-fold reduced GluN2B affinity of 4d compared with 3d. Additional removal of the benzylic OH moiety (5d) resulted in further reduced GluN2B affinity but increased σ1 and σ2 affinities. Introduction of a NO2 (6d) or NH2 moiety (7d) decreased the GluN2B affinity. 3-Benzazepin-1-ol 4i with the N-phenylcyclohexyl side chain showed the highest GluN2B affinity of this series of compounds (Ki = 2.2 nM) and, moreover, high selectivity over the PCP binding site, σ1 and σ2 receptors. In docking studies 3-benzazepines (S)-4-7 adopt the same binding poses as ifenprodil and display the same crucial interactions. Unexpectedly, the high-affinity ligands (S)-4i, (S)-4j, and (S)-6i were not able to inhibit the glutamate/glycine evoked current in two-electrode voltage clamp measurements and the cytotoxic effects of glutamate/glycine on transfected cell lines.

Lipase-catalyzed kinetic resolution as key step in the synthesis of enantiomerically pure σ ligands with 2-benzopyran structure.

In order to obtain enantiomerically pure σ1 receptor ligands with a 2-benzopyran scaffold an Oxa-Pictet-Spengler reaction with the enantiomerically pure 2-phenylethanol derivatives (R)-4 and (S)-4 was envisaged. The kinetic resolution of racemic alcohol (±)-4 using Amano Lipase PS-C II and isopropenyl acetate in tert-butyl methyl ether led to the (R)-configured alcohol (R)-4 in 42% yield with an enantiomeric excess of 99.6%. The (S)-configured alcohol (S)-4 was obtained by Amano Lipase PS-C II catalyzed hydrolysis of enantiomerically enriched acetate (S)-5 (76.9% ee) and provided (S)-4 in 26% yield and 99.7% ee. The absolute configuration of alcohol (R)-4 was determined by exciton coupled CD spectroscopy of the bis(bromobenzoate) (R)-7. The next important step for the synthesis of 2-benzopyrans 2 and 3 was the Oxa-Pictet-Spengler reaction of the enantiomerically pure alcohols (R)-4 and (S)-4 with piperidone ketal 8 and chloropropionaldehyde acetal 12. The conformationally restricted spirocyclic 2-benzopyrans 2 revealed higher σ1 affinity than the more flexible aminoethyl derivatives 3. The (R)- and (R,R)-configured enantiomers (R)-2 and (R,R)-3 represent the eutomers of this class of compounds with eudismic ratios of 4.8 (2b) and 4.5 (2c). High σ1/σ2 selectivity (>49) was found for the most potent σ1 ligands (R)-2b, (R)-2c, (R)-2d, and (S)-2d (Ki(σ1) 9-15nM).

Stereoselective synthesis and pharmacological evaluation of [4.3.3]propellan-8-amines as analogs of adamantanamines.

Amantadine (1) exerts its anti-Parkinson effects by inhibition of the NMDA associated cation channel and its antiviral activity by inhibition of the M2 protein channel of influenza A viruses. Herein the synthesis, NMDA receptor affinity and anti-influenza activity of analogous propellanamines 3 are reported. The key steps in the synthesis of the diastereomeric propellanamines syn-3 and anti-3 are diastereoselective reduction of the ketone 7 with L-Selectride to give anti-11, Mitsunobu inversion of the alcohol anti-13 into syn-13, and SN2 substitution of diastereomeric mesylates syn-14 and anti-14 with NaN3. The affinity of the propellanamines syn-3 and anti-3 to the PCP binding site of the NMDA receptor is similar to that of amantadine (Ki=11 µM). However, both propellanamines syn-3 and anti-3 do not exhibit activity against influenza A viruses. Compared to amantadine (1), the structurally related propellanamines syn-3 and anti-3 retain the NMDA antagonistic activity but loose the antiviral activity.

Diastereoselective Synthesis of Cyclic Five-Membered trans,trans-Configured Nitrodiols by Double Henry Reaction of 1,4-Dialdehydes.

Conformationally constrained perhydroquinoxalines 4 show high κ receptor affinity, selectivity over related receptors and full agonistic activity. Since the κ affinity can be correlated with the dihedral angle of the ethylenediamine pharmacophore (4a: 55°/71°), the dihedral angles of the postulated cyclopentane derivative 5a (73°/84°) and indane derivative 6a (77°/81°) were calculated. The first step of the synthesis represents a double Henry reaction of 1,4-dialdehydes 8 and 10 with nitromethane, leading predominantly to the trans,trans-configured nitrodiols 9 and 11. X-ray crystal structure analyses of 9 and 11 led to dihedral angles O2 N−C−C−OH of 73.4 and 88.3°, respectively, which reflect the calculated dihedral angles of the hypothesized final products 5a and 6a.

Synthesis and pharmacological evaluation of 5-pyrrolidinylquinoxalines as a novel class of peripherally restricted κ-opioid receptor agonists.

5-Pyrrolidinyl substituted perhydroquinoxalines were designed as conformationally restricted κ-opioid receptor agonists restricted to the periphery. The additional N atom of the quinoxaline system located outside the ethylenediamine κ pharmacophore allows the fine-tuning of the pharmacodynamic and pharmacokinetic properties. The perhydroquinoxalines were synthesized stereoselectively using the concept of late stage diversification of the central building blocks 14. In addition to high κ-opioid receptor affinity they demonstrate high selectivity over µ, δ, σ1, σ2, and NMDA receptors. In the [35S]GTPγS assay full agonism was observed. Because of their high polarity, the secondary amines 14a (log D7.4=0.26) and 14b (log D7.4=0.21) did not penetrate an artificial blood-brain barrier. 14b was able to inhibit the spontaneous pain reaction after rectal mustard oil application to mice (ED50=2.35 mg/kg). This analgesic effect is attributed to activation of peripherally located κ receptors, since 14b did not affect centrally mediated referred allodynia and hyperalgesia.

Synthesis and pharmacological evaluation of like- and unlike-configured tetrahydro-2-benzazepines with the α-substituted benzyl moiety in the 5-position.

A large set of tetrahydro-2-benzazepines with an α-hydroxy or α-(aryl)alkoxy substituted benzyl moiety in the 5-position was prepared according to the recently reported C6C1 + C3N synthetic strategy. The Heck reaction of 2-iodobenzaldehyde acetal 4 and the subsequent Stetter reaction led to the ketone 7, which was reduced diastereoselectively to form the like-configured alcohol 8. The diastereomeric unlike-configured alcohol 9 was obtained by a Mitsunobu inversion of 8. Alkylation and reductive cyclization of the diastereomeric alcohols 8 and 9 provided like- and unlike-configured 2-benzazepines 13 and 23, which allowed the introduction of various substituents at the N-atom. Analysis of the relationship between the structure and the σ1 affinity revealed that large substituents such as the butyl, benzyl or 4-phenylbutyl moiety at the benzazepine N-atom resulted in high affinity ligands. A p-methoxybenzyl ether is less tolerated by the σ1 receptor than a methyl ether or an alcohol. The unlike-configured alcohols 25d and 27d show slightly higher σ1 affinity than their like-configured diastereomers 15d and 17d. With respect to the σ1 affinity, σ1/σ2 selectivity and lipophilic ligand efficiency, like- and unlike-configured alcohols 15d and 25d represent the most promising σ1 ligands of this series. Interactions of the novel 2-benzazepines with various binding sites of the NMDA receptor were not observed.

Design, synthesis and receptor affinity of novel conformationally restricted σ ligands based on the [4.3.3]propellane scaffold.

A series of novel diastereoisomeric σ ligands 3 was designed, synthesized and pharmacologically evaluated. The highly rigid [4.3.3]propellane scaffold was used to fix the three dimensional orientation of the pharmacophoric moieties required for σ affinity. The syn,syn-configured aminocarbamate syn,syn-3a reveals the most promising σ1 affinity (Ki = 77 nM) and selectivity over the σ2 subtype (21-fold). The σ2 affinity of all four diastereomers 3 was in the low micromolar range. Analysis of the distance between the hydrophobic regions (phenyl moieties) of the diastereomers 3 led to the longest range of distances (10.3-15.2 Å) for the most potent σ1 ligand syn,syn-3a, which is in good agreement with pharmacophore models.

Microwave assisted synthesis of spirocyclic pyrrolidines -σ1 receptor ligands with modified benzene-N-distance.

Two series of σ(1) ligands with a spiro[[2]benzopyran-1,3'-pyrrolidine] (3) and a spiro[[2]benzofuran-1,3'-pyrrolidine] (4) framework were synthesized and pharmacologically evaluated. Several reaction steps were considerably improved by microwave irradiation. The σ(1) affinity of the spirocyclic ligands correlates nicely with the benzene-N-distance, i.e. 2 < 3 < 4 < 1. The σ(1) affinity of both compound classes could be increased with large N-substituents (e.g. 2-phenylethyl, octyl). Nevertheless the benzyl derivative 4a represents the most promising σ(1) ligand (K(i) = 25 nM) due to its high selectivity against the σ(2) subtype (>40-fold), the NMDA receptor and 5-HT(6) and 5-HT(7) receptors. Moreover, 4a did not inhibit the hERG channel in the heart.

Characterization of ligand binding to the σ(1) receptor in a human tumor cell line (RPMI 8226) and establishment of a competitive receptor binding assay.

The standard assay for the determination of σ(1) receptor affinities of novel compounds is a competitive binding assay using [(3)H]-(+)-pentazocine as radioligand and membrane preparations from guinea pig brain. Herein, a novel competitive binding assay was developed employing the hematopoietic cell line of human multiple myeloma (RPMI 8226), which expresses a large amount of the human σ(1) receptor. Membrane fragments of RPMI 8226 cells were prepared and characterized. A Western blot analysis confirmed the high density of σ(1) receptors in this cell line. Assay conditions were carefully optimized leading to an incubation period of 120 min, an incubation temperature of 37°C, and receptor material for each well was prepared from 300,000 cells. It was shown that a large excess (10 µM) of (+)-pentazocine, haloperidol, and di-o-tolylguanidine provided the same results during determination of the nonspecific binding. Saturation experiments with the radioligand [(3)H]-(+)-pentazocine led to a K(d)-value of 36±0.3 nM and a B(max)-value of 477±7 fmol/mg protein. These data resulted in approximately 122,000 σ(1) binding sites per cell. The assay was validated by using six known σ(1) ligands and eight σ(1) ligands prepared in our lab. The K(i)-values determined with RPMI 8226-derived receptor material are in good accordance with the K(i)-values obtained with guinea pig brain membrane preparations. Compared with guinea pig brain preparations, the RPMI 8226-derived receptor material represents a better standardized receptor material with a high density of human σ(1) receptors.

Combination of two pharmacophoric systems: synthesis and pharmacological evaluation of spirocyclic pyranopyrazoles with high σ1 receptor affinity.

The novel class of spirocyclic σ(1) ligands 3 (6',7'-dihydro-1'H-spiro[piperidine-4,4'-pyrano[4,3-c]pyrazoles]) was designed by the combination of the potent σ(1) ligands 1 and 2 in one molecule. Thorough structure affinity relationships were derived by the variation of the substituents in position 1', 1, and 6'. Whereas the small electron rich methylpyrazole heterocycle was less tolerated by the σ(1) receptor protein, the introduction of a phenyl substituent instead of the methyl group led to ligands with a high σ(1) affinity. It is postulated that the additional phenyl substituent occupies a previously unrecognized hydrophobic region of the σ(1) receptor resulting in additional lipophilic interactions. The spirocyclic pyranopyrazoles are very selective against the σ(2) subtype, the PCP binding site of the NMDA receptor, and further targets. Despite high σ(1) affinity, the cyclohexylmethyl derivative 17i (K(i) (σ(1)) = 0.55 nM) and the isopentenyl derivative 17p (K(i) (σ(1)) = 1.6 nM) showed only low antiallodynic activity in the capsaicin assay.

Expression of σ receptors of human urinary bladder tumor cells (RT-4 cells) and development of a competitive receptor binding assay for the determination of ligand affinity to human σ(2) receptors.

A selective competitive binding assay for the determination of the affinity of compounds to the human σ(2) receptor using 96-well multiplates and a solid state scintillator was developed. In the assay system, [(3)H]ditolylguanidine (DTG) was used as radioligand and membrane homogenates from human RT-4 cells physiologically expressing σ(2) receptors served as receptor material. In order to block the interaction of the unselective radioligand [(3)H]DTG with σ(1) receptors, all experiments were performed in the presence of the σ(1) selective ligand (+)-pentazocine. The density of σ(2) receptors of the cells was analyzed by a saturation experiment with [(3)H]DTG. The radioligand [(3)H]DTG was bound to a single, saturable site on human σ(2) receptors, resulting in a B(max) value of 2108±162fmol/mg protein and K(d)-value of 8.3±2.0nM. The expression of competing σ(1) receptors was evaluated by performing a saturation experiment using the σ(1) selective radioligand [(3)H](+)-pentazocine, which resulted in a B(max) value of 279±40fmol/mg protein and K(d) value of 13.4±1.6nM. For validation of the σ(2) binding assay, the K(i)-values of four σ(2) ligands (ditolylguanidine, haloperidol, rimczole and BMY-14802) were determined with RT-4 cell membrane preparations. The K(i) values obtained from these experiments are in good accordance with the K(i)-values obtained with rat liver membrane preparations as receptor material and with K(i) values given in the literature.

Development of a selective competitive receptor binding assay for the determination of the affinity to NR2B containing NMDA receptors.

A selective, rapid and efficient competitive binding assay for the determination of the affinity of compounds towards the ifenprodil binding site of NR2B subunit containing NMDA receptors has been developed. In the assay system, [(3)H]ifenprodil was used as radioligand and membrane homogenates from L(tk-) cells stably expressing recombinant human NR1a/NR2B receptors served as the receptor material. Sonication of the cells during work-up, performing all steps with 96-well multiplates and using a solid scintillator represent particular features of this assay. The binding kinetics was investigated by saturation and association/dissociation experiments. [(3)H]ifenprodil bound to a single, saturable site on human recombinant NR1a/NR2B receptors, resulting in a B(max)-value of 25.8 pmol/microg protein and K(d)-value of 7.6+/-2.3 nM (SEM). The dissociation rate constant (k(off)) was 0.03861 min(-1) and the association rate constant k(on) resulted in 0.00625 nM(-1)min(-1). The specificity of the assay was proved with cells not treated with dexamethasone, which has to be added to induce NMDA receptor synthesis of the cells. Additionally, the absence of alpha(1), sigma(1) and sigma(2) receptors was shown. The K(i)-values of the NR2B ligands ifenprodil and eliprodil determined with the new assay are in good accordance with reported data.

Conformationally constrained kappa receptor agonists: stereoselective synthesis and pharmacological evaluation of 6,8-diazabicyclo[3.2.2]nonane derivatives.

Three sets of stereoisomeric bicyclic kappa agonists with defined orientation of the pharmacophoric elements pyrrolidine and dichlorophenylacetamide were stereoselectively prepared and pharmacologically evaluated. Stereoselective reduction, reductive amination, and Mitsunobu inversions were the key steps for the establishment of the desired stereochemistry. The kappa affinity decreased in the following order depending on the N-substituent: CO(2)CH(3) > benzyl > COCH(2)CH(3). Bicyclic derivatives with (1S,2R,5R)-configuration showed the highest kappa receptor affinity, which led to dihedral angles of 97 degrees and 45 degrees for the N(pyrrolidine)-C-C-N(phenylacetamide) structural element. The most potent kappa agonist of this series was (+)-methyl (1S,2R,5R)-8-[2-(3,4-dichlorophenyl)acetyl]-2-(pyrrolidin-1-yl)-6,8-diazabicyclo[3.2.2]nonane-6-carboxylate (ent-23, WMS-0121) with an K(i) value of 1.0 nM. ent-23 revealed high selectivity against the other classical opioid receptors and related receptor systems. In the [(35)S]GTPgammaS-binding assay at human kappa-opioid receptors, ent-23 was proved to be a full agonist with the same EC(50) value (87 nM) as the prototypical full agonist U-69,593 (EC(50) = 80 nM).

Synthesis of sigma receptor ligands with unsymmetrical spiro connection of the piperidine moiety.

The symmetrically connected spiro[[2]benzopyran-1,4'-piperidines] 1 are highly potent and selective sigma(1) receptor ligands. Changing the position of the spirocyclic nitrogen atom led to the unsymmetrically connected spiro[[2]benzopyran-1,3'-piperidines] 2 with a reduced distance between the aromatic system and the basic nitrogen atom. The synthesis of 2 was performed by halogen-metal exchange at the aryl bromide 3 followed by addition to the piperidone 5 and intramolecular transacetalization. The yield of 2a was considerably improved by transmetallation of the aryllithium intermediate 4a with CeCl(3) (4c). The cis and trans diastereomers cis-2 and trans-2 were separated and characterized by nuclear Overhauser effect. After removal of the benzyl group, the secondary amine 2b was alkylated with various alkyl and arylalkyl halides. The sigma(1) and sigma(2) receptor affinity of the spirocyclic piperidines 2 were determined with receptor binding studies. Compared with the spirocyclic piperidines 1, the unsymmetrically connected piperidines 2 show remarkably reduced sigma(1) receptor affinities, whereas the selectivity over sigma(2) and NMDA receptors was retained. A stereoselective interaction of the sigma(1) receptor protein with the cis- or trans-configured spirocyclic compounds 2 was not observed. It was shown that alkyl residues at the N-atom can replace the lipophilic N-arylalkyl groups and interact with the primary hydrophobic binding site of the sigma(1) receptor protein.