7ß-Methyl substituent is a structural locus associated with activity cliff for nepenthone analogues.

With the purpose of identifying novel selective κ opioid receptor (KOR) antagonists as potential antidepressants from nepenthone analogues, starting from N-nor-N-cyclopropylmethyl-nepenthone (SLL-020ACP), a highly selective and potent KOR agonist, a series of 7ß-methyl-nepenthone analogues was conceived, synthesized and assayed on opioid receptors based on the concept of hybridization. According to the pharmacological results, the functional reversal observed in orvinol analogues by introduction of 7ß-methyl substituent could not be reproduced in nepenthone analogues. Alternatively, introduction of 7ß-methyl substituent was associated with substantial loss of both subtype selectivity and potency but not efficacy for nepenthone analogues, which was not found in 7ß-methyl orvinol analogues. Surprisingly, SLL-603, a 7ß-methyl analogue of SLL-020ACP, was identified to be a KOR full agonist. The possible molecular mechanism for the heterogeneity in activity cliff was also investigated. In conclusion, 7ß-methyl substituent was a structural locus associated with activity cliff and demonstrated as a pharmacological heterogeneity between nepenthone and orvinol analogues that warrants further investigations.

Highly selective and potent mu opioid ligands by unexpected substituent on morphine skeleton.

Unexpected substituent on the well-known morphine skeleton is described to be account for highly selective and potent mu opioid ligands, which is strongly connected to substituted aromatic groups on this omitted 8alpha-position.

3-[(3S)-3-Ethyl-1-methyl-azepan-3-yl]phenyl N-(4-fluoro-phen-yl)carbamate.

The asymmetric unit of the title compound, C(22)H(27)FN(2)O(2), a (-)-S-meptazinol derivative, contains two mol-ecules. The azepane ring adopts a similar twist chair form in both mol-ecules, while the dihedral angles between the two benzene rings are 88.17 (14) and 89.93 (14)° in the two mol-ecules. The absolute configuration of the mol-ecule was determined from the synthetic starting material. The crystal structure is stabilized by classical inter-molecular N-H⋯O hydrogen bonds.

(3S,4R)-3-Ethyl-4-hydr-oxy-3-(3-methoxy-phen-yl)-1-methyl-azepanium (2R,3R)-2,3-bis-(benzo-yloxy)-3-carboxy-propionate.

The crystal structure of the title compound, C(16)H(26)NO(2) (+)·C(18)H(13)O(8) (-), is stabilized by an extensive network of classical N-H⋯O and O-H⋯O hydrogen bonding. The crystal structure also shows an ammonium-driven diastereo-isomerism.

Novel piperazine derivative PMS1339 exhibits tri-functional properties and cognitive improvement in mice.

Amyloid-beta-induced neuroinflammation plays a central role in the extensive loss of cholinergic neurons and cognitive decline in Alzheimer's disease. The acetylcholinesterase (AChE) inhibitors are the first class of drugs used to enhance surviving cholinergic activities. However, their limited effectiveness following long-term treatment raises a need for new multi-target therapies. We report herein a novel piperazine derivative compound PMS1339 possesses multifunctional properties including anti-platelet-activating factor, AChE inhibition, Abeta aggregation inhibition and cognitive improvement. PMS1339 could significantly inhibit both mice brain AChE (IC50=4.41+/-0.63 microM) and sera butyrylcholinesterase (BuChE, IC50=1.09+/-0.20 microM). PMS1339 was also found to inhibit neuronal AChE secreted by SH-SY5Y cell line (IC50=17.95+/-2.31 microM). Enzyme kinetics experiments performed on electric eel AChE indicated that PMS1339 acts as a mixed type competitive AChE inhibitor. Molecular docking studies using the X-ray crystal structure of AChE from Torpedo californica elucidated the interactions between PMS1339 and AChE: PMS1339 is well buried inside the active-site gorge of AChE interacting with Trp84 at the bottom, Tyr121 halfway down and Trp279 at the peripheral anionic site (PAS). Thioflavin T-based fluorimetric assay revealed the ability of PMS1339 to inhibit AChE-induced Abeta aggregation. In-vivo study indicated PMS1339 (1 mg/kg i.p.) reversed scopolamine-induced memory impairment in mice. Overall, these findings indicated that PMS1339 exhibits tri-functional properties in vitro and cognitive improvement in vivo, and revealed the emergence of a multi-target-directed ligand to tackle the determinants of Alzheimer's disease.

1-Ethyl-4-hydr-oxy-9-aza-tricyclo-[7.4.1.0]tetra-deca-2,4,6-trien-8-one.

In the mol-ecule of the title compound, C(15)H(19)NO(2), the six-membered dihydro-pyridinone ring assumes a screw-boat conformation. In the crystal structure, mol-ecules are linked via O-H⋯O hydrogen bonding between hydr-oxy and carbonyl groups, forming supra-molecular chains along the a axis.

2-Methyl-1,2,3,4-tetra-hydro-isoquinolin-6-yl N-phenyl-carbamate.

In the mol-ecule of the title compound, C(17)H(18)N(2)O(2), the piperidine ring adopts a half-chair form. The two benzene rings are individually planar and make a dihedral angle of 53.90°. The crystal structure is stabilized by inter-molecular N-H⋯N hydrogen bonds and π-π stacking inter-actions (centroid-centroid distance = 3.962 Å).

(3S,4S)-3-Ethyl-4-hydr-oxy-3-(3-methoxy-phen-yl)-1-methyl-azepan-1-ium d-tartrate dihydrate.

In the title compound, C(16)H(26)NO(2) (+)·C(4)H(5)O(6) (-)·2H(2)O, a meptaz-inol derivative, three C atoms of the azepane ring are disordered over two positions, with site-occupancy factors of 0.80 and 0.20; the major disorder component adopts a twist-chair conformation, while the minor component has a chair conformation. The benzene ring is axially substituted on the heterocyclic ring, resulting in a folded conformation of the cation. The absolute configuration was determined with reference to d-tartaric acid. The crystal structure is stabilized by an extensive network of intra- and inter-molecular O-H⋯O hydrogen bonds.

(E)-6-Meth-oxy-9-methyl-1,2,3,4-tetra-hydro-9H-carbazol-4-one oxime.

The title compound, C(14)H(16)N(2)O(2), is dimerized by inversion-related inter-molecular O-H⋯O and O-H⋯N hydrogen bonding. There is also an intra-molecular C-H⋯N bond, resulting in a six-membered ring.

The Pharmacological Heterogeneity of Nepenthone Analogs in Conferring Highly Selective and Potent κ-Opioid Agonistic Activities.

To develop novel analgesics with no side effects or less side effects than traditional opioids is highly demanded to treat opioid receptor mediated pain and addiction issues. Recently, κ-opioid receptor (KOR) has been established as an attractive target, although its selective agonists could bear heterogeneous pharmacological activities. In this study, we designed and synthesized two new series of nepenthone derivatives by inserting a spacer (carbonyl) between 6α,14α-endo-ethenylthebaine and the 7α-phenyl substitution of the skeleton and by substituting the 17-N-methyl group with a cyclopropylmethyl group. We performed in vitro tests (binding and functional assays) and molecular docking operations on our newly designed compounds. The results of wet-experimental measures and modeled binding structures demonstrate that these new compounds are selective KOR agonists with nanomolar level affinities. Compound 4 from these new derivatives showed the highest affinity (Ki = 0.4 ± 0.1 nM) and the highest selectivity (µ/κ = 339, δ/κ = 2034) toward KOR. The in vivo tests revealed that compound 4 is able to induce stronger (ED50 = 2.1 mg/kg) and much longer antinociceptive effect than that of the typical KOR agonist U50488H (ED50 = 4.4 mg/kg). Therefore, compound 4 can be used as a perfect lead compound for future design of potent analgesics acting through KOR.

QSAR study of 4-phenylpiperidine derivatives as mu opioid agonists by neural network method.

A nonlinear QSAR study was conducted on a series of 4-phenylpiperidine derivatives (4PPs) acting as mu opioid agonists by three-layer back-propagation neural network (NN) method. At first a variety of molecular descriptors were calculated and then selected with two-stage least squares combining partial least squares (PLS) method. The selected four molecular descriptors, out of 292 ones, were correlated with the known analgesic activities of 38 4PPs by NN method. The established QSAR model was further validated by five additional 4PPs, as an external testing set. Moreover, a pharmacophore model was hypothesized based on the results, which would be helpful for structural optimization of 4PPs.

Inhibitory effect of intrathecal meptazinol on carrageenan-induced thermal hyperalgesia in rats.

The effect of meptazinol in the spinal cord on carrageenan-induced hyperalgesia was investigated. The latency of paw withdrawal (PWL) to a thermal stimulus was used as an index of inflammatory hyperalgesia in awake rats. Intrathecal (i.t.) injection of 10 and 100 microg meptazinol markedly increased the PWL of the carrageenan-injected paw (P<0.01). The PWL of the non-injected paw was not detectably affected by the administration of meptazinol at the doses tested. I.t. injection of naloxone (5 microg) or atropine (1 microg) alone exhibited no effect on the PWLs of either the carrageenan-injected or non-injected paw. Pretreatment with naloxone, but not atropine, completely blocked the meptazinol-induced anti-hyperalgesia. These observations suggested that mu opioid receptor rather than muscarinic acetylcholine receptor may be involved in the anti-hyperalgesia of meptazinol in the spinal cord.

Antinociceptive effects of meptazinol and its isomers on carrageenan-induced thermal hyperalgesia in rats.

Using the latency of paw withdrawal (PWL) from a noxious thermal stimulus as a measure of hyperalgesia, the effects of i.p. injection of meptazinol and its isomers, 112824 and 112825, on carrageenan-induced thermal hyperalgesia were studied in awaked carrageenan-inflamed rats. Peripheral inflammation was induced by intraplantar (i.pl.) injection of carrageenan (2 mg/100 microl) into one hindpaw in rats. Carrageenan produced marked inflammation (edema and erythema) and thermal hyperalgesia in the injected paws, which peaked at 3 h after injection and showed little change in magnitude for another 3 h. Injection of 0.1 mg/kg meptazinol (i.p.) at 3 h after carrageenan had no effect on the PWLs of either inflamed or non-inflamed hindpaw during the next 100 min (P>0.05, n=8). At the dosage of 1 and 10 mg/kg, meptazinol produced marked anti-nociception and anti-hyperalgesia in non-inflamed and inflamed hindpaw, respectively (P<0.05, n=8-11). The prolonging effect of meptazinol on PWL in inflamed hindpaw was more potent than that in non-inflamed hindpaw. Pre-administration of 1.5 mg/kg naloxone significantly antagonized meptazinol-induced anti-nociception and anti-hyperalgesia. Intraperitoneal injection of an isomer of meptazinol, 112825 (1.5 mg/kg), but not 112824 (1 mg/kg), markedly increased the PWL of the non-inflamed hindpaw. Nevertheless, both the isomers produced similar anti-hyperalgesic effect to that of meptazinol (P<0.05, n=8), which was completely reversed by naloxone (1.5 mg/mg). The results suggest that meptazinol and its isomers have anti-nociceptive and anti-hyperalgesic properties with the former more potent. The effects are mainly mediated by mu opioid receptors. This study provides an important clue for extending clinical utilization of meptazinol and its isomers.

[Binding characteristics of new synthesized opioid receptor ligands to cloned mu opioid receptors stably expressed in CHO cell].

OBJECTIVE: To determine the affinity of new opioid receptor ligands to cloned mu opioid receptors stably expressed in CHO cell. METHODS: The binding characteristics of the opioid ligand [3H] diprenorphine (3H-dip) were studied by cellular biological techniques and radioligands binding in cloned mu opioid receptors stably expressed in CHO cells in saturation binding experiments, and were followed by competition binding experiments with a variety of new synthesized opioid receptor ligands. RESULTS: The Kd and Bmax of [3H] diprenorphine bound to mu receptors were 1.06 nmol/L and 930 fmol/mg protein, respectively. Competition binding experiments revealed that ligand 3# and 12# displayed much higher affinity than DAMGO and Morphine for the cloned mu opioid receptor. However, the affinities of ligands 2#, 6#, 8# and 9# were lower than DAMGO and Morphine. CONCLUSION: The present results suggest that the new ligands 3# and 12# have higher affinity to mu opioid receptors. However, ligands 2#, 6#, 8# and 9# have lower affinity to mu opioid receptors.

Meserine, a novel carbamate AChE inhibitor, ameliorates scopolamine-induced dementia and alleviates amyloidogenesis of APP/PS1 transgenic mice.

AIMS: To investigate whether Meserine, a novel phenylcarbamate derivative of (-)-meptazinol, possesses beneficial activities against cholinergic deficiency and amyloidogenesis, the two major pathological characteristics of Alzheimer's disease (AD). METHODS: Ellman's assay and Morris water maze were used to detect acetylcholinesterase (AChE) activity and evaluate spatial learning and memory ability, respectively. Both high content screening and Western blotting were carried out to detect ß-amyloid precursor protein (APP), while RT-PCR and ELISA were conducted to detect APP-mRNA and ß-amyloid peptide (Aß). RESULTS: In scopolamine-induced dementia mice, Meserine (1 mg/kg, i.p.) significantly ameliorated spatial learning and memory deficits, which was consistent with its in vitro inhibitory ability against AChE (recombinant human AChE, IC50 = 274 ± 49 nM). Furthermore, Meserine (7.5 mg/kg) injected intraperitoneally once daily for 3 weeks lowered APP level by 28% and Aß42 level by 42% in APP/PS1 transgenic mouse cerebrum. This APP modulation action might be posttranscriptional, as Meserine reduced APP by about 30% in SH-SY5Y-APP695 cells but did not alter APP-mRNA level. And both APP and Aß42 lowering action of Meserine maintained longer than that of rivastigmine. CONCLUSION: Meserine executes dual actions against cholinergic deficiency and amyloidogenesis and provides a promising lead compound for symptomatic and modifying therapy of AD.

Bis(9)-(-)-nor-meptazinol as a novel dual-binding AChEI potently ameliorates scopolamine-induced cognitive deficits in mice.

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder which is characterized by the progressive deterioration of cognition and the emergence of behavioral and psychological symptoms in aging patients. Given that the clinical effectiveness of acetylcholinesterase inhibitors (AChEIs) has still been questioned due to dubious disease-modifying effects, the multi-target directed ligand (MTDL) design has become an emerging strategy for developing new drugs for AD treatment. Bis(9)-(-)-nor-meptazinol (Bis-Mep) was firstly reported by us as a novel MTDL for both potent cholinesterase and amyloid-ß aggregation inhibition. In this study, we further explored its AChE inhibition kinetic features and cognitive amelioration. Bis-Mep was found to be a mixed-type inhibitor on electric eel AChE by enzyme kinetic study. Molecular docking revealed that two "water bridges" located at the two wings of Bis-Mep stabilized its interaction with both catalytic and peripheral anionic sites of AChE. Furthermore, subcutaneous administration of Bis-Mep (10, 100 or 1000 ng/kg) significantly reversed the scopolamine-induced memory deficits in a typical bell-shaped dose-response manner. The maximal cognitive amelioration of Bis-Mep was achieved at 100 ng/kg, comparable with the effect of a reference drug Huperzine A at 1 mg/kg and also the relevant AChE inhibition in brain. These findings suggested that Bis-Mep might be a promising dual-binding AChE inhibitor for potential AD therapeutics.

Determination of Bis(9)-(-)-Meptazinol, a bis-ligand for Alzheimer's disease, in rat plasma by liquid chromatography-tandem mass spectrometry: application to pharmacokinetics study.

A rapid, simple and sensitive LC-MS/MS method was developed and validated for the determination of Bis(9)-(-)-Meptazinol (B9M) in rat plasma. Protein precipitation method was used for sample preparation, using five volumes of methanol as the precipitation agent. The analytes were separated by a Zorbax Extend-C18 column with the mobile phase of methanol-water (containing 5mM ammonium formate, pH 9.8) (95:5, v/v), and monitored by positive electrospray ionization in multiple reaction monitoring (MRM) mode. Retention time of IS (Bis(5)-(-)-Meptazinol) and B9M were 1.9 min and 3.3 min, respectively. The limit of detection was 0.1 ng/ml and the linear range was 1-500 ng/ml. The relative standard deviation (RSD) of intra-day and inter-day variation was 4.4-6.2% and 6.2-8.9%, respectively. The extraction recoveries of B9M in plasma were over 95%. The method proved to be applicable to the pharmacokinetic study of B9M in rat after intravenous and subcutaneous administration.

Molecular modeling and 3D-QSAR studies of indolomorphinan derivatives as kappa opioid antagonists.

Molecular modeling and 3D-QSAR studies were performed on 31 indolomorphinan derivatives to evaluate their antagonistic behaviors on kappa opioid receptor and provide information for further modification of this kind of compounds. Best predictions were obtained with CoMFA standard model (q2 = 0.693, N = 4, r2 = 0.900) and CoMSIA combined model (q2 = 0.617, N = 4, r2 = 0.904). Both models were further validated by an external test set of eight compounds with satisfactory predictions: r2 = 0.607 for CoMFA and r2 = 0.701 for CoMSIA. In addition, the 3D structure of human kappa opioid receptor was constructed based on the crystal structure of bovine rhodopsin, and the CoMSIA contour plots were then mapped into the structural model of kappa opioid receptor-GNTI complex to identify key residues, which might account for kappa antagonist potency and selectivity. The roles of nonconserved Glu297 and conserved Lys227 of human kappa opioid receptor were then discussed.

Investigation of the binding mode of (-)-meptazinol and bis-meptazinol derivatives on acetylcholinesterase using a molecular docking method.

Molecular docking has been performed to investigate the binding mode of (-)-meptazinol (MEP) with acetylcholinesterase (AChE) and to screen bis-meptazinol (bis-MEP) derivatives for preferable synthetic candidates virtually. A reliable and practical docking method for investigation of AChE ligands was established by the comparison of two widely used docking programs, FlexX and GOLD. In our hands, we had more luck using GOLD than FlexX in reproducing the experimental poses of known ligands (RMSD<1.5 A). GOLD fitness values of known ligands were also in good agreement with their activities. In the present GOLD docking protocol, (-)-MEP seemed to bind with the enzyme catalytic site in an open-gate conformation through strong hydrophobic interactions and a hydrogen bond. Virtual screening of a potential candidate compound library suggested that the most promising 15 bis-MEP derivatives on the list were mainly derived from (-)-MEP with conformations of (S,S) and (SR,RS) and with a 2- to 7-carbon linkage. Although there are still no biological results to confirm the predictive power of this method, the current study could provide an alternate tool for structural optimization of (-)-MEP as new AChE inhibitors. [Figure: see text].

Conformational re-analysis of (+)-meptazinol: an opioid with mixed analgesic pharmacophores.

AIM: To further investigate the analgesic pharmacophore of (+)-meptazinol. METHODS: Two different opioid pharmacophores, Pharm-I and Pharm-II, were established from structures of nine typical opiates and meperidine by using molecular modeling approaches according to their different structure activity relationship properties. They were further validated by a set of conformationally constrained arylpiperidines. Two conformers of (+)-meptazinol (Conformer-I and Conformer-II) detected in solution were then fitted into the pharmacophores, respectively, by Fit Atoms facilities available in SYBYL, a computational modeling tool kit for molecular design and analysis. RESULTS: Conformer-I fit Pharm-I from typical opiates well. However, Conformer-II fit none of these pharmacophores. Instead, it was found to be similar to another potent analgesic, benzofuro[2,3-c]pyridin-6-ol, whose pharmacophore was suggested to hold the transitional state between the two established pharmacophores. Unlike typical analgesics derived from 4-aryl piperidine (eg, meperidine) with one conformer absolutely overwhelming, the (+)-meptazinol exists in two conformers with similar amounts in solution. Furthermore, both conformers can not transform to each other freely in ordinary conditions based on our NMR results. CONCLUSION: (+)-meptazinol was suggested to be an opioid with mixed analgesic pharmacophores, which may account for the complicated pharmacological properties of meptazinol.