Probes for narcotic receptor mediated phenomena. Part 42: synthesis and in vitro pharmacological characterization of the N-methyl and N-phenethyl analogues of the racemic ortho-c and para-c oxide-bridged phenylmorphans

A new synthesis of N-methyl and N-phenethyl substituted ortho-c and para-c oxide-bridged phenylmorphans, using N-benzyl- rather than N-methyl-substituted intermediates, was used and the pharmacological properties of these compounds were determined. The N-phenethyl substituted ortho-c oxide-bridged phenylmorphan(rac-(3R,6aS,11aS)-2-phenethyl-2,3,4,5,6,11a-hexahydro-1H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol (12)) was found to have the highest μ-opioid receptor affinity (K(i)=1.1 nM) of all of the a- through f-oxide-bridged phenylmorphans. Functional data ([3?S]GTP-γ-S) showed that the racemate 12 was more than three times more potent than naloxone as an μ-opioid antagonist.     

Identification of a dual δ OR antagonist/μ OR agonist as a potential therapeutic for diarrhea-predominant Irritable Bowel Syndrome (IBS-d)

A small set of acyclic analogs 5 were prepared to explore their structure-activity relationships (SARs) relative to heterocyclic core, opioid receptor (OR) agonists 4. Compound 5l was found to have very favorable OR binding affinities at the δ and μ ORs (r K(i) δ=1.3 nM; r K(i) μ=0.9 nM; h K(i) μ=1.7 nM), with less affinity for the κ OR (gp K(i) κ=55 nM). The OR functional profile for 5l varied from the previously described dual δ/μ OR agonists 4, with 5l being a potent, mixed dual δ OR antagonist/μ OR agonist [δ IC(50)=89 nM (HVD); μ EC(50)=1 nM (GPI); κ EC(50)=1.6 μM (GPC)]. Compound 5l has progressed through a clinical Phase II Proof of Concept study on 800 patients suffering from diarrhea-predominant Irritable Bowel Syndrome (IBS-d). This Phase II study was recently completed successfully, with 5l demonstrating statistically significant efficacy over placebo.     

Synthesis and binding affinity of novel mono- and bivalent morphinan ligands for κ, μ, and δ opioid receptors

A novel series of homo- and heterodimeric ligands containing κ/μ agonist and μ agonist/antagonist pharmacophores joined by a 10-carbon ester linker chain were synthesized and evaluated for their in vitro binding affinity at κ, μ, and δ opioid receptors, and their functional activities were determined at κ and μ receptors in [(35)S]GTPγS functional assays. Most of these compounds had high binding affinity at μ and κ receptors (K(i) values less than 1nM). Compound 15b, which contains butorphan (1) at one end of linking chain and butorphanol (5) at the other end, was the most potent ligand in this series with binding affinity K(i) values of 0.089nM at the μ receptor and 0.073nM at the κ receptor. All of the morphinan-derived ligands were found to be partial κ and μ agonists; ATPM-derived ligands 12 and 11 were found to be full κ agonists and partial μ agonists.     

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.     

Synthesis and SAR studies of chiral non-racemic dexoxadrol analogues as uncompetitive NMDA receptor antagonists

A series of chiral non-racemic dexoxadrol analogues with various substituents in position 4 of the piperidine ring was synthesized and pharmacologically evaluated. Only the enantiomers having (S)-configuration at the 2-position of the piperidine ring and 4-position of the dioxolane ring were considered. Key steps in the synthesis were an imino-Diels-Alder reaction of enantiomerically pure imine (S)-13, which had been obtained from d-mannitol, with Danishefsky's Diene 14 and the replacement of the p-methoxybenzyl protective group with a Cbz-group. It was shown that (S,S)-configuration of the ring junction (position 2 of the piperidine ring and position 4 of the dioxolane ring) and axial orientation of the C-4-substituent ((4S)-configuration) are crucial for high NMDA receptor affinity. 2-(2,2-Diphenyl-1,3-dioxolan-4-yl)piperidines with a hydroxy moiety ((S,S,S)-5, K(i)=28nM), a fluorine atom ((S,S,S)-6, WMS-2539, K(i)=7nM) and two fluorine atoms ((S,S)-7, K(i)=48nM) in position 4 represent the most potent NMDA antagonists with high selectivity against σ(1) and σ(2) receptors and the polyamine binding site of the NMDA receptor. The NMDA receptor affinities of the new ligands were correlated with their electrostatic potentials, calculated gas phase proton affinities (negative enthalpies of deprotonation) and dipole moments. According to these calculations decreasing proton affinity and increasing dipole moment are correlated with decreasing NMDA receptor affinity.     

The influence of modifications in imide fragment structure on 5-HT(1A) and 5-HT(7) receptor affinity and in vivo pharmacological properties of some new 1-(m-trifluoromethylphenyl)piperazines

New, flexible (7, 9, 11 and 13) and rigid (8, 10, 12 and 14) imides with a 1-(m-trifluorophenyl)piperazine fragment and a tetramethylene or a 1e,4e-cyclohexylene spacer, respectively, showed very high affinity (K(i)=0.3-34 nM) and agonistic in vivo activity for 5-HT(1A) receptors. Flexible new compounds and the previously described 5 also bound to 5-HT(7) receptors (K(i)=21-134 nM). Selected glutarimide derivatives, that is, the most potent postsynaptic 5-HT(1A) receptor agonist rigid compound 8 and its flexible analogue 7, as well as the previously described full agonist-rigid compound 6 and the partial agonist-its flexible counterpart 5 exhibited moderate affinity for alpha(1)-adrenoceptors (K(i)=85 - 268 nM), but were practically devoid of any affinity for dopamine D(2) sites. Those glutarimides demonstrated anxiolytic- (5 and 7) and antidepressant-like (5, 6 and 8) activity in the four-plate and the swim tests in mice, respectively; at the same time, however, they inhibited the locomotor activity of mice. The antidepressant-like effect of 8 was significantly stronger than that induced by imipramine used as a reference antidepressant.     

Benzimidazole derivatives. Part 5: design and synthesis of new benzimidazole-arylpiperazine derivatives acting as mixed 5-HT1A/5-HT3 ligands

A series of new mixed benzimidazole-arylpiperazine derivatives were designed by incorporating in general structure III the pharmacophoric elements of 5-HT(1A) and 5-HT(3) receptors. Compounds 1-11 were synthesized and evaluated for binding affinity at both serotoninergic receptors, all of them exhibiting high 5-HT(3)R affinity (K(i)=10-62nM), and derivatives with an o-alkoxy group in the arylpiperazine ring showing nanomolar affinity for the 5-HT(1A)R (K(i)=18-150nM). Additionally, all the synthesized compounds were selective over alpha(1)-adrenergic and dopamine D(2) receptors (K(i)>1000-10,000nM). Compound 3 was selected for further pharmacological characterization due to its interesting binding profile as mixed 5-HT(1A)/5-HT(3) ligand with high affinity for both receptors (5-HT(1A): K(i)=18.0nM, 5-HT(3): K(i)=27.2nM). In vitro and in vivo findings suggest that this compound acts as a partial agonist at 5-HT(1A)Rs and as a 5-HT(3)R antagonist. This novel mixed 5-HT(1A)/5-HT(3) ligand was also effective in preventing the cognitive deficits induced by muscarinic receptor blockade in a passive avoidance learning test, suggesting a potential interest in the treatment of cognitive dysfunction.     

Synthesis and nicotinic acetylcholine receptor binding affinities of 2- and 3-isoxazolyl-8-azabicyclo[3.2.1]octanes

A series of epiboxidine homologues, 2- and 3-isoxazole substituted 8-azabicyclo[3.2.1]octane derivatives was synthesized and evaluated as potential ligands for neuronal nicotinic acetylcholine receptors in [(3)H]cytisine labeled rat brain. The 2beta-isoxazolyl-8-azabicyclo[3.2.1]octane 9b (K(i)=3 nM) was the most potent compound of the series with a binding affinity twice that of nicotine. The 3beta-isoxazolyl-8-azabicyclo[3.2.1]octane 15b (K(i)=148 nM) exhibited moderate affinity while the corresponding 2alpha- and 3alpha-isomers exhibited micromolar binding affinity.     

Binding of isotryptamines and indenes at h5-HT6 serotonin receptors

To determine if the indolic nitrogen atom is required for the binding of N(1)-benzyltryptamines at h5-HT(6) serotonin receptors, several isotryptamines and indene analogs were examined. The affinity of 3-benzyl-N(1)-(N,N-dimethylaminoethyl)indole (5, K(i)=32nM) and 1-benzyl-3-(N,N-dimethylaminoethyl)indene (11, K(i)=3nM) indicates that the indolic nitrogen atom is not essential for binding.     

Novel coumarin glycoside and phenethyl vanillate from Notopterygium forbesii and their binding affinities for opioid and dopamine receptors

Bioactivity-guided fractionation of Notopterygium forbesii has resulted in the isolation of one new coumarin glycoside and one new phenethyl vanillate, together with seventeen known compounds. The structures of these compounds were characterized by spectroscopic methods. These compounds were evaluated for their binding affinities to the opioid and dopamine receptors, and falcarindiol showed weak binding affinities to opioid receptors and moderate affinity for D1 receptor (K(i)=192+/-6 nM).     

Epiboxidine and novel-related analogues: a convenient synthetic approach and estimation of their affinity at neuronal nicotinic acetylcholine receptor subtypes

Racemic exo-epiboxidine 3, endo-epiboxidine 6, and the two unsaturated epiboxidine-related derivatives 7 and 8 were efficiently prepared taking advantage of a palladium-catalyzed Stille coupling as the key step in the reaction sequence. The target compounds were assayed for their binding affinity at neuronal alpha4beta2 and alpha7 nicotinic acetylcholine receptors. Epiboxidine 3 behaved as a high affinity alpha4beta2 ligand (K(i)=0.4 nM) and, interestingly, evidenced a relevant affinity also for the alpha7 subtype (K(i)=6 nM). Derivative 7, the closest analogue of 3 in this group, bound with lower affinity at both receptor subtypes (K(i)=50 nM for alpha4beta2 and K(i)=1.6 microM for alpha7) evidenced a gain in the alpha4beta2 versus alpha7 selectivity when compared with the model compound.     

7-Arylpiperazinylalkyl and 7-tetrahydroisoquinolinylalkyl derivatives of 8-alkoxy-purine-2,6-dione and some of their purine-2,6,8-trione analogs as 5-HT(1A), 5-HT(2A), and 5-HT(7) serotonin receptor ligands

On the basis of our earlier studies with the serotonin receptor ligands in the group of 1,3-dimethyl-3,7-dihydropurine-2,6-dione derivatives, a series of new arylpiperazinylalkyl and tetrahydroisoquinolinylalkyl analogs of 8-alkoxy-1,3-dimethyl-3,7-dihydropurine-2,6-dione (10-25) and 1,3-dimethyl-7,9-dihydro-3H-purine-2,6,8-trione (26-30) were synthesized and their 5-HT(1A), 5-HT(2A), and 5-HT(7) receptor affinities were determined. The new compounds 17, 18, 20, and 21 were found to be highly active 5-HT(1A) receptor ligands (K(i)=11-19nM) with diversified affinity for 5-HT(2A) receptors (K(i)=15-253nM). Compounds 12, 13, 15, and 19 were moderately potent 5-HT(2A) ligands (K(i)=23-57nM), whereas 17, 18, 24, and 25 showed distinct affinity for 5-HT(7) receptors (K(i)=51-83nM). Purine-2,6,8-triones showed weak affinities for 5-HT(1A) and 5-HT(7) receptors; among them, 27 and 29 were classified as 5-HT(2A) receptor ligands. The selected compounds 17 and 21 were pharmacologically evaluated to determine their functional activities at pre-(hypothermia in mice) and post-(lower lip retraction in rats) synaptic 5-HT(1A) receptors. Compound 17 showed features of a potential agonist of pre- and post-synaptic 5-HT(1A) receptors, whereas 21 was classified as a potential, weak partial agonist of postsynaptic sites. Last of all, the most interesting compound 17 tested in behavioral models showed potential anxiolytic and antidepressant activities.     

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)).     

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.     

Conformationally-restricted analogues and partition coefficients of the 5-HT3 serotonin receptor ligands meta-chlorophenylbiguanide (mCPBG) and meta-chlorophenylguanidine (mCPG)

The present investigation examined two features of arylbiguanide and arylguanidine 5-HT(3) ligands: conformation and partition coefficients. Several conformationally-constrained analogues of mCPBG (2) and mCPG (11; K(i)=32 nM) were prepared and of these only 2-amino-5-chloro-3,4-dihydroquinazoline (14; K(i)=34 nM) retained high affinity. The partition coefficient of compound 11 (LogP(app)=-0.64) was less than that of its corresponding arylbiguanide 2 (LogP(app)=-0.38). The quinazoline structure may represent a pharmacologically-active conformation of these agents, and the arylbiguanides were found more lipid soluble than their arylguanidine counterparts at physiological pH.     

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

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

3-Carboxamido analogues of morphine and naltrexone. synthesis and opioid receptor binding properties.

In response to the unexpectedly high affinity for opioid receptors observed in a novel series of cyclazocine analogues where the prototypic 8-OH was replaced by a carboxamido group, we have prepared the corresponding 3-CONH(2) analogues of morphine and naltrexone. High affinity (K(i)=34 and 1.7nM) for mu opioid receptors was seen, however, the new targets were 39- and 11-fold less potent than morphine and naltrexone, respectively.     

Design, synthesis and evaluation of novel 16-imidazolyl substituted steroidal derivatives possessing potent diversified pharmacological properties

As a part of our investigations into the structural-activity relationship studies of a novel class of medicinally active 16-substituted steroids, several new 16-imidazolyl substituted steroidal derivatives have been synthesized and pharmacologically evaluated in the current study. The new steroidal analogues 5, 6, 8, 9, 11 and 12 exhibited moderate cytotoxic effects in sixty cancer cell lines derived from nine cancers types. The imidazolyl substituted steroidal derivatives 6 (DPJ-RG-1241) and 7 (RB-401) were obtained as the powerful inhibitors of aromatase with IC50=0.18 μM and IC50=0.168 μM, respectively, approximately 1.2 and 1.4 times more potent in comparison to standard drug exemestane. The bis-quaternary steroids 13 and 14 displayed potent skeletal muscle relaxant properties. An affinity constant of 0.007 μM was observed for compound 14 on frog rectus abdominis muscle preparation and 13 displayed a very high anticholinesterase activity K(i)=25 nM, approximately 115-fold higher in comparison to standard drug galanthamine (K(i)=2.9 μM).     

gamma-Carbolines: binding at 5-HT5A serotonin receptors

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