Investigation of the N-substituent conformation governing potency and mu receptor subtype-selectivity in (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonists

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7-9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7-9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and mu receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [35S]GTP-gamma-S binding, we show that the trans-cinnamyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the mu receptor.     

Synthesis and characterization of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled fluorescent ligands for the mu opioid receptor

A series of opioid ligands utilizing the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3-propionic acid or 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- s-indacene-3-propionic acid were synthesized and characterized for their ability to act as a suitable fluorescent label for the mu opioid receptor. All compounds displaced the mu opioid receptor binding of [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol in monkey brain membranes with high affinity. The binding of fluorescent ligands to delta and kappa receptors was highly variable. 5,7-Dimethyl-BODIPY naltrexamine, "6-BNX," displayed subnanomolar affinities for the mu and kappa opioid receptors (Ki 0.07 and 0.43 nM, respectively) and nanomolar affinity at the delta (Ki 1.4 nM) receptor. Using fluorescence spectroscopy, the binding of 6-BNX in membranes from C6 glioma cells transfected with the cloned mu opioid receptor was investigated. In these membranes containing a high receptor density (10-80 pmol/mg protein), 6-BNX labeling was saturable, mu opioid specific, stereoselective (as determined with the isomers dextrorphan and levorphanol), and more than 90% specific. The results describe a series of newly developed fluorescent ligands for the mu opioid receptor and the use of one of these ligands as a label for the cloned mu receptor. These ligands provide a new approach for studying the structural and biophysical nature of opioid receptors.     

N-substituted octahydro-4a-(3-hydroxyphenyl)-10a-methyl-benzo[g]isoquinolines are opioid receptor pure antagonists

N-Methyl- and N-phenylethyl-(+/-)-1,2,3,4,4a,5,10,10a- octahydro-4a-(3-hydroxyphenyl)-10a-methyl-benzo[g]isoquinolines (4 and 5, respectively) were found to be pure opioid antagonists. These compounds were shown to share many of the characteristics identified with the N-methyl- and N-phenylethyl trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (1 and 2, respectively) including N-substituent mediated potency and a lack of N-substituent mediated antagonism. These data suggest that compounds 4 and 5 and the N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines (1 and 2) may interact with opioid receptors similarly.     

Stereoisomers of N-[1-hydroxy-(2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide: synthesis, stereochemistry, analgesic activity, and opioid receptor binding characteristics

N-[1-(2-Hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N-phenylpropanamide (ohmefentanyl,1) is an extremely potent analgesic agent with high affinity and selectivity for opioid mu receptors. There are three chiral carbons in 1, so eight optically active isomers are possible. Respective reaction of optically active 3-methyl-N-phenyl-4 -piperidinamines (5a-d) with (R)- or (S)-styrene oxide produced eight optically active intermediates which were subsequently converted to eight optically active isomers of 1 (1a-h). The absolute configurations of 1a-h were determined by X-ray analysis of (3R,4S,2'R)-(-)-cis-1a and (3R,4R,2'S)-(-)-trans-1g. The analgesic activity (mice, ip, hot plate) revealed their extreme stereodifferences; the ED50 values of (3R,4S,2'R)-(-)-cis-1a and (3R,4S,2'S)-(+)-cis-1b, which are the most potent isomers among eight isomers, were 0.004 65 (2990 times that of morphine) and 0.001 06 mg/kg (13 100 times that of morphine), respectively, while the corresponding antipodes 1d,c were the least potent compounds among the eight isomers. In agreement with pharmacological results, both 1a,b also had the highest receptor affinity and selectivity for the opioid mu receptor. The ratio of K(i)(DPDPE)&K(i)(DAMGO) was 22 800 for 1a and 22 500 for 1b. All isomers except 1c,d strongly inhibited the electrically evoked smooth muscle contraction of GPI and MVD but not that of RVD, and the inhibitory effects could be reversed by naloxone, which indicated that these compounds were potent mu agonists in GPI and MVD. There was a good linear correlation between the analgesic potencies (ED50) and the receptor affinities (K(i)(DAMGO)) or inhibitory effects (IC50) to GPI and MVD. These results suggested that the analgesic effects of ohmefentanyl are mediated by interaction between the agents and opioid mu receptors in the central nervous system and the 3R,4S configuration at the piperidine 3- and 4-carbon atoms and the S configuration at the phenylethyl 2-carbon atom are beneficial for analgesic potency and inhibitory effects in GPI and MVD and the same for an S or R configuration at the phenylethyl 2-carbon atom besides the 3R,4S configuration for receptor mu affinity and selectivity.     

The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line down-regulates without desensitizing during chronic opioid exposure

The R1.1 mouse thymoma cell line expresses a single class of kappa opioid receptors that is negatively coupled to adenylyl cyclase through a Bordetella pertussis toxin-sensitive inhibitory guanine nucleotide-binding protein. The aim of the present study was to determine whether chronic opioid treatment of R1.1 cells altered either the binding properties or the functional response associated with the kappa opioid receptor. Culturing of R1.1 cells with the kappa-selective agonist (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzeneacetamide methane-sulfonate hydrate (U50,488) for 3 hr and longer, followed by extensive washing of R1.1 cell membranes, produced a concentration- and time-dependent reduction in the binding of the kappa-selective ligand (5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1- oxaspiro(4,5)dec-8-yl) benzeneacetamide ([3H]U69,593). Culturing of R1.1 cells with 100 nM U50,488 for 24 hr produced approximately a 50% reduction in the Bmax value for [3H]U69,593 and [3H]naloxone binding. In contrast to the reduction in binding, there was no change in the inhibition of adenylyl cyclase activity by (-)-U50,488. To determine whether kappa opioid receptor function was maintained by spare receptors after agonist-induced down-regulation, membranes from untreated R1.1 cells were incubated with 400 nM of the irreversible opioid antagonist beta-chlornaltrexamine (beta-CNA) followed by extensive washing. beta-CNA produced a 50% reduction in the [3H]U69,593 binding and a 6-fold increase in the IC50 value for (-)-U50,488 inhibition of adenylyl cyclase activity, with no change in the maximal inhibition of cyclic AMP levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

(3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1-yl]chroman-4,7-diol: a conformationally restricted analogue of the NR2B subtype-selective NMDA antagonist (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)- 1-propanol

(1S,2S)-1-(4-Hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606, 1) is a recently described antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. In the present study, the optimal orientation of compounds of this structural type for their receptor was explored. Tethering of the pendent methyl group of 1 to the phenolic aromatic ring via an oxygen atom prevents rotation about the central portion of the molecule. Several of the new chromanol compounds have high affinity for the racemic [3H]CP-101,606 binding site on the NMDA receptor and protect against glutamate toxicity in cultured hippocampal neurons. The new ring caused a change in the stereochemical preference of the receptor-cis (erythro) compounds had better affinity for the receptor than the trans isomers. Computational studies suggest that steric interactions between the pendent methyl group and the phenol ring in the acyclic series determine which structures can best fit the receptor. The chromanol analogue, (3R,4S)-3-[4-(4-fluorophenyl)-4-hydroxypiperidin-1- yl]chroman-4,7-diol (12a, CP-283,097), was found to possess potency and selectivity comparable to CP-101,606. Thus 12a is a new tool to explore the function of the NR2B-containing NMDA receptors.     

N-[omega-(Tetralin-1-yl)alkyl] derivatives of 3,3-dimethylpiperidine are highly potent and selective sigma1 or sigma2 ligands

Several 3, 3-dimethyl-N-[omega-(tetrahydronaphthalen-1-yl)alkyl]piperidine derivatives and some related compounds were prepared. Their affinities and sigma-subtype selectivities were investigated by radioligand binding assays, labeling sigma1 receptors with [3H]-SKF 10047 and sigma2 receptors with [3H]-DTG. Many tested compounds bound sigma1 and/or sigma2 receptors with nanomolar or subnanomolar IC50 values. Compound (+)-22, (+)-3,3-dimethyl-1-[3-(5-methoxy-1,2,3, 4-tetrahydronaphthalen-1-yl)-n-propyl]piperidine, was the most potent (IC50 = 0.089 nM) and selective sigma1 ligand (1340-fold), showing a 10-fold enantioselectivity. Compounds 29 (3, 3-dimethyl-1-[4-(6-methoxy-1,2,3, 4-tetrahydronaphthalen-1-yl)-n-butyl]piperidine) and 31 (3, 3-dimethyl-1-[5-(1,2,3, 4-tetrahydronaphthalen-1-yl)-n-pentyl]piperidine) were highly potent (IC50 = 0.016 nM and IC50 = 0.008 nM, respectively) and highly selective sigma2 ligands (more than 100000-fold).     

The Stimulated Raman Spectrum of Symmetric 13C Cyanogen, 13C2N2

BACKGROUND: The authors examined the interaction of ketamine with recombinant mu, kappa, and delta opioid receptors and recombinant orphan opioid receptors expressed in Chinese hamster ovary cells (CHO-mu, CHO-kappa, CHO-delta, and CHO(ORL1), respectively). METHODS: CHO-mu, CHO-kappa, and CHO-delta membranes were incubated with the opioid receptor radioligand [3H]diprenorphine at room temperature. Ketamine (racemic, R(-) and S(+)) was included at concentrations covering the clinical range. CHO(ORL1) membranes were incubated with [125I]Tyr(14)nociceptin and racemic ketamine at room temperature. The effects of racemic ketamine and selective opioid receptor agonists (mu: [D-Ala2, MePhe4, Gly(ol)5] enkephalin (DAMGO); kappa: spiradoline or delta: [D-pen2, D-pen5] enkephalin (DPDPE)) on forskolin-stimulated cyclic adenosine monophosphate formation also were examined. Data are mean +/- SEM. RESULTS: Racemic ketamine increased the radioligand equilibrium dissociation constant for [3H]diprenorphine from 85+/-5 to 273+/-11, 91+/-6 to 154+/-16, and 372+/-15 to 855+/-42 pM in CHO-mu, CHO-kappa, and CHO-delta, respectively. The concentration of radioligand bound at saturation was unaffected. In CHO-mu and CHO-kappa cells, racemic ketamine did not slow the rate of naloxone-induced [3H]diprenorphine dissociation. Ketamine and its isomers also displaced [3H]diprenorphine binding to mu, kappa, and delta receptors in a dose-dependent manner, with pKi values for racemic ketamine of 4.38+/-0.02, 4.55+/-0.04, and 3.57+/-0.02, respectively. S(+)-ketamine was two to three times more potent than R(-)-ketamine at mu and kappa receptors. Racemic ketamine displaced [125I]Tyr(14)nociceptin with an estimated affinity constant of 0.5 mM. Racemic ketamine inhibited the formation of cyclic adenosine monophosphate (naloxone insensitive) in a dose-dependent manner (concentration producing 50% inhibition approximately 2 mM) in all cell lines, including untransfected CHO cells. Ketamine (100 microM) reversed DAMGO (mu) and spiradoline (kappa) inhibition of formation of cyclic adenosine monophosphate. CONCLUSIONS: Ketamine interacts stereoselectively with recombinant mu and kappa opioid receptors.     

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.     

Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with high affinity, 1 is a potent, dual inhibitor of both of the isostates of PDE 4. Kinetic and thermodynamic models describing the interactions between the nonexchangeable isostates of PDE 4 and its ligands are discussed.     

Synthesis and structure-activity relationships of a new model of arylpiperazines. 4. 1-[omega-(4-Arylpiperazin-1-yl)alkyl]-3-(diphenylmethylene) - 2, 5-pyrrolidinediones and -3-(9H-fluoren-9-ylidene)-2, 5-pyrrolidinediones: study of the steric requirements of the terminal amide fragment on 5-HT1A affinity/selectivity

In the present paper, we report the synthesis and the binding profile on 5-HT1A, alpha1 and D2 receptors of a new series of 1-[omega-(4-arylpiperazin-1-yl)alkyl]-3-(diphenylmethylene)- 2, 5-pyrrolidinediones (III) (1-4) and -3-(9H-fluoren-9-ylidene)-2, 5-pyrrolidinediones (IV) (1-4), in which the alkyl linker contains 1-4 methylenes and the aryl group is variously substituted. The results obtained are compared to those previously reported for bicyclohydantoin (I) and the related bicyclic amine (II) series. A considerable part of the tested compounds 1-4 demonstrated moderate to high affinity for 5-HT1A and alpha1 receptor binding sites but had no affinity for D2 receptors. The study of the length of the alkyl chain and the imide substructure has allowed us to suggest some differences between the 5-HT1A and the alpha1-adrenergic receptors: (i) for III and IV, affinity for the 5-HT1A receptor as a function of the length of the methylene linker decreases in the order 4 > 1 > 3 approximately 2, while for the alpha1 receptor affinity decreases in the order 3 approximately 4 > 1 approximately 2; (ii) the no-pharmacophoric steric pocket (receptor zone which does not hold the pharmacophore of the ligand but holds a nonessential fragment of the molecule) in the 5-HT1A receptor has less restriction than the corresponding pocket in the alpha1 receptor. Compounds 3a,e, which are highly selective for alpha1-adrenergic receptors, displayed antagonist activity. On the other hand, the best compromise between affinity and selectivity for 5-HT1A receptors is reached in these new series with n = 1, which is in agreement with our previous results for the bicyclohydantoin derivatives I. Two selected compounds (1d and 4e) retain agonist properties at postsynaptic 5-HT1A receptors. The same 5-HT1A agonist profile found in these compounds suggests the existence of two different no-pharmacophoric steric pockets in this receptor and a different interaction of compounds with n = 1 and n = 4. The information obtained from the interpretation of the energy minimization and 2D-NOESY experiments of compounds 1-4 together with the synthesis and binding data of new conformationally restrained analogues 4k-m is in good agreement with this working hypothesis.     

Modified ibogaine fragments: synthesis and preliminary pharmacological characterization of 3-ethyl-5-phenyl-1,2,3,4,5, 6-hexahydroazepino[4,5-b]benzothiophenes

Five phenyl-substituted derivatives and analogues of 1,2,3,4,5, 6-hexahydroazepino[4,5-b]indole, 5, a major fragment of ibogaine (1), were synthesized and tested for binding to monoamine transporters, the NMDA receptor-coupled cation channel, and dopamine and opioid receptors. All five derivatives, 9 and 17a-d, displayed 8-10-fold higher affinity at the DA transporter than ibogaine and noribogaine (4). At the serotonin transporter, two compounds (9 and 17a) exhibited higher potency than ibogaine, while the rest had weaker binding affinities than the lead compound. In keeping with their structural similarity to ibogaine, all five compounds displayed weak to poor affinity for dopamine D1 and D2 receptors. However, two compounds, 17a,c, demonstrated moderate binding affinities at dopamine D3 receptors. All five compounds displayed weak to poor affinities for mu and kappa opioid receptors and for the NMDA receptor-coupled cation channel. Despite the qualitative differences, derivatives and analogues of 5may serve as useful substitutes for ibogaine.     

De novo design, synthesis, and biological activities of high-affinity and selective non-peptide agonists of the delta-opioid receptor

On the basis of the structure-activity relationships of delta-opioid-selective peptide ligands and on a model of the proposed bioactive conformation for a potent and selective, conformationally constrained delta-opioid peptide ligand [(2S, 3R)-TMT1]DPDPE, a series of small organic peptide mimetic compounds targeted for the delta-opioid receptor have been designed, synthesized, and evaluated in radiolabeled ligand binding assays and in vitro bioassays. The new non-peptide ligands use piperazine as a template to present the most important pharmacophore groups, including phenol and phenyl groups and a hydrophobic moiety. This hydrophobic group was designed to mimic the hydrophobic character of the D-Pen residues in DPDPE, which has been found to be extremely important for increasing the binding affinity and selectivity of these non-peptide ligands for the delta-opioid receptor over the mu-opioid receptor. Compound 6f (SL-3111) showed 8 nM binding affinity and over 2000-fold selectivity for the delta-opioid receptor over the mu-opioid receptor. Both enantiomers of SL-3111 were separated, and the (-)-isomer was shown to be the compound with the highest affinity for the delta-opioid receptor found in our study (IC50 = 4.1 nM), with a selectivity very similar to that observed for the racemic compound. The phenol hydroxyl group of SL-3111 turned out to be essential to maintain high affinity for the delta-opioid receptor, which also was observed in the case of the delta-opioid-selective peptide ligand DPDPE. Binding studies of SL-3111 and [p-ClPhe4]DPDPE on the cloned wild-type and mutated human delta-opioid receptors suggested that the new non-peptide ligand has a binding profile similar to that of DPDPE but different from that of (+)-4-[((alphaR)-alpha(2S,5R)-4-allyl-2, 5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), another delta-opioid-selective non-peptide ligand.     

Synthesis and structure-activity relationships of potent and orally active 5-HT4 receptor antagonists: indazole and benzimidazolone derivatives

A series of indole-3-carboxamides, indazole-3-carboxamides, and benzimidazolone-3-carboxamides was synthesized and evaluated for antagonist affinity at the 5-HT4 receptor in the rat esophagus. The endo-3-tropanamine derivatives in the indazole and benzimidazolone series possessed greater 5-HT4 receptor affinity than the corresponding indole analogues. 5-HT4 receptor antagonist affinity was further increased by alkylation at N-1 of the aromatic heterocycle. In a series of 1-isopropylindazole-3-carboxamides, replacement of the bicyclic tropane ring system with the monocyclic piperidine ring system or an acyclic aminoalkylene chain led to potent 5-HT4 receptor antagonists. In particular, those systems in which the basic amine was substituted with groups capable of forming hydrogen bonds showed increased 5-HT4 receptor antagonist activity. While some of these compounds displayed high affinity for other neurotransmitter receptors (in particular, 5-HT3, alpha1, and 5-HT2A receptors), as the conformational flexibility of the amine moiety increased, the selectivity for the 5-HT4 receptor also increased. From this series of compounds, we identified LY353433 (1-(1-methylethyl)-N-[2-[4-[(tricyclo[3.3.1.1(3, 7)]dec-1-ylcarbonyl)amino]-1-piperidinyl]ethyl]-1H-indazole-3- carboxamide) as a potent and selective 5-HT4 receptor antagonist with clinically suitable pharmacodynamics.     

Synthesis of (+)-(1'R,2'S) and (1'S,2'R)-6,11-dimethyl-1,2,3,4,5,6 -hexahydro-3-[[2'-(alkoxycarbonyl)-2'-phenylcyclopropyl]methyl]-2 ,6 -methano-3-benzazocin-8-ol. Comparison of the affinities for sigma 1 and opioid receptors with in the diastereoisomeric MPCB and CCB

The synthesis and the in vitro receptor affinity for sigma 1 and opiod receptors of the two diastereoisomers of (+)-cis-MPCB namely, (+)-cis-(1'S,2'R)-6,11-Dimethyl-1,2,3,4,5,6 -hexahydro-3-[[2'-(methoxycarbonyl)-2'-phenylcyclopropyl]methyl]-2 ,6 -methano-3-benzazocin-8-ol, (1'S,2'R)6a and (+)-cis-(1'R,2'S)-6,11-Dimethyl-1,2,3,4,5,6-hexahydro-3- [[2-(methoxycarbonyl)-2'-phenylcyclopropyl]methyl]-2,6-methano-3-+ ++benzazocin-8 -ol, (1'R,2'S)6a are reported. Affinities of (1'S,2'R)6a and (1'R,2'S)6a were compared with those of the (-)-cis-diastereoisomers of MPCB(1), and of its p-Cl phenyl derivative CCB(2). The (+)-cis-N-normetazocine derivatives showed higher affinity for the sigma 1 sites, labeled with [3H]-(+)-pentazocine than the corresponding (-)-cis- analogs. In particular, compound (1'S,2'R)6a showed a Ki = 66.7 nM for sigma 1 receptor, associated with a good selectivity for sigma 1 with respect to kappa, mu, delta opioid receptors subtypes (Ki = > 1,000 nM). Analysis of the data seem to support the hypothesis that the (+)-cis-N-normetazocine nucleus posses a specific enantioselectivity for sigma 1 sites, when supporting bulkier N-substituents functionalized with a carboxy ester group.     

N-[2-[4-(4-Chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide: a potent and selective dopamine D4 ligand

A series of new 1-aryl-4-alkylpiperazines containing a terminal benzamide fragment or a tetralin-1-yl nucleus on the alkyl chain were synthesized and tested for binding at cloned human dopamine D4 and D2 receptor subtypes. A SAFIR (structure-affinity relationship) study on this series is herein discussed. The most relevant D4 receptor affinities were displayed by N-[omega-[4-arylpiperazin-1-yl]alkyl]-methoxybenzamides (compounds 5, 16-20), their IC50 values ranging between 0.057 and 7.8 nM. Among these, N-[2-[4-(4-chlorophenyl)piperazin-1-yl]ethyl]-3-methoxybenzamide (17) emerged since it exhibited very high affinity for dopamine D4 receptor (IC50 = 0.057 nM) with selectivity of >10 000 for the D4 versus the D2 receptor; compound 17 was also selective versus serotonin 5-HT1A and adrenergic alpha1 receptors.     

Kappa1- and kappa2-opioid receptors mediating presynaptic inhibition of dopamine and acetylcholine release in rat neostriatum

1. The effects of selective opioid receptor agonists and antagonists on N-methyl-D-aspartate (NMDA, 10 microM)-induced release of [3H]-dopamine and [14C]-acetylcholine (ACh) from superfused neostriatal slices were studied to investigate the possible occurrence of functional kappa-opioid receptor subtypes in rat brain. 2. The kappa receptor agonists (-)-ethylketocyclazocine ((-)-EKC), U69593 and the endogenous opioid peptide dynorphin A1-13 caused a naloxone-reversible inhibition of NMDA-induced [3H]-dopamine release, with pD2 values of about 9, 8.5 and 8.2, respectively, whereas both the mu agonist Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol (DAMGO) and the delta agonist D-Pen2-D-Pen5-enkephalin (DPDPE) were ineffective in this respect. The inhibitory effect of submaximally effective concentrations of dynorphin A1-13, U69593 and (-)-EKC on NMDA-induced [3H]-dopamine release were not changed by the delta1/delta2-opioid receptor antagonist naltrindole (up to a concentration of 1 microM, but reversed by the kappa receptor antagonist nor-binaltorphimine (nor-BNI), with an IC50) as low as 0.02 nM, indicating the involvement of U69593-sensitive kappa1-opioid receptors. 3. NMDA-induced [14C]-ACh release was reduced in a naloxone-reversible manner by DPDPE (pD2 about 7.2), dynorphin A1-13 (pD2 6.7) and EKC (pD2 6.2), but not by U69593 and DAMGO. The inhibitory effect of a submaximally effective concentration of DPDPE, unlike those of dynorphin A1-13 and (-)-EKC, on NMDA-induced [14C]-ACh release was antagonized by naltrindole with an IC50 of 1 nM, indicating the involvement of delta-opioid receptors in the inhibitory effect of DPDPE. On the other hand, the inhibitory effects of dynorphin A1-13 and (-)-EKC on [14C]-ACh release were readily antagonized by nor-BNI with an IC50 of about 3 nM. A 100 fold higher concentration of nor-BNI also antagonized the inhibitory effect of DPDPE, indicating the involvement of U69593-insensitive kappa2-opioid receptors in the inhibitory effects of dynorphin A1-13 and (-)-EKC. 4. Although naloxone benzoylhydrazone (NalBzoH), displaying high affinity towards the putative kappa3-opioid receptor, antagonized the inhibitory effects of dynorphin A1-13 and (-)-EKC on [3H]-dopamine and [14C]-ACh release as well as that of U69593 on [3H]-dopamine release, it displayed a low apparent affinity (IC50 about 100 nM) in each case. 5. In conclusion, whereas activation of kappa1-opioid receptors causes presynaptic inhibition of NMDA-induced dopamine release, kappa2 receptor activation results in inhibition of ACh release in rat neostriatum. As such, this study is the first to provide unequivocal in vitro evidence for the existence of functionally distinct kappa-opioid receptor subtypes in the brain.     

Expression of rat NK-2 (neurokinin A) receptor in E. coli

With the goal of obtaining sufficient functional protein for structural analysis, rat neurokinin-2 receptor was produced in Escherichia coli by linking it to the periplasmic maltose-binding protein. As a first step, we present a biochemical and pharmacological investigation of the recombinant receptor. Western-blots showed that the fusion protein was associated with the membranes. The agonist [4,5-3H-Leu9]neurokinin A and the NK-2 antagonist [3H]SR48,968 bound to the receptor in a highly specific manner. Saturation binding of the [3H]agonist demonstrated a single class of receptors (KD = 10.5 nM, Bmax = 2.5 pmol/mg protein). The [3H]antagonist bound with higher affinity to a larger receptor population (KD = 0.2 nM, Bmax = 7.2 pmol/mg protein). Competition of [3H]agonist binding with other agonists demonstrated a potency order of: neurokinin A > [Nle10]NKA(4-10) = [beta-Ala8]NKA(4-10) > substance P > senktide Against the [3H]antagonist, agonists were only partially inhibitory. Selective NK-2 antagonists inhibited binding of both [3H]ligands with an identical order of potency: SR48,968 > R396 > MEN10,376, which is consistent with NK-2 receptor pharmacology in rat tissue.