(+/-)8-Amino-5,6,7,8-tetrahydroisoquinolines as novel antinociceptive agents

Several amine-substituted 8-amino-5,6,7,8-tetrahydroisoquinolines were examined as conformationally-constrained analogs of the nicotinic cholinergic (nACh) 3-(aminomethyl)pyridines. Although these ligands failed to bind at nACh receptors, the N-ethyl-N-methyl analog 3d was found to be at least equipotent with nicotine in rodent tests of antinociception. The mechanism of action of 3d is currently unknown.     

(-)6-n-Propylnicotine antagonizes the antinociceptive effects of (-)nicotine

Several 6-alkyl analogues of nicotine were examined in radioligand binding and in vivo functional assays. Although (-)6-ethylnicotine (3) binds with high affinity at nACh receptors (Ki=5.6 nM) and produces nicotine-like actions, its n-propyl homologue (-)4 (Ki=22 nM) failed to produce such effects. In fact, (-)4 antagonized the antinociceptive effects of (-)nicotine in the tail-flick assay in mice, but not the spontaneous activity or discriminative stimulus effects of (-)nicotine. Compound (-)4 appears to selectively antagonize only one of the three effects examined and is an interesting cholinergic agent for subsequent investigation.     

Anti-amnesic properties of (±)-PPCC, a novel sigma receptor ligand, on cognitive dysfunction induced by selective cholinergic lesion in rats

Previous studies have reported that selective sigma-1 agonists may improve cognitive abilities in experimental animals possibly via a cholinergic mechanism. However, the issue of a direct action on to sigma-1 receptors in memory-related brain areas has been much less investigated. The newly synthetised compound methyl(1 R ,2 S /1 S ,2 R )-2-[4-hydroxy-4-phenylpiperidin-1-yl)methyl]-1-(4-methylphenyl) cyclopropanecarboxylate [(±)-PPCC] has recently been shown to possess high affinity for the sigma-1 receptor where it specifically acts as an agonist. Here, the functional effects of (±)-PPCC were investigated in rat models of mild or severe cognitive dysfunction based on a sub-total (≤ 70–80%) or complete (≥ 90–95%) central cholinergic depletion induced by different doses of the selective immunotoxin 192 IgG-saporin injected intraventricularly. At 5–6 weeks post-surgery, the lesioned animals exhibited dose-dependent deficits in reference memory, as assessed using the Morris water maze task, whereas working memory abilities, evaluated using the radial arm water maze task, appeared equally impaired in the two dose groups. Daily treatment with (±)-PPCC significantly improved both reference and working memory performance in all lesioned animals but it did not affect intact or sham-lesioned subjects. In a separate test, treatment with (±)-PPCC reversed the learning deficits induced by the muscarinic receptor antagonist atropine sulphate in both control and mild-lesioned rats. The effect was blocked in lesioned, but not normal animals by pre-treatment with the sigma-1 antagonist N -[2-(3,4-dichlorophenyl)ethyl]- N -methyl-2-(dimethylamino)ethylamine. The results suggest that (±)-PPCC may efficiently ameliorate perturbed cognitive abilities, and that these anti-amnesic effects most probably occur via a direct interaction of the compound with sigma-1 receptors.     

alpha,beta-Dibenzyl-gamma-butyrolactone lignan alcohols: total synthesis of (+/-)-7'-hydroxyenterolactone, (+/-)-7'-hydroxymatairesinol and (+/-)-8-hydroxyenterolactone

Two trans-alpha,beta-dibenzyl-gamma-butyrolactone lignans carrying a hydroxyl group at the beta-benzylic carbon atom and a alpha-hydroxy alpha,beta-dibenzyl-gamma-butyrolactone lignan were synthesized in racemic form using the tandem conjugate addition reaction to construct the basic lignan skeleton. Subsequent reaction steps involved either a catalytic reduction of the regenerated keto group to the alcohol, or a hydrogenolysis to benzylic methylene followed by lactone enolate formation and oxidation to give the alpha-hydroxybutyrolactones. These procedures were applied for the synthesis of 7'-hydroxyenterolactones and 7'-hydroxymatairesinols, and 8-hydroxyenterolactones, respectively. The diastereomeric mixtures of these compounds were separated either by HPLC techniques or column chromatography and the structures were elucidated using NMR spectroscopy.     

(+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone, a derivative of (+/-)-praeruptorin A, reverses P-glycoprotein mediated multidrug resistance in cancer cells

P-glycoprotein (Pgp) is an ATP-driven membrane exporter for a broad spectrum of hydrophobic xenobiotics. Pgp-overexpression is a common cause of multidrug resistance (MDR) in cancer cells and could lead to chemotherapeutic failure. Through an extensive herbal drug screening program we previously showed that (+/-)-praeruptorin A (PA), a naturally existing pyranocumarin isolated from the dried root of Peucedanum praeruptorum Dunn., re-sensitizes Pgp-mediated MDR (Pgp-MDR) cancer cells to cancer drugs. A number of PA derivatives were synthesized and one of these, (+/-)-3'-O, 4'-O-dicynnamoyl-cis-khellactone (DCK), was more potent than PA or verapamil in the reversal of Pgp-MDR. In Pgp-MDR cells DCK increased cellular accumulation of doxorubicin without affecting the expression level of Pgp. In Pgp-enriched membrane fractions DCK moderately stimulated basal Pgp-ATPase activity, suggesting some transport substrate-like function. However, DCK also inhibited Pgp-ATPase activity stimulated by the standard substrates verapamil or progesterone with decreased V(max)s but K(m)s were relatively unchanged, suggesting a primarily non-competitive mode of inhibition. While the binding of substrates to active Pgp would increase the reactivity of the Pgp-specific antibody UIC2, DCK decreased UIC2 reactivity. These results suggest that DCK could bind simultaneously with substrates to Pgp but perhaps at an allosteric site and thus affect Pgp-substrate interactions.     

Differential effects of cyanogen bromide on ligand binding by mu, delta and kappa opioid receptors

Guinea pig brain membranes treated with cyanogen bromide (CNBr) demonstrate a loss in the number of mu opioid receptors and a lower binding affinity of delta opioid receptors. These receptor changes are irreversible. Results from ligand protection experiments support the hypothesis that the location of the methionine groups, the sites at which CNBr cleaves peptides, differs between these two types of opioid receptors. Kappa receptors are significantly less sensitive to the action of CNBr than mu or delta receptors.     

(+/-)-cis-(6-Ethyl-tetrahydropyran-2-yl)-formic acid: a novel substance with antinociceptive properties

We described in this paper the first synthesis to the (+/-) cis (6-ethyl-tetrahydropyran-2-yl) formic acid (1) using the very efficient Prins cyclization reaction as strategy to construction of its tetrahydropyran skeleton. This new compound presented a significant antinociceptive property by the tail-flick model.     

The analgesic tropane analogue (+/-)-SM 21 has a high affinity for sigma2 receptors

The analgesic properties of the tropane analogue (+/-)-SM 21 have been attributed to the antagonism of presynaptic m2 receptors resulting in a potentiation of acetylcholine release. However, drugs targeting a number of other neurotransmitter receptors have been shown to enhance acetylcholine release. In the current study, in vitro studies were conducted in order to determine the affinity of (+/-)-SM 21 for serotonin 5-HT3, 5-HT4, and sigma receptors. Our results indicate that (+/-)-SM 21, and its structural congeners, have a relatively high affinity for sigma2 receptors relative to their reported affinity for muscarinic receptors. The higher affinity for sigma2 versus sigma1 receptors indicates that (+/-)-SM 21 may be a suitable lead compound for developing sigma2-selective ligands.     

(+/-)-4-[(N-allyl-cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzamide displays selective binding for the delta opioid receptor

Racemic 4-[(N-allyl-cis-3-methyl-4-piperidinyl)phenylamino]-N,N-diethylbenzam ide (3a) was synthesized and found to have good affinity and selectivity for the delta receptor. These compounds can be viewed as an analog of BW373U86 and SNC-80 where an internal piperazine nitrogen has been transposed with a benzylic carbon. Functionally, 3a behaves as an agonist at the delta receptor with no measurable stimulation of either the mu or kappa receptor subtypes and was found to be devoid of any measurable amount of antagonist activity for any opioid receptor. A comparison of 3a to SNC-80 and DPDPE in the [35S]GTPgammaS functional assay suggests that 3a may be more like the peptide DPDPE.     

A new opioid designed multiple ligand derived from the micro opioid agonist endomorphin-2 and the delta opioid antagonist pharmacophore Dmt-Tic

Opioid compounds with mixed micro agonist/delta antagonist properties could be used as analgesics with low propensity to induce tolerance and dependence. Here we report the synthesis of a new designed multiple ligand deriving from the micro selective agonist endomorphin-2 and the delta selective antagonist pharmacophore Dmt-Tic. As predicted, the resulting bivalent ligand showed a micro agonist/delta antagonist profile deriving from the corresponding activities of each pharmacophore.     

Deconstructing cytisine: The syntheses of (+/-)-cyfusine and (+/-)-cyclopropylcyfusine, fused ring analogs of cytisine

A novel fused tricyclic analog (11) of cytisine has been prepared (coined 'cyfusine') and determined to have high affinity at neuronal nicotinic acetylcholine receptors. A [3+2] cycloaddition protocol permitted entry into a 3,4-differentially difunctionalized dihydropyrrole (7). The penultimate cyclization was accomplished using the modified Van Tamelen conditions developed in our earlier synthesis of (+/-)-cytisine. Sequential ring-forming reactions ([3+2] cycloaddition/cyclopropanation/pyridone cyclization) gives a unique cyclopropyl analog (16) possessing a skeleton isoatomic with that of cytisine.     

A unique binding epitope for salvinorin A, a non-nitrogenous kappa opioid receptor agonist

Salvinorin A is a potent kappa opioid receptor (KOP) agonist with unique structural and pharmacological properties. This non-nitrogenous ligand lacks nearly all the structural features commonly associated with opioid ligand binding and selectivity. This study explores the structural basis to salvinorin A binding and selectivity using a combination of chimeric and single-point mutant opioid receptors. The experiments were designed based on previous models of salvinorin A that locate the ligand within a pocket formed by transmembrane (TM) II, VI, and VII. More traditional sites of opioid recognition were also explored, including the highly conserved aspartate in TM III (D138) and the KOP selectivity site E297, to determine the role, if any, that these residues play in binding and selectivity. The results indicate that salvinorin A recognizes a cluster of residues in TM II and VII, including Q115, Y119, Y312, Y313, and Y320. Based on the position of these residues within the receptor, and prior study on salvinorin A, a model is proposed that aligns the ligand vertically, between TM II and VII. In this orientation, the ligand spans residues that are spaced one to two turns down the face of the helices within the receptor cavity. The ligand is also in close proximity to EL-2 which, based on chimeric data, is proposed to play an indirect role in salvinorin A binding and selectivity.     

3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors

Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutagenesis experiments.     

(+/-)-2-(3-Piperidyl)-1,2,3,4-tetrahydroisoquinolines as a new class of specific bradycardic agents

A series of (+/-)-2-(3-piperidyl)-1,2,3,4-tetrahydroisoquinolines were prepared and their bradycardic activities were examined in isolated guinea-pigs' right atria and in anesthetized rats. Modifications on the benzyl moiety of the parent compound, 1, led to the identification of compound 11e as a potent and specific bradycardic agent.     

Total synthesis of phenanthroindolizidine alkaloids (+/-)-antofine, (+/-)-deoxypergularinine, and their dehydro congeners and evaluation of their cytotoxic activity

Due to their limited natural abundance and significant biochemical effects, we synthesized the alkaloids (+/-)-antofine (1a), (+/-)-deoxypergularinine (1b), and their dehydro congeners (2 and 3) starting from the corresponding phenanthrene-9-carboxaldehydes. We also evaluated their in vitro cytotoxic activity. Compounds 1a and 1b showed significant potency against various human tumor cell lines, including a drug-resistant variant, with EC(50) values ranging from 0.16 to 16ng/mL. Structure-activity correlations of these alkaloids and some of their synthetic intermediates were also ascertained. The non-planar structure between the two major moieties, phenanthrene and indolizidine, plays a crucial role in the cytotoxic activity of phenanthroindolizidines. Increasing the planarity and rigidity of the indolizidine moiety significantly reduced potency. A methoxy group at the 2-position (1a) was more favorable for cytotoxic activity than a hydrogen atom (1b).     

Effects of (+/-)-sotalol, (+/-)-propranolol, and (-)-propranolol on norepinephrine release upon hepatic nerve stimulation in the dog liver in vivo

The study was carried out to determine whether the diminished release of norepinephrine (NE) upon sympathetic activation in the presence of sotalol can be attributed to the blockade of beta-adrenoceptors in the liver. NE release from the liver was measured in hepatic venous blood collected during direct hepatic nerve stimulation in anesthetized dogs. The mean basal NE concentration in hepatic venous and aortic blood was 0.046 +/- 0.003 and 0.244 +/- 0.041 ng/mL, respectively. NE release increased significantly as stimulation frequency increased, while aortic NE concentration remained unchanged. The increasing response of NE release upon stimulation in the vehicle control group remained stable during the whole experimental period. In dogs treated with sotalol (5 mg/kg, i.v.), NE release was reduced approximately by 30-43%, and the difference was statistically significant (P less than 0.01) at 8 Hz. (+/-)-Propranolol (2.5 mg/kg, i.v.) tended to diminish it, but the difference was not significant. (-)-Propranolol (0.1 mg/kg, i.v.) did not alter NE release at any frequency tested. The beta-blocking action of these drugs in the liver, as determined by the antagonism against the hepatic arterial vasodilating response to isoproterenol, was most effective with (+/-)-propranolol (100%), followed by (-)-propranolol (90%) and sotalol (70%). The results suggest that the inhibitory effect of sotalol on NE release may be related to a mechanism other than its beta-blocking action in the dog liver.     

(+)-[C-11]-cis-N-benzyl-normetazocine: A selective ligand for sigma receptors in vivo

The in vivo biodistribution profile of the novel sigma (σ) receptor ligand (+)-[C-11]-cis-N-benzyl-normetazocine ([C-11]-(+)-NBnNM) in mouse brain was examined. This radioligand displayed high brain uptake and a distribution consistent with the density of σ receptors. Brain radioactivity levels peaked at 15 min postinjection and were largely maintained (ca. 80% of maximal values) up to 90 min postinjection. Pretreatment with several different σ ligands (haloperidol, (+)-pentazocine, DuP 734, ifenprodil) effectively inhibited [C-11]-(+)-NBnNM binding in a dose-dependent manner in all brain regions. [C-11]-(+)-NBnNM binding sites were shown to be saturable with unlabeled (+)-NBnNM (ED50 = 0.02 mg/kg) and enantioselectively inhibited by the optical isomers of pentazocine. A blocking dose of the dopamine D2 antagonist spiperone (1 mg/kg) did not significantly inhibit [C-11]-(+)-NBnNM binding. Pretreatment with the phencyclidine (PCP) blocker 1-[1-(2-thienyl)cyclohexyl] piperidine (TCP) did not significantly alter total brain tissue radioactivity. Thus, [C-11]-(+)-NBnNM binds with high specificity and selectivity to σ receptors in vivo and offers excellent potential to study σ receptors in living human brain via positron emission tomography.     

A novel opioid peptide, leumorphin, acts as an agonist at the kappa opiate receptor

The primary structure of the common precursor of porcine beta-neo-endorphin and dynorphin (preproenkephalin B) has shown the existence of a third leucine-enkephalin (leu-enkephalin) sequence with a C-terminal extension of 24 amino acids. This nonacosapeptide, named leumorphin, was approximately 70 times more potent than leu-enkephalin in inhibiting the contraction of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. This action of leumorphin, like those of beta-neo-endorphin and dynorphin, was antagonized less effectively by naloxone than that of leu-enkephalin, but more effectively by Mr2266, an antagonist relatively specific for the kappa type opiate receptor. The inhibitory action of leumorphin or beta-neo-endorphin on the contraction of the guinea pig ileum muscle strip was reduced in a dose-dependent manner by pretreatment with dynorphin and vice versa. Leumorphin as well as beta-neo-endorphin and dynorphin inhibits the contraction of the rabbit vas deferens which is known to have only the kappa type opiate receptor. This action was also effectively antagonized by Mr2266. It is concluded that leumorphin has potent opioid activity and acts at the kappa receptor, like other opioid peptides derived from preproenkephalin B.     

Derivatization of (+/-)-5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline,an imidazoline binding sites ligand,with (+)-(R)-alpha-methylbenzyl isocyanate for drug monitoring purposes

The derivatization of racemic 5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, developed as an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate was performed in chloroform. The reaction led to two pairs of diastereomers, which were separated by RP-HPLC. A kinetic study of the derivatization reaction was achieved in order to establish conditions suitable for experimental drug monitoring.     

A concise synthetic approach to beta,gamma-dehydrocurvularin: synthesis of (+/-)-di-O-methyl-beta,gamma-dehydrocurvularin

A concise synthesis of di-O-methyl-beta,gamma-dehydrocurvularin, the di-O-methylated derivative of the naturally occurring nematicidal macrolide, beta,gamma-dehydrocurvuralin, was accomplished by starting from a commercially available aromatic carboxylic acid in a three-step sequence consisting of esterification, Friedel-Crafts acylation, and microwave-promoted ring-closing metathesis.