The behavioural, but not the hypothermic or corticosterone, response to 8-hydroxy-2-(DI-n-propylamino)-tetralin, is antagonized by NAN-190 in the rat.

The 5-HT1A receptor antagonistic properties of 1-(2-methoxyphenyl)-4-[4-(2-phthalimmido)butyl] piperazine (NAN-190) were studied in rats: its effect on the 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT)-induced behavioural syndrome (flat body posture and reciprocal forepaw treading), hypothermia and secretion of corticosterone, i.e. responses mediated by 5-HT1A receptors, were examined. The drug NAN-190 (1-8 mg/kg) antagonized dose-dependently behavioural effects of 8-OH-DPAT (in both non-reserpinized and reserpine-pretreated animals); however, when administered in doses of 0.5-4 mg/kg, it did not affect the hypothermic or the hormonal response to 8-OH-DPAT. However, NAN-190 (1-8 mg/kg) given alone, produced hypothermia and increased the concentration of corticosterone in serum. The latter effects of NAN-190 were not reduced by (-)pindolol or spiperone. Moreover, the NAN-190-induced secretion of corticosterone was not affected by ketanserin, prazosin or yohimbine. The above results indicate that NAN-190 acts as a 5-HT1A receptor antagonist, only in the model of the 8-OH-DPAT-induced behavioural syndrome. The lack of effect of NAN-190 on the hypothermic or corticosterone response to 8-OH-DPAT most probably results from its own action which mimics the effects of 8-OH-DPAT. The mechanisms responsible for the NAN-190-induced hypothermia and secretion of corticosterone are still unknown, though stimulation of 5-HT1A receptors (either effect), 5-HT2 receptors and alpha 1- and alpha 2-adrenoceptors (corticosterone response) seems to be excluded.

Ketanserin analogues: the effect of structural modification on 5-HT2 serotonin receptor binding.

Ketanserin (1) is a fairly selective 5-HT2 antagonist that binds both at 5-HT2A and 5-HT2C receptors. A previous structure-affinity relationship study revealed that the structure of the piperidine-containing ketanserin molecule could be rather severely abbreviated with little effect on 5-HT2A affinity. The present investigation explores several inconsistencies identified in the earlier study and suggests that multiple modes of binding may be possible for ketanserin analogues. Perhaps the nature of the benzylic substituent is the most significant determinant of the manner in which these agents bind at 5-HT2A receptors, and it is possible that certain orientations may avail themselves of an auxiliary binding site. Depending upon the length of the piperidine N-alkyl chain, variation of the benzylic substituent from a carbonyl, to an alcohol, to a methylene group has a nonparallel influence on binding, and this may be further affected by the presence of a second ring nitrogen atom. The results of the present investigation provide evidence that although the structure of ketanserin can be abbreviated, and even modified by conversion of the piperidine ring to a piperazine, the resultant analogues may bind in more than one orientation at the receptors. A key structural feature that may play a prominent role in anchoring or orienting these compounds at 5-HT2A receptors is the benzylic carbonyl group.

Antagonism of 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane stimulus with a newly identified 5-HT2- versus 5-HT1C-selective antagonist.

DOM [i.e., 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane] is a 5-HT1C/2 serotonin agonist that exerts stimulus control of behavior in animals. In order to determine if the discriminative stimulus effect of DOM is 5-HT1C- or 5-HT2-mediated, it would be informative to conduct tests of stimulus antagonism with a 5-HT1C- or 5-HT2-selective antagonist. To date, no such agents exist. Although the neuroleptic agent spiperone binds at D2 dopamine receptors and 5-HT1A serotonin receptors, (a) it displays about a 1000-fold selectivity for 5-HT2 versus 5-HT1C sites and (b) it has been used as a "5-HT2-selective" antagonist. Because spiperone is a behaviorally disruptive agent, it is not suitable for use in drug-discrimination studies. Using the spiperone molecule as a starting point, a limited structure-affinity investigation was conducted in order to identify a suitable antagonist with high affinity and selectivity for 5-HT2 receptors, and yet an antagonist that might lack the disruptive actions of spiperone. Various modifications of the spiperone molecule were examined, but most resulted in decreased 5-HT2 affinity or in loss of selectivity. One compound, 8-[3-(4-fluorophenoxy)propyl]-1-phenyl-1,3,8-triazaspiro[4.5]de can-4-on e (26), was shown to bind at 5-HT2 sites with high affinity (Ki = 2 nM) and > 2,000-fold selectivity versus 5-HT1C sites. In tests of stimulus antagonism using rats trained to discriminate 1 mg/kg of DOM from saline vehicle, 26 behaved as a potent antagonist (ED50 = 0.003 mg/kg) and lacked the disruptive effects associated with spiperone. As such, (a) it would appear that the DOM stimulus is primarily a 5-HT2-mediated, and not 5-HT1C-mediated, phenomenon, and (b) compound 26 may find application in other pharmacologic investigations where spiperone may not be a suitable antagonist.

5-HT1 and 5-HT2 binding profiles of the serotonergic agents alpha-methylserotonin and 2-methylserotonin.

alpha-Methyl-5-hydroxytryptamine (alpha-Me-5-HT; 2) and 2-methyl-5-hydroxytryptamine (2-Me-5-HT; 3) are considered to be 5-HT2-selective and 5-HT3-selective agents, respectively. These agents were synthesized and examined at serotonin (5-HT) binding sites because there is relatively little documentation as to their selectivity and because they have not been previously examined at the newly discovered 5-HT1D and 5-HT1E sites. As previously reported, 2-Me-5-HT possesses a low affinity (Ki greater than 500 nM) for 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT2 sites; this agent also displays a low affinity for 5-HT1D (Ki = 1220 nM) and 5-HT1E (Ki greater than 10,000 nM) sites. However, alpha-Me-5-HT displays little selectivity for 5-HT1A, 5-HT1B, 5-HT1C, and 5-HT1D sites (Ki = 42, 85, 150, and 150 nM, respectively) and a very low affinity for 5-HT1E (Ki greater than 10,000 nM) sites. Depending upon the radioligand used to label the sites, alpha-Me-5-HT displays either a low affinity (Ki = 880 nM with [3H]ketanserin) or a high affinity (Ki = 3 nM with [3H]DOB) for 5-HT2 sites. These results suggest that alpha-Me-5-HT is not as selective as previously considered and that caution should be used when employing this agent in pharmacological studies because it may act as mixed 5-HT1/5-HT2 agonist.

Novel 1-phenylpiperazine and 4-phenylpiperidine derivatives as high-affinity sigma ligands.

sigma receptors may represent an exciting new approach for the development of novel psychotherapeutic agents. Unfortunately, many of the commonly used sigma ligands lack selectivity (e.g., many bind at phencyclidine or dopamine receptors) or suffer from other serious drawbacks. Recently, we described a series of 2-phenylaminoethanes that bind at sigma receptors with high affinity and selectivity. Because there is evidence that 1-phenylpiperazines can structurally mimic the 2-phenylaminoethane moiety, we prepared a series of 1-phenylpiperazines and related analogues and incorporated structural features already shown to enhance the sigma binding of the 2-phenylaminoethanes. Several of these derivatives bind at sigma receptors with high affinity (Ki = 1-10 nM) and lack appreciable affinity for phencyclidine and dopamine receptors. In as much as certain of these agents structurally resemble the high-affinity, but nonselective, sigma ligand haloperidol, and because they bind with 10 times the affinity of haloperidol, we have apparently identified what appears to be the primary sigma pharmacophore of that agent.

N-(phthalimidoalkyl) derivatives of serotonergic agents: a common interaction at 5-HT1A serotonin binding sites?

Several classes of agents are known to bind at central 5-HT1A serotonin sites In order to challenge the hypothesis that these agents bind in a relatively similar manner (i.e., share common aryl and terminal amine sites), we prepared N-(phthalimidobutyl) derivatives of examples of several such agents. With regard to arylpiperazines, we had previously shown that introduction of this functionality at the terminal amine is tolerated by the receptor and normally results in a significant (greater than 10-fold) enhancement in affinity. The results of the present study show that this bulky functionality is also tolerated by the receptor when incorporated into examples of all other major classes of 5-HT1A agents (e.g., 2-aminotetralin, phenylalklamine, indolylalkylamine, and (aryloxy)alkylamine derivatives). The length of the alkyl chain that separates the terminal amine from the phthalimido group is of major importance, and a four-carbon chain appears optimal. Alteration of the length of this chain can have a significant influence on affinity; decreasing the chain length from four to three carbon atoms can reduce affinity by an order of magnitude, and further shortening can have an even more pronounced effect.

Identification and exploitation of the sigma-opiate pharmacophore.

Certain benzomorphan "sigma-opiates" such as N-allylnormetazocine (NANM) bind at sigma receptors with modest affinity and with little selectivity (i.e., they also bind at phencyclidine or PCP sites). In order to identify the primary pharmacophore of the benzomorphans, we prepared several amine-substituted derivatives of 1-phenyl-2-aminopropane. Several simple alkyl-substituted analogues were shown to bind at sigma sites with affinities comparable to that of NANM itself; among these was the N-benzyl derivative 9 (Ki = 117 nM). Lengthening the spacer between the terminal amine and the phenyl group from one to five methylene units resulted in a significant increase in affinity (e.g. 15, Ki = 6.3 nM). In addition, unlike the benzomorphans, these phenalkylamines do not bind at PCP sites. The results of the present study reveal that (a) the 1-phenyl-2-aminopropane nucleus of the benzomorphans is sufficient for binding at sigma sites provided that the terminal amine is not a primary amine and that (b) introduction of (phenylalkyl)amine substituents affords compounds that represent a new class of high-affinity sigma-selective agents.

Structure-activity relationships for the binding of arylpiperazines and arylbiguanides at 5-HT3 serotonin receptors.

Arylpiperazines are nonselective agents that bind at 5-HT3 serotonin receptors with moderate to high affinity, whereas 1-phenylbiguanide is a low-affinity but more selective 5-HT3 agonist. In an attempt to enhance the affinity of the latter agent, and working with the assumption that similarities might exist between the binding of the two types of agents, we formulated structure-activity relationships for the binding of the arylpiperazines and then incorporated those substituents, leading to high affinity for the arylpiperazines, into 1-phenylbiguanide. A subsequent investigation examined the structure-activity relationships of the arylbiguanides and identified arylguanidines as a novel class of 5-HT3 ligands. Although curious similarities exist between the structure-activity relationships of the arylpiperazines, arylbiguanides, and arylguanidines, it cannot be concluded that all three series of compounds are binding in the same manner. Furthermore, upon investigating pairs of compounds in the three series, the arylpiperazines behaved as 5-HT3 antagonists (von Bezold-Jarisch assay) whereas the arylbiguanides and arylguanidines acted as 5-HT3 agonists.

Structural features important for sigma 1 receptor binding.

Two problems that have hampered sigma receptor research are (i) a lack of high-affinity agents and (ii) the recent identification of multiple populations of sigma receptors (i.e., sigma 1 and sigma 2 sites). Recently, several high-affinity sigma ligands have been identified, and the term superpotent sigma ligands has been coined to describe agents with Ki values of < 1 nM. We have previously shown that appropriately N-substituted phenylalkylamines bind at sigma receptors with high affinity. In the present investigation, we examine the structure-affinity relationships of these phenylalkylamine derivatives for sigma 1 binding and describe some of the first superpotent sigma 1 ligands. A binding model was developed to account for the structural features of the phenylalkylamines that appear to be important for the interaction of these agents with sigma 1 sites.

Binding of substituted and conformationally restricted derivatives of N-(3-phenyl-n-propyl)-1-phenyl-2-aminopropane at sigma-receptors.

Certain benzomorphans, such as N-allylnormetazocine, are classical "sigma-opiates" that bind both at sigma and phencyclidine (PCP) binding sites with modest affinity. Recently, we identified N-substituted 2-phenylaminoethane as being the primary sigma-pharmacophore of the benzomorphans and demonstrated that 1-phenyl-2-aminopropane (2) derivatives, depending upon their terminal amine substituents, constitute a novel class of high-affinity sigma-selective agents. With this pharmacophore, it is shown in the present investigation that the aromatic hydroxyl group (a prime feature of all the sigma-opiates) contributes little to the binding of 2 at sigma-sites. It is also demonstrated that an N-substituted aminotetralin moiety (such as 17, a conformationally restricted analogue of 2) may also be considered a sigma-opiate pharmacophore. Unlike the sigma-opiates, derivatives of 2 and 17 display no affinity for PCP sites and must consequently lack those structural features important for the binding of benzomorphans at PCP sites. Because 3-phenylpiperidines and related sigma-ligands also possess a phenylalkylamine imbedded within their structures, we propose that the 2-phenylaminoethane moiety is a common sigma-pharmacophore for derivatives of 2, the 3-phenylpiperidines, and the sigma-opiates.

2-(1-Naphthyloxy)ethylamines with enhanced affinity for human 5-HT1D beta (h5-HT1B) serotonin receptors.

Although the beta-adrenergic antagonist propranolol (1) binds at rodent 5-HT1B serotonin receptors, it displays low affinity (Ki > 10,000 nM) for its species homologue 5-HT1D beta (i.e., h5-HT1B) receptors. The structure of propranolol was systematically modified in an attempt to enhance its affinity for the latter population of receptors. Removal of the alkyl hydroxyl group, shortening of the O-alkyl chain from three to two methylene groups, and variation of the terminal amine substituent resulted in compounds, such as N-monomethyl-2-(1-naphthyloxy)-ethylamine (11; Ki = 26 nM), that display significantly higher h5-HT1B affinity than propranolol. Compound 11 was shown to bind equally well at human 5-HT1D alpha (h5-HT1D) receptors (Ki = 34 nM) and was further demonstrated to possess h5-HT1B agonist character in an adenylate cyclase assay. It would appear that such (aryloxy)alkylamines may represent a novel class of 5-HT1D receptor agonists.

NAN-190: agonist and antagonist interactions with brain 5-HT1A receptors.

NAN-190 has been reported to be a 5-HT1A antagonist in drug discrimination studies. In order to determine if the effect of NAN-190 was directly due to competitive inhibition at 5-HT1A receptors, 5-HT1A-mediated inhibition of adenylyl cyclase in hippocampal membranes was investigated. NAN-190 (10(-10)-10(-5) M), by itself, was found to have no effect on forskolin-stimulated adenylyl cyclase. NAN-190, however, did shift the 5-carboxamidotryptamine (a 5-HT1A agonist) log-concentration inhibition curve to the right in a concentration-dependent manner, typical of competitive antagonism. Schild analysis revealed a KB of 1.9 nM for NAN-190. Thus, NAN-190 appeared to be a potent competitive 5-HT1A antagonist using the in vitro adenylyl cyclase system. [3H]NAN-190 was synthesized and its 5-HT1A receptor binding properties were characterized and compared with the 5-HT1A agonist radioligand, [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT). The 5-HT1A agonists, serotonin (5-HT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) competed with equal affinities regardless of the radioligand used to label the 5-HT1A receptors. [3H]NAN-190 and [3H]8-OH-DPAT labeled the same number of sites in rat hippocampus, striatum and frontal cortex. Guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) and 5-guanylyl-imidodiphosphate (GppNHp), non-hydrolyzable analogs of GTP, inhibited specific [3H]NAN-190 binding. Adenosine-5'-O-(3-thio)triphosphate (ATP gamma S) and 5-adenylyl-imidodiphosphate (AppNHp) were ineffective. This guanylyl nucleotide-specific effect is generally associated with agonist radioligand binding to a GTP-binding protein coupled receptor. However, [3H]8-OH-DPAT was far more sensitive than [3H]NAN-190 to the Bmax reducing effects of GTP and GTp gamma S. We propose that the test for a reduction in Bmax by non-hydrolyzable guanylyl nucleotides may be more sensitive than other tests for quantifying agonist activity and may demonstrate that NAN-190 has low intrinsic activity. In summary, NAN-190 displayed antagonist-like properties in functional models of 5-HT1A receptor activity and possibly partial agonist-like properties in radioligand binding experiments.

The detoxication of nitrate by two antioxidants or a probiotic, and the effects on blood and seminal plasma profiles and reproductive function of New Zealand White rabbit bucks.

Forty-two New Zealand White male rabbits were housed individually in wire cages and randomly distributed among six experimental groups of seven rabbits each, during 16 to 61 weeks of age. There were three main nitrate groups: 0 (tap water), 350 and 700 ppm. Within the 700 ppm of nitrate, there were four subgroups, in which one group was used as control group and the other three groups were supplemented with either 200 ppm of ascorbic acid (vitamin (Vit) C), 200 ppm of Vit E with 0.2 ppm of selenium (Se) and 1000 ppm of probiotic. The nitrate was supplemented as a sodium nitrate. The aim is to test the ability of Vit C and Vit E, Se and probiotic on the deleterious effects (blood and seminal plasma biochemical constituents, semen quality and productive performance) of nitrate in drinking water. Rabbits given nitrate at 700 ppm had significantly lower plasma globulin, red blood cells (RBCs), hemoglobin (Hgb), packed cell volume % (PCV%) and total antioxidant capacity (TAC) than those given the other concentrations of nitrate. Vit C, Vit E with Se and probiotic resulted in significantly (P < 0.05) greater Hgb, RBCs, PCV% and TAC than those of bucks given water supplemented with only 700 ppm nitrate, but the aspartate aminotransferase and alanine aminotransferase concentrations in seminal plasma were lower. Testosterone in the blood plasma and the seminal plasma was significantly (P < 0.05) lower in rabbits given 700 ppm nitrate than in those given other concentrations of nitrate. Vit C, Vit E with Se and the probiotic significantly increased testosterone, fertility, number of offspring and total offspring weight of rabbits sired by bucks supplemented with 700 ppm of nitrate.

The binding of propranolol at 5-hydroxytryptamine1D beta T355N mutant receptors may involve formation of two hydrogen bonds to asparagine.

Although the beta-adrenergic receptor antagonist (-)-propranolol binds with relatively low affinity at human 5-hydroxytryptamine1D beta receptors (Ki = 10,200 nM), it displays significantly higher affinity (Ki = 17 nM) at its species homolog, 5-HT1B receptors, and at a mutant 5-HT1D beta receptor (Ki = 16 nM), where the threonine residue at position 355 (T355) is replaced with an asparagine residue (i.e., a T355N mutant). Propranolol contains two oxygen atoms, an ether oxygen atom and a hydroxyl oxygen atom, and it has been speculated that the enhanced affinity of propranolol for the T355N mutant receptor is related to the ability of the asparagine residue to hydrogen bond with the ether oxygen atom. However, the specific involvement of the propranolol oxygen atoms in binding to the wild-type and T355N mutant 5-HT1D beta receptors has never been addressed experimentally. A modification of a previously described 5-HT1D beta receptor graphic model was mutated by replacement of T355 with asparagine. Propranolol was docked with the wild-type and T355N mutant 5-HT1D beta receptor models in an attempt to understand the difference in affinity of the ligand for the receptors. The binding models suggest that the asparagine residue of the mutant receptor can form hydrogen bonds with both oxygen atoms of propranolol, whereas the threonine moiety of the wild-type receptor can hydrogen-bond only to one oxygen atom. To test this hypothesis, we prepared and examined several analogues of propranolol that lacked either one or both oxygen atoms. The results of radioligand binding experiments are consistent with the hypothesis that both oxygen atoms of propranolol could participate in binding to the mutant receptor, whereas only the ether oxygen atom participates in binding to the wild-type receptor. As such, this is the first investigation of serotonin receptors that combines the use of molecular modeling, mutant receptors generated by site-directed mutagenesis, and synthesis to investigate structure/affinity relationships.