The novel anticonvulsant loreclezole (R 72063) does not produce diazepam-like anterograde amnesia in a passive avoidance test in rats.

Rats were injected intraperitoneally with loreclezole (R 72063), diazepam, or scopolamine 60 min prior to acquisition of a passive avoidance task and tested 18 h later for retention of the learned (passive) behavior. The known impairment of performance produced by diazepam in this test is believed to be a model for the clinically observed diazepam-induced anterograde amnesia in humans. We report in this study that (1) consistent with the literature, pretreatment with diazepam (2.0-16.0 mg/kg i.p.) or scopolamine (3.0 mg/kg i.p.) produced impairment in passive avoidance performance of rats (anterograde amnesia), but (2) pretreatment with loreclezole (R 72063) (2.0-80.0 mg/kg i.p.) did not impair the acquisition, retention, or the retrieval (tested 18 h later) of passive avoidance behavior by rats at any dose. The results suggest that the anticonvulsant activity of loreclezole is mediated by a mechanism distinct from the one coupled to diazepam-like disruption of cognitive functions involved in the acquisition or posttraining information processing of passive avoidance behavior.

McN-5652: a highly potent inhibitor of serotonin uptake.

McN-5652 is one of a series of substituted pyrrolo-isoquinolines that, as a group, potently inhibit the uptake of one or more of the monoamines, norepinephrine, serotonin and dopamine. McN-5652 is characterized by exceptionally high potency as an inhibitor of the uptake of serotonin by rat brain synaptosomes in vitro (Ki approximately 0.6 nM) and ex vivo (ED50 approximately 2 mg/kg p.o.). The high potency of McN-5652 as a serotonin uptake inhibitor in vivo is indicated further by the low doses required to potentiate L-5-hydroxytryptophan-induced head twitches in mice (ED50 = 0.4 mg/kg 2 hr after p.o. dosing) and the serotonin syndrome in rats (ED50 = 1.5 mg/kg 2 hr after p.o. dosing). McN-5652 also potently inhibited the synaptosomal uptake of norepinephrine (Ki approximately 3 nM) and was a moderately potent inhibitor of the synaptosomal uptake of dopamine (Ki approximately 40 nM). McN-5652 inhibited tetrabenazine-induced ptosis in rats and mice but was much less effective in blocking the sedation caused by tetrabenazine. In rats, McN-5652 did not induce the stereotyped behavior often caused by dopamine agonists and inhibitors of dopamine uptake. Receptor binding experiments indicated that McN-5652 has a weak affinity for serotonin 5-HT2 and alpha-1 adrenergic receptors (apparent Ki approximately 200 nM) and a very low affinity for dopamine D1 and D2 receptors, serotonin 5-HT1, alpha-2 adrenergic, muscarinic and gamma-aminobutyric acid-A receptors. Experiments using the guinea pig ileum indicate that McN-5652 is a weak, noncompetitive antagonist of histamine.

Pyrroloisoquinoline antidepressants. 2. In-depth exploration of structure-activity relationships.

A series of pyrrolo[2,1-a]isoquinolines, and related compounds, were examined for antidepressant-like activity, by virtue of their antagonism of tetrabenazine-induced ptosis and sedation, and inhibition of biogenic amine uptake. Thus, we have identified some of the most potent antagonists of TBZ-induced ptosis and some of the most potent inhibitors of the uptake of dopamine, norepinephrine, and serotonin (in rat brain synaptosomes) ever reported. Compounds of particular note, in this regard, are 52b, 29b, 22b, and 48b, respectively. Biological activity was chiefly manifested by the trans isomeric class. Also, through resolution of four compounds, 7b, 24b, 37b, and 48b, biological activity was found to be associated with the (+) enantiomer subgroup (salts measured at 589 nm in MeOH), corresponding to the 6S, 10bR absolute configuration for 7b, 37b, and 48b, and the 6R,10bR configuration for 24b. An X-ray determination on (+)-24b X HBr established its absolute configuration; configurations for the other compounds were verified by enantiospecific synthesis starting with (+)-(R)-2-phenylpyrrolidine. Regarding the pendant phenyl ring, diverse substitution patterns were investigated. Those substitutions that were particularly unfavorable were 3',4',5'-trimethoxy (20b), 2',3',4',5',6'-pentafluoro (34b), 2'-trifluoromethyl (38b), 3',5'-bis(trifluoromethyl) (42b), 4'-n-butyl (44b), 2'-cyano (47b), 4'-methylsulfonyl (50b), and 2'-carboxy (58b). Exceedingly potent compounds, in one way or another, were 10b-12b, 22b, 23b, 25b, 28b, 29b, 33b, 45b, 48b, 51b-53b. The pattern of aromatic substitution had a strong impact on selectivity in the uptake tests (NE vs. DA vs. 5-HT). Activity was significantly diminished by methyl substitution of 7b at the 5 (65, 66), 6 (61b), or 10b (60b) position, by changing the phenyl group of 7b to cyclohexyl (67b), benzyl (68b), or H (72), by moving the phenyl group of 7b to the 5 (69) or 10b (70) position, by expansion of ring B to an azepine (78b), and by modification of ring C to an azetidine (77b), piperidine (75b), or azepine (74b). The interaction of selected analogues with various CNS receptors is reported. Little affinity was shown for the muscarinic cholinergic receptor, suggesting a lack of anticholinergic side effects. Interestingly, 24b and 33b displayed a high affinity for the serotonin-2 receptor, analogous to mianserin and clomipramine. After the body of data was reviewed, derivatives 24b and 48b were chosen for advanced development.

Preclinical evaluation of McN-5707 as a potential antidepressant.

Based on its activity in a variety of tests in vivo and in vitro McN-5707 [trans-6-(2-chlorophenyl)-1,2,3,5,6,10b-hexahydropyrrolo- (2,1-a)isoquinoline] is a novel potential antidepressant. McN-5707 blocked tetrabenazine-induced sedation and ptosis in mice and rats, and potently inhibited the uptake of norepinephrine by synaptosomes from rat hypothalamus (Ki approximately 2 nM), and the uptake of serotonin by synaptosomes from rat cerebral cortex (Ki approximately 10 nM). McN-5707 also inhibited the uptake of dopamine by synaptosomes from rat striatum (Ki approximately 40 nM); however, the stereotypic behavior often caused by dopamine uptake inhibitors was not evident in rats at doses of 300 mg/kg (p.o.) or less. In receptor binding assays, McN-5707 potently inhibited ketanserin binding to serotonin 5-HT2 receptors in synaptic membranes from rat cerebral cortex (apparent Ki approximately 8 nM). In mice, McN-5707 antagonized 5-hydroxytryptophan-induced head twitches. Spiperone binding to dopamine D2 receptors in synaptic membranes from rat striatum was weakly inhibited by McN-5707 (apparent Ki approximately 400 nM), as was the binding of WB4101 to alpha-1 adrenergic receptors (apparent Ki approximately 150 nM). McN-5707 was essentially inactive as an inhibitor of [3H]clonidine binding to alpha-2 adrenergic receptors and of [3H]quinuclidinyl benzilate binding to muscarinic receptors. In experiments with guinea pig ileum, McN-5707 weakly antagonized histamine-induced contractions and exhibited virtually no cholinergic or anticholinergic activity. Our observations indicate McN-5707 possesses attributes of both tricyclic and newer atypical antidepressants because it inhibits the uptake of both norepinephrine and serotonin, and blocks 5-HT2 receptors, but lacks some of the anticholinergic and behavioral properties often associated with them.

Novel inhibitors of gamma-aminobutyric acid (GABA) uptake: anticonvulsant actions in rats and mice.

SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid] and SK&F 100330A [N-(4,4-diphenyl-3-butenyl)-guvacine] represent a new series of potent, orally active inhibitors of gamma-aminobutyric acid (GABA) uptake. In vitro studies with synaptosome-rich (P2) fractions of rat brain indicated that these compounds were approximately 20 times more potent than the parent amino acids as inhibitors of [3H]GABA uptake. They did not inhibit [3H] muscimol binding at nanomolar concentrations. The present studies demonstrated that these compounds were also potent anticonvulsants when administered either orally or i.p. to rats. Both compounds attenuated the forelimb extensor component of bicuculline-induced convulsions, but had no effect on strychnine-induced convulsions, indicating that they were acting through a GABAergic mechanism in vivo. Two animal models which are known to be indicative of anticonvulsant efficacy in man are inhibition of maximal electroshock seizures (MES) and inhibition of pentylenetetrazol (PTZ) convulsions in either rats or mice. SK&F 89976A, SK&F 100330A and several related compounds were potent inhibitors of PTZ convulsions in rats. SK&F 100330A also inhibited MES convulsions in rats. In contrast, neither compound inhibited MES or electroshock seizure threshold in mice, and whereas both compounds inhibited the tonic phase of PTZ convulsions in approximately 50% of the mice tested, this inhibition was not dose-related. Thus, the rat appears to be a more suitable species for further testing of these compounds. These studies indicate that the family of compounds represented by SK&F 89976A and SK&F 100330A may have clinically relevant anticonvulsant activity.

Absolute stereochemistry and dopaminergic activity of enantiomers of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine.

Resolution of the unique dopamine receptor agonist 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine (1) was achieved by a stereospecific multistep conversion of the readily separated enantiomers of its O,O,N-trimethylated precursor 2. The absolute stereochemistry of the antipodes of 2-MeI was determined by single-crystal X-ray diffractometric analysis, thus permitting assignment of the configuration of stereospecifically related 1, as well as that of the synthetic intermediates. High-performance liquid chromatography of diastereoisomeric derivatives was utilized to determine the enantiomeric excess of the R (greater than 97%) and S (greater than 89%) isomers of 1. Examination of the isomers in several in vitro and in vivo tests for both central and peripheral dopaminergic activity revealed that activity resided almost exclusively in the R isomer. The results suggest that the properly oriented 1-phenyl substituent of 1 is important for dopamine-like activity; it may contribute to receptor binding by interaction with a chirally defined accessory site. Configurational and conformational requirements for receptor binding of 1 are considered in relationship to previously described dopaminergic agents. These studies, in accord with previous suggestions, indicate that (R)-1 interacts with dopamine receptors in a conformation in which the catecholic hydroxyls and basic nitrogen are at least nearly maximally separated.

Dopaminergic activity of substituted 6-chloro-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines.

6-Chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines were synthesized and evaluated as agonists of central and peripheral dopamine receptors. These benzazepines were prepared by cyclization of certain amino alcohols followed by demethylation of the 7,8-dimethoxy groups of the precursors to the 7,8-catecholic moiety. Preliminary evidence of dopaminergic activity was determined in anesthetized dogs by measuring the effects on renal blood flow and calculating the accompanying changes in renal vascular resistance. The most potent compounds contained an hydroxyl group on the 1-phenyl group or were substituted at the 3' position with a chloro, methyl, or trifluoromethyl group. Evidence for central dopaminergic activity was obtained by measuring rotational effects in rats lesioned in the substantia nigra and also in an in vitro assay which measured stimulation of rat striatal adenylate cyclase. The compounds with the best central dopaminergic activity were generally the benzazepines which were the most lipophilic, were substituted on the 3' position of th 1-phenyl group, and contained either a 3-N-methyl or 3-N-allyl group.

Differential effects of dopamine agonists in mature and immature rats.

It has been suggested that because of a relatively slow maturation of the central cholinergic system, that the neonatal rat (i.e. less than 2 weeks old) may be used as an in vivo model, with minimal cholinergic influence, for studying the effects of dopaminergic compounds. d-Amphetamine, 1-DOPA, and SK and F 38393-A produce a syndrome in neonatal rat similar to the stereotyped behavior characteristically produced by dopamine agonists. This neonatal syndrome includes increased motor activity and sniffing plus licking and/or biting. Apomorphine and bromocryptine do not produce this behavior in neonatal rats. The potencies of d-amphetamine, 1-DOPA, and SK and F 38393-A in producing this syndrome decrease with the age of the rat. Increases in motor activity, or sniffing plus licking and/or biting by d-amphetamine 1-DOPA, or SK and F 38393-A, in thirty and/or sixty day old rats are significantly enhanced by scopolamine pretreatment. Apomorphine- or bromocryptine-induced stereotyped behavior in thirty and sixty day old rats is not affected by scopolamine. These data suggest that the effects of certain dopaminergic agonists, which produce the neonatal syndrome, are modulated in the adult rat by cholinergic activity. The ineffectiveness of apomorphine in the neonate, however, suggest that the neonatal syndrome may not be related exclusively to dopamine, or that the dopaminergic system involved with this behavior is not sensitive to apomorphine.

Effects of GABAergic drugs on brain stimulation reward as assessed by a 'threshold' method.

Rats were trained in a two-lever intracranial self-stimulation (ICSS) paradigm. Responses at the first lever delivered brain stimulation which was decreased in magnitude after every 5 responses. Responses at the second re-set the available current to its original value. The current level at which the re-set responses occurred was defined as the 'reward threshold'. The rate of responding at each current level during the stimulate-re-set sequence was also determined. This paradigm, therefore, allowed simultaneous rate-independent and rate-dependent assessment of ICSS. Decreased reward, as demonstrated by an elevated 'reward threshold', was produced in a dose-related manner by the GABA antagonist picrotoxin. Similar effects could be produced by making each stimulation train less rewarding, i.e. by reducing the amount of charge delivered per stimulation. Conversely, increased reward, as indicated by a lower 'reward threshold', was produced in a dose-related manner by the GABA-mimetic muscimol, or by increasing the amount of charge delivered by each stimulation. Response rats were not significantly changed at any stimulation intensity following treatment with either drug. These data suggest that the effects of picrotoxin and muscimol on ICSS are due to changed perception of reward and not to altered performance of the lever pressing task. An important role for GABA in the mediation of reward needs, therefore, to be considered.

Simultaneous rate-independent and rate-dependent assessment of intracranial self-stimulation: evidence for the direct involvement of dopamine in brain reinforcement mechanisms.

A two-level intracranial self-stimulation (ICSS) paradigm was developed in which both rate-independent and dependent measures of ICSS could be obtained simultaneously. Responses at the first lever resulted in stimulation which decreased in magnitude after every fifth response, while responses at the second lever reset the current available. The current at which the reset responses occurred was defined as the 'reward threshold'. In addition, the rate of responding was determined at each current for which the animals responded during this stimulate-reset sequence. Decreased reward following treatment with the neuroleptic pimozide, a specific blocker of dopamine receptors, was demonstrated by an elevated 'reward threshold'. The same effect could be obtained in control animals by making each stimulation less rewarding, i.e., by decreasing the amount of charge per stimulation. Pimozide increased 'reward threshold' without a generalized disruption of response rates. While rates were decreased at low currents they were unchanged at high currents. 'Reward threshold' was decreased following D-amphetamine treatment, and was accompanied by a dose-related decrease in response rates at high to medium current intensities. These data suggest that neuroleptic attenuation of ICSS is due to diminished reward and not to motor deficits. Further, due to the specificity of pimozide, they suggest a direct role for dopamine in the mediation of reward.

Biochemical changes accompanying unilateral 6-hydroxydopamine lesions in the rat substantia nigra.

The biochemical consequences of a unilateral 6-hydroxydopamine injection into the substantia nigra of the rat brain were investigated. Projections of dopaminergic neurons from the A8-A9-A10 regions to a number of forebrain areas were confirmed. No innervation to the hypothalamus, including the median eminence, or to the brain stem, could be found with the present techniques. No destruction of serotonergic or GABAergic fibers could be demonstrated in the lesioned substantia nigra. Increases in glutamic acid decarboxylase activity were found restricted to the caudate and zona compacta of the substantia nigra ipsilateral to the lesion, indicating the possibility of a physiological interaction between GABAergic and dopaminergic systems. The neuroanatomical localization of the nigral dopamine-sensitive adenylate cyclase was also studied. No change in enzyme activity was found after destruction of a great proportion of the dopaminergic cells, suggesting that this enzyme has an extradopaminergic localization in the substantia nigra.

The central effects of a novel dopamine agonist.

A new peripheral dopamine agonist which causes dopaminergic renal vasodilation, was tested for central dopaminergic activity. SK & F 38393 stimulated the dopamine-sensitive adenylate cyclase in homogenates of rat caudate, as a partial agonist, and caused contralateral rotation in rats with unilateral 6-OHDA lesions of substantia nigra. Rotation was shown to be due to a direct effect on supersensitive dopamine receptors. Stimulation of cAMP formation and rotation were blocked by dopamine antagonists. In contrast to other dopamine agonists, SK & F 38393 did not cause stereotypy, emesis or inhibition of prolactin release, nor did SK & F 38393 affect dopamine turnover. The results suggest that SK & F 38393 may selectively stimulate supersensitive central dopamine receptors in vivo or may activate only a certain subclass of dopamine receptors including the receptor in the renal vasculature and the adenylate cyclase coupled postsynaptic receptor in the caudate.