Synthesis and 5-hydroxytryptamine (5-HT) activity of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines.

A series of 2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5-(6H)ones and 2,3,4,4a,5,6-hexahydro-1H-pyrazino[1,2-a]quinoxalines was shown to exhibit 5-HT2C agonist binding and functional activity. Compound 21R inhibited food intake over 2 h in fasted, male Sprague Dawley rats with ED50 values of 2 mg/kg (i.p.) and 10 mg/kg (p.o.).

The synthesis and vasopressin (AVP) antagonist activity of a novel series of N-aroyl-2,4,5,6-tetrahydropyrazolo[3,4-d]thieno[3,2-b]azepines.

Synthesis and SAR of N-[4-[(4,5-dihydropyrazolo[3,4-d]thieno[3,2-b]azepin-6(2H)-y l)carbonyl]phenyl]benzamides as arginine vasopressin (AVP) receptor antagonists are discussed. Potent orally active AVP receptor antagonists are produced when the benzamide moiety contains a phenyl group at the 2-position. Similar analogues of 4,6,7,8-tetrahydro-5H-thieno[3,2-b]azepine and VPA-985 are reported.

New generation dopaminergic agents. 7. Heterocyclic bioisosteres that exploit the 3-OH-phenoxyethylamine D2 template.

The synthesis of several bioisosteric analogs based on the 3-OH-phenoxyethylamine dopamine D2 agonist template (i.e., 3) is described. The benzimidazol-2-ones and benzthioimidazol-2-ones (7-10) and 2-trifluoromethyl-benzimidazole (13) were observed to have excellent affinity for the D2 receptor.

4,10-dihydro-5H-thieno[3,2-c][1]benzazepine derivatives and 9,10-dihydro-4H-thieno[2,3-c][1]benzazepine derivatives as orally active arginine vasopressin receptor antagonists.

Synthesis and structure-activity relationships (SAR) of arginine vasopressin receptor (AVP) antagonists are described. Potent and orally active compounds are prepared when tricyclic 10,11-dihydro-5H-pyrrolo[2,1-c][1,4]benzodiazepine moiety in VPA-985 1 is replaced with a compound 7 or 12.

5-fluoro-2-methyl-N-[5-(5H-pyrrolo[2,1-c][1,4]benzodiazepine-10(11H)-yl carbonyl)-2-pyridinyl]benzamide (CL-385004) and analogs as orally active arginine vasopressin receptor antagonists.

Synthesis and structure-activity relationships (SAR) of orally active arginine vasopressin (AVP) receptor antagonists are discussed. Potent and orally active AVP receptor antagonists are produced when ring A of VPA-985 (1) is replaced with a 3-pyridinyl unit (2b).

New generation dopaminergic agents. 2. Discovery of 3-OH-phenoxyethylamine and 3-OH-N1-phenylpiperazine dopaminergic templates.

Described in this report is a systematic study which led to the identification of two new dopamine D2 partial agonists (5 and 17). Phenols 5 and 17 represent prototypes of two new classes of D2 partial agonists as well as templates for the future design of novel dopaminergic agents.

New generation dopaminergic agents. 5. Heterocyclic bioisosteres that exploit the 3-OH-N1-phenylpiperazine dopaminergic template.

The synthesis of several bioisosteric analogs based on the 3-OH-N1-phenylpiperazine dopamine D2 agonist template (i.e., 4) is described. The indolone (5) and 2-CF3-benzimidazole (13) were observed to have excellent affinity for the D2 receptor. Several D4 selective compounds were also identified. Molecular modeling studies and a putative bioactive conformation are discussed.

Coronary artery stenosis in rats affects beta-adrenergic receptor signaling in myocytes.

OBJECTIVE: The purpose of this study was to determine whether the early chronic ischemic cardiomyopathy produced by non-occlusive coronary artery constriction was characterized by alterations in the regulation of beta-adrenoreceptor (beta-AR) signaling. METHODS: Coronary artery narrowing was surgically induced in rats and the animals sacrificed at 7 and 14 days. The changes in the biochemical properties of the multiple components of the beta-AR pathway were examined in enzymatically dissociated myocytes. RESULTS: Coronary stenosis, involving an average 55% reduction in luminal diameter, was associated with left ventricular failure and right ventricular dysfunction at both time intervals. A decrease in the quantity of beta-AR was detected at 7 days and preceded the loss of high-affinity binding sites. This regulatory modification was characterized by a reduction in beta 1 and beta 2 receptors and a shift in the isoproterenol dose response curve indicating a functional correlation between the decrease in beta-AR and attenuated inotropic support of the myocardium. The percentage of beta-AR binding agonist with high affinity decreased significantly at 14 days along with a further reduction in the density of beta 1 and beta 2 receptors. Reconstitution studies with cyc S49 lymphoma cells did not detect an impairment of Gs alpha functional activity, but the quantity of Gi alpha was increased at both intervals. Finally, activation of the catalytic unit of adenylyl cyclase by forskolin and GTP was not altered by coronary stenosis, however, basal cyclic AMP in myocytes was depressed at 14 days. CONCLUSIONS: Coronary stenosis induces distinct and progressive modifications in the beta-AR signaling cascade which may contribute to the impaired ventricular performance in this model of myocardial ischemia.

New generation dopaminergic agents. 1. Discovery of a novel scaffold which embraces the D2 agonist pharmacophore. Structure-activity relationships of a series of 2-(aminomethyl)chromans.

A series of 2-(aminomethyl)chromans (2-AMCs) was synthesized and evaluated for their affinity and selectivity for both the high- and low-affinity agonist states (D2High and D2Low, respectively) of the dopamine (DA) D2 receptor. The 7-hydroxy-2-(aminomethyl)chroman moiety was observed to be the primary D2 agonist pharmacophore. The 2-methylchroman moiety was discovered to be an entirely novel scaffold which could be used to access the D2 agonist pharmacophore. Attaching various simple alkyl and arylalkyl side chains to the 7-hydroxy 2-AMC nucleus had significant effects on selectivity for the D2High receptor vs the 5HT1A and alpha 1 receptors. A novel DA partial agonist, (R)-(-)-2-(benzylamino)methyl)chroman-7-ol [R-(-)-35c], was identified as having the highest affinity and best selectivity for the D2High receptor vs the alpha 1 and 5HT1A receptors. Several regions of the 2-AMC nucleus were modified and recognized as potential sites to modulate the level of intrinsic activity. The global minimum conformer of the 7-hydroxy-2-AMC moiety was identified as fulfilling the McDermed model D2 agonist pharmacophoric criteria and was proposed as the D2 receptor-bound conformation. Structure-activity relationships gained from these studies have aided in the synthesis of D2 partial agonists of varying intrinsic activity levels. These agents should be of therapeutic value in treating disorders resulting from hypo- and hyperdopaminergic activity, without the side effects associated with complete D2 agonism or antagonism.

Coronary artery constriction in rats affects the activation of alpha 1 adrenergic receptors in cardiac myocytes.

OBJECTIVE: To determine whether alpha 1 adrenergic receptor mediated myocyte contractility and growth are depressed acutely after non-occlusive coronary artery narrowing, the left coronary artery was constricted in rats and mechanical behaviour, cytosolic calcium, and regulation of alpha 1 adrenergic receptors were examined in myocytes seven days later. METHODS: Coronary artery stenosis was surgically induced in rats and following the estimation of global cardiac performance myocytes were enzymatically dissociated and radioligand binding studies were performed. In addition, the isotonic contractile performance, cytosolic calcium transients and noradrenaline stimulated inositol phosphate generation in myocytes were measured in the presence of WB 4101 or after chlorethylclonidine treatment. RESULTS: Estimations of cell mechanics in vitro established that peak shortening was decreased by 36% and 18% in left and right ventricular myocytes of coronary stenosed rats. Time to peak shortening was prolonged by 29% in left and 20% in right myocytes, whereas velocity of shortening was decreased by 27% in left myocytes. These alterations were associated with increases in cell length and width, indicative of myocyte hypertrophy. In addition, coronary stenosis was accompanied by reductions in the expression of alpha 1a and alpha 1b receptor subtypes in myocytes. alpha 1 Adrenergic receptor density and noradrenaline stimulated phosphoinositol turnover were decreased by 30% and 34% in left myocytes. alpha 1a Adrenergic receptor subtype mediated cytosolic calcium concentration and myocyte mechanical performance were also impaired in left myocytes only. The alpha 1a adrenergic receptor subtype antagonist WB 4101 abolished noradrenaline stimulated inositol phosphate generation in myocytes, whereas chlorethylclonidine at large doses only partially inhibited this response. CONCLUSIONS: In conclusion, coronary narrowing leads to defects in the regulation of alpha 1 adrenergic receptors on myocytes which are coupled with attenuation in the transmission of signals, possibly affecting myocyte cell function and ongoing reactive cellular hypertrophy.

Regulation of angiotensin II receptors on ventricular myocytes after myocardial infarction in rats.

To determine the effects of acute myocardial infarction on the regulation of angiotensin II (Ang II) receptors and contractile performance of left and right ventricular myocytes, coronary artery ligation was surgically induced in rats, and Ang II receptor density and affinity and the mechanical properties of surviving muscle cells were examined 1 week later. Physiological determinations of cardiac pump function revealed the presence of ventricular failure, which was associated at the cellular level with a depression in the velocity of myocyte shortening and relengthening, a prolongation of time to peak shortening, and a reduction in the extent of cell shortening. These abnormalities in single-cell function were more prominent in left than in right ventricular myocytes. Cellular hypertrophy was documented by increases in cell length and width, which were also greater in the spared myocytes of the infarcted left ventricle. Reactive hypertrophy was accompanied by a 1.84- and 1.85-fold increase in the density of Ang II receptors on left and right myocytes, respectively. On the other hand, the affinity of Ang II receptors for the radiolabeled antagonist was not altered. However, Ang II-stimulated phosphoinositol turnover was enhanced by 3.7- and 2.5-fold in left and right myocytes, respectively, after infarction. Ventricular myocytes were found to possess the AT1 receptor subtype exclusively. In conclusion, myocardial infarction leads to impairment in the contractile behavior of the remaining cells and to the activation of Ang II receptors and effector pathway associated with these receptors, which may be involved in the reactive growth adaptation of the viable myocytes.

Neuropeptide Y stimulates inositol phospholipid hydrolysis in rat brain miniprisms.

Neuropeptide Y (NPY) stimulates the hydrolysis of inositol phospholipid in rat brain miniprisms. The stimulation was two-fold in the frontal cortex and in the hippocampus, and 1.5-fold in the striatum. NPY produced no significant effects on basal inositol monophosphate levels in hypothalamic miniprisms. However, those basal levels were much higher than in the other brain regions.

Striatally-mediated response of some structurally rigid analogues of dopamine.

The potency of structurally rigid analogues of dopamine (DA) at striatal dopamine receptors was evaluated in rats using three types of assessments: (a) effectiveness in producing rotational and sniffing behaviors by intrastriatal injections (b) inhibition of [3H]-spiroperidol binding and (c) stimulation of adenylate cyclase activity. The compounds included apomorphine (APO) and its analogues, (R)-2,10,11-trihydroxyaporphine (R-THA) and (R)-2-hydroxy-10,11-methylenedioxyaporphine (MDO-APO), 2-amino-6,7-dihydroxyaminotetraline (ADTN) and its analogue, exo-2-amino-6,7-dihydroxybenzonorbornene (exo-amine). (R)-THA produced no stereotypy yet it was a potent inhibitor of [3H]-spiroperidol binding and adenylate cyclase activity. MDO-APO was quite active in inducing stereotypy and stimulating cyclase activity, but it showed low potency in displacing [3H]-spiroperidol. The exo-amine and ADTN were equally potent in enhancing rotation and sniffing intensity, however, the former was completely inactive in biochemical assessments. Except for (R)-THA, all DA analogues studied elicited dopaminomimetic behavioral activities of circling and sniffing. Relationships between the actions of these drugs in the behavioral and biochemical assessments are discussed.

Interaction of amoxapine with muscarinic cholinergic receptors: an in vitro assessment.

Amoxapine, an antidepressant with a rapid onset of therapeutic efficacy and great utility in psychotic depression, has been reported to produce anticholinergic side effects in man similar to those observed with imipramine and amitriptyline. To establish its cholinergic disposition, amoxapine and its metabolites 7-hydroxyamoxapine and 8-hydroxyamoxapine, have been evaluated by determining their effects on quinuclidinyl benzilate (QNB) binding to membrane fractions of rat and human brain, on the carbamoylcholine-stimulated accumulation of inositol phosphates in rat cerebral cortex and on the acetylcholine-induced contraction of the guinea pig ileum. In all three preparations, amoxapine was found to be a considerably weaker antagonist of muscarinic cholinergic receptors than either imipramine (4-27 fold) or amitriptyline (51-300 fold). These results indicate that for amoxapine, no correlation exists between the magnitude of muscarinic receptor inhibition and the extent of 'anticholinergic' side effects found in the clinic. Neither the metabolites of amoxapine nor species differences could account for this discrepancy.

Assay of brain calmodulin levels using high-performance liquid chromatography.

A simple, sensitive, and efficient HPLC method for the determination of calmodulin levels in brain tissue extracts is described. The assay is linear with respect to both calmodulin and protein concentrations. The specificity and validity of this assay for calmodulin is demonstrated by parallel radioimmunoassay determinations which give equivalent results. Determination of calmodulin levels in various brain regions revealed a high concentration of this protein in the hypothalamus, by comparison to other areas examined.

Hippocampal muscarinic receptor loss following trimethyl tin administration.

The effects of trimethyl tin on passive and active avoidance behavior, hippocampal muscarinic receptors and hippocampal cell destruction were examined in male rats. The animals were intubated with 18 mumoles/kg (3.5 mg/kg) of TMT hydrochloride or vehicle. When tested two weeks later treated animals exhibited marked deficits in retention of passive avoidance and extinction of active avoidance tasks. Receptor binding analysis, using 3H-QNB, revealed a significant decrease (21%) in muscarinic receptor density in the hippocampus. Histological examination of the hippocampus revealed a concomitant loss in pyramidal cells in these animals. These results suggest that muscarinic receptors reside on the hippocampal pyramidal cells and that these cells and receptors may be involved in retention of passive avoidance behavior.

The separation of 3H-benzodiazepine binding sites in brain and of benzodiazepine pharmacological properties.

In addition to anxiolytic and anticonvulsant properties, benzodiazepines (BDZ) produce sedation, ataxia, and muscular relaxation. In general, it was difficult to separate these properties within this chemical class during the search for clinically useful anxiolytics; and when BDZ's were used to characterize 3H-BDZ binding sites they indicated only a single homogenous class of receptors. A new chemical series was discovered, triazolopyridazines (TPZ, prototype CL 218,872), which showed anticonflict activity in rats and monkeys without sedation or ataxia and inhibited 3H-BDZ binding in brain membranes with kinetic characteristics suggesting the presence of multiple BDZ receptors. High affinity and low affinity sites for the TPZ were demonstrated, the former designated at Type 1 and the latter as Type 2. Anatomical and in vivo studies have supported different distributions of each receptor in brain. Lately, the physical separation of discrete proteins which bind 3H-BDZ has been reported. The multiple receptors and the variety of endogenous substances which have been proposed as modulators and ligands of the receptors might explain variability as well as selectivity in pharmacological properties in these drugs.

The effect of microwave irradiation on vasopressin in plasma and hypothalamo-neurohypophyseal system.

Radioimmunoreactive vasopressin was measured in plasma, neurohypophysis and hypothalamus of the rats after different procedures of killing: a) microwave irradiation; b) decapitation; c) decapitation following a stress induced by immobilization in a restrainer. Vasopressin content in the neurohypophysis and hypothalamus was much lower in microwave irradiated than in both decapitated and stressed decapitated rats. In addition, the data from microwave technique were inconsistent with a large scatter. Plasma vasopressin concentration was elevated in both the microwave irradiated and stressed decapitated rats, demonstrating that restraining of the animals induced an excessive stress. Microwave irradiation technique including the necessary manipulation of the animal proved to be less suitable than decapitation technique for the measurement of vasopressin. It is likely that vasopressin in the hypothalamus and neurohypophysis is relatively resistant against post-mortem proteolysis.