Preparation of some 10-[3-(dimethylamino)-1-propyl]-10H-pyrazino[2,3-b][1,4] benzothiazines as potential neuroleptics.

Chloro- and methyl-substituted 10H-pyrazino[2,3-b][1,4]benzothiazines were prepared and their structures determined by 13C NMR and X-ray crystallographic analysis. Alkylation afforded the 10-[3-(dimethylamino)-1-propyl] derivatives, which were compared to chlorpromazine in receptor-binding assays, in vivo behavioral tests, and electrochemical oxidation studies. In this series, the 2-chloro compound, 4c, proved to be the most effective derivative in displacing [3H]siperone, [3H]apomorphine, and [3H]prazosin radioligands from binding sites, being approximately as potent as chlorpromazine in this respect. However, none of the 10H-pyrazino[2,3-b][1,4]benzothiazines of this study were as active as chlorpromazine in in vivo tests predictive of neuroleptic activity.

Synthesis and receptor binding studies relevant to the neuroleptic activities of some 1-methyl-4-piperidylidene-9-substituted-pyrrolo[2,1-b][3]benzazepine derivatives.

The synthesis of a series of 1-methyl-4-(9-substituted-11H-pyrrolo[2,1-b]benzazepin-11-ylidene)piperidines (4a-f) and 1-methyl-4-(9-substituted-6,11-dihydro-5H-pyrrolo[2,1-b][3]benzazepin-11-ylidene)piperidines (4g-l) is described. As with th e 3-substituted cyproheptadine compounds 1b-e, atropisomerism exists in 4b-f, but unlike the enantiomers of 1b-e, the pyrrolobenzazepine enantiomers racemize at room temperature. Thus, the bromo compound (+)-4b has a half-life of 128 +/- 1 min at 25 degrees C, while the chloro compound (-)-4c has a half-life of 114 +/- 9 min at 25 degrees C. Compounds 4a-l have been examined for receptor binding affinities in assays that have been recognized as predictive for antipsychotic activity. The displacement of specifically bound tritiated ligands, comprising the dopamine antagonist [3H]spiperone, the dopamine agonist [3H]apomorphine, the muscarinic cholinergic antagonist [3H]quinuclidinyl benzilate (QNB), the alpha-adrenergic antagonist [3H]prazosin, the alpha-adrenergic agonist [3H]clonidine, the serotonin-1 binding agent [3H]serotonin, and the mixed serotonin agonist-antagonist [3H]lysergic acid diethylamide (LSD), by 4a-l has been measured utilizing membrane preparations of mammalian brain. Certain of the features of the receptor binding of these compounds have been shown to be common to several of the receptor sites. Data from these binding studies have been compared to corresponding data previously obtained for a series of chiral 3-substituted cyproheptadine analogues, and the receptor binding data of the two classes of compounds are discussed with respect to their molecular geometries.

A 5-hydroxytryptamine-like mode of anorectic action for 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212).

The mechanism of the reduction in food consumption elicited by 6-chloro-2-[1-piperazinyl]-pyrazine (MK-212) administered systemically was investigated in the rat. (+/-)-Fenfluramine and (+)-amphetamine were included in some studies for comparative purposes. 2 Pretreatment with methergoline, a 5-hydroxytryptamine (5-HT) antagonist, reduced the magnitude of the anorectic effect of 1.5 and 3 mg/kg of MK-212, while the anti-5-HT agents, cyproheptadine and cinanserin, were likewise effective against the 3 mg/kg dose. 3 Xylamidine, an antagonist of 5-HT that penetrates poorly into the central nervous system, completely blocked the decrease in food intake caused by 5-HT administered peripherally, while not antagonizing an equianorectic dose of MK-212. 4 Reduction of brain 5-HT by intraventricular injection of 5,6-dihydroxytryptamine, intraperitoneal administration of p-chloroamphetamine or placement of a lesion in the region of the median raphé nucleus diminished the anorectic response to 3 mg/kg of MK-212. The anorectic effect of amphetamine was reduced by p-chloroamphetamine or lesion in the raphé, but not by 5,6-dihydroxytryptamine. The decrease in food consumption produced by 1.5 mg/kg of MK-212 was antagonized by prior treatment with p-chloroamphetamine, but not by 5,6-dihydroxytryptamine. 5 Haloperidol, which blocks receptors for dopamine, antagonized the anorexigenic effect of amphetamine, but was ineffective in offsetting the action of MK-212, 3 mg/kg. 6 Pretreatment with chlorimipramine to inhibit the 5-hydroxytryptaminergic uptake mechanism did not affect the anorectic response to 3 mg/kg of MK-212, whereas the response to fenfluramine was diminished. 7 The results indicate that the anorectic action of MK-212 involves a 5-HT-like component which is more evident at the higher dose level of the compound. The anorexigenic property of MK-212 may depend, at least partly, upon the integrity of 5-HT-containing neurones in the central nervous system.

Comparison of the effects of recently developed alpha 2-adrenergic antagonists with yohimbine and rauwolscine on monoamine synthesis in rat brain.

The effects of two recently developed alpha 2-adrenergic antagonists, RX 781094 and WY 26703, on the synthesis of norepinephrine (NE), dopamine (DA) and serotonin (5-HT) in rat brain were compared to those of yohimbine, its diastereoisomer rauwolscine, and mianserin. Intraperitoneal administration of these compounds increased cortical NE synthesis with the potency order: yohimbine, RX 781094, WY 26703 greater than rauwolscine greater than mianserin. Within a similar dose range, yohimbine, rauwolscine and WY 26703 also stimulated striatal DA synthesis and decreased hypothalamic 5-HT synthesis, while RX 781094 and mianserin were very weak or inactive. Yohimbine and the structurally-related WY 26703 were also active as DA antagonists in the gamma-butyrolactone model for DA autoreceptor function. Based on the drug-induced changes in monoamine synthesis as indication of receptor-mediated events, RX 781094 has greater selectivity as an alpha 2-antagonist than compounds structurally related to yohimbine.

High affinity binding of [3H]neurotensin of rat uterus.

[3H]Neurotensin (NT) was found to bind specifically and with high affinity to crude membranes prepared from rat uterus. Scatchard analysis of saturation binding studies indicated that [3H]NT apparently binds to two sites (high affinity Kd 0.5 nM; low affinity Kd 9 nM) with the density of high affinity sites (41 fmoles/mg prot.) being about one-third that of the low affinity sites (100 fmoles/mg prot.). In competition studies, NT and various fragments inhibited [3H]NT binding with the following potencies (approximately IC50): NT 8-13 (0.4 nM), NT 1-13 (4 nM), NT 9-13 (130 nM), NT 1-11, NT 1-8 (greater than 100 microM). Quantitatively similar results were obtained using brain tissue. These findings raise the possibility of a role for NT in uterine function.

Heterogeneity of [3H]neurotensin bindings: studies with dynorphin, L-156,903 and levocabastine.

The binding of [3H]neurotensin (NT) to membranes from rat forebrain was complex, exhibiting 'high' affinity (Kd approximately 0.5 nM) and 'low' affinity (Kd approximately 5.0 nM) binding components. Dynorphin A(1-13) (DYN A(1-13] and L-156,903 (N-oxo-3-(10H-phenothiazine-10-yl)propyl-1- arginyl-1-prolyl-1-phenylalanine) potently inhibited [3H]NT binding to brain with shallow biphasic competition curves. Saturation binding studies conducted in the presence or absence of DYN A(1-13) or L-156,903 indicated that these compounds, like levocabastine, exhibited substantial selectivity for 'low' affinity NT site. Structure-activity studies indicated rigid structural requirements for the NT binding activity of DYN A(1-13) and L-156,903. In contrast to the results using brain tissue, DYN A(1-13), L-156,903 and levocabastine were very weak or inactive to inhibit [3H]NT binding to rat uterus. These studies further characterize the heterogeneity of [3H]NT binding in vitro and demonstrate clear tissue differences in binding within a given species.

Interaction of the putative dopamine autoreceptor agonists 3-PPP and TL-99 with [3H]apomorphine binding sites in rat striatal membranes.

Examination of the binding of [3H]apomorphine to rat striatal membranes in the presence and absence of the dopamine antagonist, domperidone, confirmed the previously reported presence of two classes of dopamine binding site, those designated D2 which show a high affinity for both agonist and antagonists and those designated D3 which have a high affinity for agonists and a low affinity for antagonists. In contrast to the previously reported single high affinity (KD congruent to 1 nM) D2- and D3-binding sites, two lower affinity sites (D2KD = 7-50 nM; D3KD = 41 nM) were also observed. Examination of the binding characteristics of the putative dopamine autoreceptor agonists, 3-PPP (N,N-propyl-3-(3-hydroxyphenyl)piperdine) and TL-99 (6,7-dihydroxy-2-dimethylaminotetraline) showed that they, like a number of other dopamine agonists including n-propylnorapomorphine, apomorphine and dopamine showed no preferential affinity for the D3, presynaptic binding site. It is concluded that the selectivity of dopamine agonists for the autoreceptor cannot be assessed by the in vitro radioligand binding parameters defined by the use of domperidone.

Identification of functional oxytocin receptors in lactating rat mammary gland in vitro.

The oxytocin (OT) receptor of the lactating rat mammary gland was further characterized by radioligand binding and functional assays in vitro and compared to the uterine OT receptor. In equilibrium saturation binding studies, [3H]OT bound apparently to a single site in mammary tissue with an affinity (Kd = 0.98 nM) similar to that found in the uterus (Kd = 0.68 nM). Using a variety of ligands for OT and arginine vasopressin (AVP) receptors, binding studies indicated that the recognition profile of the mammary [3H]OT binding site closely resembled that found for the uterus but was distinct from the known AVP receptor subtypes. In functional studies, OT and the highly selective OT agonist, [Thr4,Gly7]OT, were potent activators of phosphatidylinositol (PI) turnover in both mammary and uterine slices (EC50 3-5 nM). L-365,209, a novel potent and selective OT antagonist, inhibited OT-stimulated PI turnover in both tissues with similar potencies. These data provide evidence that the high-affinity [3H]OT binding site found in rat mammary tissue during lactation is a functional OT receptor coupled to PI turnover.

Tetrabenazine-induced depletion of brain monoamines: mechanism by which desmethylimipramine protects cortical norepinephrine.

Pretreatment of rats with desmethylimipramine (DMI) (5 mg/kg) significantly antagonized the tetrabenazine (TBZ)-induced (3 mg/kg) depletion of cortical norepinephrine (NE) in rats. This protective effect of DMI was fully blocked by the specific alpha 2-adrenergic antagonist, RX 781094. The doses of DMI which antagonized the depleting effects of TBZ caused rapid, maximal reductions in cortical dihydroxyphenylalanine (DOPA) accumulation, effects which were also blocked by RX 781094. Like DMI, the alpha 2-adrenergic agonist, clonidine (50 micrograms/kg) decreased cortical DOPA accumulation and rapidly prevented the TBZ-evoked loss of NE in this brain region. On the other hand, doses of RX 781094 (0.5-2.0 mg/kg) which accelerated cortical DOPA synthesis, enhanced significantly the depleting effects of a threshold dose of TBZ (0.6 mg/kg). These findings suggest that DMI reduces the rate of TBZ-induced depletion of cortical NE by lowering NE turnover through the indirect stimulation of alpha 2-adrenoceptors.

Tetrabenazine-induced depletion of brain monoamines: characterization and interaction with selected antidepressants.

The peripheral administration of tetrabenazine (TBZ) induces rapid depletion of brain regional concentrations of norepinephrine (NE), dopamine (DA) and serotonin (5-HT). With respect to both dosage and time, striatal DA was most sensitive to the effects of TBZ while hypothalamic NE was least affected. Pretreatment with the monoamine oxidase (MAO)-inhibitor, clorgyline (1-6 mg/kg) dose-dependently prevented the reduction of all three monoamines for up to 60 min after TBZ (3 mg/kg). The TBZ-induced depletion of cortical NE was also significantly antagonized by desmethylimipramine (DMI) but was of shorter duration (up to 30 min after TBZ). DMI, however, did not influence the effect of TBZ on striatal DA or hypothalamic 5-HT. The protective effects of both clorgyline and DMI were also evident under the conditions of the behavioral TBZ test utilizing high doses of TBZ (20 mg/kg).

Fenfluramine: long-term reduction in brain serotonin (5-hydroxytryptamine).

A single oral dose of 15 mg/kg of fenfluramine reduced the level of serotonin in rat brain to 48, 51 and 63% of control at 1, 15 and 30 days, respectively, after administration. 3 mg/kg p.o. of the drug caused a smaller but significant diminution in brain serotonin. At the 3 mg/kg dose level, the decreases in serotonin were, at least partially, cumulative following multiple injections spaced 24 hr apart. Brains removed 14 days after the 5th daily injection of 5 mg/kg p.o. of fenfluramine had only 60% of the concentration of serotonin found in brains from control animals. These findings demonstrate that fenfluramine has a long-lasting action on serotonin-containing neurons in brain.

Bradykinin agonist activity of a novel, potent oxytocin antagonist.

From a series of potent cyclic hexapeptide oxytocin (OT) antagonists, a compound that exhibited significant bradykinin (BK) agonist activity was identified. L-366,811 (cyclo[L-proline-D-tryptophan-L-isoleucine-D-pipecolic acid-L-piperazine-2-carboxylic acid-N-Me-D-phenylalanine]) stimulated phosphatidylinositol (PI) turnover in rat uterine slices in vitro (approximately EC50, 2 microM) with a maximal effect (15-fold increase over basal) greater than that obtained for either BK or OT. L-366,811 also elicited dose-related contractions of the isolated rat uterus, producing measurable effects at 100 nM. Several other equally potent OT antagonists from the cyclic hexapeptide structural class were either less potent or inactive as activators of uterine PI turnover or contractility. The stimulatory effects of L-366,811 on uterine PI turnover and contractions were blocked by BK antagonists but not by an arginine vasopressin (AVP)/OT antagonist. In radioligand binding studies, L-366,811 exhibited moderate affinity (IC50, 360 nM) for the [3H]BK binding site in rat uterus, consistent with its potency in the functional models. These results indicate that L-366,811 exhibits BK agonist activity in rat uterus in vitro.

[3H]L-657,743 (MK-912): a new, high affinity, selective radioligand for brain alpha 2-adrenoceptors.

L-657,743 (MK-912), a highly potent and selective alpha 2-adrenoceptor antagonist was tritiated to a high specific activity and its binding characteristics to brain tissue were determined. The specific binding of [3H]L-657,743 to rat cerebrocortex was saturable, reversible, and dependent on tissue concentration. In saturation studies, [3H]L-657,743 binding was resolved into two high affinity components exhibiting Kd values of 86 pM and 830 pM with densities of 82 fmol/mg protein and 660 fmol/mg protein, respectively. Based on the binding potencies of a variety of compounds with differing receptor selectivities, the sites labeled by [3H]L-657,743 were characteristic of alpha 2-adrenoceptors. In contrast to alpha 2-antagonists, alpha 2-agonists displayed shallow competition curves. In the presence of 100 microM GTP, Gpp(NH)p or 150 mM NaCl, the competition curve for epinephrine was shifted to the right, whereas that for yohimbine was unaffected. In studies utilizing human cerebrocortical tissue, [3H]L-657,743 also bound with high affinity to sites characteristic of alpha 2-adrenoceptors.