Synthesis and neuroleptic activity of isomeric thieno(1,4)benzothiazines.

To investigate the influence of electronic properties of the tricyclic thiazine system on neuroleptic activity, a series of the isomeric N-dimethylaminopropylthienobenzothiazines was synthesized. All compounds were screened for neuroleptic activity in mice and rats. For the active compounds lowest active doses in the antiamphetamine test were determined. Activity appeared to be dependent on the mode of annelation of the thiophene molecule: compunds bearing the same substituent and side chain with the thiophene molecule in 2,3 and 3,4 annelation were active, while those compounds with a 3,2 annelation seemed to be devoid of activity at the given dose.

Resolution of 5,6-dihydroxy-2-(N,N-di-n-propylamino)tetralin in relation to the structural and stereochemical requirements for centrally acting dopamine agonists.

The enantiomers of 5,6-dimethoxy-2-(N,N-dipropylamino)tetralin were prepared with use of (+)- and (-)-dibenzoyltartaric acid as the resolving agent. Ether cleavage with BBr3 gave the enantiomers of the dihydroxy compound 5,6-dihydroxy-2-(N,N-dipropylamino)tetralin (5,6-(OH)2-DPATN). The in vitro activities of (+)- and (-)-5,6-(OH)2-DPATN were evaluated in binding studies with rat striatal tissue with use of [3H]-N-n-propylnorapomorphine (NPA) as the ligand. IC50 (nM) values for (-)- and (+)-5,6-(OH)2-DPATN were 2.5 and 400, respectively. The in vivo efficacy of the enantiomers was evaluated by examining their effects on the metabolism of dopamine in rat striatum. After a 0.5 mumol/kg ip injection of the (-) enantiomer, the concentrations of the metabolites HVA and DOPAC were reduced to 50% of control values, whereas at this dose the (+) isomer was inactive. On the basis of these findings together with the stereochemical data of previously described DA agonists, a dopamine-receptor model has been developed which consists of two binding sites for the amine nitrogen of DA agonists in addition to a major binding sit for the m-hydroxy group. The relevance of this model with its accessory features is discussed in relation to the structure and pharmacological data of different DA agonists.

Facile syntheses of potent dopaminergic argonists and their effect on neurotransmitter release.

The facile syntheses of important intermediates used in the preparation of the two potent dopaminergic argonists, 2-amino-6,7-dihydroxytetrahydronaphthalene (11) (referred to by some authors as ADTN) and its 5,6-dihydroxyl isomer 12, are described. Thus 6,7-dimethyoxy-2-tetralone has been prepared in two steps and 5,6-dimethoxy-2-tetralone in three steps both from commercially available materials. The effects of 11, 12, and the noncatechol analogue, 2-aminotetrahydronaphthalene (ATN), on radioactive neurotransmitter release have been studied in vitro using rat brain slices. It has been shown that both 11 and 12, at a concentraiton of 2 micron, cause a release of [3H]-DA and NA, 11 being more potent than 12 in releasing [3H]-DA. ATN (2 micron) was found to be inactive in these experiments which shows the importance of the catechol function in this uptake--release process.

Synthesis of isomeric thienobenzothiazines and their effect on dopamine metabolism in rat brain.

The synthesis of the thieno[2,3-b]-, thieno[3,4-b]- and thieno[3,2-b]benzothiazines with a hydroxyethylpiperazinylpropyl side chain and various 2 substituents is described. The influence of these neuroleptic compounds on dopamine metabolism in vivo is quantitated by determining the rise in homovanillic acid concentrations in rat corpus striatum. Notable differences in activity were found among the isomers, which were useful for structure--activity correlations in the phenothiazine neuroleptics.

2-Amido-8-methoxytetralins: a series of nonindolic melatonin-like agents.

A series of unsubstituted and methoxy-substituted 2-amidotetralins (4a-q) was prepared and evaluated for their ability to compete for 2-[125I]iodomelatonin binding to chicken retinal membranes and for their potency to inhibit the calcium-dependent release of [3H]dopamine from rabbit retina. The lead compound, 2-acetamido-8-methoxytetralin (4j), showed a moderate affinity (Ki = 46 nM) and potency (IC50 = 1.4 nM) at the melatonin receptor. The structural requirements necessary for optimal agonistic activity at the melatonin receptor are as follows. First, the amido group, which should have a small, nonbranched alkyl group, is essential for affinity, and second, the methoxy substituent at the 8-position of the 2-amidotetralin ring is essential for optimal agonistic activity at the melatonin receptor. We concluded that this series of unsubstituted and methoxy-substituted 2-amidotetralins constitutes a class of nonindolic melatonin-like agents that can be used as pharmacological tools to further characterize melatonin receptors and to elucidate the mode of action of melatonin.

Synthesis and pharmacological evaluation of conformationally restricted phenothiazine analogues.

The synthesis of 3-(dimethylamino)-2,3-dihydro-4-chloro-1H-pyrido[3,2,1-kl]phenothiazine, its 10-chloro analogue, and two chloro isomers of 2[(dimethylamino)methyl]-2,3-dihydro-1H-pyrido[3,2,1-kl]phenothiazine is described. In these compounds the side chain of chlorpromazine is fixed into a certain position in order to study the dopamine-overlap theory and the role of the substituents in the phenothiazine neuroleptics. The compounds were tested for their influence on dopamine metabolism, while for the second set their ability to displace [3H]spiroperidol from dopamine receptors was assessed. No neuroleptic activity was found from the pharmacological data. The results are discussed on the basis of conformational requirements for dopamine antagonistic activity.

Synthesis and pharmacology of the enantiomers of cis-7-hydroxy-3-methyl-2-(dipropylamino)tetralin.

The enantiomers of cis-7-hydroxy-3-methyl-2-(dipropylamino)tetralin (3) have been synthesized and evaluated for activity at central dopamine (DA), 5-hydroxytryptamine, and norepinephrine (NE) receptors, by use of biochemical and behavioral tests in rats. In addition, the affinities of the compounds for striatal [3H]spiroperidol and [3H]N-propylnorapomorphine binding sites were determined. The absolute configuration of the enantiomers was determined by X-ray diffraction of (+)-3. The pharmacological effects of both enantiomers are complicated, but (2R,3S)-7-hydroxy-3-methyl-2-(dipropylamino)tetralin [(-)-3] produced biochemical effects in vivo similar to those elicited by classical DA D2-receptor antagonists.

A microdialysis study on pineal melatonin rhythms in rats after an 8-h phase advance: new characteristics of the underlying pacemaker.

This study describes the use of the microdialysis technique to elucidate specific properties of the circadian pacemaking system in the hypothalamus, by measurement of melatonin production in the pineal gland. Melatonin has appeared to be a reliable marker of the pacemaker activity, which is influenced by the light/dark cycle. A phase shift in the light/dark cycle was applied to perturb the rhythm generating system. An 8-h phase advance resulted in the disappearance of melatonin production over two days, with basal levels comparable to normal daytime levels. In the subsequent return of rhythmic melatonin production, new clock characteristics could be revealed, due to the high time-resolution measurements of microdialysis. While half of the animals still did not show any rhythmicity, the other half of the animals regained rhythmicity with entrained onset of melatonin production, while the offset was variable and not stably entrained to lights on. Ten days after the shift, the system had completely recovered and all animals regained normal rhythmicity, in phase with the new light/dark cycle. The results are interpreted in terms of the two-oscillator model, with one oscillator reacting with a phase advance and the other with a phase delay to adapt to the phase shift.

A telemetry study on the chronic effects of microdialysis probe implantation on the activity pattern and temperature rhythm of the rat.

The present study describes the effects of implantation of microdialysis probes on temperature and activity rhythms of the rat, measured with a telemetry system. For comparison two widely used types of microdialysis probes were investigated, a transcerebral probe, inserted into the pineal gland and a set of two I-shaped concentric probes, implanted bilateral into the striatum. Starting from 5 days before the operation until 8 days after surgery, activity and temperature recordings were carried out continuously with the help of previously implanted transmitters. In separate experiments the effects of two different types of anaesthesia (chloralhydrate and Hypnorm) were determined. The results show that there is a pronounced effect of surgery on amplitude and rhythmicity of the temperature and activity patterns which is still detectable 6-7 days after operation. Few differences were noticed between the transverse probe and the I-shaped probes. Anaesthesia alone induced much smaller changes, most of which had disappeared within 2 or 3 days after the treatment. The duration of action of chloralhydrate is somewhat longer compared to Hypnorm. The conclusion is that when microdialysis is used in behavioural experiments, the effects of the surgical procedure should be taken into account. If these effects are serious, the use of previously implanted guide cannulae might be necessary.

Parasympathetic inhibition of pineal indole metabolism by prejunctional modulation of noradrenaline release.

The role of the parasympathetic nervous system in rat pineal indole metabolism was investigated by transpineal in vivo microdialysis. On-line coupling to a high performance liquid chromatography system with fluorescence detection (HPLC-FD) allowed simultaneous analysis of three major indolic compounds from the pineal, i.e. serotonin, N-acetylserotonin and melatonin. Infusion of the muscarinic receptor agonists, carbachol and oxotremorine, during the dark period resulted in a marked decrease of melatonin release. This effect was suggested to be mediated by a decrease in N-acetyltransferase activity, since a similar decrease was seen in N-acetylserotonin release, while serotonin levels increased simultaneously. Nicotine did show a very slight effect on the three indoles under these circumstances. Neostigmine failed to influence pineal indole metabolism, indicating that the endogenous tonus of acetylcholine release is either absent or extremely low in the middle of the dark period. The involvement of sympathetic innervation in the muscarinic effects was investigated by measurement of noradrenaline release from the pineal by sensitive off-line HPLC-FD analysis of noradrenaline in the dialysates. Carbachol markedly decreased the noradrenaline input during the infusion. Noradrenaline release returned to baseline values immediately after infusion with carbachol. These data suggest that the in vivo inhibitory effect of muscarinic receptor agonists on pineal melatonin production is mediated by presynaptic muscarinic receptors, located on the sympathetic nerve endings. This prejunctional inhibition of noradrenaline release causes a reduced induction of N-acetyltransferase activity, resulting in decreased melatonin release.

Kinetic and pharmacological profiles of the in vitro binding of the potent dopamine agonist [3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes.

The in vitro binding of the new tritiated dopaminergic ligand [3H]N,N-dipropyl-5,6-dihydroxy-2-aminotetralin to rat striatal membranes is described. Binding assays were performed in 50 mM Tris-HCl pH 7.5 containing 1 mM EDTA and 0.01% ascorbic acid at 25 degrees C for 30 min. Specific binding at 0.5 nM [3H]DP-5,6-ADTN and 5 mg/ml membrane suspension was very high (87-93%) and was found to be linearly related with homogenate concentration over the range of 0.5-10 mg/ml (r = 0.9968). (+)Butaclamol 1 microM was used to define specific binding. Specific binding disappeared completely within 20 min when the tissue was incubated at 55 degrees C. Association and dissociation curves (obtained after addition of 1 microM (-)-DP-5,6-ADTN) were converted to linear logarithmic plots and the kinetic constants were computed (k1 = 0.054 min-1 and k-1 = 0.0188 min-1) as were the t1/2 for association (3.52 min) and dissociation (20.8 min). This resulted in an apparent KD of 0.34 nM. Increasing concentrations of [3H]DP-5,6-ADTN (0.05-3.0 nM) were found to saturate the receptor site. Linear transformation of the saturation curves and presentation of the curves as Eadie-Hofstee plots showed that only one set of receptors was labeled (nHill was 0.995 +/- 0.07) with a high affinity constant KD of 0.57 +/- 0.10 nM and a maximum number of binding sites Bmax of 18.6 +/- 2.4 pmol/g tissue. Various compounds were tested for their potency to displace the specific binding of [3H]DP-5,6-ADTN to striatal membranes (2.5 mg/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Enantiomers of monohydroxy-2-aminotetralin derivatives and their activity at dopamine autoreceptors as studied by brain dialysis.

The enantiomers of a series of dopamine (DA) agonists, monohydroxy-2-aminotetralin derivatives, were investigated using brain microdialysis. We used a 5-OH-substituted derivative, N-0437 (2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin), and three 7-OH-substituted derivatives, N-0438 (2-(N-propyl-N-2-thienylethylamino)-7-hydroxytetralin), 7-OH-DPAT (7-hydroxy-2-(N,N-di-n-propylamino)tetralin) and PHNO (4-propyl-9-hydroxynaphthoxazine; position 9 of the naphtoxazines corresponds to position 7 of the aminotetralins). We studied the activity of the enantiomers at autoreceptors regulating the release of DA following their local infusion into the striatum of the rat. We were particularly interested in the activity of R(+)-N-0437, S(-)-N-0438, S(-)-7-OH-DPAT and S(-)-PHNO, which are enantiomers that have been classified as less potent or inactive in previous studies. S(-)-N-0437, R(+)-N-0438, R(+)-7-OH-DPAT and R(+)-PHNO decreased DA release by 45-60%. Thus, these enantiomers are potent agonists at autoreceptors regulating the release of DA. The R(+) enantiomer of the 5-OH-substituted derivative N-0437 possessed antagonistic activity at autoreceptors controlling DA release, increasing DA release by 100%. This finding is consistent with reports showing that one enantiomer of other 5-OH DA agonists displays agonistic activity, while the other has antagonistic properties at DA autoreceptors. The less potent enantiomers of the 7-OH-substituted derivatives S(-)-N-0438, S(-)-7-OH-DPAT and S(-)-PHNO, however, all showed weak agonistic activity at DA autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel non-indolic melatonin receptor agonists differentially entrain endogenous melatonin rhythm and increase its amplitude.

In this study we have examined the ability of melatonin and four synthetic melatonin receptor agonists to entrain endogenous melatonin secretion in rats, free running in constant darkness. The circadian melatonin profile was measured by trans-pineal microdialysis, which not only reveals the time of onset and end of production (phase), but also the amplitude of the rhythm. Exogenous melatonin given at the onset of subjective darkness (clock time 12 h) was effective to entrain endogenous melatonin production. Only one agonist, 2-chloroacetamido-8-methoxytetralin (AH-017), mimicked this action. Two other agonists, 4-methoxy-2-(methylene propylamide)indan (GG-012) and N-[2-[2,3,7,8-tetrahydro-1H-furo(2, 3-g)indol-1-yl]ethyl]acetamide (GR196429), induced a phase-delay under free running conditions, possibly by increasing tau (tau) period. One agonist, 2-acetamido-8-methoxytetralin (AH-001) did not show any phase effect on the free running rhythm. Unexpectedly, all melatonin receptor agonists increased the amplitude of melatonin secretion. The amount of the increase varied from just below the level of significance (AH-001) to an approximately 2-fold increase (GG-012 and GR196429). This is in clear contrast to entrainment with melatonin, which significantly decreased the amplitude. It is hypothesized that entrainment and effects on amplitude of melatonin secretion are mediated by different mechanisms which can be differentially modulated using specific ligands.

Microdialysis reveals dynamics of coupling between noradrenaline release and melatonin secretion in conscious rats.

The coupling between noradrenergic innervation of the pineal gland and melatonin production was investigated. Previously, the development of a microdialysis technique was described which made it possible to study the noradrenaline (NA) input as well as the melatonin output with high time resolution. In the present study, we studied the effects of short-term changes in NA-release on melatonin secretion. A 1 min light pulse was applied around midnight and resulted in an immediate decrease of both NA and melatonin. While NA returned to basal levels in 40 min, melatonin did not reach the baseline within 2.5 h. This discrepancy in correlation between NA and melatonin indicates a rapid inactivation of N-acetyl-transferase (NAT), followed by a slow reactivation, possibly by de novo synthesis of NAT. During daytime, a perfusion with 60 mM potassium for 30 min, resulted in a rapid and short stimulation of NA release, which was not followed by an increase in melatonin production. This indicates that 30 min stimulation of NAT activation is not enough to increase the amount of melatonin produced. The combination of measuring NA input and melatonin output appears to be a valuable tool in studying the characteristics of pineal activity in great detail.

S(-)DP-5,6-ADTN as an in vivo dopamine receptor ligand: relation between displacement by dopamine agonists and their pharmacological effects.

The use of (-)DP-5,6-ADTN as a non-radioactively labeled ligand for an in vivo DA receptor assay is described and compared with racemic DP-5,6-ADTN, previously used for that purpose. The effects of four DA agonists (NPA, bromocriptine, DP-7-OH-ATN and 3-PPP) on the specific (-)DP-5,6-ADTN binding are related to their potencies to decrease striatal HVA concentrations and to induce stereotypy in rats. NPA and DP-7-OH-ATN caused a maximal decrease in HVA levels, when only a fraction of the receptors were occupied, while the occurrence of stereotypy was associated with a high receptor occupation, reflecting the higher affinity of these agonists for presynaptic than for postsynaptic receptors. Bromocriptine did not show this effect, as the dose-response relationships for HVA decrease, for induction of stereotypy and for the decrease in specific (-)DP-5,6-ADTN binding were all virtually equal to each other. While NPA and bromocriptine behaved as full postsynaptic agonists, in that maximal stereotyped behavior was observed after high doses, DP-7-OH-ATN was found to be a partial postsynaptic agonist, as it did not induce maximal stereotypy at a maximal receptor occupation. Racemic 3-PPP only caused a state of hypoactivity, but did neither affect specific (-)DP-5,6-ADTN binding nor striatal HVA levels. Our results are discussed in view of theories on the relation between receptor occupation and pharmacological effects and it is concluded that the in vivo receptor binding method using (-)DP-5,6-ADTN is a very useful tool for such investigations.

Effect of non-catecholic 2-aminotetralin derivatives on dopamine metabolism in the rat striatum.

The concentrations of dopamine (DA), dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in the striatum of rats after i.p. injection of dipropyl-2-aminotetralin and the four positional isomers of monohydroxy-dipropyl-2-aminotetralin. All compounds except 8-OH dipropylaminotetralin caused a decrease in DOPAC- and HVA-concentrations. In addition, 5-OH-dipropylaminotetralin produced a small elevation in DA concentrations. In contrast, 7-OH dipropylaminotetralin, in doses of 100 mumol/kg and more, decreased DA to 50% and initially increased HVA and DOPAC to about 200%, after which the concentrations of the metabolites fell to 30% or less. The 5-OH isomer was found to be the most potent compound, decreasing HVA concentrations to 70% at a dose of 0.14 mumol/kg. The potencies are compared to those of catechol-group containing DA-agonists such as apomorphine and N,N-dipropyl-5,6-dihydroxy-2-aminotetralin. In addition, a comparison is made with reported behavioural data. It is suggested that the more active N-alkylated 2-aminotetralins have a conformation which corresponds to that of the alpha rotamer of dopamine.

Further in vitro and in vivo studies with the putative presynaptic dopamine agonist N,N-dipropyl-7-hydroxy-2-aminotetralin.

The in vitro binding of the putative dopamine autoreceptor agonist [3H]DP-7-ATN to rat striatal membrane homogenates was investigated. The maximum number of binding sites Bmax was 497.5 +/- 50.2 fmol/mg protein and the affinity constant KD was 8.3 +/- 1.5 nM using 10 microM (+) butaclamol to define non-specific binding. Lesion of the left medium forebrain bundle by 6-hydroxydopamine resulted in an almost complete loss of dopamine in the striatum but did not affect the binding of [3H]DP-7-ATN. The binding of [3H]DP-7-ATN to the homogenates of the dopaminergic cell bodies in the substantia nigra revealed a Bmax of 542.4 +/- 40.1 fmol/mg protein and a KD of 11.1 +/- 1.3 nM. The pharmacological profile of the binding was characterized as being to D-2 receptors. No direct in vitro evidence could be found for a selective binding to DA autoreceptors. The dopamine uptake inhibitor GBR 12909 interacted in a noncompetitive manner with the in vitro binding of [3H]DP-7-ATN and the latter compounds uptake into isolated synaptosomes was not through the specific dopamine uptake system but rather through diffusion. GBR 12909 failed to reveal any agonistic or antagonistic activity in the GBL model but was able to antagonize the hypomotility in rats induced by 0.25 mg/kg DP-7-ATN. The inhibitory effect of DP-7-ATN on DA release was also demonstrated using in vivo brain dialysis in conscious rats. Based on the above results, the possibility is discussed that the release regulating DA autoreceptors, which might be coupled to the reuptake complex, and the DA biosynthesis regulating autoreceptors, are different entities.

Conformational analysis of dopamine by the INDO molecular orbital method.

The results of INDO calculations on dopamine are reported. A conformational energy map and an isodistance map for the key distances N-OH1, N-OH2 in dopamine as functions of the two main torsion angles tau1 and tau2 were constructed. In addition to the three known minima of dopamine corresponding to the trans and gauche forms, two new minima were found. The key distances of the rigid analogues of dopamine, apomorphine, isoapomorphine, 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene and isoquinoline were plotted on the isodistance map of dopamine. By taking the corresponding tau values as coordinates on the energy map, conformations of dopamine, resembling the rigid analogues, could be found. When a conformation is close to a local minimum it is assumed that this conformation is energetically favourable. The possible relation between the energy minima and the biological action of dopamine is discussed. An explanation is suggested for the lack of dopaminergic activity of isoapomorphine.

5-Oxygenated N-alkyl- and N,N-Dialkyl-2-amino-1-methyltetralins. Effects of structure and stereochemistry on dopamine-D2-receptor affinity.

The ability of a series of stereochemically well-defined 5-oxygenated 2-aminotetralins, consisting of dopamine-receptor agonists and antagonists, to displace [3H]spiperone and [3H]N-propylnorapomorphine (NPA) from calf-caudate dopamine receptor sites has been evaluated in-vitro. In addition, the partition coefficients of the compounds were determined to measure their lipophilicity. The data were compared with previously obtained in-vivo biochemical data (dopa accumulation in reserpine pretreated or non-pretreated rats). Compounds with 2S-configuration and a C5-hydroxy substituent have the highest affinity for NPA-binding sites and such derivatives also have the highest potency in-vivo. The 2R-derivatives are less efficacious and their affinity for NPA- and spiperone binding sites is influenced by their lipophilicity. On the basis of these results, a model is proposed in which the antagonists form two, and the agonists form three, strong intermolecular bonds with the D2-receptor. According to this model, the agonists, but not the antagonists, are able to donate a hydrogen bond from the phenolic hydroxyl to the receptor.