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.

Molecular modeling of the dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-OMe-BPAT.

Molecular modeling studies were undertaken in order to elucidate the possible dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1). For this purpose, a combination of indirect molecular modeling and direct construction of the seven transmembrane (7TM) domains of the receptors was employed in a stepwise, objective manner. Pharmacophore models and corresponding receptor maps were identified by superimposing selected sets of receptor agonists in their presumed pharmacologically active conformations, while taking the conformational freedom of the ligands into account. The 7TM models were then constructed around the agonist pharmacophore models, by adding the TM domains one-by-one. Initially, the relative positions of TM3, TM4, and TM5 were determined using the three-dimensional structure of bacteriorhodopsin, but subsequently the orientations of all TM domains were adjusted in order to mimic the topology of the TM domains of rhodopsin. The presumed dopamine D2 receptor binding conformations of (S)- and (R)-1 were determined by using the semirigid dopamine D2 receptor antagonist N-benzylpiquindone as a template for superposition. Similarly, the selective serotonin 5-HT1A receptor agonist flesinoxan was employed for identifying the serotonin 5-HT1A receptor binding conformations of the enantiomers of 1. After docking of the presumed pharmacologically active conformations in the 7TM models and subsequent optimization of the binding sites, specific interactions between the ligands and the surrounding amino acid residues, consistent with the structure-activity relationships, were observed. Thus, both enantiomers of 1 bound to the dopamine D2 receptor model in a similar fashion: a reinforced electrostatic interaction was present between the protonated nitrogen atoms and Asp114 in TM3; their carbonyl groups accepted a H-bond from Ser121 in TM3; their amide NH groups acted as H-bond donor to Tyr416 in TM7; and their benzamide phenyl rings were involved in a hydrophobic edge-to-face interaction with Trp386 in TM6. Differences were observed in the orientations of the 2-aminotetralin moieties, which occupied the agonist binding site. Whereas the (S)-enantiomer could form a H-bond between its 5-methoxy substituent and Ser193 in TM5, the (R)-enantiomer could not, which may account for the differences in their intrinsic efficacies at the dopamine D2 receptor. In the serotonin 5-HT1A receptor model, the benzamide phenyl rings of both enantiomers were involved in hydrophobic face-to-face interactions with Phe112 in TM3, while their protonated nitrogen atoms formed a reinforced electrostatic interaction with Asp116 in TM3. Consistent with the structure-affinity relationships of 1, the amide moieties were not involved in specific interactions. Both enantiomers of 1 could form a hydrogen bond between their 5-methoxy substituent and Thr200 in TM5, which may account for their full serotonin 5-HT1A receptor agonist properties.

Synthesis and Pharmacology of the enantiomers of the potential atypical antipsychotic agents 5-OMe-BPAT and 5-OMe-(2,6-di-OMe)-BPAT.

The optically pure enantiomers of the potential atypical antipsychotic agents 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 5) and 5-methoxy-2-{N-[2-(2,6-dimethoxy)benzamidoethyl]-N-n-propylamino}t etralin [5-OMe-(2,6-di-OMe)-BPAT, 6] were synthesized and evaluated for their in vitro binding affinities at alpha1-, alpha2-, and beta-adrenergic, muscarinic, dopamine D1, D2A, and D3, and serotonin 5-HT1A and 5-HT2 receptors. In addition, their intrinsic efficacies at serotonin 5-HT1A receptors were established in vitro. (S)- and (R)-5 had high affinities for dopamine D2A, D3, and serotonin 5-HT1A receptors, moderate affinities for alpha1-adrenergic and serotonin 5-HT2 receptors, and no affinity (Ki > 1000 nM) for the other receptor subtypes. (S)- and (R)-6 had lower affinities for the dopamine D2A and the serotonin 5-HT1A receptor, compared to (S)- and (R)-5, and hence showed some selectivity for the dopamine D3 receptor. The interactions with the receptors were stereospecific, since the serotonin 5-HT1A receptor preferred the (S)-enantiomers, while the dopamine D2A and D3 receptors preferred the (R)-enantiomers of 5 and 6. The intrinsic efficacies at the serotonin 5-HT1A receptor were established by measuring their ability to inhibit VIP-induced cAMP production in GH4ZD10 cells expressing serotonin 5-HT1A receptors. Both enantiomers of 5 behaved as full serotonin 5-HT1A receptor agonists in this assay, while both enantiomers of 6 behaved as weak partial agonists. The potential antipsychotic properties of (S)- and (R)-5 were evaluated by establishing their ability to inhibit d-amphetamine-induced locomotor activity in rats, while their propensity to induce extrapyramidal side-effects (EPS) in man was evaluated by determining their ability to induce catalepsy in rats. Whereas (R)-5 was capable of blocking d-amphetamine-induced locomotor activity, indicative of dopamine D2 receptor antagonism, (S)-5 even enhanced the effect of d-amphetamine, suggesting that this compound has dopamine D2 receptor-stimulating properties. Since both enantiomers also were devoid of cataleptogenic activity, they are interesting candidates for further exploring the dopamine D2/serotonin 5-HT1A hypothesis of atypical antipsychotic drug action.

Structural analogues of 5-OMe-BPAT: synthesis and interactions with dopamine D2, D3, and serotonin 5-HT1A receptors.

Several structural analogues of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1), a representative of a series of 2-aminotetralin-derived benzamides with potential atypical antipsychotic properties, were synthesized and evaluated for their ability to bind to dopamine D2A, D3, and serotonin 5-HT1A receptors in vitro. The structure affinity relationships revealed that the aromatic ring of the benzamide moiety of 1 contributes to the high affinities for all three receptor subtypes. Furthermore, 1 may interact with the dopamine D2 and D3 receptors through hydrogen bond formation with its carbonyl group. Investigation of the role of the amide hydrogen atom by amide N-alkylation was not conclusive, since conformational aspects may be responsible for the decreased dopaminergic affinities of the N'-alkylated analogues of 1. The effects of the amide modifications on the serotonin 5-HT1A receptor affinity were less pronounced, suggesting that the benzamidoethyl side-chain of 1 as a whole enhances the affinity for this receptor subtype probably through hydrophobic interactions with an accessory binding site. The structural requirements for the substituents at the basic nitrogen atom supported the hypothesis that the 2-aminotetralin moieties of the 2-aminotetralin-derived substituted benzamides may share the same binding sites as the 2-(N,N-di-n-propylamino)tetralins.

C5-substituted derivatives of 5-OMe-BPAT: synthesis and interactions with dopamine D2 and serotonin 5-HT1A receptors.

Eight new C5-substituted derivatives of the potential atypical antipsychotic agent 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1) have been prepared by chemical conversion of the 5-trifluoromethylsulfonyloxy (triflate) analogue 4 via various Stille-type cross-couplings, a Heck reaction, and an amidation in moderate to good yields. The 5-acetyl, 5-cyano, 5-methyl, 5-(2-furyl), 5-phenyl, methyl 5-carboxylate, and the 5-carboxamido analogues 5-11 thus obtained, the previously disclosed 5-methoxy, 5-hydroxy, and 5-unsubstituted analogues 1-3, and the 5-triflate analogue 4 were evaluated for their ability to compete for [3H]-spiperone binding to rat striatal membranes containing dopamine D2 receptors, and their ability to compete for [3H]-8-OH-DPAT binding to rat frontal cortex membranes containing serotonin 5-HT1A receptors in vitro. Compounds 1-11 displayed weak to high affinities for dopamine D2 receptors, with Ki-values ranging from 550 nM for the 5-carboxamido analogue to 4.9 nM for the 5-hydroxy analogue. The relative affinities of the 5-methoxy, 5-hydroxy, and 5-unsubstituted analogues suggested that these compounds may bind to the same site and in a similar way as the 5-oxygenated DPATs, with the 5-methoxy substituent of 1 functioning as a hydrogen bond acceptor. The serotonin 5-HT1A receptor tolerated more structural diversity at the C5-position of 1, as revealed by the higher Ki-values of 1-11, which ranged from 60 nM for the 5-carboxamido analogue to 1.0 nM for the 5-unsubstituted analogue. Partial least-squares (PLS) analysis of a set of 24 molecular descriptors, generated for each analogue, revealed no significant correlation between the dopamine D2 receptor affinities of 1-11 and their molecular properties, supporting the view that they may have different binding modes at this receptor subtype. A PLS model with moderate predictability (Q2 = 0.49) could be derived for the serotonin 5-HT1A receptor affinities of 1-11. According to the model, a relatively lipophilic, nonpolar C5-substituent should be optimal for a high affinity at this receptor subtype.

A multiway 3D QSAR analysis of a series of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-methoxybenzamides.

Recently, the multilinear PLS algorithm was presented by Bro and later implemented as a regression method in 3D QSAR by Nilsson et al. In the present article a well-known set of (S)-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-methoxybenzamides, with affinity towards the dopamine D2 receptor subtype, was utilised for the validation of the multilinear PLS method. After exhaustive conformational analyses on the ligands, the active analogue approach was employed to align them in their presumed pharmacologically active conformations, using (-)-piquindone as a template. Descriptors were then generated in the GRID program, and 40 calibration compounds and 18 test compounds were selected by means of a principal component analysis in the descriptor space. The final model was validated with different types of cross-validation experiments, e.g. leave-one-out, leave-three-out and leave-five-out. The cross-validated Q2 was 62% for all experiments, confirming the stability of the model. The prediction of the test set with a predicted Q2 of 62% also established the predictive ability. Finally, the conformations and the alignment of the ligands in combination with multilinear PLS, obviously, played an important role for the success of our model.

2-aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents.

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived substituted benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned human dopamine D2A and D3 receptors, and for [3H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (12a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM) as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.

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.

The high affinity melationin binding site probed with conformationally restricted ligand--I. Pharmacophore and minireceptor models.

The affinities of enantiomers of conformationally restricted melatonin analogues for the ML-1 and ML-2 putative melatonin receptor subtypes are reported. Most ligands exhibited reversed stereoselectivity when competing with 125I 2-iodomelatonin binding to chicken retinal (ML-1) and hamster brain (ML-2) membranes, further supporting the biochemical and pharmacological differences reported for these two sites. Based on the data for the ML-1 site and thorough conformational analyses of several ligands, two pharmacophore models were derived using the program APOLLO. The pharmacophoric elements included were putative receptor points from the amide NH, the amide CO, and the methoxy-O, together with the normal through the phenyl ring. The large drop in ML-1 affinity observed for 4-methoxy-2-acetamido-indan (6a) could not be explained from either of these models. Minireceptors were subsequently built around the two pharmacophores using Yak. Analysis of the resulting ligand-minireceptor interactions offered an explanation for the low affinity of 6a and allowed one of the pharmacophore models to be selected for use in future drug design.

The high affinity melatonin binding site probed with conformationally restricted ligands--II. Homology modeling of the receptor.

We present the first 3-D model of the melatonin receptor based on the recently published amino acid sequence of the cloned melatonin receptor. The seven trans membrane helices were positioned using the helices found in the structure of Bacterio Rhodopsine. From the results of an indirect modeling study with six melatonergic agents, an alignment of these compounds was found directing towards common interaction points. These points are suggested to be the two serines in helix three and the histidine in helix five, forming hydrogen bonds with the amide function and the methoxy-oxygen in melatonin, respectively. The ligands were docked into these binding sites and the receptor-ligand complexes were energy minimized. Considering the position of the active and inactive ligands in the receptor and their respective occupied volumes, the structure-activity relationships are rationalized by the suggested model. This model can be of use as a pharmacological test model in molecular biological studies and as a basis to develop compounds being active as synchronizing circadian agents.

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.

Norepinephrine release in the rat pineal gland: the input from the biological clock measured by in vivo microdialysis.

The sympathetic innervation of the rat pineal gland was investigated, measuring the norepinephrine (NE) release by on-line in vivo microdialysis. NE was assayed using an HPLC method with precolumn derivatization and fluorescence detection. Its high sensitivity and reliability made it very suitable to monitor the low levels of NE in the dialysates (12.5 fmol during nighttime, 3 fmol during daytime). To increase NE levels, the monoamine reuptake inhibitor cocaine was added to Ringer's solution at concentrations of 10(-6) and 10(-5) M. This resulted in increases of neurotransmitter output of 167 and 219%, respectively, but did not change the qualitative and/or quantitative outcome of other experiments. Perfusion with 10(-6) M tetrodotoxin for 1 h resulted in a decrease of the NE release by >80%, whereas perfusion with the alpha 2-receptor antagonist yohimbine caused a twofold increase. These results indicate that the NE release in the rat pineal was of neuronal origin and regulated by a negative feedback mechanism involving inhibitory presynaptic alpha 2-receptors. Long-term (i.e., 16 h) measurements are described, showing the circadian properties of NE release. A pronounced rhythm is reported, showing extremely sharp transitions between low daytime and high nighttime values. Increases and decreases are reported to occur within the duration of collecting one sample (20 min). For comparison, the rhythm of melatonin release was also recorded. The on and off switches of the sympathetic input correlated well with the circadian rhythm of melatonin release and can thus be considered as the primary clock signal, inducing the nightly production of melatonin.

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.

Exogenous melatonin entrains rhythm and reduces amplitude of endogenous melatonin: an in vivo microdialysis study.

The circadian rhythm of melatonin production was studied using on-line, in vivo microdialysis in the rat pineal gland. With this technique it was possible to record a pronounced melatonin rhythm with very high time resolution. Three phase-markers of the rhythm were calculated from the data, indicating increase (IT50), decrease (DT50) and amplitude of the rhythm. Comparing these phase markers led to several conclusions. Entrainment of the rhythm under constant darkness was performed with melatonin administration at different circadian stages [circadian time (CT) 8 and CT12] and for different periods of time (2 weeks and 4 weeks). Also, entrainment was established by applying 15 min light pulses at CT0. Entrainment of IT50 with melatonin partially uncoupled it from DT50. Four weeks entrainment in constant darkness (DD) caused a phase-delay in DT50 of 2.2 hr. Entrainment of IT50 with light at CT0 for 2 weeks in DD caused a phase-advance in DT50 of 1.3 hr. The entrainment with melatonin was restricted to a narrow window for melatonin to be applied, since injections at CT8 did not result in entrainment. Exogenous melatonin reduced the amplitude of the rhythm of endogenous melatonin. This effect was not circadian time dependent, since administration at CT8 for 2 weeks and at CT12 for 4 weeks resulted in a highly significant decrease. Light did not seem to have an effect on the amplitude. The data presented here provide us with new information about the nature of entrainment by melatonin. Since the present development of melatonergic agents for clinical use focuses on the entrainment capacity, effects of these compounds on amplitude of circadian rhythms needs to be addressed. In vivo microdialysis seems to be a good technique for that.

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.

Dopamine D2 receptor agonists: an analysis of indirect models.

To evaluate a previously suggested "dopamine (DA) D2 receptor agonist pharmacophore model" the N-propyl derivatives (1S,2S)-5-hydroxy-1-methyl-2-propylaminotetralin [(1S,2S)-3)] and trans-(+/-)-5-hydroxy-3-methyl-2-propylaminotetralin [(+/-)-4] were synthesized and their conformational preferences were studied by molecular mechanics calculations. The new compounds were evaluated for affinity to striatal DA D2 receptors labelled by [3H]spiroperidol and [3H]N-propylnorapomorphine. In comparison to the corresponding N,N-dipropyl derivatives, compounds (1S,2S)-3 and (+/-)-4 more readily adopt pharmacophore conformations and have higher affinity for DA D2 receptors. Thus, the pharmacophore model for C5-oxygenated 2-aminotetralins appears to be accurate. The DA D2 receptor binding affinities of a series of C1- or C3-methyl substituted 5- or 7-hydroxy-2-dipropylaminotetralin derivatives were also determined to enable a comparison of three different modes of superposition, of 7- and 5-hydroxylated 2-aminotetralin derivatives. The results show that the relative spatial positions of the C1- and C3-methyl groups resulting from the various superpositions of 5- and 7-hydroxylated regioisomers differ and the fact that the bulk of a methyl group may be readily accepted by the receptor does not necessarily indicate that additional bulk may be tolerated. Consequently, a definitive choice of mode of superposition of 5- and 7-hydroxylated regioisomers can not be made at present and has to wait until the binding site itself has been better defined.

Semipreparative enantiomeric separation of a series of putative melatonin receptor agents using tri-acetylcellulose as chiral stationary phase.

In order to obtain milligram amounts of the enantiomers of a series of compounds to be tested for binding to the melatonin binding site, a system for semipreparative enantiomeric separation was set up using tri-acetylcellulose as the chiral stationary phase. Interactions of this class of compounds with tri-acetylcellulose were examined on an analytical scale with a series of 20 compounds. Apparently, both steric and electrostatic interactions determine retention behavior on tri-acetylcellulose. Semipreparative separations were carried out for a subset of seven compounds. The purity of the first eluting enantiomer usually was around 99%, whereas the purity of the second eluting enantiomer was slightly less. The system described is easy to use and has the major advantage that a series of compounds can be separated with one technique. The purities obtained are sufficient for a first screen of their affinity.

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.

Microdialysis of melatonin in the rat pineal gland: methodology and pharmacological applications.

The present study describes the development of a new technique to measure melatonin contents in the pineal gland of freely moving rats, by means of on-line microdialysis. The transcerebral cannula was modified, and a sensitive assay of melatonin, using HPLC with fluorimetric detection, was set up. With this system it is possible to monitor the melatonin levels on-line in the pineal gland during day- and nighttime. The nightly increase in melatonin release was recorded. Tetrodotoxin had an inhibitory effect on nighttime levels, whereas even high concentrations did not alter the daytime level. From this we conclude that neuronal activity is necessary to synthesize melatonin and that during daytime no net neuronal activity is present. Melatonin levels could be greatly enhanced by systemic administration of the beta-agonist isoprenaline (ISO). Also, local infusion of ISO or 8-bromoadenosine 3',5'-cyclic monophosphate, an analogue of the second messenger cyclic AMP, resulted in increased melatonin levels, demonstrating the presence of beta-adrenergic receptors, coupled to a cyclic AMP-based second messenger system, on the pineal gland. Injection of phenylephrine had no effect on daytime levels. Only when administered during ISO-induced stimulation of melatonin release did it enhance this stimulated release. This proved the regulatory role of alpha 1-receptors on pinealocytes. The method presented is of special interest for investigating the innervation of the pineal gland and the biochemical processes that regulate the biosynthesis of melatonin. Also, for studies on the diurnal rhythms of melatonin release and factors that influence these rhythms in freely moving animals, this model will be of great value.

Conformational properties of melatonin and two conformationally restricted agonists: a molecular mechanics and NMR spectroscopic study.

The hormone melatonin (N-acetyl-5-methoxytryptamine) has been implicated in the regulation of several neural and endocrine processes that are cued by the daily change in photoperiod. The conformational properties of melatonin and two conformationally restricted agonists, 8-methoxy-2-acetamidotetralin and cis-1-methyl-8-methoxy-2-acetamidotetralin, were examined as a starting point for SAR studies. The conformational analysis was carried out by means of molecular mechanics calculations (MM2-85 force field) using a step-wise build-up procedure and the molecular modelling program MacroModel (which has a modified version of the MM2 force field) for Monte Carlo and systematic tree searches. NMR spectroscopy was used to evaluate the conformational behaviour in solution. The three search methods were complementary and produced a good description of the conformational characteristics. The two force fields produced different geometries of some low energy conformations and also different relative steric energies. When using the MM2-85 force field, an intra-molecular interaction between the amide-H and the aromatic system lowers the relative energies of several conformations of both melatonin and the 2-amidotetralins. However, NMR experiments indicated that these interactions, which may be quite important in vacuo, are not important in solution.