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A novel series of melatonin analogues based on the benzodioxin nucleus is described. The compounds were prepared in several steps from 2-carbethoxy-1,4-benzodioxin. Some of these derivatives competitively inhibited 2-[125I]-iodomelatonin binding to chicken brain and ovine pars tuberalis membranes, albeit with very reduced affinity compared with melatonin.

Conventional in vitro autoradiographical techniques have been used to screen a number of ovine peripheral tissues for the presence of specific 2-[ 125 I]iodomelatonin binding sites. Intense specific labelling (defined as that displaced by 1 μM melatonin) was observed in the adrenal cortex, and less intense specific binding in the spleen. Subsequent attempts to characterize these adrenal binding sites using in vitro binding assays were unsuccessful. The level of specific binding to both crude membranes and to dispersed whole cell preparations was negligible, and was not correlated with increasing protein or cell concentration. Using quantitative in vitro autoradiography, melatonin was found to competitively inhibit 2-[ 125 I]iodomelatonin binding to sections of the adrenal cortex, but with a considerably lower affinity (IC 50 of 1 μM) than that obtained for the ovine pars tuberalis (IC 50 of 150 pM) under identical conditions. These results suggest that under the conditions of in vitro autoradiography, 2-[ 125 I]iodomelatonin binds to a finite number of non- receptor sites which are restricted to the ovine adrenal gland. The ability to visualize these sites in the present study may arise through the use of an inappropriately high excess of unlabelled melatonin (1 μM). Consequently to avoid this potential problem, non-specific labelling should perhaps more appropriately be defined as binding in the presence of a concentration of melatonin at approximately 100-fold the value of the K d (i.e. 10 nM).

A high-affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [(125)|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino-acid lesions destroyed [(125)|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [(125)|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time-course of specific binding at 37 degrees C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [(125)|]iodomelatonin alone and in the presence of 1 muM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one-site binding model was consistent with a single receptor site with a K(d) of 29.3 (+/-5.9 SEM) pM and B(max) of 2.54 (+/-0.19 SEM) fmol/mg protein.

Tetrahydronaphthalenic ligands were synthesized and evaluated as melatonin receptor ligands. Biological studies show that the aromaticity of the ring bearing the side chain is not essential for affinity and activity and that replacement of the methoxy group with the bioisostere ethyl which does not offer the possibility of H-bonding, leads to antagonist or forskoline potentiating properties.

A series of N-naphthylethyl amide derivatives were synthesized and evaluated as melatonin receptor ligands. The affinity of each compound for the melatonin receptor was determined by binding studies using [2-12511iodomelatonin on ovine pars tuberalis membrane homogenates. Structure-activity relationships led to the conclusion that naphthalene is a bioisostere of the indole moiety of melatonin. Moreover it appears that the affinity is strongly affected by the size of the substituent of the nitrogen of the amidic function. Many of these ligands give biphasic dose-response curves which suggests that there may be two melatonin receptor subtypes within the ovine pars tuberalis cells. The replacement of naphthalene by benzofuran or benzothiophene did not strongly alter the affinity for the melatonin receptor. In contrast, the benzimidazole analogue was a poor ligand. Compound 7, the naphthalenic analogue of melatonin, a selective ligand of the melatonin receptor and an agonist derivative, has been selected for clinical development.

A pre-requisite to understanding the physiological mechanisms of action of melatonin is the identification of the target sites where the hormone acts. The radioligand 2-[125I]iodo-melatonin has been used extensively to localize binding sites in both the brain and peripheral tissues. In general these binding sites have been found to be high affinity, with Kd in the low picomolar range, and selective for structural analogues of melatonin. Also the affinity of these sites can generally be modulated by guanine nucleotides, consistent with the notion that they are putative G-protein coupled receptors. However, only a few studies have demonstrated that these putative receptors mediate biochemical and cellular responses. In the pars tuberalis (PT) and pars distalis (PD) of the pituitary, the amphibian melanophore and vertebrate retina, evidence indicates that melatonin acts to inhibit intracellular cyclic AMP through a G-protein coupled mechanism, demonstrating that this is a common signal transduction pathway for many melatonin receptors. However in the pars distalis the inhibition of calcium influx and membrane potential are also important mediators of melatonin effects. How many different forms or states of the melatonin receptor exist is unknown, but clearly the identification of the structure of the melatonin receptor(s) and its ability to interact with different G-proteins and signal transduction pathways are quintessential to our understanding of the physiological mechanisms of action of melatonin. In parallel the recent development of new melatonin analogues will greatly aid our understanding of the pharmacology of the melatonin receptor both in terms of the development of potent melatonin receptor antagonists and for the definition of receptor sub-types. The wide species and phylogenic diversity of melatonin binding sites in the brain has probably generated more questions than answers. Nevertheless the localization of melatonin receptors to the suprachiasmatic nucleus of the hypothalamus is at least consistent with circadian effects within the foetus and the adult. In contrast the PT of the pituitary presents an enigma in relation to the seasonal effects of melatonin. A model of how melatonin might mediate the timing of the circannual events through the PT is proposed.

Primary cultures of ovine pars tuberalis (PT) cells of the pituitary were established to investigate the mode of action of melatonin. The heterogeneous population of cells was shown to bind the radioligand 2-[125I]-melatonin over 72 h in culture, although there was a progressive decline in specific binding with time. In cells cultured for 24 h, forskolin (1 μmol/l) was found to stimulate a 12-fold increase in cyclic AMP accumulation. This response could be inhibited by melatonin in a dose-dependent manner, with an IC50 of approx. 6 pmol/l. However, melatonin did not inhibit basal levels of cyclic AMP. In homogenates of ovine PT, forskolin stimulated a dose-dependent increase in cyclic AMP, although the magnitude of this response was found to be lower than that observed in cells. This response was not inhibited by either 10 nmol or 1 μmol melatonin/l, and was also unaffected by GTP. These results provide the first evidence that the melatonin-binding site on ovine PT, recently characterized using the radioligand 2-[125I]-melatonin, functions as a physiological receptor.

The effect of guanine nucleotides and related analogues on the binding of 2-[125I]-melatonin to membranes prepared from ovine pars tuberalis was studied. Dose-dependent inhibition of 2-[125I]-melatonin binding was observed, with an order of potency of GTP gamma S much greater than Gpp(NH)p greater than GTP = GDP. GMP, cyclic GMP and ATP had negligible effects. Analysis of saturable binding revealed that GTP gamma S (1 microM) promoted an apparent reduction in receptor density of about 50%, without a concomitant change in receptor affinity. These results are consistent with a melatonin receptor existing in an equilibrium between high- and low-affinity states, with GTP and related analogues able to cause a shift in the equilibrium in favour of the lower-affinity form. The sensitivity of 2-[125I]-melatonin binding to guanine nucleotides implies the presence of a melatonin receptor on the ovine pars tuberalis, the action of which is mediated via a G protein.

A high-affinity, discretely localized melatonin receptor has been characterized and mapped within the brain and pituitary of the Syrian hamster using the high specific activity ligand [(125)|]iodomelatonin and a combination of in vitro autoradiography and membrane homogenate receptor assays. Specific binding of radioligand was found in regions of the epithalamus and hypothalamus in the brain and the pars tuberalis of the pituitary. Excitatory amino-acid lesions destroyed [(125)|]iodomelatonin binding within the brain, demonstrating that binding sites are located on neurons. Analysis of [(125)|]iodomelatonin binding to membrane homogenates of the pars tuberalis revealed a linear relationship between specific ligand binding and the amount of tissue. The time-course of specific binding at 37 degrees C reached equilibrium after 30 min and remained stable thereafter. The addition of increasing concentrations of [(125)|]iodomelatonin alone and in the presence of 1 muM melatonin showed that specific binding reached equilibrium at 80 to 100 pM. Analysis of the saturation isotherm using a one-site binding model was consistent with a single receptor site with a K(d) of 29.3 (+/-5.9 SEM) pM and B(max) of 2.54 (+/-0.19 SEM) fmol/mg protein.