6H-Isoindolo[2,1-a]indoles (5, 7, 10, 13), 5,6-dihydroindolo[2, 1-a]isoquinolines (20, 21), and 6,7-dihydro-5H-benzo[c]azepino[2, 1-a]indoles (23, 25, 27, 30) have been prepared as melatonin analogues to investigate the nature of the binding site of the melatonin receptor. The affinity of analogues was determined in a radioligand binding assay using cloned human mt(1) and MT(2) receptor subtypes expressed in NIH 3T3 cells. Agonist and antagonist potency was measured using the pigment aggregation response of a clonal line of Xenopus laevis melanophores. The 2-methoxyisoindolo[2, 1-a]indoles (7a-d) showed much higher binding affinities than the parent isoindoles (5a-e), and whereas 7a-c were agonists in the functional assay, 7d and 5a-e were antagonists. The 2-ethoxyisoindolo[2,1-a]indoles (10a-d) showed reduced binding affinities compared to their methoxy analogues, while the 5-chloro derivative 13 showed a considerable reduction in binding affinity and potency compared to 7a. The 10-methoxy-5,6-dihydroindolo[2, 1-a]isoquinolines (21a-c) had higher binding affinities than the corresponding parent indoloisoquinolines (20a-c) in the human receptor subtypes, and the parent compounds were antagonists whereas the 10-methoxy derivatives were agonists in the functional assay. The N-cyclobutanecarbonyl derivatives of both the parent (20d) and 10-methoxyl (21d) series had similar binding affinities and were both antagonists with similar potencies. The 11-methoxy-6, 7-5H-benzo[c]azepino[2,1-a]indoles (25a-d) had higher binding affinities than the corresponding parent compounds (23a-d) at the MT(2) receptor but similar affinities at the mt(1) site; all of the compounds were antagonists in the functional assay. Changing 11-methoxy for 11-ethoxy decreased the binding affinity slightly, and this was more evident at the MT(2) receptor. All of the derivatives investigated had either the same or a greater affinity for the human MT(2) receptor compared to the mt(1) receptor (range 1:1-1:132). This suggests that the mt(1) and MT(2) receptor pockets differ in their ability to accommodate alkyl groups in the indole nitrogen region of the melatonin molecule. Two compounds (7c and 25c) were tested in functional assays on recombinant mt(1) and MT(2) melatonin receptors. Compound 7c is a potent agonist with some selectivity (44-fold) for the MT(2) receptor, while 25c is an MT(2)-preferring antagonist. Increasing the carbon chain length between N-1 of indole and the 2-phenyl group from n = 1 through n = 3 leads to a fairly regular decrease in the binding affinity, but, remarkably, when n = 3, it converts the methoxy compounds from melatonin agonists to antagonists. The Xenopus melatonin receptor thus cannot accommodate an N-n-alkyl chain attached to a 2-phenyl substituent with n > 2 in the required orientation to induce or stabilize the active receptor conformation.

In earlier work we have provided evidence for the presence of a subsite within the CB1 and CB2 cannabinoid receptor binding domains of classical cannabinoids. This putative subsite corresponds to substituents on the C1'-position of the C3-alkyl side chain, a key pharmacophoric feature in this class of compounds. We have now refined this work through the synthesis of additional C1'-cycloalkyl compounds using newly developed approaches. Our findings indicate that the C1'-cyclopropyl and C1'-cyclopentyl groups are optimal pharmacophores for both receptors while the C1'-cyclobutyl group interacts optimally with CB1 but not with CB2. The C1'-cyclohexyl analogs have reduced affinities for both CB1 and CB2. However, these affinities are significantly improved with the introduction of a C2'-C3' cis double bond that modifies the available conformational space within the side chain and allows for a better accommodation of a six-membered ring within the side chain subsite. Our SAR results are highlighted by molecular modeling of key analogs.

A series of 2-, 3-, and 4-substituted phenylalkyl amides were prepared as potential melatonin analogs in order to investigate the nature of the binding site of the melatonin receptor in chicken brain. The length of the alkyl chain was systematically varied from n = 1 to 4, and methoxyl substituents were incorporated into the phenyl ring at the 2-, 3-, and 4-positions. The maximum binding affinity was found to occur when n = 3 and when the methoxyl substituent was in the 3-position, the direct analog of the carbon framework of melatonin in which the 1,2-atoms of the indole ring have been removed. Whereas there was only a relatively small decrease in binding affinity for the corresponding 2-methoxy derivatives, 4-methoxyl substitution led to a large decrease in binding affinity, suggesting that the binding sites for the side chain and methoxyl group could not now be occupied at the same time. As in the indole analogs of melatonin, replacement of the methyl group of the amide by a longer alkyl chain led to an increase in binding affinity for ethyl and propyl with a subsequent decrease in binding affinity for butyl chains. Thus N-propanoyl-3-(3-methoxyphenyl)propanamine (6f) has a binding affinity of 5.6 nM, a remarkably high affinity for so simple a compound. Substitution of halogen for 3-methoxyl in the propanamide series gave a series of compounds with lower, but still substantial, binding affinities, the 3-chloro derivative 7e showing the highest affinity, 113 nM. In the case of the 3-fluoro propanamides, a maximum in the binding affinity was not observed in the series synthesized, and these derivatives will merit further exploration. These results demonstrate the utility of simple, readily modified phenylalkylamines as a "framework" for studying the effect of changes in the nature and position of substituents on the melatonin receptor binding affinity.