Peter J. Garratt

Evidence of the Indirect Formation of the Catecholic Intermediate Substrate Responsible for the Autoactivation Kinetics of Tyrosinase

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine,N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period.N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of theortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s−1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue,N,N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived fromN,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.

Mapping the melatonin receptor. 6. Melatonin agonists and antagonists derived from 6H-isoindolo[2,1-a]indoles, 5,6-dihydroindolo[2,1-a]isoquinolines, and 6,7-dihydro-5H-benzo[c]azepino[2,1-a]indoles.

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.

Melatonin receptor pharmacology: toward subtype specificity.

The recent cloning of three distinct melatonin receptor subtypes (Mel1a, Mel1b and Mel1c) which are part of a new family of G‐protein coupled receptors, and probably mediate the physiological actions of the hormone, has spurred interest in the design of analogues with subtype selectivity. The 5‐methoxyl and N‐acetyl groups of melatonin are important for binding to and activation of the receptor. The indole nucleus serves to hold these two groups at the correct distance from one another and allows them to adopt the required orientation for interaction with the receptor binding pocket. We have investigated the subtype selectivity of a number of analogues of melatonin in which the structure has systematically been modified in order to probe the similarities and differences in the interaction of ligand and receptor subtype. At all three subtypes 5‐methoxyl and N‐acetyl groups of melatonin are important for high affinity binding. However, replacing the 5‐methoxyl group (eg with 5‐H, 5‐OH, 5‐Me or 5‐BzO) reduces affinity much less at the Mel1b receptor subtype than at either Mel1a or Mel1c cloned subtypes. This suggests differences between the Mel1b and Mel 1a/1c subtypes in the size and shape of the binding pocket or in the manner in which melatonin interacts with the receptor at this position. Further studies have revealed that analogues with longer N‐acyl carbon chains behave similarly at each subtype. These observations suggest that the ‘pocket’ into which the N‐acetyl group fits is very similar for each subtype. Substitutions at the 2‐position on the indole ring improved affinity at each receptor subtype but did not give selective analogues. The systematic ‘mapping’ of the requirements for binding at each receptor subtype should allow the design of more selective agonists and antagonists, which will be valuable tools for the characterization and classification of functional melatonin receptors.

Two novel stilbene-2-carboxylic acid phytoalexins from Cajanus cajan

Abstract Three phytoalexins were isolated from leaves of pigeon pea which had been challenged with Botrytis cinerea . One was identified as pinostrobin chalc

The design of non-peptide human bradykinin B2 receptor antagonists employing the benzodiazepine peptidomimetic scaffold

The Bradykinin B2 receptor antagonist HOE 140 (D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg) has been used as a template for the de novo design and synthesis of a small number of non-peptide lead compounds based on the 1,4-benzodiazepin-2-one framework. Two of the compounds have been found to exhibit moderate K(i) values of 8.9 and 9.2 microM at the human Bradykinin B2 receptor.

Mapping the melatonin receptor. 2. synthesis and biological activity of indole derived melatonin analogues with restricted conformations of the C-3 amidoethane side chain

Abstract A number of indole derived melatonin analogues have been prepared with the C-3 amidoethane side chain partially constrained by incorporation in a ring. The biological activity has been correlated with the conformation of the “side chain”, the nature of the N-acylating group, and the spatial distance between the methoxyl and amide functions.

Mapping the melatonin receptor. 1. the 5-methoxyl group of melatonin is not an essential requirement for biological activity

Abstract A number of indole-derived melatonin analogues have been prepared lacking the C-5 methoxyl group. Many of these analogues are melatonin-receptor agonists and the previous assumption that the 5-OMe group was required for biological activity appears to have been an artefact mainly arising from the poor binding affinities of the small range of compounds tested.

Two isoprenylated isoflavone phytoalexins from Cajanus cajan

Abstract Four phytoalexins were isolated from sliced seeds of pigeonpea which had been incubated with its native microflora. Two were identified as the known pigeonpea phytoalexins, cajanin and cajanol, and the other two were characterized as new isoprenylated isoflavones.

A dienyl stilbene phytoalexin from arachis hypogaea

Abstract The novel stilbene, 3-isopentadienyl-4,3′,5′-trihydroxystilbene, was identified as the major antifungal component elicited by slicing imbibed kernels of 11 genotypes of groundnut. Incubation for 24 hr after slicing yielded values which varied between 38.5 and 105.8 μ of the compound/g of unimbibed kernels according to cultivar. After incubation for 48 hr, yields rose to between 89.5 and 189.7 μ/g. The compound was inhibitory to both spore germination and hyphal extension of the fungus, Aspergillus flavus, at 14.0 and 11.3 μ/ml, respectively.

Synthesis of Compounds as Melatonin Agonists and Antagonists

The functions of melatonin, the hormone of the pineal gland, are of considerable current interest. Synthetic melatonin analogues as agonists and antagonists have been explored in some detail and the molecule can be considered as consisting of an indole core, acting mainly as a spacer, and the 5-methoxyl and 3-amidoethyl side chains acting as the functional components, as originally proposed by Heward and Hadley. This review focuses on the synthetic routes to these melatonin analogues, first of the core, then of the substituents that have been attached to the core, and finally those compounds with restricted conformations and those that are chiral. The importance of the various factors involved in the activity of the compounds as agonist or antagonists is indicated, as is the difference in activity of enantiomers.