2-(4,5-dihydro-1H-imidazol-2-yl)indazole (indazim) derivatives as selective I(2) imidazoline receptor ligands.

A series of variously substituted 2-(4,5-dihydro-1H-imidazol-2-yl)indazoles 3a-j and 2-(4,5-dihydro-1H-imidazol-2-yl)-4,5,6,7-tetrahydroindazole 6 were prepared by the regiospecific heteroalkylation of corresponding indazoles 1a-k with 2-chloro-4,5-dihydroimidazole (2). Their affinity to imidazoline I(2) receptors and alpha(2)-adrenergic receptors was determined by radioligand binding assay carried out on P(2) membrane preparations obtained from rat whole brains. 4-Chloro-2-(4,5-dihydro-1H-imidazol-2-yl)indazole (3f, 4-Cl-indazim) showed a 3076-fold difference in affinity for the [(3)H]2BFI-labeled imidazoline I(2) receptors relative to the [(3)H]RX821001-labeled alpha(2)-adrenergic receptors. This highly selective compound should prove to be useful tool in further understanding the functions of the imidazoline I(2) receptors.

Novel ligands for the investigation of imidazoline receptors and their binding proteins.

New ligands for imidazoline receptors are described so that these receptors can be more fully explored and understood. BU224, (2-(4,5-dihydroimidaz-2-yl)-quinoline, shows high affinity and is selective for the imidazoline-2 (I(2)) class of receptors. BU224 was tested in the rat Porsolt forced swim paradigm where it was found to decrease time spent immobile and increase the time spent swimming, consistent with an antidepressant profile. BU224 was tritiated and, in radioligand binding studies, was found to label a single population of saturable sites with high affinity. In vitro brain autoradiography with [(3)H]BU224 also showed a pattern of distribution similar to the known labeling of I(2) receptors. A new series of four 2BFI (2-(benzofuranyl)-2-imidazoline) derivatives were investigated as potential ligands for imaging brain I(2) receptors using positron emission tomography (PET). At least two, BU20012 and BU20013, retained high affinity and moderate selectivity and penetrated the brain when administered peripherally in the mouse. 2BFI has undergone the Mannich reaction to immobilized diaminodipropyl amine to fabricate an affinity column, which was used to isolate a protein from rabbit brain; this protein was sequenced and identified as the enzyme creatine kinase.

Relationship between imidazoline(2) sites and monoamine oxidase.

I(2) site-selective compounds are known to interact with and inhibit monoamine oxidase (MAO), but it remains unclear as to whether this interaction occurs through an allosteric or competitive interaction. This study used the new selective, irreversible I(2) ligand BU99006, to clarify the relationship between MAO and the I(2) binding sites (I(2)-BS). Results demonstrate that irreversible binding of BU99006 to rat brain membranes does not inhibit the enzyme or interfere with its interaction with other imidazoline enzyme inhibitors. This finding suggests that the I(2) sites that react with BU99006 are not those implicated in MAO inhibition and points to the existence of at least two distinct I(2) binding proteins.

Initial evaluation of novel selective ligands for imidazoline(2) receptors in rat whole brain.

Indazim, the indazole analogue of 2BFI, and four methyl-substituted analogues were tested for their affinity at the imidazoline(2) binding site (I(2)-BS), and this affinity was compared with their affinity at the alpha(2)-adrenoceptor to determine their structure, affinity relationship, and selectivity at the I(2)-BS. These studies showed that these ligands were highly selective for the I(2)-BS compared with 2BFI and that substitution at the 4 and 7 positions increased affinity without affecting selectivity.

Investigation of the affinities of two new beta-carbolines for rat brain imidazoline(2) receptors.

The beta-carbolines are possible endogenous ligands and modulators of the imidazoline binding sites (I-BSs). Relatively little is known about this class of compound and its interaction with the I-BS. Presented here are the binding data for two aryl ring substituted dihydro-beta-carbolines at the imidazoline(2) binding site (I(2)-BS) and alpha(2)-adrenoceptor: BG-326 (5-bromo-4,9-dihydro-3H-b-carboline) and BG-350 (5-methoxy-4,9-dihydro-3H-b-carboline). Both compounds show good affinity and selectivity for the I(2)-BS.

Identification of an I(2) binding protein from rabbit brain.

Imidazoline-2 binding proteins exist as a heterogeneous population. The aim of this study was to isolate and identify I(2) binding proteins from rabbit brain using an affinity column synthesized with a highly selective I(2) ligand, 2-(2-benzofuranyl)2-imidazoline (2BFI). The results revealed an approximately 45-kD protein to be brain creatine kinase (EC 2.7.3.2). [(3)H]-2BFI (5nM) was able to bind specifically to the purified enzyme. This study has identified brain creatine kinase as a novel I(2) binding protein.

5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) an irreversible imidazoline(2) binding site ligand: in vitro and in vivo characterisation in rat brain.

5-Isothiocyanato-2-benzofuranyl-2-imidazoline (BU99006) is an irreversible ligand based on the highly selective I(2) binding site ligand 2BFI. In competition binding assays it has been shown to have high affinity and selectivity for the I(2) binding site and to irreversibly inhibit the binding of [(3)H]2BFI. In this present study we have sought to confirm and expand on these findings both in vitro and in vivo. In vitro pre-incubation of rat whole brain membranes with BU99006 (10 microM) was shown to reduce the specific binding of [(3)H]2BFI to 10% of the control values, an effect not seen using 2BFI or BU224. Pre-treatment of rat whole brain membranes by BU99006, or by the alpha(2)-adrenoceptor antagonists RX821002 or rauwolscine had no effect on the specific binding of [(3)H]RX821002. In vivo pre-treatment of rats with BU99006 (15 mg x kg(-1), i.v.) caused a substantial loss of [(3)H]2BFI specific binding in subsequent in vitro saturation analysis and autoradiography; this loss was shown to be dose dependent. These data indicate that BU99006 is selectively and irreversibly affecting I(2) binding sites both in vitro and in vivo and that it represents an invaluable tool in the further understanding of the I(2) binding site.

Selective delta-opioid receptor ligands: potential PET ligands based on naltrindole.

Two series of delta-selective ligands related to the prototypic delta-antagonist naltrindole have been prepared and evaluated in opioid binding assays with the aim of developing new PET ligands for the delta-opioid receptor. One compound (5d) had significantly higher selectivity than naltrindole, but with substantially reduced binding affinity. For those compounds retaining similar affinity to naltrindole, those having ethyl and fluoroethyl substituents afforded the highest levels of selectivity. However, none of the compounds combined the high level of affinity and selectivity ideally suited to the development of an imaging agent.

In vitro and in vivo approaches to the characterization of the alpha2-adrenoceptor.

1. In order to more fully understand the role of the alpha2-adrenoceptor in brain function, a combination of in vitro and in vivo techniques were utilized including radioligand binding, autoradiography, brain microdialysis and antisense oligonucleotides. 2. Binding studies showed the tritiated form of the selective alpha2-adrenoceptor antagonist, RX821002 (methoxy-idazoxan) labelled an apparent single population of sites in rat brain membranes with high affinity (1 nM), for which prazosin had low affinity (1107 nM). Similar studies in rabbit brain membranes found that prazosin and oxymetazoline were able to displace [3H]-RX821002 in a biphasic manner indicating the presence of subtypes of alpha2-adrenoceptors. 3. Receptor autoradiography revealed a distribution of [3H]-RX821002 binding in rat brain consistent with the labelling of all alpha2-adrenoceptor subtypes, namely alpha(2A/D-), alpha2B and alpha2C. 4. In rat, in vivo brain dialysis experiments demonstrated peripherally administered RX821002 elevated basal noradrenaline in frontal cortex and also, although to a lesser extent, in ventral hippocampus. RX821002 was also able to elevate extracellular dopamine in the striatum. 5. A 7-day i.c.v. infusion of an antisense oligonucleotide targeting the alpha(2A/D)-adrenoceptor, resulted in a significant reduction in the autoradiographic density of [3H]-RX821002 binding in specific brain areas, notably the lateral septal nuclei and anterior hypothalamic area. 6. Several years of research by our group has extended our knowledge of the pharmacology and function of the alpha2-adrenoceptor and has provided evidence of the roles of this receptor in the control of monoamine turnover. The successful use of antisense technology to knockdown expression of the alpha(2A/D) subtype provides future opportunities to explore the physiology of this receptor subtype.

Reactions of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1 aminomutase) with vinyl and acetylenic substrate analogues analysed by rapid scanning spectrophotometry.

The reactions occurring when glutamate-1-semialdehyde amino-transferase (glutamate-1-semialdehyde 2,1 aminomutase, EC 5.4.3.8) was treated with two potential mechanism-based inactivators, namely 4-aminohex-5-enoate and 4-aminohex-5-ynoate, have been investigated by monitoring rapid transient changes in the absorption spectrum of the enzyme's prosthetic group, pyridoxal 5'-phosphate. In both cases a short-lived chromophore absorbing maximally at about 500 nm was formed in a few milliseconds. In the case of the vinyl analogue (4-aminohex-5-enoate) this chromophore, considered to be a quinonoid intermediate, converted rapidly into the pyridoxamine phosphate form of the co-enzyme in a single turnover which was accompanied by negligible inactivation. However, slow inactivation of the enzyme by this compound was observed when the enzyme was made to undergo multiple turnovers by including the efficient aldehyde substrate, succinic semialdehyde. The acetylenic compound, aminohexynoate, produced more complex spectral changes with the consecutive formation of compounds absorbing maximally at 496 nm, 450 nm, 564 nm and 330 nm. The enzyme was 90% inactivated by aminohexynoate within 10 s and thereafter lost no further activity unless aldehyde substrate was added. Mechanisms and kinetic constants consistent with the observations are proposed for each compound. The observation that the acetylenic compound is a much more potent inactivator than its vinyl analogue is attributed to the occurrence of a conjugated allene as intermediate.

Properties of the pyridoxaldimine form of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1-aminomutase) and analysis of its role as an intermediate in the formation of aminolaevulinate.

Glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1-aminomutase; EC 5.4.3.8) was converted into its pyridoxaldimine form by exhaustive replacement of endogenous pyridoxamine phosphate with pyridoxal phosphate. The isomerization of glutamate 1-semialdehyde to 5-aminolaevulinate by this form of the enzyme followed an accelerating time course which indicated that the enzyme initially had no activity but was converted into the active pyridoxamine phosphate form in an exponential process characterized by a rate constant (k) of 0.027 s-1. The pyridoxaldimine form of the enzyme was converted rapidly into the pyridoxamine form by (S)-4-aminohex-5-enoate and much more slowly by 4-aminobutyrate. The steady-state velocity of the enzyme increased in a markedly non-linear fashion with increasing enzyme concentration, indicating that the extent of dissociation of an intermediate in the reaction to free diaminovalerate and the pyridoxaldimine form of the enzyme depends upon the concentration of the enzyme.