Computer-aided molecular modeling, synthesis, and biological evaluation of 8-(benzyloxy)-2-phenylpyrazolo[4,3-c]quinoline as a novel benzodiazepine receptor agonist ligand.

Using computer-aided conformational analysis, based on molecular dynamics simulation, cluster analysis, and Monte Carlo techniques, we have designed and synthesized compounds in which a benzyloxy substituent has been incorporated into a series of pyrazoloquinoline benzodiazepine receptor (BZR) ligands. Earlier studies had shown that the benzyloxy group could act as part of the agonist pharmacophoric determinant in the beta-carboline ring system. Furthermore, the agonist beta-carboline had been correlated with a binding site orientation and volume fit for an agonist 6-phenylimidazobenzodiazepine carboxylate. The present study was undertaken to determine whether the benzyloxy substituent could be used as an agonist pharmacophoric descriptor for the phenylpyrazolo[4,3-c]quinolin-3-one BZR ligands. The results of a determination of GABA shift ratios for the synthetic ligands indicate that 8-(benzyloxy)-2-phenylpyrazolo[4,3-c]quinolin-3-one can be predicted to be an agonist at the BZR.

Structure-activity relationship studies at the benzodiazepine receptor (BZR): a comparison of the substitutent effects of pyrazoloquinolinone analogs.

The synthesis of a series of 2-phenylpyrazolo[4,3-c]quinolin-3-one derivatives and their in vitro biological evaluation as ligands for the benzodiazepine receptor are described. The in vitro activities, as determined by an analysis of GABA shift ratios, and binding affinities of these compounds to BZR are compared in terms of the electronic, lipophilic, and steric effect changes of their substituents.

Peripheral-acting benzodiazepines inhibit the growth of human melanoma cells and potentiate the antiproliferative activity of recombinant human interferons.

In this study, we evaluated the effect of a series of peripheral-acting benzodiazepines (BZDs), alone and in combination with recombinant human leukocyte (IFN-alpha A), fibroblast (IFN-beta), or immune (IFN-gamma) interferon (IFN), on the growth of human melanoma cells. Specific peripheral-acting BZDs caused a marked suppression in the proliferation of human melanoma cells. The effect on melanoma cell growth required 72 h exposure to the peripheral-acting BZDs and was not observed if the compounds were removed by 48 h. The relative potency of antiproliferative activity of a series of peripheral-acting BZDs on human melanoma cell growth did not correlate with the reported ability of these agents to bind to peripheral sites on the cell membrane of Friend erythroleukemia cells (FELC), nor did they correlate with the ability of these agents to inhibit [3H]thymidine incorporation in FELC, induce differentiation in FELC, or inhibit neurite outgrowth in nerve growth factor-treated rat pheochromocytoma (PC12) cells. The growth of human melanoma cells was also inhibited by various recombinant human IFNs, with IFN-beta displaying greater antiproliferative activity than IFN-alpha A or IFN-gamma. When the peripheral-acting BZD Ro7-3351, which displays growth inhibitory properties when used alone, was combined with IFN, the antiproliferative activity of the combination was greater than either individual compound exerted independently. The combination of IFN-beta plus Ro7-3351 was more active in suppressing HO-1 melanoma cell growth than other IFN preparations in combination with this peripheral-type BZD. Even when combined with a peripheral-acting BZD, such as Ro5-4608, which displayed only marginal antiproliferative activity against human melanoma cells when applied alone, growth suppression of the combination of this peripheral-type BZD with all three types of IFNs was more than additive. These studies suggest that specific peripheral-acting BZDs, both alone and in combination with recombinant IFNs, display novel antiproliferative activity toward human melanoma cells which may involve a different genetic locus than previously observed in other model cell culture systems.

The rational design and synthesis of haptens having specific activity as full agonists or full antagonists at the benzodiazepine receptor.

The use of computer graphics hardware, in conjunction with molecular modeling software, has allowed for a structural analysis of compounds that bind to the benzodiazepine receptor (BZR) in the nM range. The definition of additional binding requirements together with steric and/or hydrophobic limitations has been directly correlated with profiles of in vivo activity, both for full agonists and full antagonists. This information has been used for the rational design of haptens that contain the antigenic determinants necessary for the production of antibodies specific for either full agonists or for full antagonists at the BZR. The synthesis of these novel compounds has been completed.

2-benzazepines. 6. Synthesis and pharmacological properties of the metabolites of 9-chloro-7-(2-chlorophenyl)-5H-pyrimido[5,4-d] [2]benzazepine.

The 2-benzazepine 9-chloro-7-(2-chlorophenyl)-5H-pyrimido[5,4-d] [2]benzazepine (1) has been selected for development as an anxiolytic agent. In support of this program, we have confirmed by chemical synthesis the structures of three in vitro (rat liver homogenate) metabolites of 1 and confirmed the structure of the major in vivo (dog and man) metabolite of 1, compound 2. Two of the metabolites, arising from hydroxylation of the pyrimidobenzazepine ring at the 5-position (2) and N-oxide formation at the 3-position of the pyrimidobenzazepine ring (3), were found to be as active as 1 in a series of pharmacological tests. The third metabolite, formed by hydroxylation of the 7-phenyl group in the 4-position (4), was found to be inactive in the same pharmacological screens.

2-benzazepines. 5. Synthesis of pyrimido[5,4-d][2]benzazepines and their evaluation as anxiolytic agents.

A series of 5H-pyrimido[5,4-d][2]benzazepines has been synthesized, starting from the corresponding 2-benzazepin-5-ones, and evaluated as potential anxiolytic agents. Selected compounds from this series show a pharmacological profile of action different than that of diazepam. They are more potent than diazepam in the anti-pentylenetetrazole test and in the [3H]diazepam binding assay, yet show less activity in the inclined screen test. A pharmacological data profile is given for 9-chloro-7-(2-chlorophenyl)-5H-pyrimido[5,4-d] [2]benzazepine (7c). The structure-activity relationships of these potential anxiolytic agents are discussed.

Quinazolines and 1,4-benzodiazepines. 92. Conformational recognition of the receptor by 1,4-benzodiazepines.

We propose that the conformation of 1,4-benzodiazepines that is recognized by the binding site on the benzodiazepine receptor complex is one in which the planes formed by the fused benzene ring and the methylene group (and the two adjoining atoms) of the diazepine ring are in the R configuration. The derivation of this conformation was based on comparisons of computer-generated 3-dimensional structures obtained from single-crystal X-ray data for diazepam, (R)- and (S)-1,3-dimethyl-5-(2-fluorophenyl)-7-nitro-1,3-dihydro-2H-1,4-benzodiazepin-2- one, and the structurally rigid ethyl (S)-7-chloro-11,12,13,13 alpha-tetrahydro-9-oxo-9H-imidazo[1,5-alpha] pyrrolo[2,1-d][1,4]benzodiazepine-1-carboxylate. The affinity of ligands for the benzodiazepine binding site was determined using the [3H]-diazepam binding assay.

2-Benzazepines. 4. [1,2,3]Triazolo[4,5-d][2]benzazepines and dibenzo[c,f][1,2,3]triazolo[3,4-a]azepines: synthesis and evaluation as central nervous system agents.

The facile synthesis of [1,2,3]triazolo((4,5-d][2]benzazepines and dibenzo[c,f][1,2,3]triazolo[3,4-a]azepines by the addition of sodium azide to acetylenic benzophenones is described. Examination of the pharmacological data indicates that selected triazolobenzazepines are as potent as diazepam in the anti-pentylenetetrazole test and are weaker in the inclined screen and rotarod tests, suggesting that these compounds have antianxiety properties similar to diazepam with fewer deficits in motor coordination. In addition, a possible diazepam antagonist was found in the triazolo-benzazepine series. The dibenzotriazoloazepines were found to be inactive in four standard CNS screening procedures.

2-Benzazepines. 2. Thiazolo[5,4-d][2]benzazepines.

As part of a program in the area of annelated 2-benzazepines, several thiazolo[5,4-d))[2]benzazepines were prepared. Treatment of the bromo ketones 7-9 with various thio amides gave the thiazoles 10-15, which when treated with methylamine gave the title compounds. The preliminary pharmacology of these compounds showed that they had central nervous system activity similar to the 1,4-benzodiazepines, such as diazepam. The thiazolo[5,4-d][2]benzazepines were also found to bind to the benzodiazepine-receptor complex, indicating that their pharmacological actions are probably related to the 1,4-benzodiazepines.

Quinazolines and 1,4-benzodiazepines. 90. Structure-activity relationship between substituted 2-amino-N-(2-benzoyl-4-chlorophenyl)acetamides and 1,4-benzodiazepinones.

The syntheses of 2-amino-N-(2-benzoyl)-4-chlorophenyl)acetamides are reported. The pharmacological properties of these compounds were compared with data obtained from the corresponding cyclized products [5-(2,6-dichlorophenyl)-1,4-benzodiazepin-2-ones]. Evidence is presented which suggests that the central nervous system activity observed for 1,4-benzodiazepines is inherent only in the closed seven-membered ring and is not due to the ring-opened form.

Quinazolines and 1,4-benzodiazepines. LXXXIX: Haptens useful in benzodiazepine immunoassay development.

The syntheses of some 1,4-benzodiazepines potentially useful as haptens are reported. These compounds are related to chlordiazepoxide, diazepam, nitrazepam, clonazepam, and some of their metabolites. The chemistry reported here is intended to support specific immunoassay development for these drugs.

2-(2-Aminoethylamino)-1,2-diphenylethanol derivatives, a new class of topical antiinflammatory agents.

A number of analogues and derivatives of the title compound were synthesized and evaluated in a new test procedure used to detect topical antiinflammatory activity. Some general comments regarding observation on the structure-activity relationship of these compounds are made.

Quinazolines and 1,4-benzodiazepines. 75. 7-Hydroxyaminobenzodiazepines and derivatives.

7-Nitro-substituted benzodiazepines were reduced with stannous chloride in a buffered system to the corresponding 7-hydroxyamino derivatives. These compounds were alkylated, acylated, and converted to nitroso and azoxy derivatives. The rearrangement of a hydroxyamine to an aminophenol and its oxidation to an aminoquinone are also exemplified. The results of the pharmacological screening for CNS effects are given.