Antisense-designed peptides: a comparative study focusing on possible complements to angiotensin II.

A comprehensive study of antisense peptides possibly complementary to angiotensin II (AII) is described. Antisense peptides of AII were designed using two different procedures outlined by Blalock and Root-Bernstein. Also, peptide complements designed to interact as homologs of AII were investigated. Three methods were used to detect binding between these peptides and AII. Several antisense-designed peptides were studied with unprotected termini to compare the effects of protected vs. unprotected termini. It was determined that the protected antisense-designed peptides derived from Root-Bernstein's methods interacted (high micro-molar range) directly with AII, while those protected antisense peptides derived from Blalock's method interacted only with the AII receptor. Two novel AII antagonists were discovered using this technology, a Root-Bernstein derived unprotected complementary peptide (H2N-K-G-V-Y-M-H-A-L-CO2H) and a Blalock derived unprotected antisense peptide (H2N-E-G-V-Y-V-H-P-V-CO2H), which exhibited 5 microM and 70 nM affinity toward the AII receptor, respectively.

"Diversomers": an approach to nonpeptide, nonoligomeric chemical diversity.

Solid-phase chemistry, organic synthesis, and an apparatus for multiple, simultaneous synthesis have been combined to generate libraries of organic compounds ("diversomers"). Arrays of compounds were synthesized over two to three steps incorporating chemically diverse building blocks on a polystyrene-based solid support in a multiple, simultaneous manner. The generality of this approach is illustrated by the syntheses of dipeptides, hydantoins, and benzodiazepines.

Quinolone antibacterials: synthesis and biological activity of carbon isosteres of the 1-piperazinyl and 3-amino-1-pyrrolidinyl side chains.

A series of 6-fluoro-1,4-dihydro-4-oxo-3-quinoline- and 1,8-naphthyridinecarboxylic acids, substituted at the 7-position with carbon-linked side chains, was synthesized and evaluated for antibacterial activity and DNA-gyrase inhibition. Structural modifications focused on replacement of the heterocyclic nitrogen of the frequently found 1-piperazinyl and 3-amino-1-pyrrolidinyl side chains by an sp2- or an sp3-hybridized carbon. All new compounds displayed high in vitro and in vivo antibacterial activity. Potency relative to the standard nitrogenated agents was dependent on ring size and hybridization of the linking carbon atom of the side chain. Compounds with a 1,2,3,6-tetrahydro-4-pyridinyl substituent at C-7 were equipotent with their 1-piperazinyl analogs, whereas those having a 4-piperidinyl or a 3-amino-1-cyclopentenyl ring at C-7 were less active than the 1-piperazinyl or 3-amino-1-pyrrolidinyl substituted agents, respectively. This relative difference in antibacterial potency did not correlate with the observed activity against gyrase, where the majority of the new compounds were equally or more potent than their nitrogenated counterparts.

Quinolone antibacterials containing the new 7-[3-(1-aminoethyl)-1- pyrrolidinyl] side chain: the effects of the 1-aminoethyl moiety and its stereochemical configurations on potency and in vivo efficacy.

A series of stereochemically pure 7-[3-(1-aminoethyl)-1-pyrrolidinyl]-1, 4-dihydro-4-oxoquinoline and 1,8-naphthyridine-3-carboxylic acids, with varied substituents at the 1-, 5-, and 8-positions, were synthesized to study the effects of the 7-[3-(1-aminoethyl)-1- pyrrolidinyl] moiety on potency and in vivo efficacy relative to the known 7-[3-(aminomethyl)-1- pyrrolidinyl] derivatives. The antibacterial efficacies of the target compounds and their relevant reference agents were determined in vitro using an assortment of Gram-negative and Gram-positive organisms and in vivo using Escherichia coli and Streptococcus pyogenes mouse infection models. The effects of the 7-[3-(1-aminoethyl)-1-pyrrolidinyl] moiety were also examined at the level of the target enzyme by employing a DNA-gyrase supercoiling inhibition assay. Selected compounds were further evaluated for potential phototoxic and clastogenic liabilities using a phototoxicity mouse model and an in vitro mammalian cell cytotoxicity assay. It was found that the differences in in vitro antibacterial activity between the stereoisomers were significantly greater than previously reported for other optically pure 3-substituted pyrrolidinyl side chains. Relative to their 7-[3-(aminomethyl)-1-pyrrolidinyl] analogs, the (3R,1S)-3-(1-aminoethyl)pyrrolidines generally conferred a 2-4-fold increase in Gram-positive in vitro activity and an average of 10-fold improvement in oral efficacy. The level of phototoxicity and cytotoxicity of the product quinolones was ultimately determined by the combined influence of the 7-[3-(1-aminoethyl)-1-pyrrolidinyl] side chains and the other quinolone substituents. From this study, several compounds were identified with outstanding antibacterial activity and low degrees of phototoxicity and mammalian cell cytotoxicity. One such agent, 34F-R,S (PD 140248), showed the best overall blend of safety and efficacy.

A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid.

An assay for the detection of hydroperoxy derivatives of linoleic acid formed by the action of 15-lipoxygenase is described. The assay developed is based on a method first reported by Ohishi et al. (1985) Biochem. Int. 10, 205-211) with some important modifications. The assay described herein takes advantage of the ability of (9Z,11E)-13-hydroperoxyoctadecadienoic acid (13-HPODE), the product of the action of 15-lipoxygenase on linoleic acid, to oxidize N-benzoyl leucomethylene blue to methylene blue in the presence of hemoglobin. The resultant blue color is stable to light and air and can be quantified spectrophometrically at 660 nm. The linear range of the assay is 1.6-32 nmol (0.5-10 micrograms) of 13-HPODE. The utility of the assay can be extended to detect other peroxides as well as inhibitors of 15-lipoxygenase. The assay is a rapid, reliable method for the detection of lipid hydroperoxide production.

Mechanistic studies of the phototoxic potential of PD 117596, a quinolone antibacterial compound.

PD 117596 is a novel quinolone compound that is being investigated for use as an antibacterial agent. Early investigations demonstrated a significant phototoxic liability associated with this compound. These studies were undertaken to investigate the mechanism of phototoxicity using an in vitro model. In the UVA region, PD 117596 was found to be a more efficient producer of singlet oxygen than rose bengal, ciprofloxacin, nalidixic acid, or PD 118879, another quinolone under investigation. The quantum yield of photoreaction for PD 117596 was relatively low (phi = 0.021); however, it was approximately 10-fold higher than other tested quinolones. In vitro studies using a mouse erythrocyte model were used to further investigate the mechanism of phototoxicity. PD 117596-induced photohemolysis was found to be oxygen dependent with a relatively rapid onset that progressed even after removal of light. Preirradiation of the compound prevented subsequent hemolytic or photohemolytic action. BHA, BHT, alpha-tocopherol, and the iron chelator DTPA were all found to be effective at ameliorating the photohemolytic response. The photohemolytic response was markedly enhanced when D2O was substituted for H2O in the incubation medium, indicating a singlet oxygen-mediated mechanism of action. A rise in thiobarbituric acid products was noted within 1 hr of irradiation and was maximal at the time of onset of overt photohemolysis. These data suggest that singlet oxygen production by irradiated PD 117596 is responsible for secondary changes in mouse red blood cells including lipid peroxidation and ultimately results in cellular lysis.

A silica gel plate-based qualitative assay for acetylcholinesterase activity: a mass method to screen for potential inhibitors.

A procedure for the qualitative assessment of inhibitory activity towards acetylcholinesterase for a given compound is described. Solutions of the compounds of interest are spotted on silica gel TLC plates in a matrix pattern. The silica gel plate is sprayed with a solution of acetylthiocholine iodide and 5,5-dithiobis(2-nitrobenzoic acid) followed by a solution of acetylcholinesterase. The enzyme reaction produces a yellow background color with inhibitor compounds exposed as white zones where color has failed to develop. The results for a test set of compounds were compared to those obtained using the standard Ellman assay procedure and found to agree for virtually all of these compounds. The conditions of silica gel plate thickness, reagent concentration, and enzyme source under which this procedure is suitable were investigated. This represents an extremely rapid method to screen large numbers of compounds to uncover new inhibitors of acetylcholinesterase and potentially other enzymes as well.

Quinolone antibacterials: preparation and activity of bridged bicyclic analogues of the C7-piperazine.

A series of quinolone and naphthyridine antibacterial agents possessing as the C7-heterocycle bicyclic 2,5-diazabicyclo[n.2.m]alkanes, where n = 2, 3 and m = 1, 2, and a series including 4-aminopiperidine and 3-amino-8-azabicyclo[3.2.1]octanes have been prepared and evaluated in vitro and in vivo for antibacterial activity against a variety of Gram-negative and Gram-positive organisms. These compounds were also tested against the target enzyme bacterial DNA gyrase. All the examples investigated are nearly equipotent with the parent 7-piperazinyl analogues. Only endo-7-(3-amino-8-azabicyclo[3.2.1]oct-8-yl)-1-cyclopropyl-6,8-difluoro- 1,4- dihydro-4-oxo-3-quinolinecarboxylic acid displays activity that surpasses that of the piperazine parent.

New "ofloxacin" type antibacterial agents. Incorporation of the spiro cyclopropyl group at N-1.

The first example incorporating a spiro cyclopropyl group into an "ofloxacin" type of quinolone antibacterial agent has been prepared by potassium fluoride mediated ring closure of the hydroxymethyl cyclopropyl intermediate to give 9'-fluoro-7'-oxo-10'-(1-piperazinyl)spiro[cyclopropane-1,3'(2'H)-[7H] pyrido[1,2,3-de][1,4]benzoxazine]-6'-carboxylic acid. Analogues were made by substitution at C-7 by various complex amines. Evaluation of these compounds for antibacterial activity was carried out. All examples prepared and examined showed in vitro minimum inhibitory values and in vivo mouse protection results to be diminished as compared to the parent, ofloxacin.