Synthesis of three potential inhibitors of leukotriene biosynthesis.

The syntheses of 7,7-dimethyl- (1), 10,10-dimethyl- (2), and 5,6-benzoarachidonic acid (3), potential substrate analogue inhibitors of leukotriene biosynthesis, are described. Two of these compounds (1 and 2) apparently stimulated, while 3 inhibited, the activity of lipoxygenase from intact human polymorphonuclear leukocytes in vitro when stimulated with Ca2+ and calcium ionophore A23187 in the presence of BSA and arachidonic acid.

Inhibition of human leukocyte elastase, porcine pancreatic elastase, and chymotrypsin by elasnin and other 4-hydroxy-2-pyrones.

Elasnin and 15 related 4-hydroxy-2-pyrones have been assayed for in vitro inhibition of human leukocyte elastase, porcine pancreatic elastase, and bovine chymotrypsin. Inhibition constants for HL elastase range from 0.1 to 10 mM. The principal determinant of potency against the elastases is probably the substituent at position 3, which may account for the observed strong homology between the elastases in their inhibition by these compounds. Acetylation of the 4-hydroxy group has no effect on inhibition. The inhibition is noncovalent; there is no evidence of enzyme acylation by these pyrones.

Synthesis and antiallergic activity of some mono- and disubstituted xanthone-2-carboxylic acids.

A series of xanthone-2-carboxylic acids, substituted mainly with electron-withdrawing groups, has been synthesized and assayed for antiallergic activity, using the passive cutaneous anaphylaxis (PCA) reaction in the rat. The effect of substituent type and substitution pattern on PCA neutralizing capacity is presented.

Inhibition of histamine-induced gastric secretion by flavone-6-carboxylic acids.

Twenty-five flavone-6-carboxylic acids were synthesized and tested as to their ability to inhibit histamine-induced gastric acid secretion in the rat. 3-Isopropoxy-4'-methoxyflavone-6-carboxylic acid (41) showed consistent oral activity while being devoid of any other noteworthy pharmacological effects. In vitro, this compound was found to be inactive as a histamine H2 antagonist, and its mode of action remains unknown.

Synthesis and aldose reductase inhibitory activity of 7-sulfamoylxanthone-2-carboxylic acids.

A series of xanthone-2-carboxylic acids substituted in the 7 position with sulfamoyl and other groups was synthesized and assayed in vitro for inhibition of aldose reductase isolated from rabbit lenses. At a concentration of 10(-6) M, the N-methyl-N-(2-hydroxyethyl)sulfamoyl derivative 14 produced an 83% inhibition of aldose reductase. The structural requirements for this type of activity are discussed.

Dibenzotropone- and dibenzosuberonecarboxylic acids with bronchodilator activity.

The syntheses of 44 5H-dibenzo[a,d]cyclohepten-5-one derivatives bearing a carboxyl gropu at the 1, 2, 3, or 10 position and various substituents at the 7, 8, or 9 position are described. Some of the compounds showed significant bronchodilator activity in guinea pigs and protected the animals against a histamine challenge administered either by aerosol or intravenously. The most active compounds were 10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-one-2-carboxylic acids bearing a methyl or halogen substituent at the 9 position. These compounds were approximately as active as aminophylline by intraperitoneal administration.

Synthesis and biological evaluation of the enantiomers of the potent and selective A1-adenosine antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbonyl)]-xanthine.

The individual enantiomers 8 and 12 of the potent and highly selective racemic A1-adenosine antagonist 1,3-dipropyl-8-[2-(5,6-epoxynorbornyl)]xanthine (ENX, 4) were synthesized utilizing asymmetric Diels-Alder cycloadditions for the construction of the norbornane moieties. The absolute configuration of 12 was determined by X-ray crystallography of the 4-bromobenzoate 14, which was derived from the bridged secondary alcohol 13. The latter was obtained from 12 by an acid-catalyzed intramolecular rearrangement. The binding affinities of the enantiomers 8 and 12 and the racemate 4 at guinea pig, rat, and cloned human A1- and A2a-adenosine receptor subtypes were determined. The S-enantiomer 12 (CVT-124) appears to be one of the more potent and clearly the most A1-selective antagonist reported to date, with K1 values of 0.67 and 0.45 nM, respectively, at the rat and cloned human A1-receptors and with 1800-fold (rat) and 2400-fold (human) subtype selectivity. Both enantiomers, administered intravenously to saline-loaded rats, induced diuresis via antagonism of renal A1-adenosine receptors.

N-arylpiperazinyl-N'-propylamino derivatives of heteroaryl amides as functional uroselective alpha 1-adrenoceptor antagonists.

Novel arylpiperazines were identified as alpha 1-adrenoceptor (AR) subtype-selective antagonists by functional in vitro screening. 3-[4-(ortho-Substituted phenyl)piperazin-1-yl]propylamines were derivatized with N,N-dimethyl anthranilamides, nicotinamides, as well as carboxamides of quinoline, 1,8-naphthyridine, pyrazolo[3,4-b]pyridine, isoxazolo[3,4-b]pyridine, imidazo[4,5-b]pyridine, and pyrazolo[1,5-a]pyrimidines. Strips of rabbit bladder neck were employed as a predictive assay for antagonism in the human lower tract. Rings of rat aorta were used as a "negative screen" for the test antagonists. Binding to alpha 1-ARs was relatively sensitive to size and electronic features of the arylpiperazine portion of the antagonists and permissive to these features on the heteroaryl carboxamide side. These structure-affinity findings were exploited to produce nicotinamides (e.g. 13ii and 25x) and pyrazolo[3,4-b]pyridines (e.g. 37f and 37y) ligands with nanomolar affinity at the alpha 1-AR subtype prevalent in the human lower urinary tract(pA2 values: 8.8, 10.7, 9.3, and 9.9, respectively) and displaying 2-3 orders of magnitude selectivity over the alpha 1D-AR.

RS 30026: a potent and effective calcium channel agonist.

1. A series of dihydropyridine derivatives has been evaluated for calcium channel agonist activity using reversal of nisoldipine-induced inhibition of beating of aggregates of embryonic chick myocytes. This test appears to be specific for calcium channel agonists since isoprenaline and cardiac glycosides are inactive. 2. RS 30026 was the most potent of the series, was significantly more potent than CGP 28392 and of similar potency to Bay K 8644 (pEC50 = 7.45, 6.16 and 7.20, respectively). RS 30026 increased edge movement of individual aggregates, in the absence of nisoldipine, by 50% at 2 nM. 3. Compounds were also evaluated for their effects on guinea-pig papillary muscle and porcine coronary artery rings. RS 30026 displayed positive inotropism at concentrations between 10(-9) and 10(-6) M (pEC200 = 8.21), but was a much more powerful inotrope than Bay K 8644, increasing contractility to 1300% of control at 10(-6) M (compared to 350% of control for Bay K 8644). RS 30026 caused vasoconstriction at concentrations between 10(-10) and 10(-7) M. 4. Calcium channel currents in single embryonic chick myocytes were recorded by whole-cell voltage clamp techniques. RS 30026 (100 nM-500 nM) produced large increases in peak current amplitude and shifted the voltage for threshold and maximal currents to more negative values. RS 30026 (500 nM) also produced large increases in the inward tail currents evoked upon repolarization. The effects of Bay K 8644 (50 and 500 nM) were much less marked. 5. Analysis of the activation characteristics of currents showed parallel shifts in the activation curve to more negative potentials in the presence of 50 nm Bay K 8644, with a much smaller shift in the presence of 500nm Bay K 8644. RS 30026 (100 and 500nM) caused concentration-dependent shifts in the activation of the calcium channel currents with an increase of the slope of the curve. 6. RS 30026 appears to be the most potent and effective calcium channel agonist described to date.

Functional evidence equating the pharmacologically-defined alpha 1A- and cloned alpha 1C-adrenoceptor: studies in the isolated perfused kidney of rat.

1. The present study characterizes and classifies alpha 1-adrenoceptor-mediated vasoconstriction in the isolated perfused kidney of rat using quantitative receptor pharmacology and compares the results to radioligand binding studies (made in cloned alpha 1-adrenoceptor subtypes, native alpha 1A-adrenoceptors in submaxillary gland of rat, and alpha 1A-adrenoceptors in several other tissues of rat). 2. Concentration-effect curves to noradrenaline in the presence of 5-methyl-urapidil were biphasic, indicating alpha 1-adrenoceptor heterogeneity. The alpha 1-adrenoceptor subtype mediating the first phase (low affinity for 5-methyl-urapidil) could not be 'isolated' for detailed pharmacological characterization but was defined by a sensitivity to inhibition by chloroethylclonidine and an inability of methoxamine to activate the site. Additionally, vasoconstriction mediated by this alpha 1-adrenoceptor subtype or subtypes was abolished by nitrendipine (1 microM), thereby allowing characterization of the second, high affinity site for 5-methyl-urapidil. 3. The following antagonists interacted competitively with noradrenaline at the alpha 1-adrenoceptor for which 5-methyl-urapidil exhibits high affinity (pKB value): WB 4101 (10.3) > prazosin (9.5) approximately HV 723 (9.3) approximately 5-methyl-urapidil (9.2) > phenotolamine (8.6) > spiperone (pA2 = 8.1) approximately oxymetazoline (7.9). In contrast, insurmountable antagonism was seen with S(+)- and R(-)-niguldipine, the S(+)-isomer being approximately 30 fold more potent than the R(-)-isomer. Receptor protection experiments indicated that S(+)-niguldipine interacted directly with alpha 1-adrenoceptors. Dehydroniguldipine acted as a competitive antagonist (pKB = 9.0). Thus, the results with antagonists define the alpha 1-adrenoceptor as an alpha 1A-adrenoceptor. 4. An agonist 'fingerprint' was constructed in the presence of nitrendipine to define further the alpha 1A-adrenoceptor. The following order and relativity of agonist potency was obtained: cirazoline (1) approximately adrenaline (2) > noradrenaline (5) > phenylephrine (23) approximately amidephrine (31) > methoxamine (71) >> isoprenaline (1456) approximately dopamine (2210). 5. A high correlative association was shown between the affinity of antagonists obtained functionally in the isolated perfused kidney of rat and pKi values obtained from binding experiments with the cloned bovine alpha 1C-adrenoceptor (R2 = 0.85), native alpha 1A-adrenoceptors in submaxillary gland of rat (R2 = 0.79), and alpha 1A-adrenoceptors from several other tissues of rat (values taken from the literature, R2 = 0.89). 6. The present study demonstrates that the alpha 1A-adrenoceptor is the predominant alpha 1-adrenoceptor subtype mediating vasoconstrictor responses to exogenously administered noradrenaline in the isolated perfused kidney of rat. More importantly, alpha 1A-adrenoceptors mediating vasoconstrictor responses to noradrenaline exhibited a pharmacological equivalency to the cloned bovine alpha 1 c-adrenoceptor. Thus,definitive functional pharmacological data are provided for equating the two receptors and support results derived recently from molecular and radioligand binding studies.

Characterization of muscarinic receptors mediating release of epithelial derived relaxant factor (EpDRF) in guinea-pig isolated trachea.

Muscarinic receptors mediating the release of epithelial derived relaxant factor (EpDRF) have been studied by using both contractions of the guinea-pig tracheal strip (with epithelium intact or denuded) or a coaxial bioassay assembly (rat anococcygeus-recipient; guinea-pig trachea-donor tissue). Indomethacin (1 microM/1) and physostigmine (0.1 microM/1) were both present throughout the study. In the tracheal strip studies, the potencies and maximal effects of all agonists studied (acetylcholine, arecoline, bethanechol, carbachol, (+)cis-dioxolane, ethoxyethyltrimethylammonium, L-660,863, (+/-)methacholine and OXA-22) were not affected or were only slightly (but significantly) reduced by removal of the epithelium. The -log KB for the muscarinic antagonists, atropine, pirenzepine, methoctramine and 4-DAMP (4-diphenyl-acetoxy-N-methylpiperidine) were also not affected and the -log KB values were consistent with M3 muscarinic receptor function. However, the -log KB value of para-fluoro-hexahydro-siladifendol (p-F-HHSiD) was significantly (P less than 0.05) increased upon epithelial denudation (epithelium intact, 7.1; epithelium removed, 7.6). The coaxial bioassay assembly provided more convincing evidence for release of EpDRF in that all muscarinic agonists studied caused relaxations of a precontracted anococcygeus tissue. These relaxations were observed only in the presence of a tracheal tube possessing an intact epithelium. The rank order of potencies for agonists at receptors mediating EpDRF dependent relaxation were similar to those estimated at receptors causing contraction. These data suggested that a substantial receptor reserve was associated with the receptors mediating both EpDRF release and contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The action of (+/-)L-660,863 [(+/-)3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine] at muscarinic receptor subtypes in vitro.

1. The muscarinic pharmacology of a novel oxadiazole muscarinic agonist, (+/-) L-660,863, [+/-3-(3-amino-1,2,4-oxadiazole-5-yl)-quinuclidine] has been studied using pharmacological, radioligand binding and biochemical techniques, in vitro. 2. In isolated tissue experiments, (+/-)L-660,863 was a more potent agonist than carbachol in all preparations studied, being most potent at muscarinic receptors mediating negative chronotropy in guinea-pig right, spontaneously beating atria and least potent at receptors mediating contractions in canine saphenous vein and endothelial denuded rabbit aorta (-log EC50 values were 8.8, 6.6 and 6.3, respectively. The apparent affinities (-log KA) of (+/-)L-660,863) estimated by receptor inactivation, showed some selectivity toward the atrial M2 muscarinic receptor (-log KA = 7.6) in comparison to the M1 or M3 muscarinic receptors (-log KA = 5.4 and 6.2) respectively. This degree of selectivity was also observed in competition radioligand binding studies. 3. At M3 muscarinic receptors mediating inositol phosphates (IPs) accumulation in longitudinal muscle of guinea-pig ileum, the potency of (+/-)L-660,863 (-log EC50 value = 6.2) was similar to the apparent affinity calculated at M3 muscarinic receptors in the functional studies (see above). In contrast, at muscarinic receptors mediating IPs accumulation in guinea-pig atria and ventricles, the potency for (+/-)L-660,863 (-log EC50 = 6.2 and 6.4, respectively) was lower than the apparent affinity calculated at M2 muscarinic receptors from inotropic and binding studies in cardiac tissue (see above).(ABSTRACT TRUNCATED AT 250 WORDS)

RS-33295-198: a novel, potent modulator of P-glycoprotein-mediated multidrug resistance.

A novel multidrug resistance modulator, RS-33295-198, circumvented drug resistance in human, mouse, and Chinese hamster cell lines overexpressing P-glycoprotein. It enhanced the antiproliferative activity of doxorubicin, vincristine, etoposide, and paclitaxel and increased doxorubicin retention in multidrug-resistant hamster CHRC5 cells. RS-33295-198 modulated doxorubicin resistance in a murine P388/ADR leukemia model when administered ip via continuous minipump delivery, ip by bolus injection, and orally; it also improved the efficacy of vincristine toward P388/VCR leukemia when given ip or po. RS-33295-198 showed weak activity in enhancing doxorubicin efficacy against a multidrug-resistant human sarcoma xenograft.

Synthesis and bronchodilator activity of endo-2-(2-cyclopentyl-2-hydroxy-2-phenyl)acetoxy-7-methyl-7- azabicyclo-[2.2.1]heptane methobromide, a potent and long-acting anticholinergic agent.

The synthesis of the alpha-cyclopentylmandelate ester of quaternized endo-7-methyl-7-azabicyclo[2.2.1]heptan-2-ol (4, RS-11635) is described. The key step of this synthesis consists of the intramolecular trans-diaxial epoxide opening of 4-(N-methylamino)-1,2-epoxycyclohexane (8) to form the endo-azabicyclic structure 9. Evaluation of anticholinergic bronchodilator activity by intravenous administration in methacholine-challenged guinea pigs indicated 4 to be approximately twice as potent as ipratropium bromide (ED50 of 1.1 versus 2 micrograms/kg) and to have a duration of action nearly five times as long (230 versus 50 min). Evaluation of anticholinergic bronchodilator activity by aerosol administration in methacholine-challenged dogs also indicated 4 to be approximately twice as potent as ipratropium bromide and to have a duration of action nearly three times as long.

HIV seroprevalence and the acceptance of voluntary HIV testing among newly incarcerated male prison inmates in Wisconsin.

In 1986-88, voluntary and blinded HIV testing was conducted among Wisconsin male prison entrants. The HIV seroprevalence was 0.30 percent in 1986, 0.53 percent in 1987, and 0.56 percent in 1988. The seroprevalence rates among entrants tested voluntarily did not differ from those tested blindly. Voluntary HIV testing was accepted by 71 percent of male prison entrants in 1988; among entrants reporting intravenous drug use 83 percent consented to voluntary HIV testing. Voluntary HIV testing of entrants appears to be an effective screening strategy in Wisconsin prisons.

HIV-1 among childbearing women and newborns in Wisconsin.

To estimate the prevalence of antibody to human immunodeficiency virus-type 1 (HIV-1) among Wisconsin childbearing women (CBW), a continuous blinded HIV-1 seroprevalence survey is being conducted. This survey uses dried blood specimens obtained from infants as part of the Wisconsin Newborn Screening Program. From February 1989 through March 1990, 79,546 specimens from Wisconsin residents were tested for HIV-1 antibody, 21 (0.026%) were HIV-1 seropositive. Among specimens obtained from Milwaukee County CBW, 15 (0.076%) of 19,781 were HIV-1 seropositive compared to 6 (0.010%) of 59,765 obtained from other Wisconsin counties. After adjusting for maternal residence, Wisconsin minority CBW were six times more likely to be HIV-1 seropositive when compared to white CBW. The survey results underscore the need for strategies to prevent HIV-1 infection that focus on women of childbearing age in Wisconsin.

Improving estimates of HIV-1 seroprevalence among childbearing women: use of smaller blood spots.

OBJECTIVES: Nationwide, human immunodeficiency virus type 1 (HIV-1) seroprevalence surveys using dried neonatal blood specimens are critical to estimating HIV-1 seroprevalence among childbearing women. However, the noninclusion of blood specimens deemed "quantity not sufficient" (QNS) for HIV-1 antibody testing potentially introduces bias. In Wisconsin beginning in 1990, we modified the survey protocol to reduce QNS rates and assess bias introduced by QNS specimens. METHODS: The HIV-1 antibody assay was modified to use four 1/8-in blood spots when a single 1/4-in blood spot could not be obtained. Both methods obtain identical blood volumes for testing. RESULTS: During a 27-month period, 7396 (4.8%) of 154,683 specimens were deemed QNS using 1/4-in blood spots. Of these, 6590 (89%) were of sufficient quantity to be tested using four 1/8-in blood spots; 6 (0.09%) specimens tested with 1/8-in blood spots were HIV-1 Western blot assay positive compared with 44 (0.03%) of 147,287 1/4-in specimens (odds ratio = 3.0; 95% confidence interval = 1.2, 7.4). CONCLUSIONS: Because noninclusion of QNS specimens potentially introduces bias, incorporating the results of HIV-1 antibody testing of QNS specimens using four 1/8-in blood spots can improve the accuracy of HIV-1 seroprevalence estimates in these serologic surveys.

RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamine hydrochloride), a selective alpha 1A-adrenoceptor antagonist, displays low affinity for functional alpha 1-adrenoceptors in human prostate: implications for adrenoceptor classification.

Norepinephrine (NE) contracts smooth muscle cells within the human lower urinary tract (LUT) (bladder neck, prostate, and urethra). Receptor distribution and pharmacological evidence have implicated activation of alpha 1A-adrenoceptors. We disclose the pharmacological properties of the novel, selective alpha 1A-adrenoceptor antagonist N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro- alpha,alpha-dimethyl-1H-indole-3-ethanamine hydrochloride (RS-17053) and examine critically the pharmacological identity of the alpha 1-adrenoceptor mediating contractions to NE in human LUT tissues. In several tissues from rat and cloned adrenoceptors, RS-17053 displayed high affinity for the alpha 1A-adrenoceptor (pKi and pA2 estimates of 9.1-9.9) and a 30-100-fold selectivity over the alpha 1B- and the alpha 1D-adrenoceptor subtypes (pK1 and pA2 estimates of 7.7-7.8). However, in isolated smooth muscle preparations from human LUT tissues, RS-17053 antagonized responses to NE only at high concentrations. Estimates of affinity (pA2) at alpha 1-adrenoceptors mediating NE-induced contractions were 7.5 in prostatic periurethral longitudinal smooth muscle (compared with 8.6 for prazosin), 6.9 in anterior fibromuscular stroma (prazosin, 8.9), and 7.1 in bladder neck (prazosin, 8.5). These findings indicate that contractile responses to NE in human LUT tissues are mediated by a receptor displaying pharmacological properties that are clearly different from those of the defined alpha 1A-adrenoceptor and raise the possibility that multiple forms of the alpha 1A-adrenoceptor may exist in human LUT that are discriminated by RS-17053. In this regard, the affinity estimates obtained with RS-17053 and other alpha 1-adrenoceptor antagonists in human LUT tissues are identical to those described for the putative alpha 1L-adrenoceptor.