Benzophenone dicarboxylic acid antagonists of leukotriene B4. 1. Structure-activity relationships of the benzophenone nucleus.

A series of lipophilic benzophenone dicarboxylic acid derivatives was prepared which inhibited the binding of the potent chemotaxin leukotriene B4 to its receptor(s) on intact human neutrophils. With a radioligand-binding assay as a measure of receptor affinity, a structure-activity relationship for this series was investigated. Both acidic residues were required for receptor-binding activity. The relative orientation of the two acidic groups was important for optimal binding. Replacement of the carbonyl group of the benzophenone with a variety of polar and nonpolar linking groups led to only small changes in binding affinity, indicating the linking group may not be involved in receptor recognition. Further structure-activity relationships within this series are reported in an accompanying paper.

Benzophenone dicarboxylic acid antagonists of leukotriene B4. 2. Structure-activity relationships of the lipophilic side chain.

A series of lipophilic benzophenone dicarboxylic acid derivatives were found to inhibit the binding of the potent chemotaxin leukotriene B4 (LTB4) to its receptor on intact human neutrophils. Activity at the LTB4 receptor was determined by using a [3H]LTB4-binding assay. The structure-activity relationship for the lipophilic side chain was systematically investigated. Compounds with n-alkyl side chains of varying lengths were prepared and tested. Best inhibition of [3H]LTB4 binding was observed with the n-decyl derivative. Analogues with alkyl chains terminated with an aromatic ring showed improved activity. The 6-phenylhexyl side chain was optimal. Substitution on the terminal aromatic ring was also evaluated. Methoxyl, methylsulfinyl, and methyl substituents greatly enhanced the activity of the compound. For a given substituent, the para isomer had the best activity. Thus the nature of the lipophilic side chain can greatly influence the ability of the compounds to inhibit the binding of LTB4 to its receptor on intact human neutrophils. The most active compound from this series, 84 (LY223982), bound to the LTB4 receptor with an affinity approaching that of the agonist.

Leukotriene B4 (LTB4) receptor antagonists: a series of (hydroxyphenyl)pyrazoles.

A series of (hydroxyphenyl)pyrazoles was designed by molecular modeling comparison with the LTB4 structure and prepared for evaluation as LTB4 receptor antagonists, culminating in 4-ethyl-5-[[6-methyl-6-(1H-tetrazol-5-yl)heptyl]oxy]-2-(1H-pyrazol -3- yl)phenol (2). Using an assay for inhibition of specific [3H]LTB4 binding to human PMN, it was found that the pyrazole ring could be methylated at N(1) with little loss of activity while methylation at N(2) reduced activity significantly. The structure-activity relationship of the terminal acid group was investigated. Good activity was found with o- and m-phenylalkanoic acids, chromane carboxylic acid, and tetrazole groups. The best in vitro activity was realized with the pyrazole nitrogen unsubstituted and with a six-carbon chain linking the phenyl ether oxygen to the tetrazole group. Compound 2, having an IC50 of 6.4 +/- 0.8 nM in the binding assay, was selected for further preclinical evaluation.

Design, synthesis, and pharmacological evaluation of potent xanthone dicarboxylic acid leukotriene B4 receptor antagonists.

In an effort to develop increasingly potent and specific leukotriene B4 (LTB4) receptor antagonists, several xanthone dicarboxylic acids were synthesized and evaluated. Two separate synthetic routes were used to construct a xanthone nucleus containing a regiospecific orientation of each carboxylic acid pharmacophore. These compounds represent the major conformationally-restricted analogues of benzophenone dicarboxylic acids previously shown to antagonize the activation of human neutrophils by LTB4. The most potent agent was compound 32, which inhibited the specific binding of [3H]LTB4 to receptors on intact human neutrophils (IC50, 6.2 +/- 0.1 nM), LTB4-induced luminol-dependent chemiluminescence (IC50, 55 +/- 11 nM), aggregation (IC50, 133 +/- 42 nM), and chemotaxis (IC50, 899 +/- 176 nM). The compound was a poor antagonist of N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced chemiluminescence (IC50, 1599 +/- 317 nM) and aggregation (IC50, 2166 +/- 432 nM), indicating specificity in the inhibition of LTB4-stimulated events. Compound 32 (LY210073), which was completely devoid of agonist activity, appears to be one of the strongest inhibitors of LTB4 receptor binding reported so far.

Leukotriene B4 receptor antagonists: the LY255283 series of hydroxyacetophenones.

A series of hydroxyacetophenones was prepared for evaluation as leukotriene B4 (LTB4) receptor antagonists, culminating in 1-[5-ethyl-2-hydroxy-4-[[6-methyl-6-(1H-tetrazol-5- yl)heptyl]oxy]phenyl]ethanone (compound 35, LY255283). Using an assay for inhibition of specific [3H]LTB4 binding to human PMN, we found that substitution of a nonpolar substituent in the 5-position was required for activity. Best activity was realized with hydrogen in the 3-position, hydroxyl in the 2-position, short chain alkyl ketone in the 1-position, and a six- or eight-carbon chain linking the oxygen in the 4-position with an unsaturated terminal function. Compound 35, having an IC50 of 87 nM in the binding assay, was chosen for further preclinical evaluation.

Synthetic and structure/activity studies on acid-substituted 2-arylphenols: discovery of 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5- hydroxyphenoxy]-propoxy]phenoxy]benzoic acid, a high-affinity leukotriene B4 receptor antagonist.

Structural derivatives of LY255283 have been studied as receptor antagonists of leukotriene B4. Substitution of the 2-hydroxyacetophenone subunit of 1 (LY255283) with a 2-arylphenol group provided entry into several new series that feature various mono- and diacidic core functionality. These new analogues, the subject of a broad structure-activity investigation, displayed significantly increased in vitro and in vivo activity as receptor antagonists of LTB4. A series of diaryl ether carboxylic acids demonstrated especially interesting activity and led to the discovery of compound 43b, 2-[2-propyl-3-[3-[2-ethyl-4-(4- fluorophenyl)-5-hydroxyphenoxy]-propoxy]phenoxy]benzoic acid (LY293111), a 2-arylphenol-substituted diaryl ether carboxylic acid which displayed potent binding to human neutrophils (IC50 = 17 +/- 4.6 nM) and guinea pig lung membranes (IC50 = 6.6 +/- 0.71 nM), inhibition of LTB4-induced expression of the CD11b/CD18 receptor on human neutrophils (IC50 = 3.3 +/- 0.81 nM), and inhibition of LTB4-induced contraction of guinea pig lung parenchyma (pKB = 8.7 +/- 0.16). In vivo, 43b demonstrated potent activity in inhibiting LTB4-induced airway obstruction in the guinea pig when dosed by the oral (ED50 = 0.40 mg/kg) or intravenous (ED50 = 0.014 mg/kg) routes. A specific LTB4 receptor antagonist, 43b had little effect on inhibiting contractions of guinea pig lung parenchyma induced by leukotriene D4 (LTD4), histamine, carbachol, or U46619. Compound 43b has been chosen as a clinical candidate and is currently in phase I studies for a variety of inflammatory diseases.

Structure-based design of potent, amidine-derived inhibitors of factor Xa: evaluation of selectivity, anticoagulant activity, and antithrombotic activity.

To enhance the potency of 1,2-dibenzamidobenzene-derived inhibitors of factor Xa (fXa), an amidine substituent was incorporated on one of the benzoyl side chains to interact with Asp189 in the S1 specificity pocket. Lead molecule 1 was docked into the active site of fXa to facilitate inhibitor design. Subsequently, iterative SAR studies and molecular modeling led to a 1000-fold increase in fXa affinity and a refined model of the new inhibitors in the fXa active site. Strong support for the computational model was achieved through the acquisition of an X-ray crystal structure using thrombin as a surrogate protein. The amidines in this series show high levels of selectivity for the inhibition of fXa relative to other trypsin-like serine proteases. Furthermore, the fXa affinity of compounds in this series (K(ass) = 50-500 x 10(6) L/mol) translates effectively into both anticoagulant activity in vitro and antithrombotic activity in vivo.

Blockade of human neutrophil activation by 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5- hydroxyphenoxy]propoxy]phenoxy]benzoic acid (LY293111), a novel leukotriene B4 receptor antagonist.

Leukotriene B4 (LTB4), a naturally occurring pro-inflammatory product of arachidonic acid metabolism, has been associated with human inflammatory disease. This study compares the abilities of two LTB4 receptor antagonists, 2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]- propoxy]phenoxy]benzoic acid (LY293111) and 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), to displace LTB4 binding and their functional blockade of human neutrophil activation. LY293111 inhibited the binding of [3H]LTB4 with a Ki of 25 nM; SC-41930 displayed a similar potency (Ki = 17 nM). In contrast, LY293111 prevented LTB4-induced calcium mobilization with an IC50 = 20 nM, or 40 times more effectively than SC-41930 (IC50 = 808 nM). LY293111 was 300 times more potent than SC-41930 in blocking LTB4-induced CD11b up-regulation on isolated neutrophils. LY293111 also arrested LTB4-induced up-regulation of CD11b on neutrophils in whole human blood. LY293111 was not effective in blocking human neutrophil activation responses induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), platelet-activating factor (PAF), human recombinant endothelial interleukin-8 (IL-8) or human recombinant complement component 5a (C5a).

Aerosolized LTB4 produces delayed onset increases in pulmonary gas trapping.

Airway obstruction, as measured by increases in postmortem pulmonary gas trapping, and lung inflammatory changes were examined in guinea pigs exposed for up to 4 h to aerosols of leukotriene B4 (LTB4) or its non-chemotactic isomer, 6-trans-12-epi-LTB4. Airway obstruction and cytological responses in isomer-exposed animals were similar to those of unexposed control animals. LTB4-exposed animals had minimal inflammatory changes at 0.5 h but became dyspneic by 2 h and had increased airway obstruction, bronchoalveolar lavage neutrophils and eosinophils, and pulmonary tissue granulocyte scores. The LTB4-induced effects at 4 h were similar to those 2 h, except for further increase in BAL neutrophils and eosinophils. LTB4-induced airway obstructive and inflammatory changes were prevented by pretreatment with the LTB4 receptor antagonist SC-41930, but were unaffected by indomethacin. Thus, prolonged LTB4 inhalation can produce delayed onset airway obstruction that is stereospecific, cyclooxygenase-independent, and temporally associated with the influx of granulocytes into lung airways.

Pharmacologic actions of the second generation leukotriene B4 receptor antagonist LY29311: in vivo pulmonary studies.

We examined the in vivo actions of LY293111 sodium (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxyphenoxy]pro poxy]phenoxy] benzoic acid sodium salt). Guinea pigs were used to evaluate the effect of this agent on (1) acute airway obstruction produced by intravenous leukotriene B4, (2) pulmonary granulocyte infiltration and delayed onset airway obstruction resulting from a 4-h leukotriene B4 inhalation and (3) lung inflammation after aerosol challenge with the divalent cationic ionophore A23187 (6S-[6alpha(2S*,3S*),8beta(R*),9beta,11alpha]-5- (methylamino)-2-[[3,9,11-trimethyl-8-[1-methyl-2-oxo-2-(1H-pyrrol-2-yl)e thyl]-1,7-dioxaspiro[5.5]undec-2-yl]methyl]-4-benzoxazole carboxylic acid). Airway obstruction was quantitated using pulmonary gas trapping measurements and lung inflammation was evaluated by bronchoalveolar lavage (BAL) and histology. LY293111 sodium produced a dose-related inhibition of acute leukotriene B4-induced airway obstruction when administered i.v. (ED50=14 microg/kg) or p.o. (ED50=0.4 mg/kg). In contrast, LY293111 sodium did not inhibit the pulmonary gas trapping caused by aerosols of histamine, leukotriene D4, or the thromboxane mimetic U46619 (15 [(S)-hydroxy11a,9a-(epoxymethano)prosta-5Z,13E-dienoic acid]). Oral LY293111 sodium inhibited leukotriene B4-induced bronchoalveolar lavage granulocyte infiltration and delayed onset airway obstruction at doses as low as 0.3 mg/kg. In A23187-challenged animals, pulmonary inflammation was markedly inhibited at 1 h, but not 2 h and 4 h post-exposure. We conclude that LY293 11 sodium is a selective leukotriene B4 receptor antagonist with potent pulmonary anti-inflammatory activity.

Phosphatidylinositol 3-kinase activation is required for insulin-stimulated sodium transport in A6 cells.

Insulin stimulates amiloride-sensitive sodium transport in models of the distal nephron. Here we demonstrate that, in the A6 cell line, this action is mediated by the insulin receptor tyrosine kinase and that activation of phosphatidylinositol 3-kinase (PI 3-kinase) lies downstream of the receptor tyrosine kinase. Functionally, a specific inhibitor of PI 3-kinase, LY-294002, blocks basal as well as insulin-stimulated sodium transport in a dose-dependent manner (IC50 approximately 6 microM). Biochemically, PI 3-kinase is present in A6 cells and is inhibited both in vivo and in vitro by LY-294002. Furthermore, a subsequent potential downstream signaling element, pp70 S6 kinase, is activated in response to insulin but does not appear to be part of the pathway involved in insulin-stimulated sodium transport. Together with previous reports, these results suggest that insulin may induce the exocytotic insertion of sodium channels into the apical membrane of A6 cells in a PI 3-kinase-mediated manner.

Specific inhibition of leukotriene B4-induced neutrophil activation by LY223982.

LY223982, (E)-5-(3-carboxybenzoyl)-2-((6-(4-methoxyphenyl)-5- hexenyl)oxy)benzenepropanoic acid, is a newly discovered potent inhibitor of specific binding of leukotriene B4 (LTB4) to its receptor on human neutrophils. This study demonstrated that the compound is also a specific antagonist of LTB4-induced neutrophil activation under both in vitro and in vivo conditions. LY223982 was found to be 189-fold more effective in displacing [3H]LTB4 than 35S-N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) from their corresponding receptors on human neutrophils. The concentration inhibiting 50% of response (IC50) for displacement of [3H]LTB4 (13.2 nM) was only 6.8-fold higher than the value for nonradioactive LTB4. The compound inhibited the aggregation of guinea pig neutrophils caused by LTB4 more strongly than FMLP or platelet-activating factor. The IC50 for inhibition of LTB4-induced responses (74 nM) was 93- and > 135-fold lower than the IC50 for inhibition of the corresponding FMLP and platelet-activating factor-induced effects. LY223982 was also a potent antagonist of the aggregation of human neutrophils by LTB4 (IC50, 100 nM). Chemotaxis of human neutrophils induced by LTB4 was only modestly inhibited by the compound (IC50 = 6 microM) but it had even less effect on cell movement caused by FMLP. LY223982 inhibited transient leukopenia induced in rabbits with LTB4 (ED50, 3 mg/kg) but not with FMLP. It had no agonist activity in any of the test systems. In summary, the results indicate that LY223982 is a potent specific antagonist of LTB4-induced neutrophil activation.

Characterization of the spatial arrangement of the two acid-binding sites on the human neutrophil LTB4 receptor.

A series of lipophilic benzophenone dicarboxylic acids have been shown to be inhibitors of the binding of LTB4 to its receptors on intact human neutrophils (Gapinski et al. (1990). Structure-activity relationships indicated that maximum activity was achieved when an acid group was attached at the meta position of each ring. In this report, the conformation of these inhibitors that binds best to the LTB4 receptor was determined. Inhibition concentration profiles of four rigid xanthone isomers that mimicked the four major conformational states of this type of benzophenone dicarboxylic acid were compared. LY264086, 3-[4-[7-carboxy-3-[decyloxy]-9-oxo-9H-xanthene]]propanoic acid, was the most potent inhibitor. The distance between the two carboxyl groups in this isomer was found to be 9.8 A, implying that the two acid binding sites on the receptor are separated by similar dimensions. Molecular modeling studies with low energy conformers of the xanthone isomers and LTB4 suggested a configuration of the agonist when it is bound to the receptor but did not exclude all other possibilities. These experiments further support the existence of two acid-binding sites on the human neutrophil LTB4 receptor.

Binding of a novel radioiodinated thromboxane A2/prostaglandin H2 antagonist to guinea pig lung membranes.

The utilization of a novel radioiodinated TXA2/PGH2 receptor antagonist, ISAP (7-[(1R,2S,3S,5R)-6,6-dimethyl-3(4- iodobenzenesulfonylamino)-bicyclo[3.1.1]-hept-2-yl]-5(Z)-heptenoic acid) to characterize TXA2/PGH2 receptors from guinea pig lung parenchymal membranes in radioligand binding assays is described. [125I]ISAP binding was saturable, displaceable, and dependent upon protein concentration. The time course of binding yielded k1 = 2.12 x 10(8) M-1 min-1, k1 = 4.46 x 10(-3) min-1, Kd = k-1/k1 = 17.8 pM. Equilibrium binding studies indicated a single class of high affinity binding sites with a Kd of 52.7 +/- 1.9 pM and a Bmax of 92.7 +/- 7.2 fmoles/mg protein (n = 4). Binding was inhibited by a series of structurally diverse mimetics and antagonists with the rank order of potency IBOP greater than ONO11113 = SQ26655 greater than U46619 (mimetics) and (d)-S-145 greater than ISAP greater than (1)-S-145 greater than SQ29548 greater than BM13505 = I-PTA-OH (antagonists), with entantioselectivity of binding demonstrated by (d) and (1) S-145. Binding was also inhibited by prostanoids (PGD2, PGF2 alpha, and 9 alpha, 11 beta-PGF2) thought to act at the airway TXA2/PHH2 receptor, but not by histamine or carbachol, and only weakly by LTB4 and LTD4, consistent with specific binding to the lung TXA2/PGH2 receptor.

Inhibition of ex vivo neutrophil activation by oral LY293111, a novel leukotriene B4 receptor antagonist.

1. The effects of orally administered LY293111 on ex vivo neutrophil Mac-1 upregulation were determined in a total of 24 healthy male subjects within three study periods. 2. In the first period, eight volunteers received 60 mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average ex vivo Mac-1 response of the LY293111 group was 56% of the predose control (95% confidence interval (CI) 44.3 to 67.9%; P < 0.01). The inhibitory effect was maximum at the end of dosing and had disappeared by day 14. 3. In the second period, eight subjects received 120 mg LY293111 or placebo three times daily in 22 total doses over 8 days followed by a 1 week follow-up. The average response of the LY293111 group was 70% of the pre-dose control (95% CI 59.7 to 81.0%; P < 0.01). The inhibitory effect was maximum the day following the initial dose and continued throughout the dosing period. 4. In the third period, eight subjects received 200 mg LY293111 or placebo twice daily in 15 total doses over 8 days followed by a 1 week follow-up. Mac-1 upregulation was 64% of pre-dose levels (95% CI 53.8 to 75.1%; P < 0.01) over the course of the study period. The inhibition had disappeared 2 days following the final dose. Alternate neutrophil stimulation by fMLP was not inhibited. 5. No statistically significant inhibition was observed for placebo-treated subjects. 6. No statistically significant differences were apparent between the active dose regimens. 7. The results indicate that orally administered LY293111 is pharmacologically active in humans. Results from this study may be useful in determining dose selection for efficacy trials.

Pharmacologic actions of the second-generation leukotriene B4 receptor antagonist LY293111: in vitro studies.

The in vitro actions were investigated of LY293111, a potent and selective leukotriene B4 (LTB4) receptor antagonist, on human neutrophils, human blood fractions, guinea pig lung membranes, and guinea pig parenchymal and tracheal strips. The IC50 for inhibiting [3H]LTB4 binding to human neutrophils was 17.6 +/- 4.8 nM. LY293111 inhibited LTB4-induced human neutrophil aggregation (IC50 = 32 +/- 5 nM), luminol-dependent chemiluminescence (IC50 = 20 +/- 2 nM), chemotaxis (IC50 = 6.3 +/- 1.7 nM), and superoxide production by adherent cells (IC50 = 0.5 nM). Corresponding responses induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine were inhibited by 100-fold higher concentrations of LY293111. LTB4 binding to guinea pig tissues and subsequent activation were also inhibited. The Ki for inhibition of [3H]LTB4 binding to lung membranes was 7.1 +/- 0.8 nM; IC50 for preventing binding of [3H]LTB4 to spleen membranes was 65 nM. The compound inhibited LTB4-induced contraction of guinea pig lung parenchyma. At 10 nM, LY293111 caused a parallel rightward shift of the LTB4 concentration-response curve. At higher concentrations, plots were shifted in a nonparallel manner, and maximum responses were depressed. LY293111 did not prevent antigen-stimulated contraction of sensitized trachea strips. At micromolar concentrations, LY293111 inhibited production of LTB4 and thromboxane B2 by plasma-depleted human blood stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine and thrombin. In addition, at these higher concentrations, formation of LTB4 by A23187-activated whole blood and conversion of arachidonic acid to LTB4 by a human neutrophil cytosolic fraction were inhibited. In summary, LY293111 is a second-generation LTB4 receptor antagonist with much improved potency in a variety of functional assay systems.