Second-generation leukotriene B4 receptor antagonists related to SC-41930: heterocyclic replacement of the methyl ketone pharmacophore.

Our previous reports have highlighted the first-generation leukotriene B4 (LTB4) receptor antagonist SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]3,4- dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid) which has potent oral, topical, and intracolonic activity in various animal models of inflammation. Extensive structure-activity relationship studies, in which a series of heterocyclic replacements for the methyl ketone functional group of SC-41930 was explored, identified SC-50605 (7-[3-[2-(cyclopropylmethyl)-3-methoxy-4- (4-thiazolyl)phenoxy]propoxy]-3,4-dihydro-8-propyl-2H-1-benzopyran-2- carboxylic acid) as an optimized analog within a series of thiazoles. SC-50605 was found to be significantly more potent than SC-41930 in LTB4 receptor binding, chemotaxis, and degranulation assays. It also displayed very good activity in animal models of colitis and epidermal inflammation by oral, topical, intravenous, and intracolonic routes of administration. The resolved enantiomers of SC-50605 were obtained by chiral chromatography and both demonstrated good in vitro and in vivo activity. The (+)-isomer (SC-52798) is currently being evaluated as a potential clinical candidate for psoriasis and ulcerative colitis therapy.

Substituted 2-[(2-benzimidazolylsulfinyl)methyl]anilines as potential inhibitors of H+/K+ ATPase.

A series of substituted 2-[(2-benzimidazolylsulfinyl)methyl]anilines were synthesized as potential inhibitors of the acid secretory enzyme H+/K+ ATPase. Substitutions on the aniline nitrogen atom resulted in potent enzyme inhibition in vitro but weak activity in gastric fistula dogs. Electron-donating substituents on the aniline ring enhanced in vitro and in vivo potency relative to the unsubstituted analogue. The potency showed a correlation to the calculated pKa of the aniline nitrogen atom. Substitutions on the aniline and benzimidazole rings did not further enhance potency. Di- and trisubstituted aniline derivatives were potent inhibitors of the enzyme system. The preferred combination of substituents were a methoxy group on the benzimidazole ring and a single alkyl group on the aniline ring. One such compound, 76, was an effective inhibitor of acid secretion in the dog and was selected for further pharmacological study.

Genetic and developmental regulation of mouse liver alcohol dehydrogenase.

Alcohol dehydrogenase (EC 1.1.1.1; alcohol:NAD+ oxidoreductase) activity varies 2-fold in liver extracts prepared from different inbred mouse strains. The strain-specific variation is not present in kidney extracts of male mice and is developmentally specific in the liver, occurring in mice 25 days of age and older. Neither electrophoretic nor heat lability properties of the enzyme from 15-day-old animals are different from the enzyme in adult mice. Analysis of genetic crosses and recombinant inbred lines confirms that a single genetic locus, designated Adh-1-t, with additive alleles has a major effect in controlling the temporal difference in enzyme activity between strains. This enzyme has been purified from mouse liver, and antibodies to the enzyme have been produced in a goat. Quantitative immunoprecipitation reveals that a given quantity of antibody immunoprecipitates equivalent protein and enzyme activity from liver extracts prepared from high and low strain mice and from 5-day-old and adult mice. Enzyme from high and low strains can be purified to the same specific activity and has indistinguishable electrophoretic and heat denaturation properties. This evidence supports the hypothesis that the high liver activity in C57BL/6 mice is due to the presence of more enzyme molecules/g of liver than is found in low activity mice. Using radiolabeling and specific immunoprecipitation of alcohol dehydrogenase, it has been established that high level mice (C57BL/6) have a 2-fold greater relative rate of synthesis of liver alcohol dehydrogenase than is found in low strain mice (C3H). Thus, the action of the Adh-1-t locus in the mouse is to control the level of alcohol dehydrogenase protein in the liver by controlling the rate of synthesis of this enzyme.

The relationship between the activity of DNA-dependent RNA polymerase I and the rate of synthesis of rRNA in hepatoma cells in culture.

Cell culture lines were established from the transplantable mouse hepatomas H6 and H129. Both cell lines had a doubling time about 30 h when maintained in medium containing 5% foetal bovine serum. H6 cells contained about 3-4 times more DNA-dependent RNA polymerase I (Pol I; ribonucleoside triphosphate--RNA nucleotidyltransferase, EC 2.7.7.6) than did H129 cells. Moreover, the H6-cell enzyme was more heat-labile than that from H129 cells. Steady-state contents of 28S rRNA were measured in both cell lines. Exponentially growing cultures of H6 cells contained about 6.5pg of 28S rRNA/cell, and similar cultures of H129 cells contained about 5.8pg/cell. Stationary cultures of both cell lines contained about 2pg of 28S rRNA/cell. By two different techniques, the half-time for turnover of 28S rRNA was estimated to be 16-17h for both H6 and H129 cells. Knowing the turnover rate and the steady-state concentration, one may calculate that both H6 and H129 cells synthesize 28S rRNA at a rate of about 0.25 pg/h per cell. The amount of template-bound Pol I activity was similar in nuclei isolated from H6 and H129 cell cultures. These data indicate that, although H6 cells contained 3-4 times more Pol I than did H129 cells, both cell lines synthesized rRNA at about the same rate.

The leukotriene B4 receptor agonist/antagonist activities of SC-45694 in human neutrophils.

SC-45694 (7-[4-(1-hydroxy-3Z-nonenyl)phenyl]-5S-hydroxy-6Z-hept enoic acid lithium salt), a representative of a new class of leukotriene B4 (LTB4) analogs that are conformationally restricted, was evaluated for effects on human neutrophil functions. SC-45694 inhibited [3H] LTB4 binding to the high-affinity receptors on human neutrophils with a KD value of 0.76 microM, but it was a very weak inhibitor of [3H]N-formyl-methionyl-leucyl-phenylalanine binding (KD > 83 microM). SC-45694 stimulated neutrophil chemotaxis with half-maximal and maximal effects at 1 and 10 microM, respectively, and produced a maximal chemotactic response similar to that produced by LTB4. The chemotactic activity of SC-45694 was blocked by the LTB4 receptor antagonists SC-41930 and LY-255283. At concentrations that showed agonist activity for neutrophil chemotactic response, SC-45694 showed no agonist activity for degranulation, antagonized LTB4-induced degranulation and inhibited [3H] LTB4 binding to low-affinity receptors. SC-45694 inhibited LTB4-induced maximal degranulation with an IC50 value of 0.3 microM, but it did not inhibit N-formyl-methionyl-leucyl-phenylalanine-induced degranulation. The inhibition by SC-45694 of LTB4-induced degranulation was time-dependent, noncompetitive and reversible. Thus SC-45694 exhibited a specific, full LTB4 agonist activity for chemotaxis and an antagonist activity against LTB4-induced degranulation. These properties suggest that members of the new class of LTB4 analogs, such as SC-45694, may be useful in further characterizing distinct LTB4 receptor subtypes that mediate these two neutrophil functions.

SC-41930: an inhibitor of leukotriene B4-stimulated human neutrophil functions.

SC-41930 was evaluated for effects on human neutrophil chemotaxis and degranulation. At concentrations up to 100 microM, SC-41930 alone exhibited no effect on neutrophil migration, but dose-dependently inhibited neutrophil chemotaxis induced by leukotriene B4 (LTB4) in a modified Boyden chamber. Concentrations of SC-41930 from 0.3 microM to 3 microM competitively inhibited LTB4-induced chemotaxis with a pA2 value of 6.35. While inactive at 10 microM against C5a-induced chemotaxis, SC-41930 inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced chemotaxis, with 10 times less potency than against LTB4-induced chemotaxis. SC-41930 inhibited [3H]LTB4 and [3H]fMLP binding to their receptor sites on human neutrophils with KD values of 0.2 microM and 2 microM, respectively. SC-41930 also inhibited neutrophil chemotaxis induced by 20-OH LTB or 12(R)-HETE. At concentrations up to 10 microM, SC-41930 alone did not cause neutrophil degranulation, but inhibited LTB4-induced degranulation in a noncompetitive manner. SC-41930 also inhibited fMLP- or C5a-induced degranulation, but was about 8 and 10 times less effective for fMLP and C5a, respectively. The results indicate that SC-41930 is a human neutrophil LTB4 receptor antagonist with greater specificity for LTB4 than for fMLP or C5a receptors.

Characterization of prostaglandin E receptors in canine small intestine using [3H] prostaglandin E1 binding.

Prostaglandin E (PGE) receptors in canine small intestinal mucosal and muscle membrane preparations were labeled with [3H] PGE1. Saturable, high affinity binding of [3H] PGE1 was observed in both preparations. The density of binding sites (fmol/mg protein) was 39 for mucosal membranes and 60 for muscle membranes, with corresponding dissociation constants of 10.6 nM and 5.8 nM, respectively. [3H] PGE1 binding sites in both preparations showed stereospecificity and high affinity for natural PGE1 and PGE2, but not for I or F-type PGs. Synthetic PGEs such as misoprostol and enisoprost had lower affinity than PGE1 or PGE2. Several analogs of enisoprost bound weakly to the binding sites. A highly significant correlation (C.C. = 0.9) was demonstrated between mucosal and muscle binding potency for a series of enisoprost analogs. There was also a significant positive correlation between the receptor binding potency and rat diarrheagenic activity for these analogs. These results indicate that PGE receptors in canine intestinal mucosa and muscle can be directly studied with [3H] PGE1 binding. The mucosal and muscle PGE receptors may have similar ligand binding specificity. We speculate that these receptors are likely to be associated with the diarrheagenic activity of PGEs.

The in vitro pharmacology of SC-51146: a potent antagonist of leukotriene B4 receptors.

Previously, we reported that SC-41930 is a potent leukotriene B4 (LTB4) receptor antagonist. An analog of SC-41930, SC-51146, was evaluated as an antagonist of LTB4 receptors. SC-51146 was shown to bind to LTB4 high affinity binding sites on human neutrophils (PMN) with a dissociation constant (KD) value of 1.5 +/- 0.1 nM, compared to 19 +/- 1.3 nM for SC-41930. PMN chemotaxis studies and Scatchard analyses of [3H]LTB4 binding in PMN membranes indicated that SC-51146 acted as a competitive antagonist. The IC50 value of SC-51146 for the inhibition of PMN chemotaxis induced by 30 nM LTB4 was 38 +/- 12 nM. SC-51146 inhibited PMN degranulation induced by 50 nM LTB4 with an IC50 value of 29 +/- 7 nM. The antagonism by SC-51146 of LTB4-induced PMN degranulation appeared to be noncompetitive. The specificity of SC-51146 for LTB4 receptors vs. fMLP receptors was improved approximately 29 and 44 times over SC-41930. SC-51146 showed relatively weak inhibitory activity on the production of superoxide, LTB4 and/or prostaglandin E2 by human PMN or HL-60 cells. SC-51146 had little activity on ram seminal vesicle cyclooxygenase, and no activity on porcine pancreatic phospholipase A2. SC-51146 is a racemate comprised of the (+) enantiomer, SC-53228, and the (-) enantiomer, SC-53229. Both stereoisomers exhibited pharmacological profiles similar to SC-51146 in these aforementioned in vitro systems. The highly potent and specific antagonistic action of SC-51146 on LTB4 receptors should be particularly useful in elucidating the role of LTB4 in the pathogenesis of inflammatory diseases where excessive levels of LTB4 have been reported.

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions.

A leukotriene B4 (LTB4) analog, 20-trifluoromethyl LTB4 (20CF3-LTB4), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF3-LTB4 was equipotent to LTB4 as a chemoattractant (EC50, 3 nM), produced 50% of maximal activity of LTB4, and competed with [H] LTB4 for binding to intact human neutrophil LTB4 receptors. In contrast to chemotactic activity, 20CF3-LTB4 in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 microM on LTB4-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB4, 20CF3-LTB4 induced neutrophil desensitization to degranulation by LTB4. The results indicate that hydrogen atoms at C-20 of LTB4 are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF3-LTB4 is a partial agonist for chemotaxis and an antagonist for degranulation suggests that different LTB4 receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB4 can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.

Multiple actions of the leukotriene B4 receptor antagonist SC-41930.

7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro-8- propyl-2H-1-benzopyran-2-carboxylic acid (SC-41930), a leukotriene B4 (LTB4) receptor antagonist with anti-inflammatory activity in animal models of colitis, was evaluated for effects on superoxide, LTB4 and prostaglandin E2 production. SC-41930 inhibited human neutrophil (PMN) superoxide generation maximally stimulated by f-Met-Leu-Phe (IC50 4 microM) and C5a (IC50 approximately 12 microM). Moreover, postreceptor stimulation of superoxide production by NaF (a G protein activator), but not by phorbol myristate acetate, was significantly inhibited by SC-41930, indicating that SC-41930 may act via attenuation of a G protein-mediated signal transduction. SC-41930 also inhibited A23187-stimulated LTB4 production (IC50 5.3 microM) in human PMN as well as LTB4 (IC50 2.1 microM) and prostaglandin E2 (IC50 2.9 microM) production in HL-60 cells. When coinjected intradermally (400 micrograms/site), SC-41930 inhibited A23187-stimulated increases in LTB4 levels in guinea pig skin. SC-41930 inhibited human synovial phospholipase A2 (IC50 72 microM), A23187-stimulated 5-hydroxy-eicosatetranoic acid production in human PMN (IC50 8.5 microM), and rat peritoneal leukotriene A4 hydrolase (IC50 20 microM), but not ram seminal vesical cyclooxygenase. The results suggest that the anti-inflammatory activity of SC-41930 could be attributed to postreceptor inhibition of inflammatory mediator production by PMN and other cells in addition to antagonism of PMN LTB4 receptors.

Preferential binding of the novel prostaglandin SC-46275 to canine gastric versus intestinal receptors.

Prostaglandins (PGs) in the E-series exhibit potent gastric antisecretory activity, but can also cause diarrhea, which is mediated via PGE receptors. SC-46275, an omega-chain cyclopentenyl analog of the E-type PG enisoprost, was evaluated with other E-PGs for PGE receptor binding activity in gastric and intestinal tissues. SC-46275, enisoprost, misoprostol and PGE1 were first evaluated in enriched canine gastric parietal cells with [3H]misoprostol free acid binding and subsequently with [3H]PGE1 binding in canine intestinal tissues where misoprostol free acid had weak receptor binding activity. The receptor binding potency of SC-46275 (IC50, 0.013 mM) in enriched canine parietal cell preparations was found to be much greater than misoprostol and enisoprost (IC50, 10 and 8 nM), whereas PGE1 had the least potency (IC50, 37 nM). Similar relative potencies for these PGs were also obtained in the inhibition of histamine-stimulated acid secretion in enriched parietal cell preparations. In small intestinal mucosal and muscle membranes, the receptor binding potency of SC-46275 (IC50, 13 and 20 microM) was much less than misoprostol or enisoprost (IC50, 0.39-1.2 microM) and substantially less than PGE1 (IC50, 0.017 and 0.066 microM). This weak binding activity of SC-46275 in intestinal tissues is consistent with its reported weak diarrheagenic activity in the rat. These results suggest that SC-46275 binds preferentially to gastric vs. intestinal PGE receptors and is specific for the EP3 receptors.

The design and synthesis of second generation leukotriene B4 (LTB4) receptor antagonists related to SC-41930.

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)propoxy]-3,4- dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, is a selective, orally active, LTB4 receptor antagonist currently in clinical trials for psoriasis and ulcerative colitis. Exhaustive SAR studies found a potential backup compound, SC-50605, which was 7-16 times more potent than SC-41930 in LTB4 receptor binding, chemotaxis and degranulation assays. SC-50605 also inhibited LTB4-induced intradermal chemotaxis in cavine skin at an oral dose of 0.10 mg/kg and displayed good activity in animal models of colitis and epidermal inflammation both orally and topically.