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[Image: see text] IκB kinase β (IKKβ or IKK2) is a key regulator of nuclear factor kappa B (NF-κB) and has received attention as a therapeutic target. Herein we report on the optimization of a series of 3,5-disubstituted-indole-7-carboxamides for oral activity. In doing so, we focused attention on potency, ligand efficiency (LE), and physicochemical properties and have identified compounds 24 and (R)-28 as having robust in vivo activity.

Tyrosine ureas had been identified as potent muscarinic receptor antagonists with promising in vivo activity. Controlling the stereochemistry of the chiral quaternary ammonium center had proved to be a serious issue for this series, however. Herein we describe the preparation and SAR of tyrosine urea antagonists containing achiral quaternary ammonium centers. The most successful such moiety was the 2-methylimidazo[2,1-b][1,3]thiazol-7-ium group which yielded highly potent antagonists with long duration of action in an inhaled animal model of bronchoconstriction.

A novel series of N-substituted tropane derivatives was characterized as potent muscarinic acetylcholine receptor antagonists (mAChRs). Kinetic washout studies showed that the N-endosubstituted analog 24 displayed much slower reversibility at mAChRs than the methyl-substituted parent molecule darotropium. In addition, it was shown that this characteristic appeared to translate into enhanced which duration of action in a mouse model of bronchonstriction.

A series of novel biphenyl piperazines was discovered as highly potent muscarinic acetylcholine receptor antagonists via high throughput screening and subsequent optimization. Compound 5c with respective 500- and 20-fold subtype selectivity for M3 over M2 and M1 exhibited excellent inhibitory activity and long duration of action in a bronchoconstriction in vivo model in mice via intranasal administration. The novel inhaled mAChR antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease.

High throughput screening and subsequent optimization led to the discovery of novel quaternary ammonium salts as highly potent muscarinic acetylcholine receptor antagonists with excellent selectivity. Compounds 8a, 13a, and 13b showed excellent inhibitory activity and long duration of action in bronchoconstriction in vivo models in two species via intranasal or intratracheal administration. The novel inhaled muscarinic receptor antagonists are potentially useful therapeutic agents for the treatment of chronic obstructive pulmonary disease and other bronchoconstriction disorders.

In the course of our research program to develop novel muscarinic receptor antagonists for the treatment of COPD, new tropane carbamate derivatives were identified as potent anti-muscarinic agents. The synthesis, structure-activity relationships and pharmacological evaluation that led to the identification of compound 5o, are described.

Objective: The objective of this study was to investigate the renal effects of celecoxib, a cyclooxygenase-2 (COX-2) specific nonsteroidal anti-inflammatory drug (NSAID). Subjects and treatment: Six volume-depleted, conscious, chronically instrumented dogs received four treatments using a cross-over design, with at least 13 days between treatments. An i.v. bolus dose (3, 10, and 30 μmol/kg) of vehicle, indomethacin, celecoxib, or 6-MNA, the active metabolite of nabumetone, were administered with an hour between doses. Methods: Renal function was assessed at baseline and 30 min after each dose. Results: Treatment with indomethacin or celecoxib resulted in a dose-dependent reduction in renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow, sodium excretion, and fractional sodium excretion. GFR was reduced by 27% with 30 μmol/kg indomethacin (p < 0.05) and by 58% with 30 μmol/kg celecoxib (p < 0.5). Similarly, RPF was significantly (p < 0.05) reduced by 33% and 65% with 30 μmol/kg indomethacin and celecoxib, respectively. Reductions in renal function with celecoxib occurred at a therapeutic index (determined by comparison anti-edemic activities of the drugs in rat paw carrageenan assays) equal to or lower than indomethacin. In contrast, treatment with vehicle or 6-MNA resulted in no reductions in renal function. Conclusion: Administration of either celecoxib (specific COX-2 inhibitor) or indomethacin (selective COX-1 inhibitor) resulted in significant reductions in renal function. COX-2 specific inhibitors do not appear to lack the renal effects of NSAIDs.

Objective To evaluate the effects of SB 242235, a potent and selective inhibitor of p38 mitogen-activated protein (MAP) kinase, on joint integrity in rats with adjuvant-induced arthritis (AIA). Methods Male Lewis rats with AIA were orally treated either prophylactically (days 0–20) or therapeutically (days 10–20) with SB 242235. Efficacy was determined by measurements of paw inflammation, dual-energy x-ray absorptiometry for bone mineral density (BMD), magnetic resonance imaging (MRI), micro–computed tomography (CT), and histologic evaluation. Serum tumor necrosis factor α (TNFα) in normal (non-AIA) rats and serum interleukin-6 (IL-6) levels in rats with AIA were measured as markers of the antiinflammatory effects of the compound. Results SB 242235 inhibited lipopolysaccharide- stimulated serum levels of TNFα in normal rats, with a median effective dose of 3.99 mg/kg. When SB 242235 was administered to AIA rats prophylactically on days 0–20, it inhibited paw edema at 30 mg/kg and 10 mg/kg per day by 56% and 33%, respectively. Therapeutic administration on days 10–20 was also effective, and inhibition of paw edema was observed at 60, 30, and 10 mg/kg (73%, 51%, and 19%, respectively). Significant improvement in joint integrity was demonstrated by showing normalization of BMD and also by MRI and micro-CT analysis. Protection of bone, cartilage, and soft tissues was also shown histologically. Serum IL-6 levels were decreased in AIA rats treated with the 60 mg/kg dose of compound. Conclusion Symptoms of AIA in rats were significantly reduced by both prophylactic and therapeutic treatment with the p38 MAP kinase inhibitor, SB 242235. Results from measurements of paw inflammation, assessment of BMD, MRI, and micro-CT indicate that this compound exerts a protective effect on joint integrity, and thus appears to have disease-modifying properties.

Pyrimidine analogs of the pyrimidinylimidazole class of CSBP/p38 kinase inhibitors were prepared in an effort to reduce the potent inhibition of hepatic cytochrome P450 observed for the pyridinyl compounds. The substitution of pyrimidin-4-yl, 2-methoxypyrimidin-4-yl, or 2-methylaminopyrimidin-4-yl for pyridin-4-yl effectively dissociates CSBP/p38 kinase from P450 inhibition for this series and furthermore achieves an increase in oral activity.

Activation of muscarinic subtype 3 (M3) muscarinic cholinergic receptors (mAChRs) increases airway tone, whereas its blockade improves lung function and quality of life in patients with pulmonary diseases. The present study evaluated the pharmacological properties of a novel mAChR antagonist, GSK573719 (4-[hydroxy(diphenyl)methyl]-1-{2-[(phenylmethyl)oxy]ethyl}-1-azoniabicyclo[2.2.2]octane; umeclidinium). The affinity (Ki) of GSK573719 for the cloned human M1-M5 mAChRs ranged from 0.05 to 0.16 nM. Dissociation of [(3)H]GSK573719 from the M3 mAChR was slower than that for the M2 mAChR [half-life (t1/2) values: 82 and 9 minutes, respectively]. In Chinese hamster ovary cells transfected with recombinant human M3 mAChRs, GSK573719 demonstrated picomolar potency (-log pA2 = 23.9 pM) in an acetylcholine (Ach)-mediated Ca(2+) mobilization assay. Concentration-response curves indicate competitive antagonism with partial reversibility after drug washout. Using isolated human bronchial strips, GSK573719 was also potent and showed competitive antagonism (-log pA2 = 316 pM) versus carbachol, and was slowly reversible in a concentration-dependent manner (1-100 nM). The time to 50% restoration of contraction at 10 nM was about 381 minutes (versus 413 minutes for tiotropium bromide). In mice, the ED50 value was 0.02 μg/mouse intranasally. In conscious guinea pigs, intratracheal administration of GSK573719 dose dependently blocked Ach-induced bronchoconstriction with long duration of action, and was comparable to tiotropium; 2.5 μg elicited 50% bronchoprotection for >24 hours. Thus, GSK573719 is a potent anticholinergic agent that demonstrates slow functional reversibility at the human M3 mAChR and long duration of action in animal models. This pharmacological profile translated into a 24-hour duration of bronchodilation in vivo, which suggested umeclidinium will be a once-daily inhaled treatment of pulmonary diseases.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.

The antiarthritic, organogold compound auranofin was evaluated for its antiinflammatory activity in murine models of cutaneous inflammation. Initial studies involved the use of arachidonic acid-induced inflammation, an evanescent response characterized by edema and neutrophil infiltration mediated, at least in part, by leukotrienes. The edema and myeloperoxidase (MPO) activity induced by application of arachidonic acid was assayed from the inflamed ears. After application of auranofin (1 mg or 1.47 mmol/ear in solution), inhibition of the MPO response was observed but little inhibition of the edematous response occurred. The application of 50 ml of 2% auranofin ointment (∼ 1.47 mmol/ear) administered after arachidonic acid resulted in the inhibition of both the edematous (54%) and the MPO (50%) components of the inflammatory response to arachidonic acid. It was found that auranofin in vitro weakly inhibited MPO activity, suggesting that inhibition of MPO-derived oxidants might contribute to the antiinflammatory activity. In order to characterize further this antiinflammatory activity, the effect of auranofin on the response to the cutaneous application of phorbol ester (PMA) was studied. This psoriaform lesion involves intense infiltration of neurotrophils, microabcess formation and epidermal cell proliferation. The ear thickness and the MPO response were measured at 4 h after PMA application. In this model, auranofin in solution displayed marked, dose-related antiinflammatory activity. The ED50s were calculated to be 0.12 (0.18 mmol) and 0.14 mg (0.21 mmol)/ear, respectively. An investigation of the antiinflammatory activity of the ointment was limited to the edematous response because of interference with the MPO analysis; however, potent antiinflammatory activity of the auranofin ointment proved to be dose-related with an ED50 of 0.3% (0.22 mmol) at 2 h and 0.6% (0.44 mmol) at 4 h after PMA. In addition, if the response was allowed to proceed for 2 h before the application of ointment, further progression of the inflammation was essentially abolished. Despite strong inhibition of the inflammatory response, no significant alteration of PMA-induced increased DNA content was demonstrable. Overall, the completeness of inhibition and potency observed with auranofin suggest that it may be of utility in the treatment of inflammatory diseases of the skin. ©1995 Wiley-Liss, Inc.

The antiinflammatory activity of rolipram, a selective inhibitor of the cyclic AMP-specific phosphodiesterase (PDE IV), was studied. Rolipram did not inhibit 5-lipoxygenase activity but did inhibit human monocyte production of leukotriene B4 (LTB4, IC50 3.5 microM). Likewise, murine mast cell release of leukotriene C4 and histamine was inhibited. In vivo, rolipram inhibited arachidonic acid-induced inflammation in the mouse, while the low Km-cyclic-GMP PDE inhibitor, zaprinast, did not inhibit. Rolipram had a modest effect on LTB4 production in the mouse, but markedly reduced LTB4-induced PMN infiltration. Beta-adrenergic receptor activation of adenylate cyclase was important for rolipram antiinflammatory activity since beta blockade abrogated arachidonic acid-induced inflammation. Thus, the antiinflammatory profile of rolipram is novel and may result from inhibition of PMN function and perhaps vasoactive amine release and leukotriene biosynthesis. These actions may be dependent upon endogenous beta-adrenergic activity and are likely mediated through inhibition of PDE IV.

A series of N-hydroxyurea derivatives have been prepared and examined as inhibitors of 5-lipoxygenase. Oral activity was established by examining the inhibition of LTB4 biosynthesis in an ex vivo assay in the mouse. The pharmacodynamic performance in the mouse of selected compounds was assessed using an ex vivo LTB4 assay and an adoptive peritoneal anaphylaxis assay at extended pretreat times. Compounds with an extended duration of action were re-examined as the individual enantiomers in the ex vivo assay, and the (S) enantiomer of N-hydroxy-N-[2,3-dihydro-6-(phenylmethoxy)-3-benzofuranyl]urea, (+)-1a (SB 202235), was selected as the compound with the best overall profile. Higher plasma concentrations and longer plasma half-lives were found for (+)-1a relative to its enantiomer in the mouse, monkey, and dog. In vitro metabolic studies in mouse liver microsomes established enantiospecific glucuronidation as a likely mechanism for the observed differences between the enantiomers of 1a. Enantioselective glucuronidation favoring (-)-1a was also found in human liver microsomes.

Novel tropane derivatives were characterized as muscarinic acetylcholine receptor antagonists (mAChRs). Through optimization of the structure-activity relationship around the tropane scaffold, the quaternary ammonium salt 34 was identified as a very potent M(3) mAChR antagonist. The compound was functionally active and displayed greater than 24 h duration of action in a mouse model of bronchoconstriction.

A novel 4-hydroxyl(diphenyl)methyl substituted quinuclidine series was discovered as a very promising class of muscarinic antagonists. The structure-activity relationships of the connectivity of the diphenyl moiety to the quinuclidine core and around the ring nitrogen side chain are described. Computational docking studies using an homology model of the M(3) receptor readily explained the observed structure-activity relationship of the various compounds. Compound 14o was identified as a very potent, slowly reversible M(3) antagonist with a very long in vivo duration of bronchoprotection.

Design and syntheses of a novel series of muscarinic antagonists are reported. These efforts have culminated in the discovery of (3-endo)-3-(2-cyano-2,2-diphenylethyl)-8,8-dimethyl-8-azoniabicyclo[3.2.1]octane bromide (4a) as a potent and pan-active muscarinic antagonist as well as a functionally active compound in a murine model of bronchoconstriction. The compound has also displayed pharmacokinetic characteristics suitable for inhaled delivery.

SK&F 105809 {2-(4- methylsulfinylphenyl )-3-(4- pyridyl )-6,7- dihydro -[5H]- pyrrolo [l,2-a] imidazole} was determined to be a prodrug for the sulfide metabolite SK&F 105561 {2-(4- methyltniophenyl )-3-(4- pyridyl )-6,7- dihydro -[5H]- pyrrolo [1,2-a] imidazole} which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo . SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration ( ic 50 ) of 3 μM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The ic 50 was 100 μM in the absence of peroxidase activity, whereas an ic 50 of 3 μM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) production with ic 50 values of 0.1 and 2 μM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited ( ic 50 2 μM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B 2 and LTB 4 production ex vivo with a half-maximal dose ( ed 50 ) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an ic 50 of greater than 200 μM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A 2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB 4 or leukotriene D 4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

SK&F 105809 [2-(4-methylsulfinylphenyl)-3-(4-pyridyl)- 6,7-dihydro-[5H]-pyrrolo[1,2,a] imidazole] demonstrated unique antiinflammatory activities in murine models that are resistant to selective cyclooxygenase (CO) inhibitors. Both edema and inflammatory cell infiltration induced by the topical application of arachidonic acid to the mouse ear were decreased by SK&F 105809 (ED50 values of 44 mg/kg, p.o.). Polymorphonuclear leukocyte (PMN) infiltration following the intraperitoneal injection of either monosodium urate crystal or carrageenan was inhibited with ED50 values of 64 and 72 mg/kg, p.o., respectively. These inflammatory responses were unaffected by the selective cyclooxygenase inhibitor naproxen. SK&F 105809 also inhibited leukotriene B4 (LTB4) and prostaglandin E2 production in vivo in arachidonic acid-induced inflammatory exudates (ED50 values of 41 and 15 mg/kg, p.o., respectively). The inhibition of LTB4 production preceded the inhibition of PMN infiltration. The impact of inhibition of both 5-lipoxygenase (5-LO) and CO was seen with platelet-activating factor-induced vascular permeability which was inhibited markedly by SK&F 105809. However, the 5-LO inhibitor, phenidone, only strongly inhibited when coadministered with the selective CO inhibitor, indomethacin. In spite of a short half-life (14-18 min) for both SK&F 105809 and the active metabolite SK&F 105561 [2-(4- methylthiophenyl)-3-(4-pyridyl)-6,7-dihydro-[5H]-pyrrolo[1,2-a] imidazole], the pharmacological activity lasted at least 1.5 hr. The biochemical evidence of inhibition of interleukin-1 (IL-1) production and 5-LO and CO activity, in vitro, by the metabolite (SK&F 105561) seen in the companion paper (Marshall PJ, Griswold DE, Breton J. Webb EF, Hillegass LM, Sarau HM, Newton J Jr, Lee JC, Bender PE and Hanna N, Pharmacology of the pyrroloimidazole, SK&F 105809--I. Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid. Biochem Pharmacol 42: 813-824, 1991) and inhibition of the fluid and cellular phases of the inflammatory response, in vivo, by SK&F 105809 suggest that this compound possesses a unique profile of activity.

Optimization of a series of N-1-cycloalkyl-4-aryl-5-(pyrimidin-4-yl)imidazole inhibitors of p38 kinase is reported. Oral administration of inhibitors possessing a cyclohexan-4-ol or piperidin-4-yl group at N-1 in combination with alkoxy, amino(alkyl), phenoxy and anilino substitution at the 2-position of the pyrimidine was found to potently inhibit LPS-induced TNF in mice and rats. The selectivity of these new inhibitors for p38 kinase versus eight other protein kinases is high and in all cases exceeds that of SB 203580.

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [ trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole; IC50 = 44 nM vs. p38α], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3–30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC50 of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.

An analysis was conducted of the cytokine profile and inflammatory response in oxazolone sensitized mouse skin. Following exposure to oxazolone, the intralesional production of inflammatory cytokines was demonstrable at the levels of both mRNA and protein. An initial challenge led to a transient increase in tumor necrosis factor-alpha production followed predominately by the T helper (Th)1 cytokine, interferon-gamma. There was a minimal production of interleukin-4, a Th2 cytokine. Continued exposure to oxazolone led to a downregulation of interferon-gamma and an upregulation of interleukin-4 production. A strong relationship was found between interleukin-4 and the inflammatory response, as measured by ear thickness. Similar experiments conducted in mast cell-deficient mice revealed reduced neutrophil influx but only minor changes in cytokine profile. An irritant response induced by chronic exposure of mouse skin to phorbol ester did not reveal any significant interferon-gamma or interleukin-4 response but was characterized by a tumor necrosis factor-alpha response that correlated with the inflammatory response. These observations suggest that the major source of interferon-gamma and interleukin-4 in the oxazolone response may be the infiltrating lymphocytes; whereas the tumor necrosis factor-alpha may result from the local irritation seen with both oxazolone and phorbol ester. At the end of 4 wk of chronic exposure to oxazolone, it was found that serum IgE levels had significantly increased. Histologic analysis of the skin lesion revealed that a mixed infiltrate including eosinophils developed upon repeat exposure to oxazolone. These findings are consistent with an early predominate Th1 response that is reduced and largely replaced with a Th2 response upon chronic T cell activation.