Utilization of a nitrogen-sulfur nonbonding interaction in the design of new 2-aminothiazol-5-yl-pyrimidines as p38α MAP kinase inhibitors.

The design, synthesis, and structure-activity relationships (SAR) of a series of 2-aminothiazol-5-yl-pyrimidines as novel p38α MAP kinase inhibitors are described. These efforts led to the identification of 41 as a potent p38α inhibitor that utilizes a unique nitrogen-sulfur intramolecular nonbonding interaction to stabilize the conformation required for binding to the p38α active site. X-ray crystallographic studies that confirm the proposed binding mode of this class of inhibitors in p38 α and provide evidence for the proposed intramolecular nitrogen-sulfur interaction are discussed.

Potent, selective, orally bioavailable inhibitors of tumor necrosis factor-alpha converting enzyme (TACE): discovery of indole, benzofuran, imidazopyridine and pyrazolopyridine P1' substituents.

Potent and selective inhibitors of tumor necrosis factor-alpha converting enzyme (TACE) were discovered with several new heterocyclic P1' groups in conjunction with cyclic beta-amino hydroxamic acid scaffolds. Among them, the pyrazolopyridine provided the best overall profile when combined with tetrahydropyran beta-amino hydroxamic acid scaffold. Specifically, inhibitor 49 showed IC(50) value of 1 nM against porcine TACE and 170 nM in the suppression of LPS-induced TNF-alpha of human whole blood. Compound 49 also displayed excellent selectivity over a wide panel of MMPs as well as excellent oral bioavailability (F%>90%) in rat n-in-1 PK studies.

Potent, exceptionally selective, orally bioavailable inhibitors of TNF-alpha Converting Enzyme (TACE): novel 2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamide P1' substituents.

Novel ((2-substituted-1H-benzo[d]imidazol-1-yl)methyl)benzamides were found to be excellent P1' substituents in conjunction with unique constrained beta-amino hydroxamic acid scaffolds for the discovery of potent selective inhibitors of TNF-alpha Converting Enzyme (TACE). Optimized examples proved potent for TACE, exceptionally selective over a wide panel of MMP and ADAM proteases, potent in the suppression of LPS-induced TNF-alpha in human whole blood and orally bioavailable.

Alpha,Beta-cyclic-beta-benzamido hydroxamic acids: Novel oxaspiro[4.4]nonane templates for the discovery of potent, selective, orally bioavailable inhibitors of tumor necrosis factor-alpha converting enzyme (TACE).

Two novel oxaspiro[4.4]nonane beta-benzamido hydroxamic scaffolds have been synthesized in enantio- and diasteriomerically pure form. These templates proved to be exceptional platforms that have led to the discovery of potent inhibitors of TACE that are active in a cellular assay measuring suppression of LPS-induced TNF-alpha. Furthermore, these inhibitors are selective against related MMPs, demonstrate permeability in a Caco-2 assay, and display good oral bioavailability.

Discovery of beta-benzamido hydroxamic acids as potent, selective, and orally bioavailable TACE inhibitors.

Beta-benzamido hydroxamic acids were discovered as potent TACE inhibitors. A computer model was constructed to help understanding the binding activities and guiding SAR study. SAR optimization led to the discovery of compound 30 which met all in vitro and in vivo criteria for the program and was selected for further evaluation.

Benzothiazole based inhibitors of p38alpha MAP kinase.

Rational design, synthesis, and SAR studies of a novel class of benzothiazole based inhibitors of p38alpha MAP kinase are described. The issue of metabolic instability associated with vicinal phenyl, benzo[d]thiazol-6-yl oxazoles/imidazoles was addressed by the replacement of the central oxazole or imidazole ring with an aminopyrazole system. The proposed binding mode of this new class of p38alpha inhibitors was confirmed by X-ray crystallographic studies of a representative inhibitor (6a) bound to the p38alpha enzyme.

Extracellular signal-regulated kinase-dependent interstitial macrophage proliferation in the obstructed mouse kidney.

AIM: A number of growth factors have been shown to induce proliferation of renal cell types in animal models of kidney disease. In vitro studies suggest that many such growth factors induce renal cell proliferation through the extracellular signal-regulated kinase (ERK) pathway. The aim of this study was to determine the functional role of ERK signalling in cell proliferation in the obstructed kidney. METHODS: Unilateral ureteric obstruction was induced in C57BL/6J mice which then received an ERK inhibitor drug (U0126 100 mg/kg t.i.d.), vehicle (DMSO) or no treatment, starting at day 2 after unilateral ureteric obstruction surgery and continuing until animals were killed on day 5. Cell proliferation was assessed by uptake of bromodeoxyuridine (BrdU). RESULTS: In normal mice, phosphorylation (activation) of ERK (p-ERK) was restricted to collecting ducts. Western blotting identified a marked increase in p-ERK in the obstructed kidney in the no-treatment and vehicle-treated groups. Immunostaining showed strong p-ERK staining in many tubules and in interstitial cells. U0126 treatment inhibited ERK phosphorylation as assessed by western blot and immunostaining. The number of BrdU+ cortical tubular cells was reduced by vehicle treatment but was not further changed by U0126 treatment. In contrast, interstitial cell proliferation in the obstructed kidney was unaltered by vehicle treatment, but this was significantly inhibited by U0126. This was associated with a reduction in interstitial macrophage accumulation, but no effect was seen upon interstitial accumulation of alpha-SMA+ myofibroblasts. Renal fibrosis, as assessed by collagen deposition, was unaffected by U0126 or vehicle treatment. CONCLUSION: These studies show that accumulation of interstitial macrophages in the obstructed kidney is, in part, dependent upon the ERK signalling pathway.

Role of the ERK pathway in psychostimulant-induced locomotor sensitization.

BACKGROUND: Repeated exposure to psychostimulants results in a progressive and long-lasting facilitation of the locomotor response that is thought to have implications for addiction. Psychostimulants and other drugs of abuse activate in specific brain areas extracellular signal-regulated kinase (ERK), an essential component of a signaling pathway involved in synaptic plasticity and long-term effects of drugs of abuse. Here we have investigated the role of ERK activation in the behavioral sensitization induced by repeated administration of psychostimulants in mice, using SL327, a brain-penetrating selective inhibitor of MAP-kinase/ERK kinase (MEK), the enzyme that selectively activates ERK. RESULTS: A dose of SL327 (30 mg/kg) that reduced the number of activated ERK-positive neurons by 62 to 89% in various brain areas, had virtually no effect on the spontaneous locomotor activity or the acute hyperlocomotion induced by cocaine or D-amphetamine. Pre-treatment with SL327 (30 mg/kg) prior to each drug administration prevented the locomotor sensitization induced by repeated injections of D-amphetamine or cocaine. The SL327 pre-treatment abolished also conditioned locomotor response of mice placed in the context previously paired with cocaine or D-amphetamine. In contrast, SL327 did not alter the expression of sensitized response to D-amphetamine or cocaine. CONCLUSION: Altogether these results show that ERK has a minor contribution to the acute locomotor effects of psychostimulants or to the expression of sensitized responses, whereas it is crucial for the acquisition of locomotor sensitization and psychostimulant-conditioned locomotor response. This study supports the important role of the ERK pathway in long-lasting behavioral alterations induced by drugs of abuse.

Long-term depression in the adult hippocampus in vivo involves activation of extracellular signal-regulated kinase and phosphorylation of Elk-1.

Protein kinase cascades likely play a critical role in the signaling events that underlie synaptic plasticity and memory. The extracellular signal-regulated kinase (ERK) cascade is suited well for such a role because its targets include regulators of gene expression. Here we report that the ERK cascade is recruited during long-term depression (LTD) of synaptic strength in area CA1 of the adult hippocampus in vivo and selectively impacts on phosphorylation of the nuclear transcription factor Elk-1. Using a combination of in vivo electrophysiology, biochemistry, pharmacology, and immunohistochemistry, we found the following: (1) ERK phosphorylation, including phosphorylation of nuclear ERK, and ERK phosphotransferase activity are increased markedly, albeit transiently, after the induction of NMDA receptor-dependent LTD at the commissural input to area CA1 pyramidal cells in the hippocampus of anesthetized adult rats; (2) LTD-inducing paired-pulse stimulation fails to produce lasting LTD in the presence of the ERK kinase inhibitor SL327, which suggests that ERK activation is necessary for the persistence of LTD; and (3) ERK activation during LTD results in increased phosphorylation of Elk-1 but not of the transcription factor cAMP response element-binding protein. Our findings indicate that the ERK cascade transduces signals from the synapse to the nucleus during LTD in hippocampal area CA1 in vivo, as it does during long-term potentiation in area CA1, but that the pattern of coupling of the ERK cascade to transcriptional regulators differs between the two forms of synaptic plasticity.

Regulation of extracellular signal-regulated kinase by cannabinoids in hippocampus.

Endocannabinoids form a novel class of intercellular messengers, the functions of which include retrograde signaling in the brain and mediation or modulation of several types of synaptic plasticity. Yet, the signaling mechanisms and long-term effects of the stimulation of CB1 cannabinoid receptors (CB1-R) are poorly understood. We show that anandamide, 2-arachidonoyl-glycerol, and Delta9-tetrahydrocannabinol (THC) activated extracellular signal-regulated kinase (ERK) in hippocampal slices. In living mice, THC activated ERK in hippocampal neurons and induced its accumulation in the nuclei of pyramidal cells in CA1 and CA3. Both effects were attributable to stimulation of CB1-R and activation of MAP kinase/ERK kinase (MEK). In hippocampal slices, the stimulation of ERK was independent of phosphatidyl-inositol-3-kinase but was regulated by cAMP. The endocannabinoid-induced stimulation of ERK was lost in Fyn knock-out mice, in slices and in vivo, although it was insensitive to inhibitors of Src-family tyrosine kinases in vitro, suggesting a noncatalytic role of Fyn. Finally, the effects of cannabinoids on ERK activation were dependent on the activity of glutamate NMDA receptors in vivo, but not in hippocampal slices, indicating the existence of several pathways linking CB1-R to the ERK cascade. In vivo THC induced the expression of immediate-early genes products (c-Fos protein, Zif268, and BDNF mRNAs), and this induction was prevented by an inhibitor of MEK. The strong potential of cannabinoids for inducing long-term alterations in hippocampal neurons through the activation of the ERK pathway may be important for the physiological control of synaptic plasticity and for the general effects of THC in the context of drug abuse.

Sultam hydroxamates as novel matrix metalloproteinase inhibitors.

In this communication we describe the design, synthesis, and evaluation of novel sultam hydroxamates 4 as MMP-2, -9, and -13 inhibitors. Compound 26 was found to be an active inhibitor (MMP-2 IC(50) = 1 nM) with 1000-fold selectivity over MMP-1 and good oral bioavailability (F = 43%) in mouse. An X-ray crystal structure of 26 in MMP-13 confirms the key hydrogen bonds and prime side binding in the active site.

Design, synthesis, and evaluation of benzothiadiazepine hydroxamates as selective tumor necrosis factor-alpha converting enzyme inhibitors.

Elevated levels of tumor necrosis factor-alpha (TNF-alpha) have been associated with several inflammatory diseases, and therefore, strategies for its suppression have become important targets in drug discovery. Our efforts to suppress TNF-alpha have centered on the inhibition of TNF-alpha converting enzyme (TACE) through the use of hydroxamate inhibitors. Starting from broad-spectrum matrix metalloproteinase (MMP) inhibitors, we have designed and synthesized novel benzothiadiazepines as potent and selective TACE inhibitors. The benzothiadiazepines were synthesized with variation in P1 and P1' in order to effect potency and selectivity. The inhibitors were evaluated versus porcine TACE (pTACE), and the initial selectivity was assessed with counterscreens of MMP-1, -2, and -9. Several potent and selective inhibitors were discovered with compound 41 being the most active against pTACE (K(i) = 5 nM) while still maintaining good selectivity versus the MMP's (at least 75-fold). Most compounds were assessed in the human peripheral blood mononuclear cell assay (PBMC) and the human whole blood assay (WBA) to determine their ability to suppress TNF-alpha. Compound 32 was the most potent compound in the PBMC assay (IC(50) = 0.35 microM), while compound 62 was the most active in the WBA (IC(50) = 1.4 microM).

Receptor density dictates the behavior of a subset of steroid ligands in glucocorticoid receptor-mediated transrepression.

By co-expressing glucocorticoid receptor (GR) and transcriptional reporter systems in GR-deficient Cos-7 cells, we profiled potency and efficacy of a panel of GR ligands as a function of GR expression levels (density). Our results show that potency and efficacy for GR full agonists, such as dexamethasone, in these transrepression assays are affected by receptor density. Intriguingly, receptor density dramatically influenced the behavior of the GR antagonist RU486 or the GR agonist medroxyprogesterone acetate (MPA). At high receptor density, both MPA and RU486 behaved as full agonists in transrepression: reducing GR density, however, resulted in conversion of these ligands from full agonist to full antagonists. In contrast, varying GR density could not convert cortisol and budesonide from GR agonists to antagonists. These results have clearly demonstrated, for the first time, an effect of receptor density on the agonist and antagonist properties of RU486 and MPA in GR-mediated transrepression.

The map kinase ERK regulates renal activity of cyclin-dependent kinase 2 in experimental glomerulonephritis.

BACKGROUND: In vitro, the extracellular signal-regulated kinase (ERK) is an intracellular convergence point of multiple stimuli, which affect the cell cycle. However, the role of ERK in cell cycle regulation in vivo is unknown. METHODS: To address this issue, ERK activity was blocked both in vitro in mesangial cells (MC) and in vivo in experimental glomerulonephritis (GN) by a pharmacological inhibitor (U0126) of the ERK-activating kinase. RESULTS: In stimulated MC, inhibition of ERK reduced cyclin-dependent kinase 2 (CDK2) phosphorylation, CDK2 activity and cyclin E/A expression, whereas downregulation of CDK inhibitor p27(Kip1) expression was inhibited. In vivo, U0126 was given to rats in the acute phase of anti-Thy 1.1 GN. We previously showed that glomerular cell proliferation was reduced by 67% upon treatment with the inhibitor compared to nephritic controls. Now, we detected a significant increase in renal CDK2-activity/phosphorylation in the nephritic controls, that was significantly and dose-dependently reduced by ERK inhibition. CDK2 activation was accompanied by an increase in renal expression of cyclins E/A and the enhanced binding of these cyclins to CDK2 in the nephritic controls. These changes were blunted by U0126 treatment. Finally, we noted an increased expression and CDK2-binding of p27(KIP1) protein in the nephritic controls which was decreased in U0126 treated rats. CONCLUSIONS: Our observations provide the first evidence that ERK is an intracellular regulator of renal CDK2 activity in vivo in a glomerulonephritis model.

Pharmacokinetics and pharmacodynamics of DPC 333 ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)), a potent and selective inhibitor of tumor necrosis factor alpha-converting enzyme in rodents, dogs, chimpanzees, and humans.

DPC 333 ((2R)-2-((3R)-3-amino-3{4-[2-methyl-4-quinolinyl) methoxy] phenyl}-2-oxopyrrolidinyl)-N-hydroxy-4-methylpentanamide)) is a potent and selective inhibitor of tumor necrosis factor (TNF)-alpha-converting enzyme (TACE). It significantly inhibits lipopolysaccharide-induced soluble TNF-alpha production in blood from rodents, chimpanzee, and human, with IC(50) values ranging from 17 to 100 nM. In rodent models of endotoxemia, DPC 333 inhibited the production of TNF-alpha in a dose-dependent manner, with an oral ED(50) ranging from 1.1 to 6.1 mg/kg. Oral dosing of DPC 333 at 5.5 mg/kg daily for 2 weeks in a rat collagen antibody-induced arthritis model suppressed the maximal response by approximately 50%. DPC 333 was distributed widely to tissues including the synovium, the site of action for antiarthritic drugs. Pharmacokinetic and pharmacodynamic studies in chimpanzee revealed a systemic clearance of 0.4 l/h/kg, a V(ss) of 0.6 l/kg, an oral bioavailability of 17%, and an ex vivo IC(50) for the suppression of TNF-alpha production of 55 nM (n = 1). In a phase I clinical trial with male volunteers after single escalating doses of oral DPC 333, the terminal half-life was between 3 and 6 h and the ex vivo IC(50) for suppressing TNF-alpha production was 113 nM. Measurement of the suppression of TNF-alpha production ex vivo may serve as a good biomarker in evaluating the therapeutic efficacy of TACE inhibitors. Overall, the pharmacological profiles of DPC 333 support the notion that suppression of TNF-alpha with TACE inhibitors like DPC 333 may provide a novel approach in the treatment of various inflammatory diseases including rheumatoid arthritis, via control of excessive TNF-alpha production.

A new 4-(2-methylquinolin-4-ylmethyl)phenyl P1' group for the beta-amino hydroxamic acid derived TACE inhibitors.

A new P1' group for TACE inhibitors was identified by eliminating the oxygen atom in the linker of the original 4-(2-methylquinolin-4-ylmethoxy)phenyl P1' group. Incorporation of this 4-(2-methylquinolin-4-ylmethyl)phenyl group onto different beta-aminohydroxamic acid cores provided compound 18, which demonstrated potent porcine TACE (p-TACE) and human whole blood activity, excellent PK properties, and good selectivity against a variety of MMPs.

Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe.

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.

Synthesis and structure-activity relationship of a novel, achiral series of TNF-alpha converting enzyme inhibitors.

A novel series of achiral TNF-alpha converting enzyme (TACE) inhibitors has been discovered. These compounds exhibited activities from 0.35 to 11nM in a porcine TACE assay and inhibited TNF-alpha production in an LPS-stimulated whole blood assay with an IC(50) value of 23nM for the most potent one. They also have excellent selectivities over related metalloproteases including aggrecanases.

Selective inhibition of eosinophil influx into the lung by small molecule CC chemokine receptor 3 antagonists in mouse models of allergic inflammation.

CC chemokine receptor (CCR) 3 is a chemokine receptor implicated in recruiting cells, particularly eosinophils (EPhi), to the lung in episodes of allergic asthma. To investigate the efficacy of selective, small molecule antagonists of CCR3, we developed a murine model of EPhi recruitment to the lung. Murine eotaxin was delivered intranasally to mice that had previously received i.p. injections of ovalbumin (OVA), and the effects were monitored by bronchoalveolar lavage. A selective eosinophilic influx was produced in animals receiving eotaxin but not saline. Furthermore, the number of EPhi was concentration- and time-dependent. Although anti-CCR3 antibody reduced the number of EPhi, the effect of eotaxin in OVA-sensitized mice was not a direct chemotactic stimulus because mast cell deficiency (in WBB6F1-Kitw/Kitw-v mice) significantly reduced the response. Two representative small molecule CCR3 antagonists from our program were characterized as being active at mouse CCR3. They were administered p.o. to wild-type mice and found to reduce eotaxin-elicited EPhi selectively in a dose-dependent manner. Pump infusion of one of the inhibitors to achieve steady-state levels showed that efficacy was not achieved at plasma concentrations equivalent to the in vitro chemotaxis IC90 but only at much higher concentrations. To extend the results from our recruitment model, we tested one of the inhibitors in an allergenic model of airway inflammation, generated by adoptive transfer of OVA-sensitive murine T helper 2 cells and aerosolized OVA challenge of recipient mice, and found that it inhibited EPhi recruitment. We conclude that small molecule CCR3 antagonists reduce pulmonary eosinophilic inflammation elicited by chemokine or allergenic challenge.