Design and synthesis of a series of (2R)-N(4)-hydroxy-2-(3-hydroxybenzyl)-N(1)- [(1S,2R)-2-hydroxy-2,3-dihydro-1H-inden-1-yl]butanediamide derivatives as potent, selective, and orally bioavailable aggrecanase inhibitors.

A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.

Discovery of macrocyclic hydroxamic acids containing biphenylmethyl derivatives at P1', a series of selective TNF-alpha converting enzyme inhibitors with potent cellular activity in the inhibition of TNF-alpha release.

SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of

Biology of TACE inhibition.

Studies conducted over the past decade have demonstrated a central role for tumour necrosis factor alpha (TNFalpha) in inflammatory diseases. As a result of this work, a number of biological agents that neutralise the activity of this cytokine have entered the clinic. The recent clinical data obtained with etanercept and infliximab highlight the relevance of this strategy. TNFalpha converting enzyme (TACE) is the metalloproteinase that processes the 26 kDa membrane bound precursor of TNFalpha (proTNFalpha) to the 17 kDa soluble component. Although a number of proteases have been shown to process proTNFalpha, none do so with the efficiency of TACE. A series of orally bioavailable, selective, and potent TACE inhibitors are currently in clinical development. These inhibitors effectively block TACE mediated processing of proTNFalpha and can reduce TNF production by lipopolysaccharide stimulated whole blood by >95%. Through a series of studies it is shown here that >80% of the unprocessed proTNFalpha is degraded intracellularly. The remainder appears to be transiently expressed on the cell surface. Although, in vitro, TACE inhibition has also been implicated in shedding of p55 and p75 surface TNFalpha receptors, the in vivo data cast doubt on the consequences of this finding. In a mouse model of collagen-induced arthritis, the inhibitors are efficacious both prophylactically and therapeutically. The efficacy seen is equivalent to strategies that neutralise TNFalpha. In many studies greater efficacy is observed with the TACE inhibitors, presumably owing to greater penetration to the site of TNFalpha production.

P1, P2'-linked macrocyclic amine derivatives as matrix metalloproteinase inhibitors.

A novel series of 13- and 14-membered macrocyclic amines was developed by linking the P1 and P2' groups. The synthesis entails stereoselective Frater alkylation to install the anti-succinate configuration and macrocyclic amination via nucleophilic displacement. This strategy resulted in a new class of conformationally constrained inhibitors that are potent and selective for MMP-8 and 9 over MMP-1 and 3.

Macrocyclic hydroxamate inhibitors of matrix metalloproteinases and TNF-alpha production.

Several macrocyclic, hydroxamate derivatives were synthesized and evaluated as inhibitors of matrix metalloproteinases (MMPs) and tumour necrosis factor-alpha (TNF-alpha) production. These macrocycles are anti-succinate based inhibitors linked from P1 to P2'. A variety of functionality was installed at the P1-P2' linkage, which gave inhibitors that displayed excellent MMP inhibition and good TNF-alpha suppression.

Terphenyl cyclooxygenase-2 (COX-2) inhibitors: optimization of the central ring and o-biphenyl analogs.

The discovery of terphenyl derivatives as highly selective COX-2 inhibitors resulted from our efforts to overcome poor pharmacokinetics demonstrated by the COX-2 selective diarylthiophene DuP 697 [2-bromo-4-(4'-sulfonylmethyl)phenyl-5-(4'-fluoro)phenylthiophe ne]. Detailed SAR related to the ortho-biphenyls and variants of the central ring are described herein.

Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes.

Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However, the importance of this event for monocyte function is not clear. To address this issue, we used the novel MAP/ERK kinase (MEK) inhibitor, U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK, Jun NH2-terminal kinase (JNK), and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However, the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound, confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function, we measured production of the cytokines IL-1, IL-8, and TNF, as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1, IL-8, TNF, or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS.

Structure-based design and synthesis of a series of hydroxamic acids with a quaternary-hydroxy group in P1 as inhibitors of matrix metalloproteinases.

Examination of the S1 area of the active site of pro-stromelysin has led us to the design of novel and potent inhibitors of matrix metalloproteinases containing constrained quaternary-hydroxy group at P1. The synthesis and biological activity of these compounds with variations at P1', P2', and P3' will be described.

The fate of pro-TNF-alpha following inhibition of metalloprotease-dependent processing to soluble TNF-alpha in human monocytes.

Human monocytes rapidly produce TNF-alpha following activation by bacterial LPS. The message for TNF-alpha encodes a 26-kDa protein that is proteolytically processed to the secreted 17-kDa form. Sequencing of the N terminus of the protein secreted by monocytes shows processing of the 26-kDa pro-TNF-alpha to a mature form at the projected metalloprotease cleavage site to generate 17-kDa TNF-alpha with the N terminus VRSSSR-. The addition of hydroxamic acid-based metalloprotease inhibitors to the cell culture is capable of blocking >95% of the production of soluble TNF-alpha and leads to a transient, but reproducible, increase in cell surface TNF-alpha as measured by FACS analysis. The cell surface TNF-alpha was demonstrated to increase the cell's ability to kill L929 tumor targets and induce PG production from human gingival fibroblasts. The buildup of cell surface TNF-alpha is unable to account for the TNF-alpha that is not secreted when inhibitor is present. Pulse-chase analysis of the cells demonstrates rapid degradation of the pro-TNF-alpha that remains unprocessed in the monocytes. Through inhibition of processing and secretion by brefeldin A, processing was shown to occur at a postendoplasmic reticulum site and is closely associated with movement to the cell surface.

Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II.

Prostaglandins have wide-ranging effects in the body and are thought to be important mediators of inflammation. Cyclooxygenase (COX) plays a key regulatory role in prostaglandin synthesis, and occurs in both constitutive (COX-1) and inducible (COX-2) isoforms. COX-1 is thought to provide cytoprotective effects, whereas COX-2 is both inducible and the major isoform of inflammatory cells. Reduction of prostaglandin production by inhibition of cyclooxygenases appears to be the main mechanism of action of most non-steroidal anti-inflammatory drugs (NSAIDS). Here we present an animal model of COX-2 deficiency that was generated by gene targeting. Defects in null mice correlating with reduced viability included renal alterations, characteristic of renal dysplasia (100% penetrance), and cardiac fibrosis (50% penetrance). Female Cox-2-/- mice were infertile. COX-2 deficiency failed to alter inflammatory responses in several standard models, but striking mitigation of endotoxin-induced hepatocellular cytotoxicity was observed.

2'-substituted chalcone derivatives as inhibitors of interleukin-1 biosynthesis.

A series of 2'-substituted chalcone derivatives has been found to show potent inhibition of the production of IL-1 beta from human peripheral blood monocytes stimulated with lipopolysaccharide (LPS), with IC50 values in the 0.2-5.0-microM range. Some members of the series have also shown inhibition of septic shock induced in mice by injection of LPS, although with low potency. Qualitative structure-activity relationships have shown that the enone is required for activity, which may be mediated by conjugate addition of a biological nucleophile to the chalcone. Electron-poor aromatic rings beta to the ketone give enhanced potency. Although electronic effects in the other ring (directly attached to the ketone) are minimal, this ring must possess an ortho substituent for good activity without cytotoxicity, suggesting a degree of selectivity which would not be expected for simple, nonspecific alkylating agents.