The discovery of sulfonylated dipeptides as potent VLA-4 antagonists.

Directed screening of a carboxylic acid-containing combinatorial library led to the discovery of potent inhibitors of the integrin VLA-4. Subsequent optimization by solid-phase synthesis afforded a series of sulfonylated dipeptide inhibitors with structural components that when combined in a single hybrid molecule gave a sub-nanomolar inhibitor as a lead for medicinal chemistry. Preliminary metabolic studies led to the discovery of substituted biphenyl derivatives with low picomolar activities. SAR and pharmacokinetic characterization of this series are presented.

Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Inhibition of matrix metalloproteinases by N-carboxyalkyl peptides.

An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.

Characterization of zinc-binding sites in human stromelysin-1: stoichiometry of the catalytic domain and identification of a cysteine ligand in the proenzyme.

A determination of the zinc stoichiometry of the catalytic domain of the human matrix metalloproteinase stromelysin-1 has been carried out using enzyme purified from recombinant Escherichia coli that express C-terminally truncated protein. Atomic absorption spectrometry revealed that both the proenzyme (prostrom255) and the mature active form (strom255) contained nearly 2 mol of Zn/mol of protein. Full-length prostromelysin purified from a mammalian cell culture line also contained zinc in excess of 1 equiv. While zinc in prostrom255 could not be removed by dialysis against o-phenanthroline, similar treatment of mature strom255 resulted in the loss of one-half of the original zinc content. The peptidase activity of the zinc-depleted protein was reduced by greater than 85% but could be restored upon addition of Zn2+ or Co2+. Addition of a thiol-containing inhibitor to a CoZn hybrid enzyme resulted in marked spectral changes in both the visible and ultraviolet regions characteristic of sulfur ligation to Co2+. This direct evidence for an integral role in catalysis and inhibitor binding confirms the location of the exchangeable metal at the active site. To examine the environment of zinc in the proenzyme, a fully cobalt-substituted proenzyme was prepared by in vivo metal replacement. The absorbance features of dicobalt prostrom255 were consistent with metal coordination by the single cysteine present in the propeptide, although the data do not allow assignment to a particular zinc site.(ABSTRACT TRUNCATED AT 250 WORDS)

Antioxidant-based inhibitors of leukotriene biosynthesis. The discovery of 6-[1-[2-(hydroxymethyl)phenyl]-1-propen-3-yl]-2,3-dihydro-5- benzofuranol, a potent topical antiinflammatory agent.

The leukotrienes, metabolites of arachidonic acid produced through the action of the enzyme 5-lipoxygenase, are important mediators of immediate hypersensitivity and inflammation. Among the variety of diseases in which the leukotrienes may play a symptomatic or causative role is the dermatological condition psoriasis, a chronic proliferative disease of the skin. This study reports the synthesis and comparative biological activities of various ortho-substituted phenols including 4-methoxyphenols, 6-hydroxy-1,2,3,4-tetrahydrobenzopyrans, 2,3-dihydro-5-benzofuranols, and 5-benzofuranols. The phenols prepared in this study were evaluated for their ability to inhibit the production of leukotriene B4(LTB4) in isolated human polymorphonuclear leukocytes (PMNs) and to inhibit a topical inflammatory response in the topical mouse ear (TME) model. In the former case, when the log IC50 was plotted versus the log of the octanol/water partition coefficient (log P), to eliminate the effect of lipophilicity, the 2,3-dihydro-5-benzofuranol ring system was shown to be more potent than the other ring systems examined throughout the range of partition coefficients studied. The ability to inhibit leukotriene production in vitro in human PMNs can be rationalized on the basis of a model that suggests that the observed inhibition is dependent on the kinetic ability of the inhibitor to reduce a radical species and on the fraction of inhibitor that is partitioned into the cell membrane. While the in vivo antiinflammatory activity as measured by the TME did not correlate with the in vitro data, it was felt that the TME represented a valuable measure of the ability of a compound to penetrate the skin to the site of an ongoing inflammatory response. Of the compounds synthesized in this study, 6-[1-[2-(hydroxymethyl)phenyl]-1-propen-3-yl]-2,3-dihydro-5-benzof uranol (1, L-651896) was chosen for further development.

2,3-Dihydro-5-benzofuranols as antioxidant-based inhibitors of leukotriene biosynthesis.

The enzymes that catalyze the oxidative metabolism of arachidonic acid have provided fertile ground for the development of useful therapeutic agents for nearly a quarter century. Inhibitors of the enzyme cyclooxygenase prevent the formation of the prostaglandins and thromboxanes and are clinically useful antiinflammatories and peripheral analgesics. More recently it has been discovered that the enzyme 5-lipoxygenase is the first step in the formation of a series of biologically important metabolites of arachidonic acid, the leukotrienes. Evidence suggests that an inhibitor of 5-lipoxygenase may be a useful therapeutic agent in the treatment of asthma, immediate hypersensitivity, and inflammation. Various antioxidants have been examined as inhibitors of 5-lipoxygenase in vitro. We were intrigued by recent reports that the 2,3-dihydro-5-benzofuranol ring system maximizes the stereoelectronic effects necessary for efficient hydrogen atom abstraction by peroxyl radicals. In this study we describe the synthesis of over 50 new 2,3-dihydro-5-benzofuranols and their biological evaluation as inhibitors of leukotriene biosynthesis in isolated human polymorphonuclear leukocytes. We show that the 2,3-dihydro-5-benzofuranol ring system, although not a potent inhibitor of leukotriene biosynthesis in itself, can provide a useful template for the design of antioxidant-based inhibitors of leukotriene biosynthesis. Furthermore, within a structural class the potency of a given analogue can be predicted on the basis of its overall calculated lipophilicity (log P). The data are interpreted in terms of a model in which the observed inhibition by this class of inhibitors is dependent on the intrinsic ability of the antioxidant to reduce the enzyme and on the fraction of the inhibitor that is partitioned into the membrane.