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An alpha4beta1/alpha4beta7 dual antagonist, 35S-compound 1, was used as a model ligand to study the effect of divalent cations on the activation state and ligand binding properties of alpha4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both alpha4beta1 and alpha4beta7, but 2S-[(1-benzenesulfonyl-pyrrolidine-2S-carbonyl)-amino]-4-[4-methyl-2S-(methyl-[2-[4-(3-o-tolyl-ureido)-phenyl]-acetyl]-amino) pentanoylamino]-butyric acid (BIO7662), a specific alpha4beta1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that alpha4beta1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly alpha4beta7 with a KD of 1.9 nM in the presence of 1 mM Mn2+, and binding was inhibited only 29% by BIO7662, suggesting that the probe is a potent antagonist of activated alpha4beta7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily alpha4beta1 with a KD of 18 nM. In contrast, the binding of 35S-compound 1 to Mn2+-activated Jurkat cells occurred slowly, reaching equilibrium by 60 min, and failed to dissociate within another 60 min. The ability of four alpha4beta1/alpha4beta7 antagonists to block binding of activated alpha4beta1 or alpha4beta7 to vascular cell adhesion molecule-1 or mucosal addressin cell adhesion molecule-1, respectively, or to 35S-compound 1 was measured, and a similar rank order of potency was observed for native ligand and probe. Inhibition of 35S-compound 1 binding to alpha4beta1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that alpha4beta1 and alpha4beta7 have distinct binding properties for the same ligand, and binding parameters are dependent on the state of integrin activation in response to different divalent cations.

The SAR of 1-sulfonyl-cyclopentyl carboxylic acid amides, ligands for the VLA-4 integrin, was investigated. This effort resulted in the identification of N-(3-phenylsulfonyl-3-piperidinoyl)-(L)-4-(2',6'-dimethoxyphenyl)phenylalanine 52 as a potent, selective VLA-4 antagonist (IC(50)=90 pM). Expansion of the SAR demonstrated that this structural unit can be used to identify a diverse series of sub-nanomolar antagonists.

A series of potent N-(aralkyl-, arylcycloalkyl-, and heteroaryl-acyl)-4-biphenylalanine VLA-4 antagonists was prepared by rapid analogue methods using solid-phase chemistry. Further optimization led to several highly potent compounds (IC(50) <1 nM). Evaluation of rat pharmacokinetic revealed generally high clearance.

The design, synthesis, and biological evaluation of N-arylprolyl-dipeptide derivatives as small molecule VLA-4 antagonists is described. Potency against VLA-4 and alpha(4)beta(7) and rat pharmacokinetic evaluation revealed some advantages over the related N-(arylsulfonyl)-prolyl-dipeptide analogues.

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to alpha(4)beta(1) or alpha(4)beta(7) on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human alpha(4)beta(7)-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC(50) values of 0.1 and 1 microM, respectively. Intravital microscopy was used to quantitate alpha(4)-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn(2+) to activate alpha(4)beta(7) binding to ligand, anti-alpha(4) monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-beta(1) mAb did not block adhesion, demonstrating that this interaction was dependent on alpha(4)beta(7). TR14035 blocked adhesion to HEVs [ED(50) of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, alpha(4)beta(7)/alpha(4)beta(1) antagonists blocked alpha(4)beta(7)-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.

Given the proposed involvement of VLA-4 in inflammatory processes, a program to identify orally active VLA-4 antagonists was initiated. Herein, we report the discovery of a N-tetrahydrofuroyl-(L)-phenylalanine derivative (17) and related analogues as potent VLA-4 antagonists with good oral bioavailability.

Acylated beta-amino acids are described as potent, specific and orally bioavailable antagonists of VLA-4. The initial lead was identified from a combinatorial library. Subsequent optimization using a traditional medicinal chemistry approach led to significant improvement in potency (up to 8-fold) while maintaining good pharmacokinetic properties.

A combinatorial library of 28 pools of 180 compounds (345 diastereomers) was designed and prepared in support of the delineation of the SAR of two prototypical VLA-4 antagonists. Deconvolution of the active pools led to the identification of three novel series of VLA-4 antagonists with low nanomolar potencies.

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.

We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.

The SAR of 2-pyridyl-3,5-diaryl pyrroles, ligands of the human glucagon receptor and inhibitors of p38 kinase, were investigated. This effort resulted in the identification of 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrr ole 49 (L-168,049), a potent (Kb = 25 nM), selective antagonist of glucagon.

A series of carbohydroxamido-oxazolidine inhibitors of UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase, the enzyme responsible for the second step in lipid A biosynthesis, was identified. The most potent analog L-161,240 showed an IC50 = 30 nM in the DEACET assay and displayed an MIC of 1-3 microg/mL against wild-type E. coli.

Investigation of furans, pyrroles and pyrazolones identified 3-pyridyl-2,5-diaryl-pyrroles as potent, orally bioavailable inhibitors of p38 kinase. 3-(4-pyridyl-2-(4-fluoro-phenyl)-5-(4-methylsulfinylphenyl)-pyrrol e (L-167307) reduces secondary paw swelling in the rat adjuvant arthritis model: ID50 = 7.4 mg/kg/b.i.d.

The design of P2-P3 conformational restrictions in renin inhibitors by the use of a renin computer graphic model led to the synthesis of inhibitors containing N-Boc, N-acetyl, and N-phthalyl derivatives of 3(S)-amino-4(R,S)-2-piperidones and 4(S)-amino-2-benzazepinones in place of phenylalanine in the control compound N-acetyl-L-phenylalanyl-N-[4(S)-[(butylamino)carbonyl]-1(S)- (cyclohexylmethyl)-2(S)-hydroxy-5-methylhexyl]-L-norleuci namide (32). The piperidone inhibitors were prepared by utilization of the Evans chiral auxilliary to introduce the amino group with enantioselectivity and also to act as a leaving group in an intramolecular cyclization to the piperidone. The most potent inhibitor, 3(S)-(acetylamino)-alpha(S)-butyl-N-[4(S)- [(butylamino)carbonyl]-1(S)-(cyclohexylmethyl)-2(S)-hydroxy-5- methylhexyl]-2-oxo-4(R)-phenyl-1-piperidineacetamide (18, IC50 = 21 nM), was 25-fold less potent than the acyclic control 32. Considerable dependence of potency with the size of the P4 derivative was observed as had been expected based on the presynthetic modeling studies. Attempts to rationalize the observed potencies on the basis of further molecular modeling studies suggested that the loss in inhibitor potency was due to the conformational restrictions distorting the 3S center from the geometry present in the putative extended conformation present when the inhibitor is bound within the renin active site.

In order to investigate the hypotensive mechanisms of action of peptide renin inhibitors, blood pressure responses to five renin inhibitors were compared with those to the angiotensin converting enzyme inhibitor, enalaprilat, in conscious African green and rhesus monkeys. (3S-4S)-4-amino-5-cyclohexyl-3-hydroxy pentanoic acid (ACHPA)-containing renin inhibitory peptide (ACRIP) and enalaprilat both decreased blood pressure in euvolemic and volume-depleted African green monkeys. However, while a maximum dose of enalaprilat reduced blood pressure to 80 +/- 4 and 56 +/- 4 mmHg in the euvolemic and volume-depleted monkeys, respectively, ACRIP lowered pressure to life-threatening levels (less than 40 mmHg) under both conditions. The relative potencies of ACRIP and four other renin inhibitors for inhibiting in vitro plasma renin activity (PRA; IC50) were compared with their potencies in reducing blood pressure by 15 mmHg (ED15 mmHg) and lowering blood pressure more than enalaprilat in volume-depleted rhesus monkeys. All renin inhibitors lowered blood pressure significantly beyond the maximal response to enalaprilat. Despite a significant correlation (r = 0.99, P less than 0.05) between the in vitro PRA inhibitory potency and the in vivo ED15 mmHg, doses which lowered blood pressure beyond the maximal responses to enalaprilat were not significantly correlated (r = 0.53, P greater than 0.05) with the in vitro PRA IC50 values. Furthermore, the profound depressor responses to renin inhibitors in rhesus monkeys were accompanied by increases in the heart rate and decreases in pulse pressure.(ABSTRACT TRUNCATED AT 250 WORDS)