An α4β1/α4β7 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 α4 integrins. In the presence of 1 mM each Ca2+/Mg2+, 35S-compound 1 bound to several cell lines expressing both α4β1 and α4β7, but 2 S -[(1-benzenesulfonyl-pyrrolidine-2 S -carbonyl)-amino]-4-[4-methyl-2 S -(methyl-{2-[4-(3- o -tolyl-ureido)-phenyl]-acetyl}-amino) pentanoylamino]-butyric acid (BIO7662), a specific α4β1 antagonist, completely inhibited 35S-compound 1 binding, suggesting that α4β1 was responsible for the observed binding. 35S-Compound 1 bound RPMI-8866 cells expressing predominantly α4β7 with a K D 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 α4β7. With Ca2+/Mg2+, 35S-compound 1 bound Jurkat cells expressing primarily α4β1 with a K D 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 α4β1/α4β7 antagonists to block binding of activated α4β1 or α4β7 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 α4β1 in Ca2+/Mg2+ was used to identify nonselective antagonists among these four. These studies demonstrate that α4β1 and α4β7 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 4 integrin, 4 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 4 1 and 4 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 4 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 4 7 to an I-MAdCAM-Ig fusion protein with IC50 values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to 4 1 or 4 7 on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human 4 7-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC50 values of 0.1 and 1 M, respectively. Intravital microscopy was used to quantitate 4-dependent adhesion of fluorescent murine lymphocytes in Peyer’s patch HEVs. When cells were prestimulated with 2 mM Mn to activate 4 7 binding to ligand, anti4 monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti1 mAb did not block adhesion, demonstrating that this interaction was dependent on 4 7. TR14035 blocked adhesion to HEVs [ED50 of 0.01–0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, 4 7/ 4 1 antagonists blocked 4 7-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow. The ability of lymphocytes to arrest under conditions of vascular flow enables their movement into both normal lymphoid tissues and sites of inflammation. Lymphocyte recruitment in the vasculature is regulated by the differential expression and activation of homing receptors (selectins and integrins) on lymphocytes that interact with counter-receptors of the Ig superfamily on high endothelial venules (HEVs) (Bargatze and Butcher, 1993; Bargatze et al., 1995). This interaction mediates a multistep process, involving rolling and tethering of leukocytes to endothelial ligands, rapid activation of integrins by locally released chemokines, stable adhesion of activated integrins to endothelial ligands, and transendothelial migration through the vessel wall (Bargatze et al., 1995; Warnock et al., 2000). Although all integrins expressed on leukocytes can mediate firm adhesion, 4 7 and 4 1 are members of a small subset of integrins that can also mediate rolling (Berlin et al., 1995). Mucosal addressin cell adhesion molecule-1 (MAdCAM-1), expressed on HEVs of Peyer’s patch and other gut-associated lymphoid tissues (GALTs), is the principal ligand for 4 7, an integrin highly expressed on gut-homing memory lymphocytes (Berlin et al., 1993; Shyjan et al., 1996; Briskin et al., 1997). 4 7 binds to MAdCAM-1 with higher affinity than to VCAM-1 or the CS-1 subdomain of human fibronectin. Although both 4 7 and 4 1 can bind VCAM-1 and CS-1, 4 1 does not bind MAdCAM-1 (Berlin et al., 1993). Both L-selectin and 4 7 mediate the initial attachment and rolling of lymphocytes by interacting with MAdCAM-1, whereas 4 7 ABBREVIATIONS: HEV, high endothelial venule; MAdCAM-1, mucosal addressin cell adhesion molecule-1; GALT, gut-associated lymphoid tissue; mAb, monoclonal antibody; mpk, milligrams per kilogram; VCAM-1, vascular cell adhesion molecule-1; PCR, polymerase chain reaction; DMSO, dimethyl sulfoxide; bp, base pair(s); GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; MLN, murine mesenteric lymph node; HBSS, Hanks’ balanced salt solution; Ab, antibody; AUC, area under the curve; LFA-1, lymphocyte function antigen-1; ICAM, intercellular adhesion molecule; compound 1, N-{N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl}-(D)-phenylalanine; compound 2, N-{N-[(3chlorobenzene)sulfonyl] azetidine-2-(S)-carboxyl}-(L)-4-(2 ,6 -bis-methoxyphenyl)phenylalanine; TR14035, N-(2,6-dichlorobenzoyl)-(L)-4-(2 ,6 bis-methoxyphenyl)phenylalanine. 0022-3565/02/3021-153–162$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 302, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4788/988060 JPET 302:153–162, 2002 Printed in U.S.A. 153 at A PE T Jornals on N ovem er 3, 2017 jpet.asjournals.org D ow nladed from also mediates the firm adhesion of lymphocytes via this ligand (Rott et al., 1996). Lymphocyte trafficking in the GALT not only enables normal immune responses (Butcher and Picker, 1996) but also contributes to unwanted inflammation (Podolsky and Fiocchi, 2000). Gut inflammation can induce dramatic changes in the extent and selectivity of lymphocyte recruitment to the gut wall (Briskin et al., 1997; Picarella et al., 1997). For example, the expression of MAdCAM-1 can be up-regulated by as much as 5-fold on blood vessels at sites of intestinal inflammation (Briskin et al., 1997), and proinflammatory cytokines facilitate the recruitment of lymphocytes and other leukocytes to sites of active inflammation (Podolsky and Fiocchi, 2000). The tissue-specific distribution of MAdCAM-1 and selective interaction with gut-homing memory lymphocytes expressing 4 7 suggest a contributing role of this ligandreceptor pair to inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis. Blockade of 4 7 and MAdCAM-1 with antibodies defines their role in models of inflammatory bowel disease. Monoclonal antibodies (mAbs) directed against 4 or 4 7 block lymphocyte homing to intestinal sites in naı̈ve mice (Hamann et al., 1994), and mAbs against 7 or MAdCAM-1 reduce inflammation in mouse models of colitis (Picarella et al., 1997; Kato et al., 2000). In the cotton-top tamarin, which spontaneously develops colitis, an anti4 7 mAb effectively resolved the established colitis (Hesterberg et al., 1996). Perhaps the strongest argument for the importance of 4 integrins in mediating inflammation of the gut is derived from recent clinical trials with Antegren (anti4; Elan/Biogen, Cambridge, MA). In a blinded placebo-controlled phase II trial of 248 patients with moderate-to-severe Crohn’s disease, Antegren administered at a single dose of 3 mpk i.v. resulted in a 46% remission rate after 6 weeks, versus 27% remission with placebo (Ghosh et al., 2001). We have identified a dual 4 7/ 4 1 antagonist by evaluating the ability of the compound to block the binding of human or murine 4 7-expressing cells to soluble human or murine MAdCAM-Ig. Small molecule antagonists of 4 7 that block the static adhesion of human 4 7-expressing cells to the CS-1 subdomain of human fibronectin, human VCAMIg, human MAdCAM-Ig, or murine MAdCAM-Ig have been described previously (Shroff et al., 1996, 1998; Carson et al., 1997; Harriman et al., 1999; Martin et al., 1999). The ability of 4 7 antagonists to block the binding of murine 4 7expressing cells to soluble murine MAdCAM-Ig under static conditions has also been reported (Martin et al., 1999), but the ability of compounds to block ligand binding under in vitro or in vivo shear flow conditions has not been examined. We used in vitro shear flow assays to quantitate the adhesion of both human and murine 4 7-expressing cells to human and murine MAdCAM-Ig, and we characterized the ability of compounds to inhibit adhesion to HEVs in an in vivo model. Materials and Methods Antibodies and Cell Lines The following purified monoclonal antibodies were obtained from BD PharMingen (San Diego, CA): 4B4 (mouse anti-human 1), FIB27 (rat anti-mouse 7 that cross-reacts with human 7), DATK32 (rat anti-mouse 4 7), Ha2/5 (hamster anti-rat 1 that cross-reacts with murine 1; Mendrick and Kelly, 1993), MEL-14 (rat anti-mouse L-selectin), and isotype controls (hamster IgM, rat IgG2b, and rat IgG2a). HP2/1 (mouse anti-human 4) was obtained from Beckman Coulter, Inc. (Fullerton, CA). PS/2 (rat anti-mouse 4) (Miyake et al., 1991) and a matched isotype control (rat anti-human Ras Ab) were supplied by LigoCyte (Bozeman, MT). The following cell lines were used: RPMI-8866 cells (human B cell line) obtained from J. Wilkins (University of Manitoba, Winnipeg, MB, Canada), TK-1 cells (murine T cell line) obtained from I. Weissman (Stanford University, Stanford, CA) (Holzmann and Weissman, 1989), and Jurkat (human T cell line) and RBL-2H3 cells (rat mucosal-type mast cell line) from American Type Culture Collection (Manassas, VA). Expression and Purification of Cellular Adhesion Molecule-Immunoglobulin Fusion Proteins Human MAdCAM-Ig. Domains 1 and 2 of human MAdCAM-1 (GenBank no. U43628) were amplified by PCR using human small intestinal cDNA (Invitrogen, Carlsbad, CA) as a template and the following primer sequences: 5 -PCR primer, 5 -ATTAGGAATTCGCCACCATGGATTTCGGACTGGCCCTCCTGCTGG-3 ; and 3 -PCR primer, 5 AATTGGGATCCACTTACCTGTGGAGGTCGGGCTGTGCAGGACGGGGATG-3 . PCR was performed in the presence of 10% DMSO with KlenTaq (CLONTECH, Palo Alto, CA) in a thermocycler (MJ Research, Waltham, MA) by using 40 cycles with the following parameters: 45 s at 94°C, 45 s at 60°C, and 90 s at 72°C. The resulting PCR product of 660 bp was digested with EcoRI and BamHI and ligated into a pIg (R & D Systems, Minneapolis, MN) expression vector. The pIg vector contains the genomic fragment that encodes the hinge region, CH2 and CH3 of human IgG1 (GenBank no. Z17370), and the fragment encoding human MAdCAM-1 (hMAdCAM-1) was ligated proximal to the IgG1 region. The sequence of the resulting hMAdCAM-1 fragment fused to human IgG1 was verified using Sequenase (U.S. Biochemical Corp., Cleveland, OH). The fragment encoding the entire MAdCAM-Ig fusion was subsequently excised from the pIg vector with EcoRI and NotI and ligated to pcDNA3.1/neo (Invitrogen). The resulting vector, pcDNA3.1/neo-MAdCAM-Ig, was transfected into CHOKI cells (CCL61; American Type

The α4 integrin, α4β7, 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 α4β1 and α4β7,N-(2,6-dichlorobenzoyl)-(l)-4-(2′,6′-bis-methoxyphenyl)phenylalanine (TR14035) andN-{N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl}-(d)-phenylalanine (compound 1), were tested for their ability to block the binding of α4β7-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 α4β7to an 125I-MAdCAM-Ig fusion protein with IC50values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to α4β1 or α4β7 on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human α4β7-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC50 values of 0.1 and 1 μM, respectively. Intravital microscopy was used to quantitate α4-dependent adhesion of fluorescent murine lymphocytes in Peyer9s patch HEVs. When cells were prestimulated with 2 mM Mn2+ to activate α4β7 binding to ligand, anti-α4 monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-β1 mAb did not block adhesion, demonstrating that this interaction was dependent on α4β7. TR14035 blocked adhesion to HEVs [ED50 of 0.01–0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, α4β7/α4β1antagonists blocked α4β7-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.

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Three heterocyclic series of nonpeptide angiotensin II receptor antagonists (quinazolinones, imidazo[4,5-b]pyridines, and triazolinones) have been transformed in parallel from agents highly selective for the AT1 receptor subtype into ligands with equal and high affinity for the AT1 and AT2 subtypes. These compounds are orally active with long duration of action in animal models of hypertension. They should be useful for investigating potential therapeutic advantages of dual AT1/AT2 receptor blockade.

Modification of the 6-N-alkyl-N-acyl groups of L-159,689, 6-(N-benzoyl-N-pentyl)-amino-2-propyl-3-[(2'-(tetrazol-5-yl)biphen-4-yl)methyl] led to the identification of the 6-(N-benzoyl-N-(3-pyridylmethyl)) analog(L-162,537). L-162,537 had improved aqueous solubility and oral bioavailability in the dog. The SAR of this class of AT(1) and AT(2) Ligands is discussed.

The quinazolinone sulfonylcarbamate L-163,579 (9) is a potent, balanced antagonist of the binding of angiotensin II (Ang II) to human AT(1) and AT(2) receptors. This antagonist produces a long-lasting blockade of Ang II-induced presser response in both rats and dogs after oral administration.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA potent, orally active, balanced affinity angiotensin II AT1 antagonist and AT2 binding inhibitorStephen E. de Laszlo, Carol S. Quagliato, William J. Greenlee, Arthur A. Patchett, Raymond S. L. Chang, Victor J. Lotti, Tsing Bau Chen, Stacey A. Scheck, Kristi A. Faust, and Cite this: J. Med. Chem. 1993, 36, 21, 3207–3210Publication Date (Print):October 1, 1993Publication History Published online1 May 2002Published inissue 1 October 1993https://pubs.acs.org/doi/10.1021/jm00073a024https://doi.org/10.1021/jm00073a024research-articleACS PublicationsRequest reuse permissionsArticle Views315Altmetric-Citations105LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The synthesis of lactone 1 from the phenylalanine derived precursor 2 via Wadsworth-Emmons reaction followed by reduction and isomerization is described.