ABSTRACT
VLA-4 (alpha(4)beta(1), very late activating antigen-4), a key cell surface integrin plays an important role in inflammation by promoting leukocyte attachment and extravasation from the vasculature into the peripheral tissues. As such, VLA-4 antagonists may be useful in the treatment, prevention, and suppression of diseases where cell adhesion and migration are important such as asthma, rheumatoid arthritis, and multiple sclerosis. Herein, we report on the discovery, synthesis, and biological evaluation of amidines as small molecule antagonists of VLA-4.
Subject(s)
Amides/chemistry , Amidines/chemistry , Integrin alpha4beta1/antagonists & inhibitors , Area Under CurveABSTRACT
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.
Subject(s)
Cations, Divalent/metabolism , Dipeptides/pharmacology , Integrin alpha4beta1/antagonists & inhibitors , Integrins/antagonists & inhibitors , Phenylalanine/pharmacology , Phenylurea Compounds/pharmacology , Binding Sites , Cell Line , Dipeptides/chemistry , Humans , Integrin alpha4beta1/metabolism , Integrins/metabolism , Jurkat Cells , K562 Cells , Kinetics , Ligands , Phenylalanine/analogs & derivatives , Phenylalanine/chemistry , Phenylurea Compounds/chemistry , Protein Binding , Radioligand Assay , Sulfur Radioisotopes , Tumor Cells, Cultured , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
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.
Subject(s)
Integrin alpha4beta1/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Amides/chemistry , Amides/metabolism , Amides/pharmacology , Cell Adhesion Molecules , Humans , Immunoglobulins , Inhibitory Concentration 50 , Integrin alpha4beta1/metabolism , Jurkat Cells , Mucoproteins/antagonists & inhibitors , Phenylalanine/pharmacokinetics , Radioligand Assay , Receptors, Lymphocyte Homing/antagonists & inhibitors , Structure-Activity Relationship , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
A series of substituted N-(3,5-dichlorobenzenesulfonyl)-(L)-prolyl- and (L)-azetidyl-beta-biaryl beta-alanine derivatives was prepared as selective and potent VLA-4 antagonists. The 2,6-dioxygenated biaryl substitution pattern is important for optimizing potency. Oral bioavailability was variable and may be a result of binding to circulating plasma proteins.
Subject(s)
Integrin alpha4beta1/antagonists & inhibitors , Propionates/chemical synthesis , Administration, Oral , Alanine/chemical synthesis , Alanine/pharmacokinetics , Alanine/pharmacology , Biological Availability , Humans , Inhibitory Concentration 50 , Jurkat Cells , Metabolic Clearance Rate , Propionates/pharmacokinetics , Propionates/pharmacology , Protein Binding , Structure-Activity Relationship , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
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.
Subject(s)
Dipeptides/chemical synthesis , Dipeptides/pharmacology , Integrin alpha4beta1/antagonists & inhibitors , Animals , Dipeptides/blood , Dipeptides/pharmacokinetics , Half-Life , Metabolic Clearance Rate , Molecular Structure , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
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.
Subject(s)
Integrins/antagonists & inhibitors , Phenylalanine/pharmacology , Receptors, Lymphocyte Homing/antagonists & inhibitors , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antineoplastic Combined Chemotherapy Protocols/immunology , Cell Adhesion/drug effects , Cell Adhesion Molecules , Cyclophosphamide/immunology , Doxorubicin/immunology , Endothelium, Vascular/cytology , Endothelium, Vascular/drug effects , Etoposide/immunology , Female , Flow Cytometry , Humans , Immunoglobulin G/isolation & purification , Immunoglobulin G/pharmacology , Immunoglobulins , Integrin alpha4beta1 , Ligands , Lymphocytes/drug effects , Methotrexate/immunology , Mice , Mice, Inbred BALB C , Mucoproteins/antagonists & inhibitors , Peyer's Patches/cytology , Peyer's Patches/drug effects , Phenylalanine/analogs & derivatives , Recombinant Fusion Proteins/pharmacology , RheologyABSTRACT
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.
Subject(s)
Integrin alpha4beta1/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Administration, Oral , Animals , Binding Sites , Biological Availability , Dose-Response Relationship, Drug , Furans/chemical synthesis , Furans/chemistry , Furans/pharmacokinetics , Furans/pharmacology , Half-Life , Inhibitory Concentration 50 , Macaca mulatta , Phenylalanine/chemical synthesis , Phenylalanine/pharmacokinetics , Phenylalanine/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , Sulfonamides/pharmacology , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
A series of substituted tetrahydrofuroyl-1-phenylalanine derivatives was prepared and evaluated as VLA-4 antagonists. Substitution of the alpha carbon of the tetrahydrofuran with aryl groups increased the specificity for VLA-4 versus alpha(4)beta(7) while amide substitution increased the potency of the series without increasing the specificity. Substitution of the beta carbon of the tetrahydrofuran with keto or amino groups slightly improved the specificity for VLA-4 versus alpha(4)beta(7) but with a significant loss in binding affinity for VLA-4.
Subject(s)
Integrin alpha4beta1/antagonists & inhibitors , Phenylalanine/analogs & derivatives , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Administration, Oral , Animals , Furans/chemical synthesis , Furans/chemistry , Furans/pharmacokinetics , Furans/pharmacology , Humans , Inhibitory Concentration 50 , Ligands , Molecular Weight , Phenylalanine/chemical synthesis , Phenylalanine/pharmacokinetics , Phenylalanine/pharmacology , Rats , Stereoisomerism , Structure-Activity Relationship , Substrate Specificity , Sulfonamides/blood , Sulfonamides/chemistry , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
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.
Subject(s)
Amino Acids/chemical synthesis , Inflammation Mediators/chemical synthesis , Integrin alpha4beta1/antagonists & inhibitors , Acylation , Administration, Oral , Amino Acids/metabolism , Amino Acids/pharmacokinetics , Animals , Biological Availability , Combinatorial Chemistry Techniques , Drug Evaluation, Preclinical , Inflammation Mediators/metabolism , Inflammation Mediators/pharmacokinetics , Metabolic Clearance Rate , Protein Binding , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity RelationshipABSTRACT
A series of substituted N-(3,5-dichlorobenzenesulfonyl)-L-prolyl- and alpha-methyl-L-prolyl-phenylalanine derivatives was prepared as VLA-4/VCAM antagonists. The compounds showed excellent potency with a wide variety of neutral, polar, electron withdrawing or donating groups on the phenylalanine ring (IC50 approximately 1 nM). Heteroaryl ring substitution for phenylalanine was also well tolerated. Pharmacokinetic studies in rat were performed on a representative set of compounds in both series.
Subject(s)
Dipeptides/pharmacokinetics , Integrin alpha4beta1/antagonists & inhibitors , Animals , Biological Availability , Dipeptides/chemical synthesis , Dipeptides/chemistry , Dogs , Haplorhini , Inhibitory Concentration 50 , Metabolic Clearance Rate , Phenylalanine , Rats , Rats, Sprague-Dawley , Sheep , Structure-Activity Relationship , Sulfones , Vascular Cell Adhesion Molecule-1/drug effectsABSTRACT
N-(3,5-Dichlorophenylsulfonyl)-(R)-thioprolyl biarylalanine 10a has been identified as a potent and specific antagonist of the alpha(4)beta(1) integrin. Altering the configuration of thioproline from R to S led to a series of dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), and the N-acetyl analogue 8b was found to be the most potent dual antagonist. A binding site model for alpha(4)beta(1) and alpha(4)beta(7) is proposed to explain the structure-activity relationship.
Subject(s)
Alanine/pharmacology , Integrins/antagonists & inhibitors , Receptors, Lymphocyte Homing/antagonists & inhibitors , Sulfhydryl Compounds/pharmacology , Alanine/analogs & derivatives , Alanine/chemistry , Integrin alpha4beta1 , Structure-Activity Relationship , Sulfhydryl Compounds/chemistryABSTRACT
The alpha(4)beta(1) and alpha(4)beta(7) integrins are implicated in several inflammatory disease states. Systematic SAR studies of an alpha(4)beta(1)-specific arylsulfonyl-Pro-Tyr lead led to the identification of a new alpha(4)beta(7) binding site, best captured by O-carbamates of Tyr for this structural class. Several compounds showed a 200- to 400-fold improvement in alpha(4)beta(7) binding affinity while maintaining subnanomolar alpha(4)beta(1) activity, for example 2l, VCAM-Ig alpha(4)beta(1) IC(50)=0.13 nM, VCAM-Ig alpha(4)beta(7) IC(50)=1.92 nM.
