Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part III: synthesis of potent antagonists with alpha(v)beta3/alpha(IIb)beta3 dual activity and improved water solubility

In order to optimize our novel integrin alpha(v)beta3/alpha(IIb)beta3 dual antagonists, spatial screening at the N-terminus was performed. The alpha(v)beta3 antagonistic activity varied depending on the space that was occupied by the N-terminus, but high potency against alpha(IIb)beta3 was well maintained. The (3S)-aminopiperidine analogue had the strongest activity against alpha(v)beta3, and the S isomer at piperidine was more potent than the R isomer. Compounds selected on the basis of SAR analysis of a novel lead compound showed acceptable early absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles and sufficient water solubility for use as infusion drugs. Docking studies with the alpha(v)beta3 receptor were performed to confirm the SAR findings.     

Tricyclic pharmacophore-based molecules as novel integrin alpha(v)beta3 antagonists. Part 1: design and synthesis of a lead compound exhibiting alpha(v)beta3/alpha(IIb)beta3 dual antagonistic activity

In order to generate novel compounds with integrin alpha(v)beta3-antagonistic activity together with antiplatelet activity, tricyclic pharmacophore-based molecules were designed and synthesized. Although piperazine-containing compounds initially prepared were selective alpha(IIb)beta3 antagonists, replacement of piperazine with piperidine furnished a potent alpha(v)beta3/alpha(IIb)beta3 dual antagonist. Structure-activity relationship (SAR) studies provided clues for further development of tricyclic pharmacophore-based integrin antagonists.     

Tricyclic pharmacophore-based molecules as novel integrin alphavbeta3 antagonists. Part IV: preliminary control of alphavbeta3 selectivity by meta-oriented substitution

To establish the in vivo efficacy of alphavbeta3/alphaIIbbeta3 dual antagonists possessing a tricyclic pharmacophore, a corresponding alphavbeta3-selective antagonist was required as a control. We initially took two synthetic approaches to obtain alphavbeta3-selective antagonists based on the RGD recognition pattern or on modification of the dihedral angle between the central benzene ring and the adjacent heterocycle, but both proved unsuccessful. However, synthesis of novel antagonists with meta-substitution of the central benzene ring generated weak selectivity for alphavbeta3 over alphaIIbbeta3 for the first time in the family of compounds with the tricyclic pharmacophore. Optimization of meta-oriented antagonists furnished an alphavbeta3-selective antagonist exhibiting inhibitory activity not only in a receptor-binding assay, but also in a cell adhesion assay.     

Substituted benzocyloheptenes as potent and selective alpha(v) integrin antagonists

A novel series of potent and specific alpha(v) integrin antagonists has been obtained by aminoalkyl substitutions on benzocyloheptene acetic acids as a rigid GD bioisostere. The preferred compounds 1-2, 1-3 and 1-8, showed nano- to subnanomolar IC(50) values on alpha(v)beta(3) and alpha(v)beta(5) integrins, with favorable pharmacokinetics.     

Non-peptide alpha(v)beta(3) antagonists. Part 3: identification of potent RGD mimetics incorporating novel beta-amino acids as aspartic acid replacements.

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared incorporating various beta-amino acids as aspartic acid mimetics. Modification of the beta-alanine 3-substituents alters the potency and physicochemical properties of these receptor antagonists and in some cases provides orally bioavailable alpha(v)beta(3) inhibitors.     

Non-peptide alpha(v)beta(3) antagonists. Part 4: potent and orally bioavailable chain-shortened RGD mimetics

Potent non-peptidic alpha(v)beta(3) antagonists have been prepared where deletion of an amide bond from an earlier series of linear RGD-mimetics provides a novel series of chain-shortened alpha(v)beta(3) antagonists with significantly improved oral pharmacokinetics. These chain-shortened alpha(v)beta(3) antagonists represent structurally novel integrin inhibitors.     

The discovery of small molecule carbamates as potent dual alpha(4)beta(1)/alpha(4)beta(7) integrin antagonists.

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.     

Synthesis of pyrazoles and isoxazoles as potent alpha(v)beta3 receptor antagonists

We describe a series of pyrazole and isoxazole analogs as antagonists of the alpha(v)beta3 receptor. Compounds showed low to sub-nanomolar potency against alpha(v)beta3, as well as good selectivity against alpha(IIb)beta3. In HT29 cells, most analogs also demonstrated significant selectivity against alpha(v)beta6. Several compounds showed good pharmacokinetic properties in rats, in addition to anti-angiogenic activity in a mouse corneal micropocket model. Compounds were synthesized in a straightforward manner from readily available glutarate precursors.     

Non-peptide alpha(v)beta(3) antagonists. Part 5: identification of potent RGD mimetics incorporating 2-aryl beta-amino acids as aspartic acid replacements

A series of novel, highly potent alpha(v)beta(3) receptor antagonists with favorable pharmacokinetic profiles has been identified. In this series of antagonists, 2-aryl beta-amino acids function as potent aspartic acid replacements.     

Synthesis and biological evaluation of non-peptide alpha(v)beta(3)/alpha(5)beta(1) integrin dual antagonists containing 5,6-dihydropyridin-2-one scaffolds

Small constrained non-peptidic molecules consisting of a polyfunctionalized rigid core, carrying appendages corresponding to arginine and aspartic acid side chains, have been recently reported to be promising for drug development. In this work, the 5,6-dihydropyridin-2-one was envisaged as a scaffold to turn into potential integrin ligands, introducing a carboxylic acid and a basic appendage. The synthesis and the antiadhesion activity of a small library of peptidomimetics capable to recognize alpha(v)beta(3) and alpha(5)beta(1) integrins has been herein reported.     

Synthesis of highly potent and selective hetaryl ureas as integrin alpha(V)beta3-receptor antagonists

Solid-phase synthesis and SAR of integrin alpha(V)beta3-receptor antagonists containing a urea moiety as non-basic guanidine mimetic are described. The most potent compounds exhibited IC(50) values towards alpha(V)beta3 in the nanomolar range and high selectivity versus related integrins like alpha(IIb)beta3. For selected examples efficacy in functional cellular assays is demonstrated.     

Synthesis of 2,5-thiazole butanoic acids as potent and selective alpha(v)beta3 integrin receptor antagonists with improved oral pharmacokinetic properties

We describe a series of 2,5 thiazole containing compounds, which are potent antagonists of the integrin alpha(v)beta3 and show selectivity relative to the other integrins, such as alpha(IIb)beta3 and alpha(v)beta6. These analogs were demonstrated to have high bioavailability relative to other relative heterocyclic analogs.     

Non-peptide alpha v beta 3 antagonists. Part 7: 3-Substituted tetrahydro-naphthyridine derivatives

A series of 3-substituted tetrahydro-[1,8]naphthyridine containing alpha(v)beta(3) antagonists was prepared. A comparison of their in vitro IC(50) values to the electron properties of the 3-substituents revealed a good linear Hammett correlation (rho=-1.96, R(2)=0.959). Electron-withdrawing groups at the 3-position of the tetrahydro-[1,8]naphthyridine decreased potency while electron-donating groups enhanced potency.     

Solid-phase synthesis of dual alpha4beta1/alpha4beta7 integrin antagonists: two scaffolds with overlapping pharmacophores

Two structural classes of dual alpha4beta1/alpha4beta7 integrin antagonists were investigated via solid-phase parallel synthesis. Using an acylated amino acid backbone, lead compounds containing biphenylalanine or tyrosine carbamate scaffolds were optimized for inhibition of alpha4beta1/VCAM and alpha4beta7/MAdCAM. A comparison of the structure-activity relationships in the inhibition of the alpha4beta7/MAdCAM interaction for substituted amines employed in both scaffolds suggests a similar binding mode for the compounds.     

Protein phosphatase 2A negatively regulates integrin alpha(IIb)beta(3) signaling

Integrin alpha(IIb)beta(3) activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin alpha(IIb)beta(3) signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin alpha(IIb)beta(3) in resting platelets and in human embryonal kidney 293 cells expressing alpha(IIb)beta(3). The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin alpha(IIb) is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to alpha(IIb)beta(3) during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac(alpha) in 293 cells decreased alpha(IIb)beta(3)-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac(alpha) expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated alpha(IIb)beta(3) adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac (alpha)-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A(calpha) expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac (alpha) can negatively regulate integrin alpha(IIb)beta(3) signaling by suppressing the ERK1/2 signaling pathway.     

Nonpeptide alpha(v)beta(3) antagonists. Part 2: constrained glycyl amides derived from the RGD tripeptide.

Mimetics of the RGD tripeptide are described that are potent, selective antagonists of the integrin receptor, alpha(v)beta(3). The use of the 5,6,7,8-tetrahydro[1,8]naphthyridine group as a potency-enhancing N-terminus is demonstrated. Two 3-substituted-3-amino-propionic acids previously contained in alpha(IIb)beta(3) antagonists were utilized to enhance binding affinity and functional activity for the targeted receptor. Further affinity increases were then achieved through the use of cyclic glycyl amide bond constraints.     

Transmembrane signal transduction of the alpha(IIb)beta(3) integrin

Integrins are composed of noncovalently bound dimers of an alpha- and a beta-subunit. They play an important role in cell-matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low-affinity and a high-affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix-helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the alpha(IIb)beta(3) integrin, this structural motif represents the high-affinity state. A second conformation of the TM domain of alpha(IIb)beta(3) is identified as the low-affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three-state mechanism.     

Discovery and evaluation of piperidinyl carboxylic acid derivatives as potent alpha(4)beta(1) integrin antagonists.

Piperidinyl carboxylic acid-based derivatives were prepared as antagonists of the leukocyte cell adhesion process that is mediated through the interaction of the alpha(4)beta(1) integrin (VLA-4, very late antigen 4) and the vascular cell adhesion molecule 1 (VCAM-1). Compounds 2a-h inhibited the adhesion in a cell-based assay in the low and sub micromolar range, a pharmacokinetic study of 2d is reported.     

Nonpeptide alpha(v)beta3 antagonists: identification of potent, chain-shortened 7-oxo RGD mimetics

Potent, novel 7-oxo alpha(v)beta3 antagonists have been prepared. These antagonists offer decreased plasma protein binding and excellent pharmacokinetic profiles.     

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa)

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.